BLAST XII MEETING
HILTON TUCSON EAST
TUCSON, ARIZONA
JANUARY 20-25, 2013
Meeting Chairperson:
Dr. Urs Jenal – University of Basel, Basel, Switzerland
Meeting Vice-Chairperson:
Dr. Karen Ottemann – University of California, Santa Cruz, CA
Program Committee:
Dr. John S. Parkinson (Chairperson) – University of Utah, Salt Lake City, UT
Dr. Rasika Harshey – The University of Texas, Austin, TX
Meeting Review Committee:
Dr. Alan Wolfe (Chairperson) – Loyola University, Maywood, IL
Dr. Christine Josenhans – Medical University Hannover, Hannover, Germany
Dr. Kirsten Jung – Ludwig-Maximilians-Universitaet Muenchen, Munich, Germany
Dr. Christopher Rao – University of Illinois at Urbana-Champaign, Urbana, IL
Poster Awards Committee:
Dr. John Kirby (Chairperson) – University of Iowa, Iowa City, IA
Dr. Brian Crane – Cornell University, Ithaca, NY
Dr. Sean Crosson – University of Chicago, Chicago, IL
Dr. Kylie Watts – Loma Linda University, Loma Linda, CA
Speaker Award Committee:
Dr. Urs Jenal – University of Basel, Basel, Switzerland
Dr. Karen Ottemann – University of California, Santa Cruz, CA
Board of Directors – BLAST, Inc.:
Dr. Robert Bourret (Chairperson) – University of North Carolina, Chapel Hill, NC
Dr. Joe Falke – University of Colorado, Boulder, CO
Dr. Michael Manson – Texas A&M University, College Station, TX
Dr. John S. Parkinson – University of Utah, Salt Lake City, UT
Dr. Phillip Matsumura (Chair Emeritus) – Molecular Biology Consortium, Chicago, IL
Conference Coordinators:
Ms. Tarra Bollinger – Molecular Biology Consortium, Chicago, IL
Ms. Peggy O’Neill – Molecular Biology Consortium, Chicago, IL
ii
TABLE OF CONTENTS
COMMITTEE INFORMATION ........................................................................................ii
TABLE OF CONTENTS ................................................................................................. iii
AWARDS INFORMATION.............................................................................................. iv
MEETING SCHEDULE ...................................................................................................v
SPEAKER PROGRAM ................................................................................................... vi
POSTER LIST ................................................................................................................ix
SPEAKER ABSTRACTS .................................................................................................1
POSTER ABSTRACTS ................................................................................................. 51
PARTICIPANT LIST .................................................................................................... 119
INDEX ......................................................................................................................... 135
iii
AWARDS INFORMATION
Robert M. Macnab Award for an Outstanding Poster Presentation by a Postdoctoral Scientist
This award was established at BLAST VIII (2005) and is named in memory of the late
Robert M. Macnab, Ph.D., who was an integral member of the Bacterial Locomotion
and Signal Transduction Community. Dr. Macnab spent his 30 year career studying
the assembly, structure and function of the bacterial flagellum. Bob actively
participated in the BLAST meetings and served on the Program and Review
Committees for BLAST IV. At the time of his death in 2003, Bob was a professor in
the Department of Molecular Biophysics and Biochemistry at Yale University.
Robert M. Macnab Memorial Travel Awards
We are pleased to announce the establishment of the first travel awards for the BLAST meeting,
remembering our colleague Dr. Robert M. Macnab on the 10th anniversary of his death. The intent of
the awards is to help young scientists from outside the country that hosts BLAST to attend the
meeting. The recipients chosen by the Board of Directors for BLAST XII are graduate students
Ana Martinez del Campo from George Dreyfus' lab at the Universidad Nacional Autónoma de México
and Ambroise Lambert from Mathieu Picardeau's lab at the Institut Pasteur - Paris.
(The Macnab poster and travel awards are sponsored by generous donations from
Mrs. May K. Macnab)
Robert J. Kadner Award for an Outstanding Poster Presentation by a Graduate Student
This award was established at BLAST IX (2007) and is named in memory of the late
Robert J. Kadner, Ph.D., who was an integral member of the Bacterial Locomotion and
Signal Transduction Community. Dr. Kadner spent his career studying microbial
physiology of E. coli transport systems. Bob actively participated in the BLAST
meetings and served as Chair of the Review Committee for BLAST V, Vice-Chair of
BLAST VII and Meeting Chair of BLAST VIII. At the time of his death in 2005, Bob was
the Norman J. Knorr Professor of Basic Sciences in the Department of Microbiology at
the University of Virginia, School of Medicine. This award is sponsored by BLAST.
Nucleic Acids Research Award for an Outstanding Poster Presentation by a Young
Investigator
This award was established at BLAST XI (2011) and is presented to a
young investigator whose research is in on regulation of transcription.
The award is sponsored by Nucleic Acids Research (NAR), an Oxford
University Press Journal, which publishes the results of leading edge
research into physical, chemical, biochemical and biological aspects of nucleic acids and proteins
involved in nucleic acid metabolism and/or interactions (http://nar.oxfordjournals.org).
BLAST Board of Directors' Award for an Outstanding Talk
This award, the first for speakers, was established at BLAST XI (2011) by Phil
Matsumura, Founding Chair of the BLAST Board of Directors. In contrast to the
poster awards, which are open only to graduate students and/or postdoctoral
scientists, all speakers are eligible for the BLAST Board of Directors Award.
(This award is sponsored by a generous donation from Dr. Philip Matsumura)
iii
BLAST XII MEETING SCHEDULE
TIME
EVENT
LOCATION
Sunday, January 20, 2013
4:00 pm
4:00 pm – 7:00 pm
7:00 pm – 8:30 pm
9:00 pm – 11:00 pm
Poster room available for poster setup
Meeting Registration
Dinner
Welcome Reception
Gala Rooms A & B
Outer Vistas
Private Dining Room
Private Dining Room
Monday, January 21, 2013
7:30 am
8:45 am
9:00 am
10:15 am
12:00 pm
2:00 pm
6:00 pm
7:30 pm
8:30 pm
– 8:30 am
– 9:00 am
– 12:00 pm
– 10:30 am
– 1:30 pm
– 4:00 pm
– 7:30 pm
– 10:00 pm
– 8:45 pm
Breakfast
Welcome & Announcements
Meeting Session – “Flagella 1”
Coffee Break
Lunch
Poster Session – even numbered posters
Dinner
Meeting Session – “Two-Component Signaling”
Coffee Break
Private Dining Room
Salon B & C
Salon B & C
Salon A
Private Dining Room
Gala Rooms A & B
Private Dining Room
Salon B & C
Salon A
Tuesday, January 22, 2013
7:30 am
8:45 am
10:15 am
12:00 pm
2:00 pm
6:00 pm
7:30 pm
8:30 pm
– 8:30 am
– 12:00 pm
– 10:30 am
– 1:30 pm
– 4:00 pm
– 7:30 pm
– 10:00 pm
– 8:45 pm
Breakfast
Meeting Session –“Chemoreceptor Arrays & Signaling”
Coffee Break
Lunch
Poster Session – odd numbered posters
Dinner
Meeting Session – “Swarming & Gliding”
Coffee Break
Private Dining Room
Salon B & C
Salon A
Private Dining Room
Gala Rooms A & B
Private Dining Room
Salon B & C
Salon A
Wednesday, January 23, 2013
7:15 am
8:30 am
9:55 am
12:00 pm
12:15 pm
– 8:15 am
– 11:30 am
– 10:10 am
–
1:30 pm
Breakfast
Meeting Session – “Receptors”
Coffee Break
Tours Depart
Lunch
Private Dining Room
Salon B & C
Salon A
Atrium Lobby
Private Dining Room
Thursday, January 24, 2013
7:30 am
8:45 am
10:15 am
12:00 pm
4:00 pm
5:30 pm
7:00 pm
7:30 pm
8:30 pm
9:40 pm
10:00 pm
–
–
–
–
–
–
–
–
–
–
–
8:30 am
12:00 pm
10:30 am
1:30 pm
5:00 pm
7:00 pm
7:30 pm
9:40 pm
8:45 pm
10:00 pm
12:00 am
Breakfast
Meeting Session – “Flagella 2”
Coffee Break
Lunch
Town Hall Meeting for students & postdocs
Dinner
Business Meeting for all attendees
Meeting Session – “Regulation”
Coffee Break
Awards Presentation
Reception
Private Dining Room
Salon B & C
Salon A
Private Dining Room
Salon B & C
Private Dining Room
Salon B & C
Salon B & C
Salon A
Salon B & C
Private Dining Room
Friday, January 25, 2013
7:00 am –
8:30 am
Breakfast
Private Dining Room
v
BLAST XII PROGRAM
January 21, 2013
Flagella 1
Chair – Howard Berg
Monday Morning (8:45 am – 12:00 pm)
PRESENTER
Morgan Beeby
Lewis Evans
Milana Fraiberg
Hajime Fukuoka
BREAK
Lele, Pushkar
ABSTRACT
PAGE NO.
TITLE
Structural dissection of bacterial flagellar motors in live bacteria by
electron cryo-tomography
How does the flagellum grow at a constant rate?
CheY acetylation in bacterial chemotaxis: How does it generate flagellar
clockwise rotation?
Direct imaging of CheY-binding to a functioning bacterial flagellar motor
Mechanism for adaptive remodeling of the bacterial flagellar switch
Flagella stator homologues power bacterial gliding motility by moving in
Beiyan Nan
helical trajectories
Jonathan Partridge The FliL protein increases flagellar motor output in Salmonella
Xiaowei Zhao
3-D visualization of bacterial flagellar assembly in Borrelia burgdorferi
January 21, 2013
Lindsie Goss
Daniela Keilberg
BREAK
Shonna McBride
Tarek Msadek
Hendrik Szurmant
4
5
6
7
8
9
Two-Component Signaling
Chair – Steven Porter
Monday Evening (7:30 pm – 10:00 pm)
Rachel CreagerAllen
2
3
Autophosphorylation of the response regulator PhoB receiver domain is
cooperative and saturable
Ser/Thr phosphorylation regulates two-component systems involved in
cell wall metabolism
LinkedIn – finding connections: A response regulator links the Frz system
to the MglA/MglB GTPase/GAP module to regulate polarity of motility
systems in M. xanthus
Resistance of Clostridium difficile to bacterial antimicrobial peptides
through a disjoined two-component system that responds to multiple
substrates
The WalKR system controls major staphylococcal virulence genes and is
involved in triggering the host inflammatory response
What sequence diversity teaches us about bacterial signaling
vi
10
11
12
13
14
15
January 22, 2013
Chemoreceptor Arrays and Signaling
Chair – Yuhai Tu
Tuesday Morning (8:45 am – 12:00 pm)
PRESENTER
Ariane Briegel
Christopher Jones
Milena Lazova
John S. Parkinson
BREAK
Kene Piasta
Lynmarie
Thompson
Ady Vaknin
Kylie Watts
ABSTRACT
PAGE NO.
TITLE
Structural changes of bacterial chemoreceptor arrays underlie different
activation states
Formation and segregation of the cytoplasmic chemosensory cluster in
Rhodobacter sphaeroides
Network-level properties of Salmonella typhimurium chemotaxis
How HAMP domains and methylation bundles control stimulus response
and sensory adaptation in chemoreceptor signaling
Testing and refining the receptor-CheA regulatory domain interface in
functional, membrane-bound arrays using disulfide mapping and TAMIDS
Differences between signaling states of chemotaxis receptors revealed by
hydrogen exchange mass spectrometry
Prolonged stimuli alter the bacterial chemosensory clusters
The sensing and signaling roles of multiple PAS-HEME domains in Aer-2
type chemoreceptors
January 22, 2013
16
17
18
19
20
21
22
23
Swarming and Gliding
Chair – Larry Shimkets
Tuesday Evening (7:30 pm – 10:00 pm)
Functions of Proteus mirabilis FliL in swarming and responding to surface
viscosity
The AAA+ protease LonA regulates SwrA levels and swarming motility in
Sampriti Mukherjee Bacillus subtilis
Daisuke Nakane
Helical flow of surface protein required for Flavobacterium gliding motility
BREAK
Bacterial transitions: Optimization of biological and physical factors by
Joshua Shrout
Pseudomonas aeruginosa during swarming
Structure of proteins involved in Mycoplasm mobile gliding revealed by
Yuhei Tahara
electron microscopy and high speed atomic force microscopy (AFM)
Bacteriophytochromes 1 and LOV-HK work in a signaling network to
Liang Wu
regulate swarming motility of Pseudomonas syringae strain B728a
Yi-Ying Lee
vii
24
25
26
27
28
29
January 23, 2013
Wednesday Morning (8:30 am – 11:30 am)
PRESENTER
Tino Krell
Caitlin Brennan
Matthew Sears
Andreas Möglich
BREAK
Michael Morse
Christopher Rao
Ben Webb
Laurence Wilson
Receptors
Chair – Birgit Prüβ
ABSTRACT
PAGE NO.
TITLE
Multiple agonistic and antagonistic signals control the action of the
complex sensor kinase TodS
Determining chemoreceptor-ligand interactions in the light-organ
symbiont Vibrio fischeri to understand niche specificity
Chemotaxis to norepinephrine (Ne) requires the Tsr chemoreceptor and
conversion enzymes induced by QseC
Structure and function of a blue-light-regulated sensor histidine kinase
Effects of molecular adsorption on the trajectories and accumulation of
motile bacteria at the air/water interface
Towards an integrated model of the chemotaxis pathway in Bacillus
subtilis
Methyl accepting chemotaxis protein U of Sinorhizobium meliloti binds the
seed exudate component, proline
Time-dependent chemotactic response in a large population
January 24, 2013
Detecting a conformational change in the periplasmic region of the
sodium-driven stator protein PomB by the disulfide crosslink
Coordinated switching of bacterial flagellar motors: Evidence for direct
Bo Hu
motor-motor coupling?
Helicobacter pylori flagellar motor: Structural insight into the mechanism
Anna Roujeinikova of stator assembly and activation
Shahid Khan
Correlated mutation analysis of the flagellar motor protein FliG
BREAK
Ria Sircar
Structure and activity of Thermotoga maritima FliY
Borrelia burgdorferi flagella export apparatus and virulence: insight into
Tao Lin
type III secretion system
Masayoshi
Dynamic conformational changes of flagellar filament observed by highNishiyama
pressure microscopy
Novel Leptospira protein is essential factor in determining the flagellar
Elsio Wunder
sheath, translational motility, and virulence phenotype
Shiwei Zhu
January 24, 2013
Birgit Prüβ
Ching Wooen Sze
32
33
34
35
36
37
38
39
40
41
42
43
44
45
Regulation
Chair – Tarek Msadek
Thursday Evening (7:30 pm – 9:40 pm)
Chakib Mouslim
BREAK
31
Flagella 2
Chair – Kelly Hughes
Thursday Morning (8:45 am – 12:00 pm)
Susanne Gebhard
Jürgen Michael
Lassak
30
Interactions between a transporter and a sensor kinase mediate signal
transduction in antimicrobial peptide detoxification modules
A novel translational regulation mechanism mediated by elongation factor
EF-P allows tight adjustment of protein copy numbers
Transcriptional activation and repression of six flhDC promoters: An
intricate promoter configuration for flhDC in Salmonella typhimurium
Escherichia coli biofilms may favor a mixture of motile and non-motile
bacteria
Rrp1 regulates chitobiose utilization of the Lyme disease spirochete
Borrelia burgdorferi
viii
46
47
48
49
50
POSTERS - BLAST XII
Poster # Lab
Presenter
Title
Structure and function of chemoreceptors and the role
of multiple CheW homologues in Rhodobacter
sphaeroides
Structure-function studies of the response regulator
CheY6 from Rhodobacter sphaeroides
Deciphering the role of the periplasmic domain of PilJ:
Mechanisms of signal transduction
Photosensory protein BphP1 initiates an unknown
signal transduction pathway through phosphotransfer
to Psyr_2449 in Pseudomonas syringae B728A
A novel inducer of Roseobacter motility is also a
disruptor of algal symbiosis
Does the C-ring rotate?
1
Judith
Armitage
James Allen
2
Judith
Armitage
Sonia
Bardy
Gwyn
Beattie
Matthew Smith
Robert
Belas
Howard
Berg
Robert
Bourret
Robert Belas
8
Robert
Bourret
Ruth Silversmith
9
Robert
Bourret
Sean
Crosson
Stephani Page
Frederick
Dahlquist
Georges
Dreyfus
Georges
Dreyfus
Robert Levenson
Marc
Erhardt
Joseph
Falke
Toshio
Fukuda
Rasika
Harshey
Marc Erhardt
Penelope
Higgs
Michio
Homma
Michio
Homma
Penelope Higgs
Michio
Homma
Seiji Kojima
3
4
5
6
7
10
11
12
13
14
15
16
17
18
19
20
21
Sonia Bardy
Regina McGrane
Basarab Hosu
Bob Bourret
Sean Crosson
Ana Martinez-del Campo
Manuel Osorio-Valeriano
Joseph Falke
Hirotaka Tajima
Hyo Kyung Kim
Mizuki Gohara
Norihiro Takekawa
A non-conserved active site residue influences
response regulator reaction kinetics, phosphodonor
specificity, and partner kinase interactions
Nonconserved active site residues modulate CheY
autophosphorylation kinetics and phosphodonor
preference
Experimental analysis of receiver domain
autodephosphorylation kinetics guided by
The Brucella abortus general stress response system
regulates chronic mammalian infection,
and exhibits non-canonical post-translational regulation
Studies of flagellar motor protein FliG and its
interactions with FliF and FliM
Chemotaxis signaling pathway of the flagellar system 2
from Rhodobacter sphaeroides
The C terminus of the flagellar muramidase SltF
modulates the interaction with FlgJ in Rhodobacter
spaheroides
Profound effect of hook length on motility and virulence
in Salmonella
Structural and functional disulfide mapping of the CheACheW interface in the bacterial chemosensory array
Analysis of the repellent-sensing mechanisms of
Escherichia coli
Identification four new GGDEF/EAL domain proteins
that regulate motility in Salmonella , one of which
localizes to the mid-cell and inhibits cell division
Signalling systems controlling developmental cell fate
segregation in Myxococcus xanthus
Attempt to investigate the interaction between the rotor
and the stator by using solution NMR
Importance of charged residues of PomA and FliG for
rotor-stator interaction in the sodium-driven flagellar
motor of Vibrio alginolyticus
Regulation of the flagellar number in Vibrio
alginolyticus : Role of ATP binding motif of FlhG and
the novel protein SflA
Page #
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
POSTERS - BLAST XII
Poster # Lab
Presenter
Title
Kelly
Hughes
Kelly
Hughes
Masahiro
Ito
Kelly Hughes
Effect of codon context on translation speed in vivo
73
Kelly Hughes
Salmonella fliC mRNA translation
74
Masahiro Ito
75
Urs
Jenal
Barry
Taylor
Christine
Josenhans
Isabelle Hug
Kirsten
Jung
Ikuro
Kawagishi
Daniel
Kearns
Keng Hwee
Chiam
John
Kirby
Stefan Behr
Koji
Nakayama
Chunhao
Li
Keiko Sato
Effect of a single motP mutation for motility at neutral
pH of the Na+-driven flagellar motor of alkaliphilic
Bacillus pseudofirmus OF4
Towards a mechanism for surface mechano-sensation
in Caulobacter crescentus
Surface accessibility study identifies domain
interactions in the aerotaxis receptor Aer
Role of energy sensor TlpD of Helicobacter pylor i in
gerbil colonization and whole genome analyses after
adaptation in the gerbil
Interconnectivity between two LytTR-like twocomponent systems in Escherichia coli
Localization control of chemotaxis-related signaling
components of V. cholerae
The role of FlgM in regulating flagellar assembly in
Bacillus subtilis
Role of the Pseudomonas aeruginosa flagellar motor
in swimming motility and chemotaxis
Binding affinity provides kinetic preference, specificity
and signaling fidelity for two-component systems
regulating development in Myxococcus xanthus
Secretion and gliding of Bacterodetes phylum
Jun
Liu
Jun
Liu
Michael
Manson
Jun Liu
38
Mark
McBride
Yongtao Zhu
39
Makoto
Miyata
Makoto
Miyata
Makoto
Miyata
MD
Motaleb
Dianne
Newman
Akihiro Tanaka
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
40
41
42
43
Darysbel Perez
Wiebke Behrens
Geetha Hiremath-Mendez
Rebecca Calvo
Chui Ching Wong
John Kirby
Chunhao Li
Jun Liu
Christopher Adase
Hiroki Yamamoto
Isil Tulum
Syed Sultan
Naomi Kreamer
The response regulator Rrp1 regulates chitobiose
utilization and virulence of the Lyme disease spirochete
Borrelia burgdorferi
Cryo-electron tomography of Escherichia coli minicells
reveals molecular architecture of intact flagellar motor
The torque generator of bacterial flagellar motor
revealed in Borrelia burgdorferi
The cytoplasmic aromatic anchor of TM2 regulates
transmembrane signal transduction by the Tar
chemoreceptor
Comparative analysis of Flavobacterium johnsoniae
and Cellulophaga algicola gliding motility and protein
secretion
Gliding of Mycoplasma mobile can be explained by
directed binding
Gliding machinery of Mycoplasma mobile observed by
electron microscopy
Gene manipulation of gliding bacterium, Mycoplasma
mobile
Is motility or possession of periplasmic flagella crucial
for the pathogenesis of Lyme disease?
Fe(II) sensing in Pseudomonas aeruginosa
Page #
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
POSTERS - BLAST XII
Poster # Lab
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
Presenter
Title
Takayuki
Nishizaka
Karen
Ottemann
Yoshiaki Kinosita
John
Parkinson
John
Parkinson
John
Parkinson
Mathieu
Picardeau
Steven
Porter
Germán Piñas
Simon
Rainville
Simon
Rainville
Christopher
Rao
Susan
Rosenberg
Edward
Ruby
Florian
Schubot
Guillaume Paradis
Direct observation of unitary step of gliding machinery
in Mycoplasma mobile
The Helicobacter pylori TlpD chemoreceptor is critical
for multiple stages of normal stomach colonization and
mediates a response to iron and other reactive oxygen
species-generating conditions
The role of receptor-kinase interactions in array
signaling
Transmembrane signaling via synthetic control cables
in the Tsr chemotaxis receptor
Tsr-E502: An unorthodox chemoreceptor sensory
adaptation site
Characterization of a chemotaxis locus in Leptospira
involved in motility and virulence
Stimulatory interactions between hybrid histidine
protein kinases in the virulence signalling network of
Pseudomonas aeruginosa
Taking control of the bacterial flagellar motor
Karen Ottemann
Smiljka Kitanovic
Xuesheng Han
Ambroise Lambert
Steven Porter
Ismael Duchesne
The motility of bacteria in an anisotropic liquid
environment
Santosh Koirala
Phenotypic variation and vistability within flagellar gene
network in Salmonella enterica serovar Typhimurium
Abu Amar Al Mamun
Identity and function of a large gene network underlying
mutagenic repair of DNA breaks under stress
Marie-Stephanie Aschtgen Is bacterial twitching motility required to establish the
squid/Vibrio symbiosis?
Florian Schubot
The Pseudomonas aeruginosa type three secretion
system transcriptional activator ExsA interacts with the
anti-activator protein ExsD in a temperature-dependent
manner
Liz
David Milner
A surface-regulated orphan ParA-like protein has an
Sockett
impact on gliding motility in Bdellovibrio bacteriovorus
Liz
Sarah Basford
The role of polyphosphate related genes in the
Sockett
predatory bacteria Bdellovibrio bacteriovorus
Lotte
Beata Jakobczak
Identification of a novel cell-envelope subcomplex
Søgaard-Andersen
involved in gliding motility in Myxococcus xanthus
Lotte
Carmen Friedrich
Deciphering the assembly pathway of type IV pili in
Søgaard-Andersen
Myxococcus xanthus
Lotte
Kristin Wuichet
Diversity of small Ras-like G-proteins in prokaryotes
Søgaard-Andersen
Claudia
Claudia Studdert
An engineered serine chemoreceptor with symmetric
Studdert
insertions mimics natural transducers of the 40H class
Claudia
Claudia Studdert
Functional and structural analysis of homologous
Studdert
conserved residues CheW-R62 and CheA-R555 within
the chemotaxis ternary complex
Tom
Francois Anquez
Effect of chemoeffector on the stability of
Shimizu
chemoreceptors assembly studied by photo-activated
localization microscopy
Alan
Alaa Abouelfetouh
The flip of a coin: Acetylation or phosphorylation by
Wolfe
acetyl phosphate in biofilms
Page #
95
96
97
98
99
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
POSTERS - BLAST XII
Poster # Lab
66
67
Alan
Wolfe
Zhihong
Xie
Presenter
Title
Alan Wolfe
Acetyl phosphate is a potent regulator of bacterial
protein acetylation
A novel chemoreceptor couples degradation of
aromatic pollutants with chemotaxis
Zhihong Xie
Page #
117
118
SPEAKER ABSTRACTS
1
BLAST XII
_____Mon. Morning Session
STRUCTURAL DISSECTION OF BACTERIAL FLAGELLAR MOTORS IN LIVE BACTERIA BY
ELECTRON CRYO-TOMOGRAPHY
Morgan Beeby1, David R. Hendrixson2, Deborah Ribardo2, Patrizia Abrusci3, Steven Johnson3, Susan
M. Lea3, Songye Chen4, and Grant J. Jensen4
1
Imperial College London, South Kensington Campus, London SW7 2AZ, UK
2
Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, Texas 75390
3
Sir William Dunn School of Pathology, Oxford University, South Parks Road, Oxford, OX1 3RE, UK
4
Division of Biology, California Institute of Technology, Pasadena, CA, USA, and Howard Hughes
Medical Institute, California Institute of Technology, Pasadena, CA, USA
The bacterial flagellar motor spins the flagellar filament to propel the cell to favourable
environments. While considerable insights into many facets of the motor's function and origins are
promised by understanding the structure and assembly of the motor, such studies have historically
been hampered by the intact motor's intimate association with the cell envelope and the lack of tools to
image it there. The recent development of electron cryo-tomography remedies this problem by
producing 3-D images of frozen, living bacteria at a resolution sufficient to resolve individual
macromolecules. Because electron cryo-tomography can be used to image whole bacteria, and thus
avoids the need for purification procedures, imaging the motor in situ is now routine and dependent
only upon our ability to culture bacteria. We reasoned that a comparative study of a range of flagellar
motors from phylogenetically diverse bacteria, including alpha-, gamma- and epsilon- bacteria,
firmicutes and spirochaetes, would provide information on motor function, assembly, and evolution not
apparent from studies focusing on a single model organism. Results revealed widespread elaboration
upon the 'normal' Salmonella enterica or Escherichia coli motor that enabled us to combine our
comparative electron cryo-tomography approach with genetic screens and bioinformatics to pinpoint the
location of multiple constituent proteins in the 3-D structure of the motor. Some of these proteins are
ubiquitous members of the flagellar export apparatus, while others are specific adaptations found only
in a subset of the bacteria. In combination, these comparative results propose further testable
hypotheses regarding universal flagellar motor function and suggest reasons why some bacteria have
evolved elaborate structures around their motors that are not found in model organisms.
Lab: Morgan Beeby
________________
2
BLAST XII
_____Mon. Morning Session
HOW DOES THE FLAGELLUM GROW AT A CONSTANT RATE?
Lewis D.B. Evans, Simon Poulter, Eugene M. Terentjev*, Colin Hughes & Gillian M. Fraser
Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, UK.
*Cavendish Laboratory, University of Cambridge, JJ Thomson Avenue, Cambridge CB3 OHE, UK.
Bacteria swim by means of long flagella on the cell surface. Flagella are assembled from
thousands of subunits that are translocated across the cell membrane by a dedicated export machinery
at the base of each flagellum. Subunits then travel through a central channel in the flagellum to reach
their assembly site at the tip. Remarkably, as the flagellum lengthens outside the cell the rate of
flagellum growth does not appear to change, precluding the possibility that subunits diffuse passively
through the external channel. Several studies have focused on the energy source for growth of flagella,
but the mechanisms proposed appear incompatible with the observation that the rate of flagellum
growth is independent of flagellum length. So the question remains, how do subunits move through the
long inert channel in the external flagellum and, in particular, what is the energy source? We propose a
simple physical mechanism that engenders a constant rate of subunit delivery to the flagellum tip
independent of channel length.
Lab: Colin Hughes
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3
BLAST XII
_____Mon. Morning Session
CheY ACETYLATION IN BACTERIAL CHEMOTAXIS: HOW DOES IT GENERATE FLAGELLAR
CLOCKWISE ROTATION?
Milana Fraiberg1, Oshri Afanzar1, Yishai Levin2 and Michael Eisenbach1
1
Department of Biological Chemistry, The Weizmann Institute of Science, 76100 Rehovot, Israel.
2
Proteomics and Mass Spectrometry Teaching Unit, The Weizmann Institute of Science, 76100
Rehovot, Israel.
N-acetylation of proteins (on lysine residues) is highly prevalent, from bacteria to mammals.
One of the best-studied proteins that undergo this covalent modification is CheY, the excitatory
response regulator of bacterial chemotaxis, whose acetylation is required for normal chemotaxis. In my
talk I will focus on our studies aimed at resolving the enigma of how CheY is activated by acetylation to
shift flagellar rotation from the default direction, counterclockwise, to clockwise, even though its binding
to the switch of the flagellar motor does not increase.
Lab: Michael Eisenbach
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4
BLAST XII
_____Mon. Morning Session
DIRECT IMAGING OF CheY-BINDING TO A FUNCTIONING BACTERIAL FLAGELLAR MOTOR
Hajime Fukuoka, Yuichi Inoue, Hiroto Takahashi, Akihiko Ishijima
Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, 2-1-1 Katahira, Aobaku, Sendai, 980-8577, Japan
Escherichia coli cell swims by rotating flagellar motors and migrates toward favorable
environment by controlling the rotational direction of the flagellar motors. The rotational direction of
flagellar motor is controlled by chemotaxis signaling system. When the chemoreceptors sense the
chemotacitc signals, they modulate the auto-phosphorylation activity of a histidine protein kinase,
CheA. The phosphoryl group on CheA is rapidly transferred to a response regulator, CheY. From the
previous biochemically and genetically investigations, the binding of phosphorylated CheY (CheY-P) to
the FliM, which is one of the components of flagellar motor, is believed to change the rotational
direction of the motor from counterclockwise (CCW) to clockwise (CW). However, it is not directly
observed the rotational switching induced by the binding of CheY-P. In this study, we tried to directly
observe the rotational switching induced by CheY-binding in a single functioning flagellar motor.
In order to observe the binding of CheY to the functioning flagellar motor, we constructed the
expression system of GFP-fusion protein of CheY and observed the localization of CheY-GFP in a
tethered cell under TIRF microscopy. In our microscopic system, the localization of CheY-GFP
(fluorescence image) and the rotational motion of tethered cell (bright field image) were recorded
simultaneously by using EMCCD and high-speed CCD cameras, respectively.
By using our microscopic system, the fluorescent spot derived from CheY-GFP was observed at
the rotational center of the tethered cell during CW rotation. The fluorescence intensity at the rotational
center of the tethered cell coordinately changed with the rotational switching of the motor; the
fluorescence intensity increased during CW rotation and decreased during CCW rotation. A cross
correlation analysis between rotational switching and the change of the fluorescence intensity showed a
major peak nearly 0s. The lag time of rotational switching after the binding or dissociation of CheYGFP, which was estimated from the peak-time of correlation, was within our sampling rate (50
ms/frame). The number of CheY-GFP molecules, which was estimated by the comparison with the
fluorescence intensity of the motor constructed by the FliM-GFP, was smaller than that of FliM
molecules in a motor. These results indicate that the conformational change of one of the FliM subunit
by the binding of CheY-P should be spread to neighboring FliM subunits as proposed in previous
theoretical models, and suggest that there is a strong cooperativity in the binding and dissociation of
CheY-P to the flagellar motor in a rotational switching. The highly binding cooperativity would
accelerate the conformational change of the basal body and contribute to the rapid response of motor
to the intracellular signal.
Lab: Akihiko Ishijima
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5
BLAST XII
_____Mon. Morning Session
MECHANISM FOR ADAPTIVE REMODELING OF THE BACTERIAL FLAGELLAR SWITCH
Pushkar P. Lele, Richard W. Branch, Vedhavalli S. J. Nathan and Howard C. Berg
Department of Molecular and Cellular Biology, Harvard University, 16 Divinity Ave, Cambridge MA
02138
The bacterial flagellar motor has been shown to adapt to changes in the steady-state
concentration of the chemotaxis signaling molecule, CheY-P, by changing the FliM content [Yuan J,
Branch RW, Hosu BG, & Berg HC (2012) Adaptation at the output of the chemotaxis signalling
pathway. Nature 484(7393):233-236]. We show here that the number of FliM molecules in the motor
and the fraction of FliM molecules that exchange depend on the direction of flagellar rotation, not on
CheY-P binding per se. Our results are consistent with a model in which the structural differences
associated with the direction of rotation modulate the strength of FliM binding. When the motor spins
CCW, FliM binding strengthens, the fraction of FliM molecules that exchanges decreases, and the ring
content increases. The larger number of CheY-P binding sites enhances the motor’s sensitivity, i.e.,
the motor adapts. An interesting unresolved question is how additional copies of FliM might be
accommodated.
Lab: Howard Berg
_________________
6
BLAST XII
_____Mon. Morning Session
FLAGELLA STATOR HOMOLOGUES POWER BACTERIAL GLIDING MOTILITY BY MOVING IN
HELICAL TRAJECTORIES
Beiyan Nan1, Jigar N. Bandaria1,2, Amirpasha Moghtaderi1, Im-Hong Sun1, Ahmet Yildiz1,2* and David
R. Zusman1*
1
Department of Molecular and Cell Biology
2
Department of Physics, University of California, Berkeley, CA 94720, USA.
Many bacterial species use gliding motility in natural habitats since external flagella function
poorly on hard surfaces. However, the mechanism(s) of gliding remain elusive since surface motility
structures are not apparent. Here, we characterized the dynamics of the Myxococcus xanthus gliding
motor protein AglR, a homologue of the Escherichia coli flagella stator protein MotA. We observed that
AglR decorated a helical structure and the AglR helices rotated when cells were suspended in liquid or
when cells moved on agar surfaces. We found that single molecules of AglR, unlike MotA/MotB, can
move laterally within the membrane in helical trajectories. AglR slowed down transiently at gliding
surfaces, accumulating in clusters. Our work shows that the untethered gliding motors of M. xanthus, by
moving within the membrane, can transform helical motion into linear driving forces that push against
the surface.
Lab: David Zusman
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7
BLAST XII
_____Mon. Morning Session
THE FliL PROTEIN INCREASES FLAGELLAR MOTOR OUTPUT IN SALMONELLA
Jonathan D. Partridge* and Rasika M. Harshey
Section of Molecular Genetics and Microbiology, University of Texas at Austin, Austin, TX 78712
The fliL gene is present in numerous flagellated bacteria, often co-expressed with genes
encoding the switch complex or stator components. The precise function of FliL is not known, but its
absence is associated with defects in both swimming and swarming motility. In Salmonella, absence of
FliL leads to small defects in swimming but complete elimination of swarming, the latter being known to
correlate with rod fracture1. Here we show a role for FliL beyond the structural. Using a variety of
assays we show that FliL interacts strongly with itself, the MS ring protein (FliF) and also MotA and
MotB proteins. Single motor experiments show that motor speed is slower in the absence of FliL. These
and other experiments show that FliL increases PMF through the MotAB stators either by recruiting, by
stabilizing, or by increasing proton flow through the stators. From this analysis, we place FliL outside
and around the MS ring, in proximity to the stators and expand its previous remit beyond structural
support at the rod. Overexpression of FliL in conjunction with the stator proteins MotA and MotB
overcomes surface friction to allow swarming on harder agar surfaces.
1. U. Attmanspacher, B. Scharf and R. M. Harshey. 2008. Mol. Microbiol. 68: 328-341.
Lab: Rasika Harshey
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8
BLAST XII
_____Mon. Morning Session
3-D VISUALIZATION OF BACTERIAL FLAGELLAR ASSEMBLY IN BORRELIA burgdorferi
Xiaowei Zhao1*, Kai Zhang2*, Tristan Boquoi3, Bo Hu1, Chunhao Li2, Md A. Motaleb3, Kelly A. Miller4,
Milinda E. James4, Nyles W. Charon4, Steven J. Norris1, Jun Liu1
1. Department of Pathology and Laboratory Medicine, Medical School, University of Texas Health
Science Center at Houston, Houston, TX 77030
2. Department of Oral Biology, State University of New York at Buffalo, Buffalo, NY 14214
3. Department of Microbiology and Immunology, East Carolina University, Greenville, NC 27834
4. Department of Microbiology, West Virginia University, Morgantown, WV 26506
The bacterial flagellum is a remarkable self-assembled molecular machine, and is composed of
the flagellar motor, the rod, the hook and the filament. The flagellar assembly in Escherichia and
Salmonella, which has been a well-studied paradigm model, is a finely orchestrated biochemical
process from highly regulated gene expression to protein assembly. The flagellum-specific type III
secretion system (T3SS) is responsible for the translocation and the assembly of key proteins of the rod
(FliE, FlgB, FlgC, FlgF and FlgG), the hook (FlgE) and the filament (FliC). Despite the recent progress
in understanding overall flagellar structure and assembly pathway, direct visualization of the
intermediates of this assembly process has not been achieved at the molecular level. In this study,
cryo-electron tomography was utilized to determine three-dimensional (3-D) structures of flagellum from
several key flagellar mutants (fliE, flgB, flgC, flhO (a homolog to E. coli flgF), flgG, flgE, flgI, and flaB (a
homolog to E. coli fliC), which were all constructed in Borrelia burgdorferi, a Lyme disease spirochete.
A complete set of 3-D intermediate structures of the T3SS mediated flagellar assembly indicated a
clear progression of added densities. Evidently, the products of each rod gene form a small but distinct
portion of the rod. This observation differs from the prior hypothesis that the lack of any of the rod
proteins causes an overall failure of rod construction. Surprisingly, the export channel, which traverses
the axial center of the MS ring, appears to be closed until the start of rod assembly. The modular
composite structure of the rod may help to explain the intrinsic flexibility and robustness of the rod,
which play a critical role in transmitting the motor rotation to the filament.
Lab: Jun Liu
______________________
9
BLAST XII
_____ Mon. Evening Session
AUTOPHOSPHORYLATION OF THE RESPONSE REGULATOR PHoB RECEIVER DOMAIN IS
COOPERATIVE AND SATURABLE
Rachel Creager-Allena, Ruth Silversmithb, Robert Bourretb
Departments of aBiochemistry & Biophysics and bMicrobiology & Immunology, University of North
Carolina, Chapel Hill, NC 27599-7290
Two component systems, comprised of histidine kinases and response regulators, are a
common means of signal transduction in microorganisms. Response regulators can autophosphorylate
using small-molecule phosphodonors. To date, comprehensive kinetic characterization of response
regulator autophosphorylation is limited to CheY, which exhibits non-saturable autophosphorylation
kinetics with respect to phosphodonor concentration. Non-saturable kinetics presumably reflect weak
substrate binding. We characterized autophosphorylation of the receiver domain of the Escherichia coli
response regulator PhoB (PhoB N ) by the small molecule phosphodonor phosphoramidate. The quench
in intrinsic fluorescence of a tryptophan in the active site of PhoB N was used to monitor
autophosphorylation time courses.
In contrast to CheY, the rate of autophosphorylation of PhoB N was saturable and cooperative
with respect to phosphoramidate concentration. Binding of a phosphoryl group analog was also
cooperative. Saturability of autophosphorylation suggests that phosphodonors bind tighter to PhoB N
than to CheY. Cooperativity indicates communication between PhoB N active sites and suggests that
PhoB N dimerization might play a role in autophosphorylation kinetics. Consistent with such a possibility,
disruption of the PhoB N dimerization interface by mutation1 led to autophosphorylation kinetics that
were not cooperative and were much slower than wild type PhoB N . Furthermore, the rate of PhoB N
autophosphorylation increased with protein concentration, as expected if dimerization enhances
autophosphorylation.
Mathematical modeling showed that the cooperativity of PhoB N autophosphorylation could be
explained by formation of a heterodimer (one phosphorylated monomer and one unphosphorylated
monomer of PhoB N ). Essential to the model was that the dimerization constants for the heterodimer
and for the dimer consisting of two phosphorylated monomers of PhoB N were on the same order of
magnitude (5 μM). Another essential feature of the model was that the autophosphorylation rate of the
PhoB N monomer was at least 10-fold slower than the heterodimer.
Phosphorylation-mediated dimerization allows many response regulators to bind to tandem sites
on DNA and regulate transcription. Our data challenge the notion that response regulator dimers
primarily form between two phosphorylated monomers, and raise the possibility that response regulator
heterodimers containing one phosphoryl group may participate in gene regulation.
1. Mack, T.R., Gao, R., Stock, A.M. Probing the roles of the two different dimers mediated by the
receiver domain of the response regulator PhoB. J Mol Biol, 2009. 389(2): p: 349-364.
Lab: Robert Bourret
_______________
10
BLAST XII
_____ Mon. Evening Session
Ser/Thr PHOSPHORYLATION REGULATES TWO-COMPONENT SYSTEMS INVOLVED IN CELL
WALL METABOLISM
Lindsie Goss & Jonathan Dworkin
Columbia University, 701 W 168th St., HHSC 1218, New York, NY 10032
Bacteria must be able to quickly respond and adapt to changing environments. The ability to
sense and transduce these signals historically has been studied in the context of classical twocomponent systems (TCS), composed of histidine kinases and response regulators. Bacteria also
contain eukaryotic-like Ser/Thr kinases (eSTKs) and eukaryotic-like Ser/Thr phosphatases (eSTPs) but
the role of Ser/Thr phosphorylation has been unclear. My projects focus on the regulation of two
different TCSs by Ser/Thr phosphorylation and suggest that these two types of signal transduction
pathways convergently regulate physiological responses in bacteria involving the cell wall.
Bacillus subtilis PrkC, a member of a well-conserved eukaryotic-like Ser/Thr kinase family,
contains extracellular PASTA domains that bind cell wall peptidoglycan fragments. PrkC regulates
expression of the autolysin YocH, which is also a primary target of the WalRK TCS, the only essential
TCS in B. subtilis and a number of other Gram-positive bacteria. It is sensitive to cell wall perturbations
but how it senses these effects is unclear. We find that PrkC phosphorylates WalR, the response
regulator, both in vitro and in vivo, on T101. Allelic replacement strains that make a conservative serine
substitution mimic phenotypes resulting from deletion of the threonine kinase, emphasizing the
physiological relevance of this modification. This modification is conserved in S. aureus and we are
examining whether the phenotypic consequences are also conserved. Finally, we are trying to
understand the mechanistic implications of this modification for WalR.
Some Enterococci contain a TCS (VanRS) that responds to vancomycin and induces the
expression of genes that modify the cell wall such that the normally sensitive bacteria are now resistant
to vancomycin. While it is clear that vancomycin induces VanRS activation, how it is detected remains
mysterious. Vancomycin treatment results in the production of peptidoglycan fragments that are sensed
by the enterococci PrkC homolog IreK. Thus, IreK could act as the activator of the VanRS system. We
have both in vitro and in vivo data suggesting that threonine phosphorylation of VanS, the histidine
kinase, is required for full induction of the VanRS operon. We find that application of staurosporine, an
IreK inhibitor, blocks induction of the VanRS operon in response to vancomycin. Thus, the convergent
action of eSTKs and TCS may have important clinical ramifications as well as implications for
understanding the physiological regulation of TCS in bacteria.
Lab: Jonathan Dworkin
____________
11
BLAST XII
_____ Mon. Evening Session
LINKEDIN – FINDING CONNECTIONS: A RESPONSE REGULATOR LINKS THE Frz
CHEMOSENSORY SYSTEM TO THE MglA/MglB GTPase/GAP MODULE TO REGULATE POLARITY
OF MOTILITY SYSTEMS IN M. xanthus
Keilberg, Daniela, Wuichet, Kristin, Drescher, Florian, Søgaard-Andersen, Lotte
Max Planck Institute for Terrestrial Microbiology, Marburg, 35043 Germany
M. xanthus cells possess two independent gliding machines: the adventurous (A) system and
the social (S) system. A-motility allows movement of single cells, while S-motility is cell-cell contact
dependent. Mutations which abolish both of these systems lead to non-motile cells, while mutations in
only one of them allow bacterial cells to move by means of the intact system. While S-motility depends
on extension and retraction of Type-4-pili powered by ATP, A-motility requires focal adhesion
complexes connected to a MotAB-like motor powered by proton motive force. Furthermore, M. xanthus
cells can reverse the direction of movement, which is accompanied by an inversion of the polarity of
both motility systems. Reversals are induced by the Frz chemosensory system, acting upstream of a
small GTPase, MglA and its cognate GTPase activating protein, MglB.
RomR is a response regulator, which is required for motility and reversals and localizes in a
bipolar asymmetric pattern with a large cluster at the lagging cell pole. We found that RomR can
directly interact with MglA and MglB. Furthermore, RomR, MglA and MglB affect the localization of each
other in all pair-wise directions suggesting that RomR stimulates motility by promoting correct
localization of MglA and MglB in MglA/RomR and MglB/RomR complexes at opposite poles. Moreover,
localization analyses suggest that the two RomR complexes mutually exclude each other from their
respective poles. We further show that RomR interfaces with FrzZ, the output response regulator of the
Frz chemosensory system, to regulate reversals. Thus, RomR serves at the interface to connect a
classic bacterial signalling module (Frz) to a classic eukaryotic polarity module (MglA/MglB). This
modular design is paralleled by the phylogenetic distribution of the proteins suggesting an evolutionary
scheme in which RomR was incorporated into the MglA/MglB module to regulate cell polarity followed
by the addition of the Frz system to dynamically regulate cell polarity.
Lab: Lotte Søgaard-Andersen
_______
12
BLAST XII
_____ Mon. Evening Session
RESISTANCE OF CLOSTRIDIUM difficile TO BACTERIAL ANTIMICROBIAL PEPTIDES IS
MEDIATED THROUGH A DISJOINED TWO-COMPONENT SYSTEM THAT RESPONDS TO
MULTIPLE SUBSTRATES
Jose M. Suárez, Adrianne N. Edwards and S. M. McBride*
Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA U.S.A.
Clostridium difficile is the major causative agent of antibiotic-associated diarrhea. To persist in
the intestine the bacteria must cope with host innate defenses, including cationic antimicrobial peptides
(CAMPs) made by the host and the indigenous microbial flora. We previously showed that C. difficile
responds to CAMPs by inducing expression of genes that lead to CAMP resistance. The first such C.
difficile gene cluster identified (cprABCK) encodes an ABC-type transporter and a sensor kinase typical
of bacterial two-component systems (TCS). These genes are highly related to genes that encode
immunity proteins in lantibiotic producer species. Using qRT-PCR and directed mutagenesis, we
showed that the transporter and kinase genes are directly involved in resistance to CAMPs. However,
no apparent response regulator is encoded in the vicinity of cprABCK. Here, we identify an orphan
response regulator, CD3320, that encodes the response regulator that controls the cprABCK genes in
response to antimicrobial peptides. We cloned the CD3320 gene and upstream region in a vector that
replicates in C. difficile and demonstrated that cells adapted faster to CAMPs and expression of the
cprABCK operon increased. By expressing these regulators and a Pcpr::lacZ reporter fusion in a
heterologous host, Bacillus subtilis, we were able to show how CprK and CD3320 (renamed CprR)
interact to activate expression of the cpr operon. In addition, we were able to identify putative residues
of both the lantibiotics and of CprK that are involved in sensing and activation of this system. These
results demonstrate how the Cpr ABC-transporter and regulators allow for substrate recognition that is
broader than the typicallly narrow specificity of lantibiotic producer systems, resulting in resistance to
multiple bacterial CAMPs through a single mechanism. The cpr lantibiotic immunity system represents
a novel antimicrobial peptide resistance mechanism that has not been described in any other species.
Lab: Shonna McBride
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13
BLAST XII
_____ Mon. Evening Session
THE WalKR SYSTEM CONTROLS MAJOR STAPHYLOCOCCAL VIRULENCE GENES AND IS
INVOLVED IN TRIGGERING THE HOST INFLAMMATORY RESPONSE
Aurélia Delauné1, Sarah Dubrac1, Charlène Blanchet2, Olivier Poupel1, Ulrike Mäder3, Aurélia Hiron1,
Aurélie Leduc4, Catherine Fitting2, Pierre Nicolas4, Jean-Marc Cavaillon2, Minou Adib-Conquy2 and
Tarek Msadek1
1
Institut Pasteur, Biology of Gram-Positive Pathogens, Department of Microbiology, Paris, France
2
Institut Pasteur, Cytokines and Inflammation, Department of Infection and Epidemiology, Paris,
France
3
Interfaculty Institute for Genetics and Functional Genomics, Department for Functional Genomics,
Ernst Moritz Arndt University, Greifswald, Germany
4
Mathématique, Informatique et Génome, INRA, UR1077, Jouy-en-Josas, France
The WalKR two-component system is essential for viability of Staphylococcus aureus, playing a
central role in controlling cell wall metabolism. We produced a constitutively active form of the WalR
response regulator in S. aureus through a phosphomimetic amino acid replacement (D55E). The strain
displayed significantly increased biofilm formation and α-hemolytic activity. Using transcriptome
analysis, we showed that transcription of 108 genes was increased in the presence of the walRc allele,
including eleven involved in cell wall metabolism. Several of the upregulated genes encode major
virulence proteins involved in host matrix interactions (efb, emp, fnbA, fnbB), cytotoxicity (hlgACB, hla,
hlb), innate immune defense evasion (scn, chp, sbi) and signal transduction (saePQRS). For many of
these, we showed that control by WalR occurs through increased activation of the SaeSR twocomponent system. The impact on pathogenesis of varying cell envelope dynamics through WalR
activity was studied using a murine sepsis model, showing that strains producing constitutively active
WalRc are strongly diminished in their virulence. We show that this is due to early and enhanced
triggering of the host inflammatory response, associated with higher levels of released peptidoglycan
fragments. Indeed, neutrophil recruitment and proinflammatory cytokine production (TNF-α, IL-6, IL-1β)
were significantly increased when the walRc allele was expressed, leading to enhanced bacterial
clearance. Taken together, our results indicate that WalKR play an important role in virulence and
eliciting the host inflammatory response by controlling autolytic activity, suggesting that the precise finetuning of WalKR activity may be critical in the switch between S. aureus commensal and pathogenic
lifestyles.
References:
1. Delauné, A., S. Dubrac, C. Blanchet, O. Poupel, U. Mader, A. Hiron, A. Leduc, C. Fitting, P. Nicolas, J.
M. Cavaillon, M. Adib-Conquy, and T. Msadek. 2012. The WalKR system controls major staphylococcal
virulence genes and is involved in triggering the host inflammatory response. Infect. Immun. 80:3438–
3453.
2. Delauné, A., O. Poupel, A. Mallet, Y. M. Coic, T. Msadek, and S. Dubrac. 2011. Peptidoglycan
crosslinking relaxation plays an important role in Staphylococcus aureus WalKR-dependent cell
viability. PLoS One 6:e17054.
Lab: Tarek Msadek
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14
BLAST XII
_____ Mon. Evening Session
WHAT SEQUENCE DIVERSITY TEACHES US ABOUT BACTERIAL SIGNALING
Hendrik Szurmant1, Alexander Schug2, Martin Weigt3
1
Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla,
California, USA; 2 Steinbuch Centre for Computing, Karlsruhe Institute of Technology, Karlsruhe,
Germany; 3 Laboratoire de Génomique des Microorganismes, Université Pierre et Marie Curie, Paris,
France
Two-component signal transduction mechanisms are woven in the fabric of cellular regulation in
bacteria, plants and lower eukaryotic forms of life. These innate systems play a wide variety of roles
from sensing the environment to controlling the cell cycle, cellular behavior and development. Twocomponent systems comprise a sensor protein, the histidine kinase and an output protein, the response
regulator, which typical serves as a transcription factor. Since the completion of the first bacterial
genome-sequencing project in 1995 more than 3000 bacterial genomes have been sequenced in part
thanks to the rapid development of high throughput sequencing technology. Encoded by these
genomes are more than 105 two-component system proteins, whose sequences have been sampled in
evolutionary space to accomplish perfect functionality. Hidden within this sequence variation is a wealth
of information that can inform about the molecular mechanisms underlying the signal transduction
cascade, namely how the kinase modulates its activity in response to signals, how the kinase identifies
its target response regulator from other response regulators of identical structure in the cell and how
the kinase physically interacts with its target response regulator. This talk will discuss a computational
method, called Direct Coupling Analysis that we developed in order to answer the questions of
molecular activation and interaction specificity in two-component signaling. Going beyond the field of
signal transduction, our technology proves promising in identifying protein interaction surfaces and
protein complex structures formed by interacting proteins.
Lab: Hendrik Szurmant
15
BLAST XII
_____ Tue. Morning Session
STRUCTURAL CHANGES OF BACTERIAL CHEMORECEPTOR ARRAYS UNDERLIE DIFFERENT
ACTIVATION STATES
Ariane Briegel1, Peter Ames2, James C. Gumbart3, Catherine Oikonomou1, John S. Parkinson2 and
Grant J. Jensen3
1
California Institute of Technology, 1200 E. California Blvd., Pasadena, CA 91125
2
University of Utah, 257 South 1400 East Salt Lake City, UT 84112
3
Argonne National Laboratory, 9700 S. Cass Avenue, Argonne, IL 60439
Chemotactic bacteria utilize a highly sensitive and adaptable sensory system to swim towards
attractants and away from repellents. A polar, highly organized sensory patch of transmembrane
receptor proteins detects changes in nutrient concentrations. Attractants and repellents bind to the
sensory domains of the receptors, thereby regulating the activity of the histidine kinase CheA, which
phosphorylates a soluble messenger protein. This messenger protein in turn diffuses through the
cytoplasm to the flagellar basal body, where it modulates the direction of flagellar rotation.
EM maps generated by sub-volume averaging of wild-type chemoreceptor arrays in the adapted
state, together with a new crystal structure, revealed the protein arrangement of the core chemotaxis
proteins in the array. The trimers of receptor dimers lie at each vertex of a hexagon, arranged such that
one dimer points towards the center of the ring. The receptors surround rings of alternating regulatory
subdomains (P5) of CheA and the coupling protein CheW. The CheA kinase subdomains (P4) project
toward the cytoplasm and away from the receptors, while the dimerization subdomains (P3) link
neighboring rings together to form a stable, extended lattice.
To get insight to the structural changes of the arrays during activation, we have now analyzed
sub-volume averages from various E.coli mutants with chemoreceptor arrays locked in either constantly
CheA-activating or inactivating states. Our results indicate that the chemoreceptors remain in a
hexagonal arrangement with identical center-to-center spacing in all activation states. The EM maps do
reveal a difference in the CheA packing, however: in the physiological CheA inactive state, both P1
and P2 subdomains of the CheA dimer are stably packed in a ‘rudder-like’ density beneath two
adjacent receptor trimers. In the constantly CheA activating states, this rudder-like density is less
prominent, suggesting more dynamic P1 and P2 subdomains.
Lab: Grant Jensen
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16
BLAST XII
_____ Tue. Morning Session
FORMATION AND SEGREGATION OF THE CYTOPLASMIC CHEMOSENSORY CLUSTER IN
RHODOBACTER sphaeroides
Christopher Jones, Nelly Dubarry, Mark Roberts and Judith Armitage
Department of Biochemistry, University of Oxford, South Parks Road, Oxford, UK, OX1 3QU
Rhodobacter sphaeroides, like many bacterial species, has a complex chemosensory network
containing multiple homologues of each component of the paradigm chemosensory pathway of
Escherichia coli. In R. sphaeroides this network is located to spatially distinct regions of the cell with
proteins making complete pathways found at the poles with membrane spanning chemoreceptors and
at around midcell with cytoplasmic chemoreceptors. New clusters develop during cell elongation so that
upon cell division each daughter cell inherits one polar cluster and one cytoplasmic cluster.
Recent work has shown that the segregation of the cytoplasmic cluster is dependent on a ParA
ATPase homologue PpfA. Par systems are used by bacteria to segregate chromosomes and plasmids
during cell division, and consist of ParA proteins which polymerise and ParB proteins which associate
with DNA and activate ParA depolymerisation. An increasing number of ParA homologues have been
shown to be involved in partitioning protein complexes. PpfA is encoded within the operon encoding
the cytoplasmic chemosensory pathway of R. sphaeroides and interacts with one of the cytoplasmic
chemoreceptors TlpT with the N-terminus of this protein acting as the ParB. Together these proteins
use the chromosome to segregate the chemosensory clusters on division. PpfA localises over the
chromosome, but forms foci around the cytoplasmic chemosensory cluster.
Using two colour fluorescence time lapse microscopy cytoplasmic cluster components are
shown to co-localise with PpfA foci throughout the cell cycle. Static images suggested that the cluster
was positioned at midcell, and then once a new cluster was formed they were positioned at ¼ and ¾
positions but current results show that the chemosensory cluster and associated PpfA foci are highly
mobile within the cell.
Results will be presented on the pattern of chemosensory cluster positioning through the cell
cycle and the possible mechanisms used for forming and segregating the chemosensory clusters.
Lab: Judith Armitage
______________
17
BLAST XII
_____ Tue. Morning Session
NETWORK-LEVEL PROPERTIES OF SALMONELLA TYPHIMURIUM CHEMOTAXIS
Milena D. Lazova1, Bob Rosier1, Filippo Menolascina2, Roman Stocker2, Thomas S. Shimizu1*
FOM Institute for Atomic and Molecular Physics (AMOLF), Amsterdam, The Netherlands.
2
Ralph M. Parsons Laboratory, Massachusetts Institute of Technology, Cambridge, MA, USA
1
Input-output relationships of the bacterial chemotactic response have been characterized in the
model species Escherichia coli, using in vivo experiments (1-3) and theoretical modeling (4, 5).
However, genomic studies have revealed that the E. coli system represents a streamlined example of
bacterial chemotaxis: the inferred topologies of the chemotactic signaling networks demonstrate a great
diversity across prokaryotes (6, 7). The dynamical features of chemotactic signaling, mediated by these
varied topologies remains largely unknown.
We examine here the chemotaxis system of the pathogen Salmonella typhimurium. It is a close
relative to E. coli: chemotactic genes of S. typhimurium and E. coli are similar enough that deletions of
chemotactic genes in one species are readily complemented by the orthologous genes from the other
(8). However, differences in the chemoreceptor structure and chemoreceptor types (9, 10) suggest that
the chemotactic signaling has diverged between the two species.
We have used a quantitative physiology approach to characterize the chemotactic signaling
dynamics of S. typhimurium in response to different chemoeffectors. Real-time fluorescence resonance
energy transfer (FRET) measurements (11), combined with time-varying stimuli allowed us to
characterize the chemotactic transfer functions. We have identified conserved features of E. coli and S.
typhimurium chemotactic signaling, and highlighted differences in the receptor-kinase responses, as
well as in the adaptation kinetics and frequency response. We probed how these physiological
differences influence the performance of cells executing chemotaxis in controlled microenvironments,
by observing the migration of bacterial populations in steady gradients created in microfluidic devices
(12). We find striking differences between the transient drift velocity, as well as the steady state
behavior of S. typhimurium and E. coli populations, which we explain in terms of the underlying control
physiology.
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
Sourjik V & Berg HC (2002) Proc Natl Acad Sci U S A 99, 123-127.
Shimizu TS, Tu Y, & Berg HC (2010) Mol Syst Biol 6, 382.
Lazova MD, Ahmed T, Bellomo D, Stocker R, & Shimizu TS (2011) Proc Natl Acad Sci U S A
108, 13870-13875.
Tu Y, Shimizu TS, & Berg HC (2008) Proc Natl Acad Sci U S A 105, 14855-14860.
Jiang L, Ouyang Q, & Tu Y (2010) PLoS Comput Biol 6, e1000735.
Szurmant H & Ordal GW (2004) Microbiol Mol Biol Rev 68, 301-319.
Wuichet K & Zhulin (2010) IB Sci Signal 3, ra50.
DeFranco AL, Parkinson JS, & Koshland DE, Jr. (1979) J Bacteriol 139, 107-114.
Biemann HP & Koshland DE, Jr. (1994) Biochemistry 33, 629-634.
Yamamoto K & Imae Y (1993) Proc Natl Acad Sci U S A 90, 217-221.
Sourjik V, Vaknin A, Shimizu TS, & Berg HC (2007) Methods Enzymol 423, 365-391.
Ahmed T, Shimizu TS, & Stocker R (2010) Nano Lett 10, 3379-3385.
Lab: Tom Shimizu
_________________
18
BLAST XII
_____ Tue. Morning Session
HOW HAMP DOMAINS AND METHYLATION BUNDLES CONTROL STIMULUS RESPONSE AND
SENSORY ADAPTATION IN CHEMORECEPTORS
Runzhi Lai, Peter Ames and John S. Parkinson
Biology Department, University of Utah, Salt Lake City, Utah 84112
In transmembrane chemoreceptors of the methyl-accepting chemotaxis protein (MCP) family, a
HAMP domain at the cytoplasmic face of the membrane accepts stimulus signals from the periplasmic
sensing domain and converts them to conformational changes that modulate activity of a CheA kinase
at the hairpin tip of the MCP molecule. The dynamic bundle model proposes that chemoreceptor
signaling involves opposed shifts in packing stability of the four-helix HAMP bundle and the adjacent
methylation helix (MH) bundle, which contains the covalent modification sites for sensory adaptation.
To test aspects of the dynamic bundle model, we measured the signaling behaviors of serine (Tsr)
receptors that had structural alterations in the HAMP and/or MH bundles, using the FRET-based in vivo
kinase assay pioneered by Sourjik & Berg (2002).
The Tsr homodimer contains five modification sites in each subunit: E residues correspond to
unmethylated sites; Q residues mimic methylated E sites. In a host strain lacking the CheR and CheB
adaptation enzymes, we measured the serine sensitivities of Tsr variants that had all possible
combinations of E and Q residues at sites 1-4, in combination with an E at site 5. We observed an
exponential relationship between the K 1/2 for serine and the number of Q residues in the receptor, with
little site specificity. This finding implies that each methylated (or Q) position makes an equivalent free
energy contribution to transitions between kinase-on and kinase-off output states.
Amino acid replacements in the HAMP domain can also alter Tsr response sensitivity by shifting
the equilibrium between kinase-on and kinase-off output states. For example, Tsr-S255A is shifted
toward the off state (K 1/2 = 2.7 µM, compared to 17 µM for wild-type in the QEQE modification state),
whereas S255G (K 1/2 = 1.6 mM) is greatly shifted toward the on state. Yet, both mutant receptors
mediate robust chemotactic responses in adaptation-proficient cells, suggesting that the adaptation
system can offset S255 lesions by adjusting receptor modification state. Consistent with this
interpretation, we found that increased Q:E ratios at the methylation sites shifted S255A to higher K 1/2
values (e.g., QQQE = 7 µM), whereas reduced Q:E ratios shifted S255G to lower K 1/2 values (e.g.,
EEEE = 33 µM).
Despite their opposite shifts in response sensitivity, both S255A and S255G suppress the
signaling defects of I229A and I229S lesions, which alone shift output to the kinase-on state. For
example, in an adaptation-proficient background K 1/2 of I229S = 250 µM, whereas I229S+S255A =
11 µM and I229S+S255G = 27 µM). I229 and S255 reside in the same packing layer of the four-helix
HAMP bundle. Structural interactions between these positions could restore response sensitivity by
matching HAMP packing stability to the opposing structural effects of receptor methylation, thereby
poising the receptor to respond to small attractant stimuli. This and other signaling implications of the
structural interplay between the HAMP and MH bundles will be discussed in the talk.
Lab: John S. Parkinson
____________
19
BLAST XII
_____ Tue. Morning Session
TESTING AND REFINING THE RECEPTOR-CheA REGULATORY DOMAIN INTERFACE IN
FUNCTIONAL, MEMBRANE-BOUND ARRAYS USING DISULFIDE MAPPING AND TAM-IDS
Kene N. Piasta and Joseph J. Falke
Department of Chemistry and Biochemistry, University of Colorado at Boulder, Jennie Smoly Caruthers
Biotechnology Building, 596 UCB, Boulder, CO 80309
The bacterial chemotactic signaling array is composed of repeating core contacts between a
receptor trimer-of-dimers, a homodimeric CheA kinase, and the CheW coupling protein. CheA
incorporation into the array activates its kinase activity, while receptor-mediated attractant signals
inhibit kinase activity. Structures of receptor fragments, CheA, CheW, and various combinations have
been solved, but a molecular view of protein-protein contacts in the active, membrane-bound signaling
array has proved elusive. The CheA regulatory domain, P5, shares an SH3-like domain fold with
CheW, and NMR findings from the Dahlquist lab indicate P5 and CheW possess similar receptor
binding sites. Two recent crystal structures solved in the Crane Lab reveal two distinct possibilities for
the receptor-SH3-like domain interface. However, currently available information does not ascertain
whether one of these two interfaces, or a different interface, most accurately describes the native
contacts in chemotactic arrays. Here we construct two models of the receptor-P5 interface based on
the two Crane lab structures, and then we test the predictions of each model in vitro utilizing a disulfide
mapping approach in the active, membrane-bound signaling array. This approach can also reveal
signaling events that move through the receptor-P5 interface as changes in disulfide bond formation
rate. Finally, we utilize Tryptophan and Alanine Mutagenesis to Identify Docking Sites (TAM-IDS) to test
predicted receptor-P5 contacts in vivo. Together these approaches are yielding an improved model of
receptor-CheA-CheW contacts in the active, membrane-bound signaling array as well as providing new
insights into signal transduction from receptor to CheA.
Lab: Joseph Falke
_________________
20
BLAST XII
_____ Tue. Morning Session
DIFFERENCES BETWEEN SIGNALING STATES OF CHEMOTAXIS RECEPTORS REVEALED BY
HYDROGEN EXCHANGE MASS SPECTROMETRY
Seena S. Koshy, Stephen J. Eyles, Robert M. Weis, Lynmarie K. Thompson
Department of Chemistry, University of Massachusetts, Amherst MA 01003
Bacterial chemotaxis receptors provide the sensory input needed to direct swimming towards
favorable environments. Ligand binding to the receptor periplasmic domain modulates activation of a
kinase CheA bound at the cytoplasmic tip of the receptor. Receptors operate in complexes with CheA
and a coupling protein CheW that together form extended arrays in the membrane. Understanding the
differences in conformation and dynamics between the kinase-activating and kinase-deactivating
signaling states of the receptor will provide insight into the mechanism of transmembrane signaling.
We have used hydrogen exchange mass spectrometry to probe the differences between the
kinase-activating and kinase-deactivating signaling states of the receptor cytoplasmic domain
assembled on membrane vesicles in functional complexes with CheA and CheW. Local exchange
measurements reveal several differences between these states, including differences in the exchange
pattern (EX1 vs. EX2) and in the fraction that are protected from exchange at long time (16 hours).
Preliminary analysis shows that peptides corresponding to the adaptation region undergo EX1
exchange (long-lived unfolded state with complete exchange during each unfolding event) in the
kinase-activating state and EX2 exchange (short-lived unfolded state with partial exchange) in the
kinase-deactivating state. This suggests the adaptation region is destabilized in the kinase-activating
state. Peptides corresponding to the signaling domain cytoplasmic tip of the receptor show greater
protection from exchange in the kinase-activating state, which could be due to changes in interactions
with CheA and CheW. This hydrogen exchange mass spectrometry approach is a promising means of
detecting changes in structure and dynamics of a functional membrane-bound, multi-protein complex.
This research supported by GM 47601, GM085288, and a Fellowship to Seena Koshy from the
University of Massachusetts as part of the Chemistry-Biology Interface Training Program (NRSA T32
GM08515).
Lab: Lynmarie Thompson
__________
21
BLAST XII
_____ Tue. Morning Session
PROLONGED STIMULI ALTER THE BACTERIAL CHEMOSENSORY CLUSTERS
Vered Frank and Ady Vaknin
The Racah Institute of Physics, The Hebrew University, Jerusalem 91904, Israel.
While the principal structure of these receptor arrays is becoming clear, their dynamic nature
and operation are not yet understood. By measuring the fluorescence-polarization of tagged receptorclusters in live Escherichia coli cells, we show that during a stimulus, the packing of the receptors in
clusters slowly changes. Consistent with these physical changes, we find that the regulation of kinase
activity by these clusters slowly evolves, altering the effective cooperativity of the response. Timelapse fluorescence imaging indicated that despite these changes, the clusters do not generally
dissociate upon ligand binding. These data suggest that prolonged stimuli induce changes in receptor
packing within chemosensory clusters, which, in turn, alters the coupling between the receptors, and
thus leading to non-stationary signal transduction.
Lab: Ady Vaknin
__________________
22
BLAST XII
_____ Tue. Morning Session
THE SENSING AND SIGNALING ROLES OF MULTIPLE PAS-HEME DOMAINS IN Aer-2 TYPE
CHEMORECEPTORS
Magi B. Ishak Gabra, Kahryl G. Bennett, Adwoa O. Wiafe, Andrew J. Hong, Lana S. Haddad, Mark S.
Johnson and Kylie J. Watts
Division of Microbiology and Molecular Genetics, Loma Linda University, Loma Linda, CA, 92350
Aer-2 is a soluble PAS-HAMP chemoreceptor that is found in a number of pathogens including
Pseudomonas aeruginosa (PaAer-2) and Vibrio cholerae (VcAer-2). We previously showed that PaAer2 binds heme and can signal in response to various oxy-gases. The recently solved PaPAS structure
revealed a uniquely placed histidine residue (H234 on E5η) that coordinates heme, and an unusual
tryptophan residue (W283 on I8β) that may stabilize heme-oxygen binding.
Unlike PaAer-2, which contains a single PAS domain, VcAer-2 contains two PAS domains. Here
we show that both VcPAS domains bind heme and have spectra similar to PaPAS. The hemecoordinating His and oxygen-stabilizing Trp residues of PaPAS are both conserved in VcPAS-1 (H101,
W151) and VcPAS-2 (H226, W276). We found that the VcAer-2 receptor signals only in response to
oxygen and not in response to other oxy-gases. To identify which of the two VcPAS domains sense
oxygen and to determine the importance of the His and Trp residues for Aer-2 function, we engineered
amino acid substitutions into both truncated and full-length Aer-2 receptors for spectral and behavioral
studies.
The His substitutions, H234A in PaPAS, H101A in VcPAS-1, and H226A in VcPAS-2, each
lowered the extent of heme bound to each PAS domain (24%, 15% and 2% of wild-type heme,
respectively), indicating that the E5η histidine coordinates heme in Aer-2 type PAS domains. However,
full-length receptors with heme-binding defects in PaPAS or VcPAS-2 retained partial function. In
contrast, the heme-binding defect in VcPAS-1 caused a stronger response to oxygen, suggesting that
VcPAS-1 is not required for oxygen sensing and may in part function to inhibit VcPAS-2 signaling.
The Trp substitutions, W283L in PaPAS, and W276L in VcPAS-2, yielded aberrant PAS-heme
peptides that by spectral analyses appeared to bind imidazole so tightly it was not removed by
dithionite or by dialysis. The bound imidazole prevented the association of oxygen or carbon monoxide.
Thus, the I8β tryptophan may be required for oxygen sensing by PaPAS and VcPAS-2. Notably, the
Trp substitutions in PaPAS or VcPAS-2 created signal-on receptors, a state that typically requires gas
binding. In contrast, the Trp substitution, W151L in VcPAS-1, significantly inhibited the ‘on state’ of
VcAer-2, even though VcPAS-1 was still able to bind oxygen. This supports our hypothesis that VcPAS1 does not signal oxygen binding, and further suggests that although VcPAS-1 can bind oxygen, it does
not stabilize its binding via the Trp-mediated mechanism that is used by PAS-heme ‘sensing’ domains.
The results of this study have begun to reveal the similarities and differences between Aer-2 receptors
from different species and the differences between PAS-heme domains from Aer-2 and those of other
well-studied proteins such as FixL or DOS. Further studies are needed to elucidate the unique signaling
mechanisms used by Aer-2 type receptors.
Lab: Kylie Watts
23
BLAST XII
_____ Tue. Evening Session
FUNCTIONS OF PROTEUS MIRABILIS FliL IN SWARMING AND RESPONDING TO SURFACE
VISCOSITY
Yi-Ying Lee and Robert Belas
Department of Marine Biotechnology, University of Maryland, Baltimore County, and Institute of Marine
and Environmental Technology, 701 East Pratt Street, Baltimore, Maryland 21202
Proteus mirabilis is a dimorphic Gram-negative enterobacterium and well-known for its ability of
move over agar surfaces by flagellar-dependent swarming motility. In liquid culture, P. mirabilis cells
are uniformly 1.5-2.0 μm rods with 4-6 flagella, called swimmer cells. When P. mirabilis encounters a
highly viscous environment or a solid surface, swimmer cells differentiate into elongated (10-80 μm),
highly flagellated swarmer cells that lack of septa and contain multiple nucleoids. The bacteria detect a
surface by monitoring the rotation of their flagellar motors. Conditions that prevent rotation of swimmer
cell flagella trigger production of swarmer cells. This process involves a ubiquitous but functionally
enigmatic flagellar basal body-associated protein called FliL. fliL is the first gene in an operon
(fliLMNOPQR) that encodes proteins of the flagellar rotor switch complex and flagellar export
apparatus. We used a fliL knock out mutant to gain further insight into the function of FliL. Loss of FliL
results in cells that cannot swarm (Swr-), but do swim (Swm+), and in liquid produces cells that look like
wild-type swarmer cells, “pseudoswarmer cells”, which are elongated, contain multiple, evenly spaced
nucleoids, and lack septa. Unlike swarmer cells, pseudoswarmer cells are not hyperflagellated due to
reduced expression of flaA (the gene encoding flagellin), despite an increased transcription of both flhD
and fliA, two positive regulators of flagellar gene expression. We found that defects in fliL prevent
viscosity-dependent sensing of a surface and viscosity-dependent induction of flaA transcription.
Studies with fliL cells unexpectedly revealed a requirement of the fliL promoter, fliL coding region, and a
portion of fliM DNA to complement the Swr- phenotype. The data support a dual role for FliL, as a
critical link in sensing a surface and in the maintenance of flagellar rod integrity.
Lab: Robert Belas
_________________
24
BLAST XII
_____ Tue. Evening Session
THE AAA+ PROTEASE LonA REGULATES SwrA LEVELS AND SWARMING MOTILITY IN BACILLUS
subtilis
Sampriti Mukherjee*, Joyce E. Patrick and Daniel B. Kearns
Indiana University – Bloomington, IN, USA
Bacillus subtilis exhibits robust swarming motility atop semi-solid media (1). Swarming motility is
facilitated by secretion of a lipopeptide surfactant (surfactin) and powered by rotating flagella. Flagella
are complicated, membrane-anchored, proteinaceous nanomachines that propel bacteria in their milieu.
Each flagellum is built from over 30 proteins and has three architectural domains: basal body, hook and
filament. Swarming motility is correlated with an apparent increase in flagellar number in swarm cells
when compared to cells propagated in liquid media (swim cells). The mechanism and relevance of
hyper-flagellation during swarming motility in B. subtilis is poorly understood.
One way in which flagellar number could be increased is by an increase in the expression of
the flagella and chemotaxis operon (fla/che) that encodes for the proteins that comprise the hook and
basal body, and the alternate sigma factor σD required for the expression of the filament protein. The
putative master regulatory protein SwrA increases transcription of the fla/che operon and is required for
swarming in B. subtilis (2). Here we find that the increase in flagella number was correlated with an
increase in the intracellular level of the SwrA. SwrA protein levels significantly increase in swarm cells
when compared to swim cells and overexpression of SwrA results in hyperflagellation in swim cells.
However, swrA gene transcription and translation do not change between swarm and swim cells
implying post-translational regulation on SwrA.
Swarming motility is regulated by Lon protease of the AAA+ protease family in diverse species
such as Proteus mirabilis and Vibrio parahaemolyticus (3). We find that a lonA mutant exhibits increase
in SwrA protein levels in swim cells. We therefore hypothesize that LonA potentially targets SwrA in
swim cells but under conditions favorable to swarming LonA is inhibited and SwrA levels accumulate to
allow hyper-flagellation and swarming motility. Thus although the master regulators of flagella
biogenesis are distinct in different bacteria, the mechanism to regulate them appears to be similar.
1. Kearns, D.B., and Losick, R. (2003) Swarming motility in undomesticated Bacillus subtilis. Mol
Microbiol 49:581-590.
2. Kearns, D.B., Chu, F., Rudner, R., and Losick, R. (2004) Genes governing swarming in Bacillus
subtilis and evidence for a phase variation mechanism controlling surface motility. Mol Microbiol
52:357-369.
3. Patrick, J.E., and Kearns, D.B. (2012) Swarming motility and the control of master regulators of
flagellar biosynthesis. Mol. Microbiol. 83:14-23.
Lab: Daniel Kearns
________________
25
BLAST XII
_____ Tue. Evening Session
HELICAL FLOW OF SURFACE PROTEIN REQUIRED FOR FLAVOBACTERIUM GLIDING MOTILITY
Daisuke Nakane, Keiko Sato, Hirofumi Wada, Mark J. McBride and Koji Nakayama
Department of Molecular Microbiology and Immunology, Graduate School of Biomedical Sciences,
Nagasaki University, 852-8588 Nagasaki, Japan
Cells of Flavobacterium johnsoniae and of many other members of the phylum Bacteroidetes
exhibit rapid gliding motility over surfaces by a unique mechanism. These cells do not have flagella or
pili, and instead rely on a novel motility apparatus comprised of Gld and Spr proteins. SprB, a 669 kDa
cell-surface adhesion, is required for efficient gliding. SprB was visualized by electron microscopy as
thin 150 nm long filaments extending from the cell surface. Fluorescence microscopy revealed
movement of SprB proteins toward the poles of the cell at approximately 2 μm/s. The fluorescent
signals appeared to migrate around the pole and continue at the same speed toward the opposite pole
along an apparent left-handed helical closed loop. Movement of SprB, and of cells, was rapidly and
reversibly blocked by the addition of CCCP, which dissipates the proton gradient across the
cytoplasmic membrane. In a gliding cell, some of the SprB protein appeared to attach to the
substratum. The cell body moved forward and rotated with respect to this point of attachment. Upon
reaching the rear of the cell, the attached SprB was often released from the substratum, and apparently
recirculated to the front of the cell along a helical path. The results suggest a model for Flavobacterium
gliding, supported by mathematical analysis, in which adhesins such as SprB are propelled along a
closed helical loop track, generating rotation and translation of the cell body.
Lab: Koji Nakayama
_______________
26
BLAST XII
_____ Tue. Evening Session
BACTERIAL TRANSITIONS: OPTIMIZATION OF BIOLOGICAL AND PHYSICAL FACTORS BY
Pseudomonas aeruginosa DURING SWARMING
Morgen Anyan, Huijing Du, Oleg Kim, Zhiliang Xu, Mark Alber, and Joshua Shrout
University of Notre Dame, 156 Fitzpatrick Hall, Notre Dame, IN 46556 USA
Pseudomonas aeruginosa is one of several bacteria that swarms in groups. Often, but not
always, P. aeruginosa forms branched tendril patterns during swarming; we find that cells propagate as
high density waves that spread symmetrically as rings within swarms towards the extending tendrils.
These marvelous tendril patterns only form when P. aeruginosa produces sufficient rhamnolipid at
advancing swarm edge. During optimal swarm conditions we find that both cells and rhamnolipid
propagate as high density waves that move symmetrically as rings within swarms towards the
extending tendrils. Simulations conducted using a multi-scale model suggest that wave propagation
and tendril formation depend upon competition between the changing viscosity of the bacterial liquid
suspension and the liquid film boundary expansion caused by Marangoni forces. We have found that P.
aeruginosa swarms spread due to oscillations between biological and physical factors. Essentially, we
find that P. aeruginosa efficiently colonizes surfaces by controlling the physical forces responsible for
expansion of thin liquid films and by propagating towards the tendril tips.
We find that type IV pili interfere with optimal flagellar swarming; a hyperpiliated mutant (pilU) is
swarm deficient. Alternatively, for bacteria devoid of type IV pili, these bacteria spread more easily than
the wild type. A pilA mutant shows improved swarming and this phenotype can be complemented with
PilA expressed in trans. We are studying this phenotype as part of our combined experimentalmodeling approach to study how the cells optimally align and coordinate during swarming.
Lab: Joshua Shrout
_______________
27
BLAST XII
_____ Tue. Evening Session
STRUCTURE OF PROTEINS INVOLVED IN MYCOPLASMA mobile GLIDING REVEALED BY
ELECTRON MICROSCOPY AND HIGH SPEED ATOMIC FORCE MICROSCOPY (AFM)
Yuhei Tahara1, Noriyuki Kodera2, Toshio Ando2, Makoto Miyata1
1
Department of Biology, Graduate School of Science, Osaka City University, 3-3-138 Sugimoto
Summiyoshiku, Osaka-shi, Japan
2
Department Physics, Kanazawa University, 920-1192 Kakuma-machi, Kanazawa, Japan
Mycoplasma mobile, a fish pathogen, glides on solid surfaces with a unique mechanism. The
gliding machinery is assembled on the protrusion mainly from three huge proteins, designated Gli521,
Gli349, and Gli123, weighing 521k, 349k, 123kDa, respectively. In this study, we analyzed molecular
shapes of these proteins essential for gliding, by negative-staining electron microscopy and high speed
AFM. Gli521 has the role of “crank”, transmitting movements from motor to leg. Previously, rotary shadowing electron microscopy showed that the Gli521 molecule is shaped, “ ? ” ,120 nm long, and
featured with a “hook” at the C terminus and an “oval” at the N terminus. In the present study, we have
shown that the molecule is shaped, “ω”, and featured with a central hinge, based on better resolution of
negative-staining electron microscopy. Analyses by high speed AFM have shown that the central hinge
bends to only one direction. Gli349 is shaped, “♪ “, 100 nm long and function as “leg”. Analyses by
high speed AFM have shown that the Gli349 molecule consists of a large and a small domains
connected by a thin elastic filament. Gli123 has the role of “mount” essential for Gli349 and Gli521
subcellular localization. Analyses by electron microscopy have shown that the molecule of this protein
is an ellipse of 12.5 nm and 15 nm axes, featured with a small protrusion and two dimples, reminiscent
of “Japanese covered bowl”. These results give us better images for the machinery and the
mechanism of M. mobile gliding.
Lab: Makoto Miyata
________________
28
BLAST XII
_____ Tue. Evening Session
BACTERIOPHYTOCHROME 1 AND LOV-HK WORK IN A SIGNALING NETWORK TO REGULATE
SWARMING MOTILITY OF PSEUDOMONAS SYRINGAE STRAIN B728a
Liang Wu, Regina S. McGrane and Gwyn A. Beattie
Department of Plant Pathology and Microbiology, Iowa State University, Ames, IA, 50010
Photosensory proteins have long been studied in plants, algae, fungi and photosynthetic
prokaryotes. Genome sequencing has indicated the presence of photosensory protein-encoding genes
in non-photosynthetic bacteria, including the foliar plant pathogen Pseudomonas syringae. P. syringae
has three genes that are predicted to encode two classes of photosensory proteins: the
bacteriophytochromes, BphP1 and BphP2, and a LOV protein histidine kinase, LOV-HK. BphP1 is a
red/far-red light receptor that uses biliverdin as a chromophore, whereas Lov-HK is a blue light receptor
that uses a FMN (flavin mononucleotide)-binding LOV domain to sense light. BphP2 has eluded
characterization because of its recalcitrance to purification. The biological roles of
bacteriophytochromes and LOV proteins have been identified for a few non-photosynthetic bacterial
species; these include light-mediated regulation of pigment production by a bacteriophytochrome in
Deinococcus radiodurans, adhesion by LOV-HK in Caulobacter cresentus and Xanthomonas
axonopodis, and cellular proliferation by LOV-HK in macrophages by Brucella abortus. The signal
transduction pathways for these biological functions are unknown, as are the biological roles and signal
transduction pathways for these proteins in P. syringae. Here we report that swarming motility of P.
syringae strain B728a is regulated by both BphP1 and LOV-HK. Moreover, we discovered a novel
regulatory network in which BphP1 functions to repress swarming motility in response to red/far-red
light and blue light, and LOV-HK suppresses this unusual BphP1-mediated blue light repression.
Lab: Gwyn Beattie
_________________
29
BLAST XII
____ Wed. Morning Session
MULTIPLE AGONISTIC AND ANTAGONISTIC SIGNALS CONTROL THE ACTION OF THE
COMPLEX SENSOR KINASE TodS
Hortencia Silva-Jiménez, Jesús Lacal, Andreas Busch, M. Eugenia Guazzaroni, Juan Luis Ramos and
Tino Krell
Department of Environmental Protection, Estación Experimental del Zaidín, Consejo Superior de
Investigaciones Científicas, C/ Prof. Albareda, 1, 18008 Granada, Spain
The TodS/TodT two component system (TCS) controls the expression of the toluene
dioxygenase pathway in Pseudomonas putida for the metabolisation of the toxic compounds toluene,
benzene and ethylbenzene into Krebs cycle intermediates. The TodS sensor kinase is characterized by
a complex architecture and harbors two PAS domains, two transmitter modules and a receiver domain.
Microcalorimetric studies of purified TodS have shown that a wide variety of aromatic signal molecules
bind to the N-terminal PAS domain with high affinity (1,2). Interestingly, these molecules can be
classified as agonists and antagonists (3). Only the binding of agonists (toluene for example) causes an
upregulation of the autokinase activity of the N-terminal transmitter module and consequently gene
expression in vivo. In contrast, antagonist binding has no effect on the autokinase activity and gene
expression. We show that TodS employs an internal phosphorlay prior to the phosphorylation of the
TodT of which its phosphorylated form stimulates gene expression (2). Using in vivo gene expression
studies we show that the presence of antagonists (o-xylene for example) reduces the magnitude of
agonist-mediated gene expression (2). Recent studies show that the activity of other TCSs is also
based on the concerted action of agonists and antagonists (4). Crude oil can be understood as a
mixture of agonists and antagonists and our data might provide an explanation for the reduced pathway
expression observed in biodegradation experiments of complex mixtures such as petrol. In addition to
aromatic compounds the TodS autokinase activity in vitro and consequently the in vivo expression is
modulated specifically by the oxidative stress agent menadione (5). The amino acids necessary for the
sensitivity to menadione have been identified using site-directed mutagenesis. Experiments with
varying concentration of toluene and menadione show that the menadione-mediated reduction of TodS
autokinase activity dominated over toluene-mediated stimulation. The physiological relevance of sensor
kinases with a complex, multidomain architecture is still poorly understood. In this context, this work is
one of the first demonstrations that multiple types of signal molecules regulate the action of complex
sensor kinases.
References:
Lacal et al. (2006) Proc. Natl. Acad. Sci. USA 103, 8191-8196
Busch et al. (2009) J. Biol. Chem. 284, 10353-10360
Busch et al. (2007) Proc. Natl. Acad. Sci. USA 104, 13774-13779
Silva-Jiménez et al. (2012) Microb. Biotechnol. 5, 489-500.
Silva-Jiménez et al., manuscript in preparation
Lab: Tino Krell
____________________
30
BLAST XII
____ Wed. Morning Session
DETERMINING CHEMORECEPTOR-LIGAND INTERACTIONS IN THE LIGHT-ORGAN SYMBIONT
VIBRIO fischeri TO UNDERSTAND NICHE SPECIFICITY
Caitlin A. Brennan1,*, Run-zhi Lai2, Cindy R. DeLoney-Marino3, Mark J. Mandel4, John S. Parkinson2,
and Edward G. Ruby1
*Presenting author
1
Department of Medical Microbiology and Immunology, University of Wisconsin, Madison, WI
2
Department of Biology, University of Utah, Salt Lake City, UT
3
Department of Biology, University of Southern Indiana, Evansville, IN
4
Department of Microbiology-Immunology, Northwestern University Feinberg School of Medicine,
Chicago, IL
The bioluminescent bacterium Vibrio fischeri enters into a monospecific association with the
Hawaiian bobtail squid, Euprymna scolopes, that begins anew each generation. Symbionts from the
surrounding seawater form mucus-bound aggregates on the light-organ surface, and then migrate to
epithelium-lined crypts deep within its tissue, a distance of less than 100 micrometers. Despite this
short distance, the initiation process is still exquisitely specific and complex. By using confocal
microscopy to observe the behavior of a GFP-labeled cheA mutant, we have identified three stages at
which chemotaxis likely mediates symbiotic initiation. To fully understand how V. fischeri senses and
responds to the environment presented by the host, we sought to characterize the chemoreceptorligand pairings that underlie this behavior. Similar to other bacteria within the Vibrionaceae, the
genome of V. fischeri encodes dozens of predicted methyl-accepting chemotaxis proteins (MCPs),
none of which have been previously studied and cannot be well-characterized using bioinformatics.
To identify the chemoattractants sensed by these MCPs, we used two complementary
approaches: reverse genetics in V. fischeri, and heterologous expression of chimeric MCPs in
Escherichia coli. We first generated 19 individual MCP mutants and screened for altered responses to
six known V. fischeri chemoattractants using soft-agar assays, followed by capillary-assay analysis.
Because functional redundancy by other V. fischeri MCPs could hide potential interactions in this
approach, we also constructed chimeric proteins in which V. fischeri ligand-binding domains were fused
to E. coli Tar, immediately upstream of the HAMP domain. These chimeras were expressed in a ΔMCP
E. coli strain and analyzed by the FRET (Förster resonance energy transfer)-based in vivo kinase
assay (adapted from Sourjik and Berg, 2002), in which pools of potential chemoattractants were used
as stimuli. Using these two approaches, we have begun to clarify the chemotactic repertoire of V.
fischeri, including the characterization of VfcA (Vibrio fischeri chemoreceptor A), which mediates
chemotaxis towards multiple amino acids, and VfcB, which senses several sugars, including the chitinbreakdown products N-acetyl-glucosamine and chitobiose. Taken together, these approaches are
providing insight into the role of chemotaxis in this symbiosis, as well as defining a system by which to
identify chemoattractants in bacteria with large numbers of predicted MCPs.
Lab: Edward Ruby
_________________
31
BLAST XII
____ Wed. Morning Session
CHEMOTAXIS TO NOREPINEPHRINE (NE) REQUIRES THE Tsr CHEMORECEPTOR AND
CONVERSION ENZYMES INDUCED BY QseC
S. Pasupuleti1, M. R. Sears2, A. Jayaraman1, M. D. Manson2
Departments of 1Chemical Engineering and 2Biology
Texas A&M University
College Station, TX 77840
It has been reported that norepinephrine (NE) is an attractant sensed by E. coli and S.
typhimurium. We have found that the actual attractant is the NE metabolite dihydroxymandelic acid
(DHMA). NE is metabolized into DHMA in a two-step pathway involving the TynA tyramine oxidase and
the FeaB aromatic aldehyde dehydrogenase. NE induces the synthesis of TynA and FeaB via the
QseC sensor kinase. The peak response to DHMA is seen at concentrations of ~100 nM, a potentially
physiological range in the gastrointestinal tract. Current data suggests that DHMA is sensed via Tsr and
serves as an attractant at low concentrations and as an antagonist at higher concentrations. We
present a model for how this behavior might arise through differential responses to DHMA mediated by
the two serine-binding sites of the Tsr homodimer.
Lab: Michael Manson
_______________
32
BLAST XII
____ Wed. Morning Session
STRUCTURE AND FUNCTION OF A BLUE-LIGHT-REGULATED SENSOR HISTIDINE KINASE
Ralph P. Diensthuber1, Martin Bommer2,3, Andreas Möglich1
Humboldt-Universität zu Berlin, Institut für Biologie, Biophysikalische Chemie.
2
Humboldt-Universität zu Berlin, Institut für Biologie, Strukturbiologie/Biochemie.
3
Helmholtz-Zentrum Berlin für Materialien und Energie, BESSY-II.
1
Two-component systems (TCS) which comprise sensor histidine kinases (SHK) and reponseregulator proteins represent the predominant strategy by which prokaryotes sense and respond to a
changing environment. To date, no atomic structure of an intact SHK containing both sensor and
effector (catalytic) modules has been elucidated. Here, we report the full-length crystal structure of the
dimeric, blue-light-regulated SHK YF1 at 2.3 Å resolution in which two N-terminal light-oxygen-voltage
(LOV) photosensors are connected by a coiled coil to the C-terminal effector modules. A second
coaxial coiled coil derived from the N-termini of the LOV photosensors and inserted between them
crucially modulates light regulation: single mutations within this coiled coil attenuate or even invert the
signal response of the TCS. Structural motifs identified in YF1 recur in signal receptors, and the
underlying signaling principles and mechanisms may be widely shared between soluble and
transmembrane, prokaryotic and eukaryotic signal receptors of diverse biological activity.
Lab: Andreas Möglich
______________
33
BLAST XII
____ Wed. Morning Session
EFFECTS OF MOLECULAR ADSORPTION ON THE TRAJECTORIES AND ACCUMULATION OF
MOTILE BACTERIA AT THE AIR/WATER INTERFACE
Michael Morse and Jay X. Tang
Department of Physics, Brown University, Providence RI
Motility allows bacteria to move through its environment in order to acquire nutrients, avoid
danger, and perform other biological tasks. Cells can sometimes actively control their movement
through processes such as chemotaxis. However, the environment itself plays a vital role determining
cellular behavior due to the underlying interactions between the cell and its surroundings. For instance,
micro-swimmers such as Escherichia coli, Salmonella enterica, and Vibrio cholerea are able to propel
themselves through liquids by rotating their helical flagella. The cell's motility is dependent on many
factors such as the viscosity of the medium and concentration of various chemicals. Any enhancements
or obstacles to proper bacterial motility can affect cell lifetimes, reproduction rates, etc.
We are particularly interested in bacterial swimming near an interface with other media (either a
solid or a second fluid). Interfaces have interesting physical characteristics that affect cell motility. We
are studying how different interfaces, including interfaces with different chemical compositions, affect
bacterial motility and accumulation. In our experiments, we find that monotrichous bacterium
Caulobacter crescentus travels in unique swimming trajectories near the air/water interface.
Additionally, we find that the swimming behavior of a large population of swarmer cells is highly
sensitive to adsorption of different classes of molecules. It is unclear whether this behavior has
evolutionarily beneficial impacts on cell motility and growth or if near interface forces hinder certain
biological activities. The wide variety of individual swimming and motility methods used by different
bacterial species could be partially accounted for as coping methods for these interactions. We are also
able to explain the large-scale behavior of a population of cells through examining the forces and
torques acting on individual cells. The goal of our study is to quantitatively define these forces and their
consequences on bacterial motility and accumulation near different surfaces.
Lab: Jay Tang
_____________________
34
BLAST XII
____ Wed. Morning Session
TOWARDS AN INTEGRATED MODEL OF THE CHEMOTAXIS PATHWAY IN BACILLUS subtilis
Christopher Rao, George Ordal, Hanna Walukiewicz, George Glekas, Mathew Plutz, and Payman
Tohidifar
Departments of Biochemistry and Chemical & Biomolecular Engineering, University of Illinois at
Urbana-Champaign
Bacillus subtilis employs three adaptation mechanisms for chemotaxis: chemoreceptor
methylation, the CheC/CheD/CheYp system, and the enigmatic CheV system. While there is some
redundancy between these three systems, we now know that they do not function independently of one
of another. Rather, they may function as integrated unit. E. coli, on the other hand, employs just one
adaptation mechanism involving chemoreceptor methylation. Even then, this system functions
differently in the two bacteria: methylation is selective and antagonistic in B. subtilis whereas it additive
and synergistic in E. coli.
During the past year, we have significantly advanced our understanding of the methylation and
the CheC/CheD/CheYp systems. These results have enabled us to revise and extend our previous
model of the B. subtilis chemotaxis pathway. In particular, our new model is now supported by
extensive biochemical data.
In this talk, I will discuss our recent progress modeling the chemotaxis pathway in B. subtilis.
Using this model we have explored how the selective methylation system functions in B. subtilis. While
the results are still preliminary, our models demonstrate that the receptor complexes must adapt at
least three distinct conformations. A key advance was developing a robust in vitro receptor-coupled
kinase assay. This assay enabled us to characterize the methylation sites on McpB, the paradigm
chemoreceptor for B. subtilis chemotaxis. In addition, this assay has enabled us to characterize
interactions between the methylation and CheC/CheD/CheYp adaptation systems. In particular, we
have determined how CheD affects receptor-coupled kinase activity as a function of the methylation
state. Collectively, these experimental data allowed us to extend our model to account for CheC and
CheD. This new model offers fresh insights on the ultimate question: why does B. subtilis employ
multiple adaptation systems when a single one would suffice?
Lab: Christopher Rao
______________
35
BLAST XII
____ Wed. Morning Session
METHYL ACCEPTING CHEMOTAXIS PROTEIN U OF SINORHIZOBIUM meliloti BINDS THE SEED
EXUDATE COMPONENT, PROLINE
Benjamin A. Webb, Richard Helm, Sherry Hildreth, Angelica Thapa, Birgit E. Scharf.
Virginia Tech, Blacksburg, Va. 24060
Sinorhizobium meliloti can exist as a free-living bacterium using chemotaxis to navigate through
the soil and it can also live an endosymbiotic life with particular host-legume species. Host specificity
has been shown to be determined mainly at the site of root hair infection, involving the exchange of
host plant secreted flavonoids and rhizobial derived nodulation factors [1, 2]. In this study, we
investigate chemotactic signals preceding the established line of communication and show that the
seed exudate profiles of select host and non-host legumes differ significantly. We also demonstrate
that S. meliloti shows strong positive chemotaxis toward the seed exudate of the agriculturally important
host, Medicago sativa (alfalfa). S. meliloti has 8 chemoreceptors that contribute to chemotaxis
including two cytosolic receptors [3]. Methyl accepting Chemotaxis Protein U (McpU) is one of the
transmembrane receptors and plays a major role in the positive chemotaxis toward the seed exudate
studied. McpU is maximally expressed during peak of cellular motility and localizes at the cell pole [4]. It
is predicted to have a cytosolic domain for signal conversion, adaptation, CheA/CheW interaction, and
a relatively large periplasmic region composed of 245 amino acids residues. Previous studies have
shown it to be important for positive chemotaxis toward a wide range of nutrient attractants [3]. Here,
we draw a correlation between the presence of proline in host seed exudates and the strong positive
chemoattraction elicited by this amino acid. Amongst single mcp knock-out mutants, an mcpU deletion
strain exhibits greatly diminished positive response toward seed exudateand proline. Complementation
analyses confirmed that the response of S. meliloti to proline is mediated by McpU. Using isothermal
titration calorimetry, we show that L-proline binds to the periplasmic domain of McpU with a binding
constant of 148μM. These results show that McpU has a positive role in sensing the components of
host seed exudate, suggesting that chemotaxis toward the germinating seed is a key step in the early
establishment of the endosymbiosis.
References
1.
Goedhart J, Bono JJ, Gadella TW, Jr.: Rapid colorimetric quantification of lipochitooligosaccharides from Mesorhizobium loti and Sinorhizobium meliloti. Mol Plant
Microbe Interact 2002, 15(9):859-865.
2.
Phillips DA, Tsai SM: Flavonoids as plant signals to rhizosphere microbes. Mycorrhiza
1992, 1(2):55-58.
3.
Meier VM, Muschler P, Scharf BE: Functional analysis of nine putative chemoreceptor
proteins in Sinorhizobium meliloti. J Bacteriol 2007, 189(5):1816-1826.
4.
Meier VM, Scharf BE: Cellular localization of predicted transmembrane and soluble
chemoreceptors in Sinorhizobium meliloti. J Bacteriol 2009, 191(18):5724-5733.
Lab: Birgit Scharf
__________________
36
BLAST XII
____ Wed. Morning Session
TIME-DEPENDENT CHEMOTACTIC RESPONSE IN A LARGE POPULATION
Laurence G. Wilson*, Rongjing Zhang
The Rowland Institute at Harvard, 100 Edwin H Land Blvd., Cambridge, MA. 02142, USA
We have previously reported techniques for quantifying motility, in terms of straight-line
swimming speed (DDM) and motor speed (DFM) in free-swimming cells. These methods can be used
with any standard microscope equipped with a fast camera (ideally capturing between 100-500 frames
per second). Both techniques have recently benefitted from equipment modifications that have
extended their applicability to faster-swimming organisms; we show data from a number of different
species including E. coli, R. sphaeroides, S. typhimurium. In an extension to one of these techniques
(DDM) we show how the spatial and temporal dependence of chemotactic behavior may be observed.
Although much is now known about the biochemical signaling pathways that allow bacteria to respond
to fluctuating chemostimulant concentration, the implications for the group behavior of cells are
unknown. Previous microscopic studies have focused on single-cell behavior to build up a picture of
how cells adapt to changing environmental conditions. We use our new methods to provide a
comprehensive and highly automated characterization of collective bacterial behavior - around 5000
cells simultaneously - in the presence of a chemical stimulus.
Lab: Laurence Wilson
______________
37
BLAST XII
___ Thurs. Morning Session
DETECTING A CONFORMATIONAL CHANGE IN THE PERIPLASMIC REGION OF THE SODIUMDRIVEN STATOR PROTEIN PomB BY THE DISULFIDE CROSSLINK
Shiwei Zhu1, Masato Takao2, Na Li1, Michio Homma1, Katsumi Imada2 and Seiji Kojima1
1
Division of Biological Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku,
Nagoya 464-8602, Japan; 2Graduate School of Science, Osaka University, 1-1 Machikaneyama,
Toyonaka, Osaka 560-0043, Japan
Bacterial flagellar motor consists of the rotor and stator, and their interaction that is coupled with
the ion flux through the stator generates torque. The ion flux activity of the stator is activated only when
it assembled around the rotor. Based on the crystal structure and functional analysis of the periplasmic
region of the H+-driven stator protein MotB from Salmonella, a hypothesis of the stator activation is
proposed: during the assembly step, a stator will undergo conformational change in the periplasmic
region of the B subunit to activate the proton channel (Kojima et al, 2009). To test this model, we
adopted disulfide crosslinking approach for the Na+-driven stator protein PomB from Vibrio, whose
crystal structure of its periplasmic region (PomB C ) has recently been solved. If an intramolecular
disulfide bridge is formed between predicted movable elements, it will suppress this conformational
change, and consequently inhibit the function of the stator. Based on the structural information of
PomB C , we introduced several pairs of cysteine replacements into cysteine-less PomB, one at the α1helix (residues, 154-169) and the other at the core domain (residues, 170~). As a result, we found that
the pair of cysteine replacements M157C-I186C and that of I164C-L217C inhibited the function of the
stator without affecting its stability. Addition of the reducing agent DTT in the medium rescued quickly
the function of these stators. These results support the model that a large conformational change in the
PomBC is required for stator activation.
Kojima S, Imada K, Sakuma M, Sudo Y, Kojima C, Minamino T, Homma M, Namba K. Mol. Microbiol.
(2009) 73, 710-718.
Lab: Michio Homma
________________
38
BLAST XII
___ Thurs. Morning Session
COORDINATED SWITCHING OF BACTERIAL FLAGELLAR MOTORS: EVIDENCE FOR DIRECT
MOTOR-MOTOR COUPLING?
Bo Hu and Yuhai Tu
IBM T.J. Watson Research Center, Yorktown Heights, NY, 10598, USA
The swimming of Escherichia coli is propelled by its multiple flagellar motors. Each motor spins
either clockwise (CW) or counterclockwise (CCW), under the control of an intracellular regulator, CheYP. A long standing question is whether these motors work independently or not. There can be two
mechanisms (extrinsic and intrinsic) to coordinate the switching of bacterial motors. The extrinsic one
arises from the fact that different motors in the same cell sense a common input (CheY-P) which
fluctuates near the motors' response threshold. An alternative, intrinsic mechanism is direct motormotor coupling which makes synchronized switching energetically favorable. Here, we develop simple
theoretical models for both mechanisms and uncover their different hallmarks. A quantitative
comparison to the recent experiments suggests that the direct coupling mechanism may be responsible
for the observed sharp correlation between motors in a single E. coli. We discuss possible origins of
this coupling (e.g., hydrodynamic interaction) and present some feasible experimental suggestions.
Lab: Yuhai Tu
_____________________
39
BLAST XII
___ Thurs. Morning Session
H. pylori FLAGELLAR MOTOR: STRUCTURAL INSIGHT INTO THE MECHANISM OF STATOR
ASSEMBLY AND ACTIVATION
Daniel A. Andrews, Jenna O'Neill, Matthew C. Wilce and Anna Roujeinikova
Department of Biochemistry and Molecular Biology and Department of Microbiology, Monash
University, Clayton, Victoria 3800, Australia.
Carcinogenic bacterium Helicobacter pylori uses the flagellar motor to drill into the mucus layer
of the stomach and move towards the epithelial surface, where it colonizes [1]. Flagellar rotation is
powered by the proton influx through the stator ring MotA/MotB [2,3]. The work presented at the
meeting will inform the audience about the latest discoveries of novel factors that mediate stator/rotor
interaction [4,5] and our latest findings that establish the relationship between the structure, dynamics
and function of MotB. We have determined the first crystal structure of the protein domain that anchors
the proton-motive-force-generating mechanism of the bacterial flagellar motor to the cell wall, and
formulated a model of how the stator attaches to peptidoglycan (PG) [6,7]. Analysis of the accumulated
structural, biochemical and mutagenesis data suggested a mechanism by which MotB’s cell wall
binding activity is inhibited until the stator is incorporated into the motor [8]. Putting this in the
perspective of the stator assembly and activation, we propose that in the pre-assembled stator units
with the closed channel the linker is partially folded, stabilizing the dimer in the conformation that
cannot insert into the PG mesh. This inhibition occurs via a dual mechanism: distance constraint and
suboptimal geometry of two PG-binding sites. When the diffusing stator unit collides with the rotor, a
drastic conformational change in MotB is induced, resulting in unwinding of the two beta strands at the
edge of the beta sheet, extension of the linker, re-arrangement of the dimer geometry and insertion of
the Pal-like conserved core domain into PG mesh. Implications for stator activation via rotational
displacement of the transmembrane helices of MotB will be discussed.
References:
[1] Yoshiyama, H. & Nakazawa, T. (2000). Microbes Infect. 2, 55-60.
[2] Kojima, S. & Blair, D.F. (2004) Biochemistry 42, 26-34.
[3] Blair, D.F. & Berg, H.C. (1988) Science 242, 1678-1681.
[4] Blair, K.M., Turner, L., Winkelman, J.T., Berg, H.C. & Kearns, D.B. (2008) Science 320, 1636-1638.
[5] Boehm, A., Kaiser, M., Li, H., Spangler, C., Kasper, C.A. et al. (2010) Cell 141, 107-116.
[6] Roujeinikova, A. (2008). Proc. Natl. Acad. Sci. USA, 105, 10348-10353.
[7] Reboul, C. F., Andrews, D. A., Nahar, M. F., Buckle, A. M. & Roujeinikova, A. (2011). PLoS ONE, 6,
e18981.
[8] O'Neill, J., Xie, M., Hijnen, M. & Roujeinikova, A (2011). Acta Cryst. D67, 1009-1016.
Lab: Anna Roujeinikova
____________
40
BLAST XII
___ Thurs. Morning Session
CORRELATED MUTATION ANALYSIS OF THE FLAGELLAR MOTOR PROTEIN FliG
Shahid Khan%, Jens Kleinjung# and William R. Taylor#
%
Molecular Biology Consortium @ Lawrence Berkeley National Laboratory, Berkeley, CA 94720 USA
#
Division of Mathematical Biology, MRC National Unit for Medical Research, London MW7 1AA, UK
We are applying correlated mutation analysis (CMA), an emerging technique for the analysis of
protein-protein interactions, to the architecture of the bacterial flagellar basal body. The flagellar basal
body is a multi-component, multi-subunit assembly embedded in the cytoplasmic membrane. It harbors
machinery for flagellar assembly, energization and switching of flagellar rotation. The membrane
protein FliF forms rings (MS-rings) that subsequently direct the assembly of the cytoplasmic C ring.
Binding of the chemotaxis signal protein CheY to the C ring protein FliM switches rotation sense. There
is a mismatch between the MS and C ring subunit stoichiometry. The mismatch is accommodated by
the protein FliG that lies at the interface between the MS and C rings. FliG interacts with the force
generating Mot membrane protein complexes, in addition to FliF and FliM. An atomic model of FliG and
its binding partners within the MSC ring will be important for elucidation of the motility mechanism. CMA
is one approach towards this goal.
FliG is the most, well characterized protein of the flagellar basal body. FliG subunit
stoichiometry has approximated from purified basal bodies. Membrane permeable cross-linkers, affinity
chromatography and suppressor mutations have identified FliG interaction surfaces responsible for selfassociation and binding to FliF, FliM and MotA.
FliG has been localized to basal bodies by
immunoelectron microscopy. Native and mutant FliG X-ray crystal structures are available, including a
complex of the FliG and FliM interaction domains. The structures have been fit to low resolution
electron cryomicroscopy maps of native basal bodies and overproduced intact MSC rings. These data
have mainly been obtained in the enteric bacteria Escherichia coli and Salmonella typhimurium; with Xray protein structures from the thermophiles Aquifex aeolicus and Thermatoga maritima.
Flagellar motility is widespread in prokaryotes. A phylogenetic analysis is useful for revealing
conserved design features. The FliG phylogenetic tree, based on over two thousand sequences,
revealed several instances of horizontal gene transfer, particularly in spirochetes. Representative
species covering phylogenetic spread were used to seed multiple sequence alignments. Where
available, Pfam alignments were used. The alignments were used to generate model structures based
on secondary structure prediction and correlation analysis for identification of residue contacts1. The
top ranked models were validated by comparison with the structures of the two essential domains; the
middle and carboxy-terminal domain. There are a number of reasons for false positives. These include
artifacts arising from partial correlation as well as coupling due to function or self-association. Algorithm
development has entailed elimination of partial correlations, utilization of filters based on the
biochemical data and bootstrap analysis to assess variation. A model of FliG interaction surfaces and
conformational transitions based on our CMA will be presented as well as ongoing developments of the
method for its application to flagellar basal body architecture.
------------------------------------------------------------------------------------------------------------------1.
Taylor, W. R. & Sadowski, M. I. (2011). Structural constraints on the covariance matrix derived
from multiple aligned protein sequences. PLoS One 6, e28265.
Lab: Shahid Khan
41
BLAST XII
___ Thurs. Morning Session
CORRELATED STRUCTURE AND ACTIVITY OF THERMOTOGA maritima FLIY
Ria Sircar, Anna R. Greenswag, Alexandrine M. Bilwes, Gabriela G. Bonet, and Brian R. Crane
Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14850
In bacterial chemotaxis, phosphorylated CheY levels dictate the direction of flagellar rotation.
Switching between the counter-clockwise and clockwise directions dictates whether the bacteria will
swim smoothly or tumble. For robust chemotaxis, the CheY-P signal must be terminated so that the
cells can return back to the prestimulus level. In E.coli this function is accomplished by the phosphatase
CheZ. However, in Thermotoga maritima, Bacillus subtilis and many other bacteria the CYX family of
phosphatases, comprising of CheC, CheX and FliY, fulfills this role. Here we report the crystallographic
structure of the last member of this family to have its structure determined: FliY. FliY, unlike CheC and
CheX is an integral component of the flagellar rotor. FliY is a multidomain protein with a N-terminal
CheY binding domain, a central CheC like domain and a C-terminal domain homologous to the
conserved rotor protein FliN. The structure of the FliY middle domains is similar to CheC and the rotor
protein FliM. Previous studies have identified two putative active sites on FliY to involve E35/N38 and
E132/N135. We mutated these residues to assess their contribution to phosphatase activity in T.
maritima FliY. We find that both the active sites are individually capable of dephosphorylating CheY but
the second active site (E132/N135) is more reactive. Interactions of FliY with other rotor proteins were
investigated and shown to have implications for rotor structure and function in FliY-containing bacteria.
Lab: Brian Crane
__________________
42
BLAST XII
___ Thurs. Morning Session
BORRELIA burgdorferi FLAGELLA EXPORT APPARATUS AND VIRULENCE: INSIGHT INTO TYPE
III SECRETION SYSTEM
Tao Lin, Lihui Gao, Xiaowei Zhao, Jun Liu, and Steven J. Norris
UT Medical School at Houston, PO Box 20708, Room MSB 2.124, Houston, TX 77225-0708
The mechanisms of pathogenesis in Borrelia burgdorferi are largely unknown. The ability of this
spirochete to migrate to distant sites in the tick and mammalian host is likely dependent on a robust
chemotaxis response and motility. B. burgdorferi contains multiple copies of the chemotaxis genes
cheA, cheB, cheR, cheW, cheX, and cheY. Multiple chemotaxis proteins may provide diversity in terms
of function and/or structural location, or may be differentially expressed under varied physiological
conditions. Proteins involved in motility are encoded by 38 genes including 36 flagellar & motor genes
and 2 flagellin genes, whereas chemotaxis proteins are encoded by 20 genes including 15 chemotaxis
genes and 5 chemotaxis receptor genes. To dissect the mechanism of Borrelia chemotaxis and motility
and the relationship between the virulence and chemotaxis/motility, we examined Himar1 transposon
mutants in 24 chemotaxis/motility genes including 10 motility genes, 10 chemotaxis genes and 4
chemotaxis receptor genes in the transformable and infectious B. burgdorferi strain 5A18NP1. The
infectivity of these mutants was determined using a signature-tagged mutagenesis (STM) procedure in
C3H/HeN mice and a newly developed, high throughput Luminex procedure. Among the mutants in 24
genes tested, mutations in flgI, flgJ, and cheW-3 exhibited reduced infectivity. Mutants in the 21 genes
(cheW-1, fliG-1, fliZ, fliH, fliI, flgB, flaA, flbA, cheA-1, cheA-2, cheB-1, cheB-2, cheY-1, cheR-2, cheW-2,
cheX, cheY-2, mcp-1, mcp-3, mcp-4, and mcp-5) showed no infectivity, indicating that these genes are
required for full infectivity in B. burgdorferi. In examining morphology under dark-field microscopy, 18
mutants did not exhibit obvious morphologic defects. Seven mutants (fliH, fliI, flbA, flaA, cheA-2, cheB2, and cheR-2) exhibited elongated, string-like and/or rod-shaped morphology. In terms of motility,
mutations in 13 genes did not induce obvious motility defects, whereas 7 mutants (flaA, flgI, fliG-1, fliW1, cheA-2, cheR-2, and mcp-5) showed a reduced motility, and string-like mutants (flbA, fliH, and fliI)
were nearly non-motile, ‘trembling’ in a few sites of the cell. The string-shaped or rod-shaped mutants
often bend at the cell center. The swimming ability of these mutants was evaluated by measuring their
velocity in a highly viscous medium containing 1% methylcellulose. Seven mutants (fliH, fliI, flaA, flbA,
flgI, cheX, and mcp-4) appeared to be incapable of translating motion, 8 mutants (fliG1, fliW-1, cheA-2,
cheR-2, cheW-2, cheW-3, cheY-2, and mcp-5) exhibited reduced motility, and inactivation of 6 genes
(flgJ, fliZ, cheA-1, cheB-1, cheB-2, mcp-1, and mcp-3) did not induce decreased motility in 1%
methylcellulose medium. FliI and FliH, which are central to export apparatus function, appear to be
required for mouse infection. fliH and fliI mutants also had reduced motility, growth and division defects
(resulting in elongated organisms), and structural changes in the flagellar motor. Disruption of either fliH
and fliI also resulted in altered cellular protein profiles; we are in the process of identifying the proteins
that are underrepresented in the mutants. Inactivation of fliI and fliH genes resulted in the loss of the
FliH/FliI complex as determined by Cryo-EM. These results represent the initial characterization of the
flagellar export apparatus of B. burgdorferi, and continued studies will provide insight into export
apparatus functions in this organism.
Lab: Steven Norris
________________
43
BLAST XII
___ Thurs. Morning Session
DYNAMIC CONFORMATIONAL CHANGES OF FLAGELLAR FILAMENT OBSERVED BY HIGHPRESSURE MICROSCOPY
Masayoshi Nishiyama1,2 and Yoshiyuki Sowa3
1
The Hakubi Center, Kyoto University, Kyoto 606-8302, Japan
2
WPI-iCeMS Complex2, Kyoto University, Kyoto 606-8501, Japan
3
Department of Frontier Bioscience, Hosei University, Tokyo 184-8584, Japan
The bacterial flagellar motor is a molecular machine that rotates a flagellum in both directions.
CCW rotation allows the left-handed helical filaments to form a bundle that propels the cell smoothly,
whereas CW rotation of a filament leads to change the shape of filament in right-handed helix and
break the bundle, and inhibits smooth swimming of the cell, called a tumble. The switching in the helical
structure is thought to be caused by directional mechanical actions arising from abrupt change of
exerted torque by the motor rotation. Here, we show that application of pressure can also change the
helical structure of flagellar filaments. The flagellar filaments in E. coli cells were fluorescently labeled,
and then the images were acquired by using a high-pressure microscope [1, 2] with some
modifications. We measured the diameter and pitch of the individual filaments and then classified them
into 11 possible waveforms which are predicted from structural data. At 0.1MPa (ambient pressure), all
flagellar filaments formed left-handed helical structure (normal form). At 40 MPa, we found left-handed
forms (normal and coiled forms) and right- (curly I (or II)). At 80 MPa, 80% flagellar filaments took curly
I (or II) forms. After the pressure was released, most filaments returned to the initial left-handed
structures. The application of pressure is thought to enhance the structural fluctuation and/or
association of water molecules with the exposed regions of flagellin molecules, and results in switching
the helical from left- to right-handed structure.
[1] Nishiyama M. and Y. Sowa. 2012. Microscopic Analysis of Bacterial Motility at High Pressure.
Biophys. J. 102:1872-1880.
[2] Nishiyama M. and S. Kojima. 2012. Bacterial motility measured by a miniature chamber for highpressure microscopy. Int. J. Mol. Sci. 13:9225-9239.
Lab: Masayoshi Nishiyama
_______________
44
BLAST XII
___ Thurs. Morning Session
NOVEL LEPTOSPIRA PROTEIN IS ESSENTIAL FACTOR IN DETERMINING THE FLAGELLAR
SHEATH, TRANSLATIONAL MOTILITY, AND VIRULENCE PHENOTYPE
Elsio A. Wunder Jr.1,2, Cláudio P. Figueira2, Nadia Benaroudj3, Mitermayer G. Reis2, Nyles Charon4,
Mathieu Picardeau3, and Albert I. Ko1,2
1
Yale School of Public Health and Medicine, New Haven, USA; 2Gonçalo Moniz Research Center,
Oswaldo Cruz Foundation, Brazilian Ministry of Health, Salvador, Brazil; 3Unité de Biologie des
Spirochètes, Institute Pasteur, Paris, France; 4West Virginia University, Department of Microbiology and
Immunology, Morgantown, USA
Background: Leptospires, the causative agent of leptospirosis, are unique among spirochetes and
bacteria as a whole in that they have hooked-shaped ends and coiled periplasmic flagella, both of
which are required for translational motility. However, the factors which produce these morphological
phenotypes and the role of motility plays in leptospiral virulence have not been evaluated.
Methods and Findings: We identified small and large colonies on agar when plating a Leptospira
interrogans serovar Copenhageni isolate from a Brazilian patient with pulmonary hemorrhage
syndrome. Leptospires from small colonies had straight terminal ends, and although their ends gyrated
and produced spiral waves, they were unable to produce translational motility. Furthermore, purified
periplasmic flagella from motility-deficient clones were straight in contrast to the coiled structure
observed in those from motile clones and wild-type (wt) strains. LC-MS analysis of purified flagella and
genomic DNA sequencing identified that the motility-deficient clones had a mutation that disrupted the
gene encoding a novel 36kDa protein, which we called Flagella coil protein 1 (Fcp1). Fcp1 protein was
distributed throughout the length of wt flagella. Cryo-electron tomography determined that the flagellar
diameter in situ of motility-deficient clones were significantly smaller than that of motile clones
(17.6±0.97 vs 22.8±1.99mm). We produced fcp1- mutants by performing allelic exchange in a wt strain
and found that these mutants showed similar loss of terminal hook end morphology, coiled periplasmic
flagellar structure, and translational motility, as did motility-deficient clone. We rescued these
phenotypes by complementing motility-deficient clone and fcp1- mutants with the wt fcp1 gene. Finally
disruption of fcp1 was associated with inability to penetrate epithelial tissue barriers and loss of
virulence during experimental infection of hamsters while complementation of the gene restored these
phenotypes.
Conclusion: In summary, we identified a novel flagella structural protein unique to Leptospira and
required for the formation of the flagellar sheath, and to maintain its coiled structure of periplasmic
flagella and hook-shaped morphology, which allows the pathogen to perform translational motility.
Moreover our studies provide the first evidence which use genetic knockout and complementation
approaches to demonstrate that translational motility, a classic attribute of leptospires and pathogenic
spirochetes, is essential for virulence.
Support: NIAID 5 U01 AI088752, and 5 R01 AI052473, and FIC 7 D43 TW00919
Lab: Albert Ko
____________________
45
BLAST XII
___ Thurs. Evening Session
INTERACTIONS BETWEEN A TRANSPORTER AND A SENSOR KINASE MEDIATE SIGNAL
TRANSDUCTION IN ANTIMICROBIAL PEPTIDE DETOXIFICATION MODULES
Sebastian Dintner, Susanne Gebhard
Ludwig-Maximilians-University Munich, Department Biology I, Microbiology, Grosshaderner Str. 2-4,
82152 Martinsried, Germany
Resistance of many Firmicutes bacteria against antimicrobial peptides is mediated by so-called
detoxification modules that are comprised of a two-component regulatory system (TCS) and an ATPbinding-cassette (ABC) transporter. Strikingly, the transporters and TCSs have an absolute and mutual
requirement for each other in both sensing of and resistance against their respective antimicrobial
compounds: expression of the transporter is regulated by the TCS, yet the sensor kinase is unable to
detect a stimulus in the absence of an active transporter. These findings suggest a novel mode of
signal transduction where the transporter constitutes the actual sensor of antimicrobial peptides.
Database searches revealed the wide-spread occurrence of such modules among Firmicutes bacteria,
and parallel phylogenetic analysis showed that transporters and TCSs have co-evolved. Based on
these findings, we hypothesize the formation of a sensory complex between both components. In vivo
and in vitro approaches studying the bacitracin-resistance module BceRS-BceAB of Bacillus subtilis
support this hypothesis by indicating direct physical contacts between the sensor kinase, BceS, and the
transport permease, BceB. Furthermore, random mutagenesis of BceB revealed that resistance and
signaling are genetically separable traits of the transporter, showing that signal transduction is indeed
mediated by the transporter directly and is not a secondary effect of substrate translocation. Taken
together, our results show that Bce-type ABC-transporters and TCSs have co-evolved to form selfsufficient detoxification modules against antimicrobial peptides, and suggest a novel signaling
mechanism involving formation of a sensory complex between transporter and sensor kinase.
Lab: Susanne Gebhard
_____________
46
BLAST XII
___ Thurs. Evening Session
A NOVEL TRANSLATIONAL REGULATION MECHANISM MEDIATED BY ELONGATION FACTOR
EF-P ALLOWS TIGHT ADJUSTMENT OF PROTEIN COPY NUMBER
Lassak, J.1,2,5, Ude, S.1,2,5, Starosta, A.L.1,3, Kraxenberger, T.1,2,4, Wilson, D.N.1,3, and Jung, K.1,2*
1
Center for integrated Protein Science Munich (CiPSM)
2
Department of Biology I, Microbiology, Ludwig-Maximilians-Universität München, Großhaderner
Strasse 2-4, 82152 Martinsried, Germany
3
Gene Center and Department of Biochemistry, Ludwig-Maximilians-Universität München, FeodorLynen-Str. 25, 81377 Munich, Germany
4
Present address: AMSilk GmbH, Am Klopferspitz 19, 82152 Martinsried, Germany
5
These authors contributed equally to this work
Elongation factor P (EF-P) binds to the ribosome and stimulates peptide-bond formation. It has
a ubiquitous distribution, being conserved in all bacteria and orthologous to archaeal and eukaryotic
initiation factor 5A (a/eIF-5A). E. coli EF-P is post-translationally β-lysinylated by the sequential action
of YjeK, YjeA. Inactivation of the efp, yjeA or yjeK genes results in defects in bacterial growth, fitness
and membrane integrity, as well as stress resistance and virulence. Despite the conservation and
importance of EF-P, the biological function has remained enigmatic. We found that EF-P relieves
ribosome stalling at polyproline stretches. We further demonstrate the relevance of EF-P in vivo to
control the protein copy number and stress response of the polyproline-containing pH-receptor CadC.
The E. coli proteome contains 95 proteins with a polyproline stretch, including the histidine kinases
EnvZ and PhoR and the flagella master regulator FlhC. Our new findings now rationalize the observed
defects in motility and attenuated virulence in efp mutants. Bacterial, archaeal and eukaryotic cells
contain 100-1000’s of polyproline-containing proteins of diverse function and we suggest that EF-P
plays a central role for the tight copy number adjustment of various pathways across all kingdoms of
life.
Lab: Kirsten Jung
_________________
47
BLAST XII
___ Thurs. Evening Session
TRANSCRIPTIONAL ACTIVATION AND REPRESSION OF SIX flhDC PROMOTERS: AN INTRICATE
PROMOTER CONFIGURATION FOR flhDC IN SALMONELLA typhimurium
Chakib Mouslim and Kelly Hughes
University of Utah
In Salmonella typhimurium, the transcriptional factor FlhDC is involved in coordinating the
expression of flagellar genes and pathogencity island 1 (Spi1). Transcription of flhDC is controlled
negatively and positively by multiple regulatory factors including, RcsB, LrhA, SlyA, DskA, EcnR, HNS,
cAMP/CRP, CsrA, PefI-SrgD, HilD, RtsB and QseB. In Salmonella, the flhDC promoter region contains
6 transcriptional start sites that drive the transcription of the flhDC operon. The detailed mechanism of
this regulation and the mechanism by which these transcriptional factors integrate signals to regulate
the expression of flhDC are largely unknown. We characterized individual promoters within the flhDC
promoter region. We investigated the effect of the six individual promoters and their role in flhDC
transcription in addition to the effect of the different transcriptional regulators on each promoter. These
promoters can respond differently to growth conditions that activate specific regulators leading to the
flexibility in the adjustment of flhDC expression. Depending on their arrangement these multiple
promoters drive transcription on their own or interact with each other. These interactions particularly
coupled with the effect of selective transcriptional regulators generate regulatory circuits between
expression of flagellar genes and Spi1.
Lab: Kelly Hughes
_________________
48
BLAST XII
___ Thurs. Evening Session
ESCHERICHIA COLI BIOFILMS MAY FAVOR A MIXTURE OF MOTILE AND NON-MOTILE
BACTERIA
Shelley M. Horne, Joseph Sayler, Ty Lynnes, Priyankar Samanta, & Birgit M. Prüβ*
Department of Veterinary and Microbiological Sciences, North Dakota State University, Fargo ND
To shed light into the ongoing controversy on motility constituting an advantage or disadvantage
for Escherichia coli in a biofilm, we performed two long-term experiments. First, we recovered colonies
from 7 and 14 days old biofilms, formed by the partially motile and good biofilm forming E. coli K-12
strain AJW678 and the highly motile, poor biofilm former MC1000. Up to 5% of the colonies that were
recovered from either parental strain had altered motility. In the case of AJW678, the motility phenotype
changed to hyper motile. For MC1000, motility changed to entirely or partially non-motile. Many of the
MC1000 derived non-motile colonies contained insertions of IS1 in their flhD promoter or the open
reading frame of FlhC. Some of these colonies produced slightly more biofilm biomass than their
MC1000 parent.
To further investigate the moderate inverse correlation between motility and biofilm biomass, we
performed a competitive experiment, where MC1000 and its isogenic flhD::kn mutant were inoculated
at a 1:1 ratio. Biofilms maintained a mixture of both bacterial populations over the first two weeks post
inoculation. Towards the end of week 4, the flhD mutant took over the population. In a spatial
experiment, flhD was expressed higher at the top layer of the biofilm than at its bottom. Altogether, we
will come to the conclusion that E. coli biofilms may need both, parent bacteria and flhD mutants for
optimal functionality, at least during the first two weeks of their formation. Motility may constitute an
advantage in certain areas of the biofilm.
Lab: Birgit. Prüβ
__________________
49
BLAST XII
___ Thurs. Evening Session
Rrp1 REGULATES CHITOBIOSE UTILIZATION OF THE LYME DISEASE SPIROCHETE BORRELIA
burgdorferi
Ching Wooen Sze1, Alexis Smith3, Young Hee Choi2, Xiuli Yang3, Utpal Pal3, Aiming Yu2, and
Chunhao Li1*
Department of Oral Biology, The State University of New York at Buffalo, Buffalo, New York1,
Department of Pharmaceutical Sciences, The State University of New York at Buffalo, Buffalo, New
York2,
Department of Veterinary Medicine, University of Maryland and Virginia-Maryland Regional College of
Veterinary Medicine, College Park, Maryland3
Life cycle alternating between arthropod and mammals forces the Lyme disease spirochete,
Borrelia burgdorferi, to adapt to different host milieus by utilizing diverse carbohydrates. B. burgdorferi
lacks the de novo biosynthesis pathway of N-acetylglucosamine (GlcNAc) but encodes a set of
chitobiose metabolic genes that allows conversion of chitobiose to GlcNAc. The chbC gene, a key
chitobiose transporter, is dually regulated by RpoD and RpoS. Rrp1 is a response regulator that
synthesizes cyclic diguanylate (c-di-GMP) in B. burgdorferi. In this report, we found that the rrp1 mutant
had growth defects and formed membrane blebs when GlcNAc was replaced by chitobiose in growth
medium. The expression level of chbC was significantly repressed in the mutant and constitutive
expression of chbC successfully reversed the phenotype. Immunoblotting and transcriptional studies
revealed that Rrp1 is required for the activation of BosR and its impact on chbC is likely mediated by
the BosR-RpoS signaling pathway. Although the rrp1 mutant was unable to be transmitted via tick bite,
exogenous supplementation of GlcNAc into unfed ticks partially rescued the transmission. Based on
these results, we propose that Rrp1 governs the chitobiose metabolism of B. burgdorferi via a BosRRpoS-ChbC regulatory network essential for the transmission of the spirochete.
Lab: Chunhao Li
__________________
50
POSTER ABSTRACTS
51
BLAST XII
________________ Poster #1
STRUCTURE AND FUNCTION OF CHEMORECEPTORS AND THE ROLE OF MULTIPLE CheW
HOMOLOGUES IN RHODOBACTER SPHAEROIDES
James R. Allen and Judith P. Armitage
Department of Biochemistry, South Parks Road, Oxford, OX1 3QU
Rhodobacter sphaeroides is a model organism for investigating complex chemosensory
pathways now identified in many bacterial species. The proteins that make up the complex chemotactic
system of R. sphaeroides are encoded over 3 distinct major operons as well as additional other smaller
loci. 13 chemoreceptors are encoded in the genome; 9 localise to a membranous cluster much like that
seen in Escherichia coli (known as Methyl-accepting Chemotaxis Proteins or MCPs) while 4 have no
transmembrane regions and localise to a cytoplasmic chemosensory cluster (these are designated
Transducer-like proteins or Tlps). Previous analysis of the roles of individual MCP homologues as has
proved difficult. A new approach is needed if the complexity of signalling through multiple receptors is to
be analysed in full.
R. sphaeroides also contains 4 homologues of the scaffold protein CheW and 4 homologues of
the scaffold and phosphotransfer protein CheA. CheW 2 , CheW 3 and CheA 2 have been shown to be
required to localise the MCP receptors into a tight, cooperative signalling array while CheW 1 and
CheA 1 are not involved. An additional CheW, CheW4, localises to the cytoplasmic cluster, but not to
the membrane cluster, even when over expressed. All R. sphaeroides CheWs are able to complement
E. coli deletions. Why are 3 CheW homologues required in R. sphaeroides? Structural analysis of the
MCPs suggests that most fall into the 34H category (designating the number of heptad repeats and
thus length of cytoplasmic domain) with MCPA being 36H and MCPM being 38H.
Previous deletion studies of the R. sphaeroides MCPs also showed that once a certain subset
had been deleted, no tight receptor cluster could be observed with CheWs still present. This not only
prevented individual MCPs from being tested for their activity to various substrates but also suggests
complex interactions with to the scaffold.
In order to analyse the binding of the MCPs to individual components of the scaffold and, in
turn, analyse their substrate specificity, a new approach is needed. The E. coli receptor deletion mutant
UU2612 (courtesy of S Parkinson) contains all of the E. coli scaffold proteins but has been deleted for
all known E. coli MCPs. Plasmid-based expression of individual MCPs has shown that E. coli scaffold
proteins are insufficient for full localisation of R. sphaeroides MCPs tagged with RFP. Co-expression
with R. sphaeroides CheW proteins shows that MCPB and MCPJ can localise into a tight cluster using
CheW3, indicative of that seen in the wild type system. CheW2 is unable to localise these MCPs. This
shows that UU2612 containing the R. sphaeroides CheW scaffold is a viable approach to assess CheW
specificity and cluster formation. We will describe recent data on the structure and function of the
R. sphaeroides chemoreceptors and the roles of the CheWs.
Lab: Judith Armitage
______________
52
BLAST XII
STRUCTURE-FUNCTION STUDIES
RHODOBACTER SPHAEROIDES
________________ Poster #2
OF
THE
RESPONSE
REGULATOR
CheY 6
FROM
Matthew W Smith, Nick Delalez, Christina Redfield, Judy Armitage
University of Oxford Oxford Centre for Integrative Systems Biology New Biochemistry Building South
Parks Road Oxford OX3 1QU
Rhodobacter sphaeroides has a complex chemosensory network which comprises multiple
homologous genes and involves metabolic sensing (also known as energy taxis). R. sphaeroides has
two different flagellar motors but only one is expressed under laboratory conditions (Fla1). Unlike most
bacteria, the expressed motor displays a stop-and-go phenotype. R. sphaeroides genome also
encodes six different CheY proteins but only three (CheY 3 , CheY 4 and CheY 6 ) have been shown to
interact with the Fla1 motor. Deletions of CheY 3 , CheY 4 and CheY 6 result in the flagellar motor being
unable to stop (smooth swimming). CheY 6 has been shown to be essential for chemotaxis whereas
CheY 3 and CheY 4 have some functional redundancies. Although CheY 6 can stop the motor alone, R.
sphaeroides requires the presence of either CheY 3 or CheY 4 to be chemotactic. To date, not much is
known about how these three CheY proteins interact to stop the flagellar motor.
Structural studies of CheY 6 using NMR highlighted a flexible loop region (residues G109-K118)
that is not present in the other CheYs. It was hypothesised that this flexible loop might confer to CheY 6
a different mechanism compared to other CheY proteins. Deletion mutants were made to investigate
the role of this loop in CheY 6 function. Phenotypical studies were performed using swim plates and
tethering assays. The mutants showed smooth swimming but wild type phenotype was restored upon
expression of wild type CheY 6 from plasmid, indicating that CheY 6 ∆loop is unable to stop the motor.
However, results also revealed that CheY 6 ∆loop is able to compete with wild type CheY 6 for
phosphorylation by the kinase protein CheA 3 . Current studies using phosphotransfer assays and NMR
aim at further characterising the mechanism of CheY 6 function in the Fla1 chemotaxis pathway. The
results of these studies will be presented and the importance of the findings discussed.
Lab: Judith Armitage
___________
53
BLAST XII
________________ Poster #3
DECIPHERING THE ROLE OF THE PERIPLASMIC DOMAIN OF PilJ: MECHANISMS OF SIGNAL
TRANSDUCTION
Vishwakanth Y. Potharla, Geoff T. Riddell, and Sonia L. Bardy
Department of Biological Sciences, University of Wisconsin-Milwaukee, Milwaukee WI 53211
The Chp chemosensory cluster is one of four chemosensory signal transduction systems
in Pseudomonas aeruginosa. While this signal transduction pathway has long been recognized to
regulate twitching motility (via type IV pili) (1), it has recently been discovered to regulate intracellular
levels of cAMP, by modulating activity of the adenylate cyclase CyaB (2). Intracellular levels of cAMP
correlate with levels of pilin on the cell surface, but not pilin function. Encoded within the chp gene
cluster is one MCP, two CheW-like proteins, one CheA-CheY hybrid, a methyltransferase and
methylesterase and two response regulator proteins. Mutational analysis of the sole MCP (PilJ) was
used to investigate the role of PilJ in signal transduction through the Chp chemosensory system.
Deletion of the putative periplasmic domain of PilJ resulted in a reduction of intracellular cAMP levels.
This reduction was equivalent to the reduction of cAMP seen in a ΔpilJ strain, which is expected to
have a non-functional Chp system. In contrast, deletion of the periplasmic domain reduced, but did not
inhibit twitching motility, although strain dependent differences were observed for this phenotype.
Furthermore, deletion of the periplasmic domain did not prevent directional twitching in response to the
chemoattractant (18:1) phosphatidylethanolamine (PE).
Replacement of PilJ periplasmic and
transmembrane domains with those from Tsr resulted in only a minor reduction in twitching motility and
surface piliation. Our results suggest that neither the periplasmic domain nor the transmembrane
domains of PilJ are essential for twitching motility, or directional twitching towards PE, but that the
periplasmic domain/protein conformation has a significant effect on cAMP levels.
(1) Darzins, A. 1994. Characterization of a Pseudomonas aeruginosa gene cluster involved in pilus
biosynthesis and twitching motility: sequence similarity to the chemotaxis proteins of enterics
and the gliding bacterium Myxococcus xanthus. Mol. Microbiol. 11:137-53.
(2) Fulcher, N., P. Holliday, E. Klem, M. Cann, and M. Wolfgang. 2010. The Pseudomonas
aeruginosa Chp chemosensory system regulates intracellular cAMP levels by modulating
adenylate cyclase activity. Mol. Microbiol. 76:889-904.
Lab: Sonia Bardy
________________________
54
BLAST XII
________________ Poster #4
PHOTOSENSORY PROTEIN BphP1 INITIATES AN UNKNOWN SIGNAL TRANSDUCTION
PATHWAY THROUGH PHOSPHOTRANSFER TO Psyr_2449 IN PSEUDOMONAS SYRINGAE
B728A
Regina S. McGrane, Liang Wu, and Gwyn A. Beattie
Department of Plant Pathology and Microbiology, Iowa State University, Ames, IA 50010
Many organisms perceive and respond to light and thus require photosensory proteins.
Photosensory proteins can be grouped into six families based on the specific wavelength that induces a
conformational change in the chromophore. Until recently, the study of light-induced signaling pathways
has been limited to plants, animals, fungi and cyanobacteria. However, with the availability of
sequenced bacterial genomes, photoreceptors have been identified in a wide range of heterotrophic
bacteria, but their functional roles remain unknown. The plant pathogen Pseudomonas syringae is
among these heterotrophs; it encodes two bacteriophytochromes (BphPs) and a LOV (light-oxygenvoltage)-histidine kinase protein (LOV-HK). Bacteriophytochromes are the most abundant photosensory
proteins found in bacteria and are responsive to red/far-red light through an association with bilin
chromophores. They convert between two stable conformations, a red light-absorbing Pr form and a
far-red light-absorbing Pfr form. P. syringae expresses bphP1 in an operon with bphO, which encodes a
heme oxygenase that breaks down heme into the chromophore biliverdin. It expresses bphP2 in an
operon with bphR, which encodes a response regulator, BphR. Both BphP1 and BphP2 are histidine
kinases that are likely to be involved in two-component systems. Little is known of BphP2 because of its
resistance to purification. Previous studies demonstrated that BphP1 from P. syringae absorbs red light
(698nm) to convert to its Pfr form, which is the active kinase, and also that biliverdin binding is required
for kinase activity. In addition to providing the chromophore, BphO has been shown to directly interact
with BphP1 and to likely assist in folding. Although biochemical analysis of BphP1 is available, the
target(s) of its kinase activity have not been identified, and the potential biological impacts of the BphP1
signaling pathway remain unknown. Identification of a BphP1-mediated signaling pathway is especially
difficult because it is an orphaned histidine kinase with no associated response regulator. To further
elucidate
this
pathway
we
utilized
a
tool
to
predict
kinase/regulator
interactions, http://www.swissregulon.unibas.ch/cgi-bin/TCS.pl, which uses Bayesian network methods
applied to multiple sequence alignment. We tested the predicted response regulators using yeast-twohybrid analyses and phosphotransfer assays and identified Psyr_2449 as a target for BphP1-mediated
phosphotransfer.
Lab: Gwyn Beattie
______________
55
BLAST XII
________________ Poster #5
A NOVEL INDUCER OF ROSEOBACTER MOTILITY IS ALSO A DISRUPTOR OF ALGAL
SYMBIOSIS
Robert Belas and Preeti Sule
Dept Marine Biotechnology, University of Maryland Baltimore County, IMET, 701 East Pratt St.,
Baltimore, MD 2102
Silicibacter sp. TM1040, a member of the Roseobacter clade, forms a symbiosis with unicellular
phytoplankton, which is inextricably linked to the biphasic “swim or stick" lifestyle of the bacteria.
Mutations in flaC bias the population towards the motile phase. Renewed examination of the FlaCstrain (HG1016) uncovered that it is composed of two different cells: a pigmented type, PS01, and a
non- pigmented cell, PS02, each of which has an identical mutation in flaC. While monocultures of
PS01 and PS02 had few motile cells (0.6% and 6% respectively), co-culturing the two strains resulted
in a 10-fold increase in the number of motile cells. Cell-free supernatants from co-culture or wild-type
cells were fully capable of restoring motility to PS01 and PS02, which was due to increased fliC3
(flagellin) transcription, FliC3 protein levels per cell, and flagella synthesis. The motility-inducing
compound has an estimated mass of 226 Da, as determined by mass spectrometry, and is referred to
as Roseobacter Motility Inducer (RMI). Mutations affecting genes involved in phenyl acetic acid
synthesis significantly reduced RMI, while defects in tropodithietic acid (TDA) synthesis had marginal or
no effect on RMI. RMI biosynthesis is induced by p-coumaric acid, a product of algal lignin degradation.
When added to algal cultures, RMI caused loss of motility, cell enlargement, and vacuolization in the
algal cells. RMI is a new member of the roseobacticide family of troponoid compounds whose activities
affect roseobacters, by shifting their population toward motility, as well as their phytoplankton hosts,
through an algicidal effect.
Lab: Robert Belas
______________
56
BLAST XII
________________ Poster #6
DOES THE C-RING ROTATE?
Basarab G. Hosu, Veda Nathan, Howard C. Berg
Department of Molecular and Cellular Biology, Harvard University, 16 Divinity Ave, Cambridge, MA
02138
E. coli is propelled by a set of rotary motors. Each motor drives a helical filament, which rotates
in the external aqueous environment. Although the rotation of the extracellular components of the
flagellum (hook and filament) is well documented, the actual mechanism of rotation of the intracellular
components remains to be clarified.
We investigate the rotation of the C-ring, believed to be part of the rotor, by polarized
microscopy. We expect to learn whether the C-ring rotates with respect to the cell body, at what rate
and whether a vernier mechanism is involved in driving the filament.
Lab: Howard Berg
______________
57
BLAST XII
________________ Poster #7
A NON-CONSERVED ACTIVE SITE RESIDUE INFLUENCES RESPONSE REGULATOR REACTION
KINETICS, PHOSPHODONOR SPECIFICITY, AND PARTNER KINASE INTERACTIONS
Robert M. Immormino and Robert B. Bourret
Department of Microbiology and Immunology, University of North Carolina, Chapel Hill, NC 27599-7290
USA
Two-component regulatory systems, minimally composed of a sensor kinase and a partner
response regulator protein, are common mediators of signal transduction in microorganisms. All
response regulators share a receiver domain with conserved active site residues that catalyze
phosphorylation and dephosphorylation reactions. To understand the factors that endow different
response regulators with their distinctive characteristics, our laboratory investigates the consequences
of altering non-conserved portions of receiver domains, particularly the impact on reaction kinetics and
specificity.
In this study, we explored the effects of various amino acids at non-conserved position T+1 (one
residue C-terminal of the conserved Thr/Ser) in the receiver domain active site. Position T+1 is most
commonly occupied by the small residues Ala or Gly, sometimes by the slightly larger Ser or Thr, and
occasionally by the much larger Val or Met. Karl Volz proposed 20 years ago (Volz, 1993) that the
prevalence of small amino acids at position T+1 serves to allow access to the site of phosphorylation.
Distinctive differences in amino acid preference at position T+1 between different response regulators
suggest the T+1 residue has functional consequences. We chose Escherichia coli CheY, CheB, and
NarL as representative of the Ala/Gly, Ser/Thr, and Val classes respectively and then used wildtype
and mutant response regulators carrying various substitutions at position T+1 to assess (1) selfcatalyzed dephosphorylation; (2) self-catalyzed phosphorylation with phosphoramidate, acetyl
phosphate, or monophosphoimidazole; and/or (3) phosphotransfer from the CheA or NarX sensor
kinases. To help clarify the molecular mechanisms underlying the observed effects on reaction kinetics
and phosphodonor specificity, we determined the X-ray crystal structures of four CheY mutants
containing substitutions at position T+1 and constructed molecular models of phosphodonors in the
active site.
Our results help explain the distinctive distributions of amino acids at position T+1 in the CheB,
CheY, and NarL response regulator families and may contribute to prediction of response regulator
self-catalyzed reaction rates based on amino acid sequence.
Reference
Volz, K. (1993) Structural conservation in the CheY superfamily. Biochemistry 32, 11741-11753.
Lab: Robert Bourret
____________
58
BLAST XII
________________ Poster #8
NONCONSERVED ACTIVE SITE RESIDUES MODULATE CheY AUTOPHOSPHORYLATION
KINETICS AND PHOSPHODONOR PREFERENCE
Stephanie A. Thomas, Robert M. Immormino, Robert B. Bourret, and Ruth E. Silversmith
Department of Microbiology and Immunology, University of North Carolina, Chapel Hill, NC 27599-7290
In two-component signal transduction, response regulator proteins contain the catalytic
machinery for their own covalent phosphorylation and can catalyze phosphotransfer from a partner
sensor kinase or autophosphorylate using various small molecule phosphodonors. Although response
regulator autophosphorylation is physiologically relevant and a powerful experimental tool, the kinetic
determinants of the autophosphorylation reaction and how those determinants might vary for different
response regulators and phosphodonors are largely unknown.
We characterized the
autophosphorylation kinetics of 21 variants of the model response regulator Escherichia coli CheY that
contained substitutions primarily at nonconserved active site positions D+2 (CheY residue 59) and T+2
(CheY residue 89), two residues C-terminal to conserved D57 and T87, respectively. Overall, the CheY
variants exhibited a >105-fold range of rate constants (k phos /K S ) for reaction with phosphoramidate,
acetyl phosphate, or monophosphoimidazole, with the great majority of rates enhanced over wild type
CheY. Although phosphodonor preference varied substantially, nearly all the CheY variants reacted
faster with phosphoramidate than acetyl phosphate. Correlations between charge of the D+2/T+2 side
chains and rate indicated electrostatic interactions are a kinetic determinant. Moreover, the effect of
ionic strength varied with phosphodonor but not active site composition, suggesting at least two types
of electrostatic interactions influence autophosphorylation kinetics. Nonpolar surface area of the
D+2/T+2 side chains also correlated with autophosphorylation rate, especially for reaction with
phosphoramidate and monophosphoimidazole. Computer docking strongly suggested that highly
accelerated monophosphoimidazole autophosphorylation rates for CheY variants with a tyrosine at
position T+2 likely reflect structural mimicry of phosphotransfer from the sensor kinase histidyl
phosphate.
Lab: Robert Bourret
____________
59
BLAST XII
________________ Poster #9
EXPERIMENTAL ANALYSIS OF RECEIVER DOMAIN AUTODEPHOSPHORYLATION KINETICS
GUIDED BY BIOINFORMATICS
Stephani Page1, Robert Immormino2, Robert Bourret2
Departments
of
Biochemistry
&
Biophysics1
University of North Carolina Chapel Hill, NC 27599
and
Microbiology
&
Immunology2
Receiver domains of response regulators contain the site of phosphorylation, which mediates
responses by switching output function on and off. Receiver domains have highly conserved active site
residues that catalyze phosphorylation and dephosphorylation. Despite the conserved active site and
similarities in structure and sequence amongst receiver domains, self-catalyzed dephosphorylation of
receiver domains reportedly occurs over a nearly million-fold range of reaction rates. Receiver domain
autodephosphorylation rates appear to be tuned to the timescales of the different biological processes
in which response regulators participate. For example, receiver domains involved in chemotaxis
typically autodephosphorylate faster than those involved in transcriptional regulation.
We want to understand what factors influence receiver domain autodephosphorylation rates and
the mechanisms by which those factors exert influence. Sequence, structural, and kinetic analyses
have drawn our focus to variable residues at the active site of receiver domains, in particular, residues
at positions D+2 and T+2, two positions C-terminal to a conserved Asp and Thr/Ser, respectively, that
mediate dephosphorylation chemistry. Specifically, residues at D+2 and T+2 (i) have the strongest coevolutionary relationship amongst residues found at any two positions in receiver domains, (ii) are
positioned appropriately to influence catalysis, and (iii) affect autodephosphorylation rates when
changed. For a more comprehensive assessment of the influence of D+2 and T+2 on receiver domain
autodephosphorylation, we used bioinformatics to select D+2/T+2 pairs representing a large fraction of
naturally occurring receiver domain sequences. Our collection of 48 mutants contains combinations of
amino acids at D+2 and T+2 that are found in 60% of wild type receiver domain sequences in a nonredundant database of nearly 14,000 response regulators.
Using E. coli response regulators CheY and PhoB as scaffolds, we assessed the effects of changing
residues at positions D+2 and T+2 on autodephosphorylation rates. Autodephosphorylation rates of 42
CheY mutants spanned a 120-fold range. Six mutants of PhoB had autodephosphorylation rates
spanning a 60-fold range. The range of autodephosphorylation rates observed was the result of both
enhancing and diminishing substitutions.
By changing just two variable active site residues, we have been able to “tune” CheY and PhoB
to a range of different autodephosphorylation timescales, thus accounting for two out of six orders of
magnitude in the reported range of receiver domain autodephosphorylation rates. We are currently
exploring the mechanistic basis by which positions D+2 and T+2 influence receiver domain
autodephosphorylation rates.
Lab: Robert Bourret
____________
60
BLAST XII
_______________ Poster #10
THE BRUCELLA ABORTUS GENERAL STRESS RESPONSE SYSTEM REGULATES CHRONIC
MAMMALIAN
INFECTION,
AND
EXHIBITS
NON-CANONICAL
POST-TRANSLATIONAL
REGULATION
Hye-Sook Kim1,2 , Clayton C. Caswell3, Robert Foreman2,4, R. Martin Roop II3, and Sean Crosson1,2,4,*
1
Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL 60637,
USA.
2
Howard Taylor Ricketts Laboratory, Argonne National Laboratory, Argonne, IL, 60439, USA.
3
Department of Microbiology and Immunology, East Carolina University School of Medicine,
Greenville, NC 27834, USA.
4
The Committee on Microbiology, The University of Chicago, Chicago, IL 60637, USA.
Brucella spp. are adept at establishing a chronic infection of mammalian host cells, though the
mechanisms by which Brucella maintain long-term host residence remain poorly defined. We
demonstrate that core components of the α-proteobacterial general stress response (GSR) system,
PhyR and σE1, are top-level regulators of Brucella abortus virulence that are specifically required for
maintenance of chronic animal infection. ∆phyR and ∆rpoE1 null mutants exhibit decreased survival in
the face of oxidative and acid stress in vitro, but are not defective in infection of primary murine
macrophages or initial colonization of BALB/c mice. However, ∆phyR and ∆rpoE1 mutants are cleared
from a murine host beginning one month post-infection. Thus, the B. abortus GSR system affects a
transcriptional program that is dispensable for initial host colonization, but is required to maintain longterm infection. We experimentally define the B. abortus GSR transcriptional regulon and show that it
contains genes previously linked to virulence including genes required for oxidative and acid stress
survival, and genes that affect immunomodulatory components of the cell envelope. These data
support a model in which the GSR system regulates stress survival, and affects how B. abortus
interfaces with the host immune system. We further present a molecular-level analysis of purified GSR
proteins that defines unique features of regulation in B. abortus. In particular, we demonstrate that the
cellular level of PhyR is controlled post-translationally by regulated proteolysis. PhyR proteolysis
provides a mechanism to attenuate spurious PhyR protein interactions that would inappropriately
activate the GSR transcriptional program. We conclude that the B. abortus GSR system regulates
transcription of genes required for acute stress survival and long-term interaction between the
bacterium and its mammalian host, and that PhyR proteolysis is a novel regulatory feature in B. abortus
that ensures proper control of the GSR.
Lab: Sean Crosson
_____________
61
BLAST XII
_______________ Poster #11
STUDIES OF FLAGELLAR MOTOR PROTEIN FliG AND ITS INTERACTIONS WITH FliF AND FliM
Robert Levenson, Armand Vartanian, Susan Gelman, Hongjun Zhou, Frederick W. Dahlquist
Department of Chemistry and Biochemistry, University of California at Santa Barbara, Santa
Barbara, CA, 93106
The cytoplasmic ring (C-ring) of the flagellar motor is composed of three proteins: FliG, FliM,
and FliN, each present in different copy numbers. These proteins perform the function of transmitting
torque from the stators to the basal body, as well as regulating the rotational direction of the flagellum.
FliG is an entirely alpha-helical protein composed of three domains, FliG N , FliG M , and FliG C . There
remain significant questions regarding the organization of the FliG domains within the C-ring. To gain
insight into these questions, we have been studying these proteins using a variety of biophysical
methods.
The N-terminal domain of FliG, FliG N , has been known to interact with the membrane protein
FliF in one of the first interactions during the assembly of the flagellar motor. We have previously
studied the strong interaction of T. maritima FliG N with an NMR-unlabeled synthetic peptide
corresponding to a 38 amino acid C-terminal portion of FliF (FliF C ). We have now extended the study of
this complex by producing a fusion protein that coexpresses the two proteins in one polypeptide, and
we present preliminary structural NMR data of this fusion protein. We also present NMR data from the
FliF C -bound full-length FliG protein. This data, together with the recently-published crystal structure of
the FliG MC -FliM complex from our lab as well as other results, combine to support a structural model of
the C-ring that agrees very well with available cryo-EM data.
We have also used NMR spectroscopy to study the interplay between FliG MC , FliM, and CheY.
We present experiments using methyl-labelled FliG MC that indicate that FliG C and CheY competitively
bind to FliM, extending previous in vitro results that used individual FliG M or FliG C domains to the full
FliG MC protein.
Lab: Frederick Dahlquist
________
62
BLAST XII
_______________ Poster #12
CHEMOTAXIS SIGNALING PATHWAY OF THE FLAGELLAR SYSTEM 2 FROM RHODOBACTER
SPHAEROIDES
Ana Martínez del Campo1, Teresa Ballado1, Javier de la Mora1, Laura Camarena2 and Georges
Dreyfus1.
1
Departamento de Genética Molecular, Instituto de Fisiología Celular, Universidad Nacional Autónoma
de México. Circuito Exterior S/N Ciudad Universitaria, 04510 México D.F.
2
Departamento de Biología Molecular y Biotecnología, Instituto de Investigaciones Biomédicas,
Universidad Nacional Autónoma de México.
Rhodobacter sphaeroides posses several reiterated chemotactic genes: 4 cheW, 2 cheB, 3
cheR, 4 cheA and 6 cheY, which are encoded in three loci cheOp1, cheOp2 and cheOp3. Moreover,
R. sphaeroides genome contains two flagellar gene clusters. The genes of the first cluster are
constitutively expressed under laboratory conditions and are required for the synthesis of the wellcharacterized single subpolar flagellum (Fla1); which allows the swimming of the WS8 wild-type strain.
The genes of the second flagellar cluster are not expressed in the wild-type strain, however strains that
express it have been isolated. When expressed, the Fla2 genes of R. sphaeroides produce polar
flagella that are required for swimming. When the cell is swimming with the Fla1 flagellum only cheOp2
and cheOp3 are essential. In contrast CheY1, CheY2 and CheY5, which are encoded in cheOp1, are
required for the chemotactic control of the Fla2 flagella. Here we report that when R. sphaeroides
swims with Fla2 flagella, cheOp1 controls its chemotactic behavior. Additionally, the molecular
dissection of cheOp1 allowed us to identify a novel component of chemotaxis signaling pathway of the
Fla2 flagella from R. sphaeroides; which we are currently investigating.
This project is supported by grants IN-206811 from DGAPA and 106081 from CONACyT.
A.M.-D.C. is a recipient of a Robert M. Macnab Memorial Grant to attend BLAST XII.
Lab: Georges Dreyfus
__________
63
BLAST XII
_______________ Poster #13
THE C TERMINUS OF THE FLAGELLAR MURAMIDASE SltF MODULATES THE INTERACTION
WITH FlgJ IN RHODOBACTER SPHAEROIDES
Manuel Osorio-Valeriano1, Javier de la Mora1, Teresa Ballado1, Laura Camarena2 y Georges
Dreyfus1*.
Instituto de Fisiología Celular1, Instituto de Investigaciones Biomédicas2, Universidad Nacional
Autónoma de México. Ap. Postal 70-243, 04510 México, D.F., México. Tel. 56225618.
Macromolecular structures such as the bacterial flagellum must traverse the cell wall, for this
reason, the assembly of some of these structures requires the activity of peptidoglycan degrading
enzymes. In Salmonella enterica, FlgJ has been characterized as a bifunctional protein, acting as a
scaffold for rod assembly and also acting as a muramidase degrading the PG layer to facilitate rod
penetration. The FlgJ homologue in R. sphaeroides lacks the muramidase domain. In a previous study
we have characterized a flagellum specific muramidase SltF, whic interacts with the scaffold protein
FlgJ. We proposed that this interaction occurs in the periplasm during rod formation and helps localize
SltF at the flagellar assembly site.
In this study, we demostrate that the export of SltF to the periplasm is dependent on the SecA
pathway. Additionally we carried out a deletion analysis of the C-terminal portion of SltF, the resulting
truncated mutants were unable to restore wild-type swimming in a Δsltf genetic background. These
mutants retained their catalytic activity, however they lost the SltF-SltF interaction and also show a
higher affinity binding FlgJ, when compared to the wild-type protein. We propose that this region
modulates the interaction with the scaffold protein FlgJ during the flagellum biogenesis.
This study was supported by grants from CONACyT (106081) and DGAPA/UNAM (IN206811-3).
Lab: Georges Dreyfus
__________
64
BLAST XII
_______________ Poster #14
PROFOUND EFFECT OF HOOK LENGTH ON MOTILITY AND VIRULENCE IN SALMONELLA
Christian Hotz1, Hanna M. Singer1, Carole Bourquin2 and Marc Erhardt1
1
Département
de
Médecine,
Université
de
Fribourg,
1700
2
Helmholtz Centre for Infection Research, 38124 Braunschweig, Germany
Fribourg,
Switzerland
Introduction and Aims
Bacteria swim through liquid environments by rotating a rigid, helical organelle, the flagellum.
The flagellum enables bacteria to swim towards nutrients and away from harmful substances. In
addition, flagellar motility contributes to bacterial pathogenesis by promoting bacteria-host interactions,
biofilm formation, adherence and invasion of epithelial cells.
The structure of the bacterial flagellum consists of a long external filament connected to a
membrane-embedded basal-body at the cell surface by a short curved structure, the hook. The hook is
a flexible universal joint and transmits the motor torque to the helical propeller over a wide range of its
orientation for swimming and tumbling.
In Salmonella enterica the hook structure extends 55 nm beyond the cell surface. The length of
the hook is controlled via intermittent secretion of a molecular ruler molecule. To address the question
why length of the hook structure is tightly controlled, we analyzed various hook length variation mutants
for their effects in motility and invasion assays.
Material and Methods
Various deletion and insertions mutants of FliK, the molecular ruler protein responsible for hook
length control, were analyzed in a battery of swimming and swarming motility assays, as well as for
their capability to infect macrophages. Deletions and insertions in FliK give rise to shorter and longer
hook structures, respectively.
Key Results
We demonstrate that the variation of flagellar hook length has a profound effect on motility and
virulence in Salmonella. Deletion and insertion variants of the FliK molecular ruler were secreted and
resulted in induced the substrate specificity switch as shown by assembly of flagellar filaments. Long
hook mutants displayed a defect in swarming and swimming motility, as well as a profound defect in
their capability to invade both macrophages and epithelial cells in a Gentamicin protection assay.
Conclusion
Our results suggest that the flagellar hook length of 55 nm in Salmonella enterica has evolved to
match the requirements for optimal motility. The ability to efficiently move in a directed manner confers
distinct advantages. Potential benefits of efficient motility in Salmonella include the ability to search for
nutrients, to avoid toxic substances and facilitate contact with eukaryotic host cells for invasion. The
costs of motility are significant, including the metabolic burden of flagellar biosynthesis and the
energetic expenses for flagellar rotation. Thus, it is not surprising that the flagellar structure is subject to
strict control and has evolved to provide the optimal requirements for efficient motility.
Lab: Marc Erhardt
______________
65
BLAST XII
_______________ Poster #15
STRUCTURAL AND FUNCTIONAL DISULFIDE MAPPING OF THE CheA-CheW INTERFACE IN THE
BACTERIAL CHEMOSENSORY ARRAY
Andrew M. Natale and Joseph J. Falke
Molecular Biophysics Program, and Department of Chemistry and Biochemistry, University of Colorado
at Boulder, UCB 596, Boulder, CO 80309-0596
The bacterial chemosensory array, which senses gradients of attractant and repellent ligands
and accordingly modulates cellular swimming behavior, is composed of a highly ordered, ultrastable
protein lattice. The repeating core unit within the lattice consists of a membrane-spanning trimer of
receptor dimers, a dimeric histidine kinase CheA bound at the receptor tips, and two scaffolding CheW
monomers. Signals are transduced from the periplasmic ligand binding receptor domains to the
cytoplasmic receptor tips where they control the activity of the kinase. An increasing body of recent
evidence has begun to paint a picture of the large scale architecture of the lattice, while other studies
have illuminated more of its specific protein-protein contacts. While the picture of the array is becoming
clearer, questions remain about the identities of the packing interfaces between core components and
about the nature of signal transduction through these interfaces. To address some of these questions,
the present study examines the CheA kinase – CheW scaffolding protein interface using a strategy of
disulfide mapping. While the structure of isolated CheA regulatory domain in complex with CheW has
been solved, we aim to probe the interface structure in active, membrane bound arrays reconstituted in
vitro from the three full length core proteins. Using combinations of single cysteine CheA and CheW
mutants we are able to form interfacial disufides and map contact points. We can use similar
experimental techniques to identify signaling induced changes in packing or dynamics at the interface,
as well as to test whether this interface plays a role in mediating the significant cooperativity observed
in the array. Our latest findings will be presented at the meeting.
Lab: Joseph Falke
______________
66
BLAST XII
_______________ Poster #16
ANALYSIS OF THE REPELLENT-SENSING MECHANISMS OF ESCHERICHIA COLI
Hirotaka Tajima1, Takaya Inui2, Eri Iijima3, Ken Konemura3, Hisako Matsuzawa3, Toshio Fukuda1,
Michio Homma4, Ikuro Kawagishi2,3
1: Graduate School of Engineering, Nagoya Univesity, 2: Graduate School of Engineering and 3:
Faculty of Bioscience and Applied Chemistry, Hosei University, Koganei 184-8584, and 4: Graduate.
School of Science, Nagoya University Chikusa-ku, Nagoya 464-8603, Japan
E. coli recognizes the environmental factors such as chemical compounds, temperature, and
osmotic pressure, and migrates toward or from the factors by altering the direction of flagellar rotation.
Above all, behavior of to the chemical compounds, including some amino acids, sugars, metal ions,
termed as chemotaxis, is the most famous and best characterized. All components of chemotaxis have
been identified and studied about them. Four chemoreceptors of E. coli (Tar, Tsr, Trg and Tap) can
sense variety of factors such as amino acids, sugars, metal ions, indole, pH and temperature.
Chemotactic signals are transferred by phosphor-relay of histidine kinase CheA and response regulator
CheY. Chemoreceptors produce signals by the regulation of CheA, however, the mechanism is poorly
understood. Especially, the sensing mechanisms of repellents, such as, Ni2+ and indole, are less
understood than that of attractants. Then we analyzed the repellent-sensing mechanism of
chemoreceptors.
kcal/mol
µcal/sec.
time (min.)
time (min.)
time (min.)
Tar of E. coli mediates
0
50
100
0
50
100
0
50
100
repellent responses to the divalent
0
0
0
cations, Ni2+ and Co2+. NikA was
-0.03
-0.025
presumed to serves as a primary
-0.06
-0.3
receptor. However, Englert et al. (2010)
-0.05
-0.2
0
reported that E. coli requires neither the
0
-0.3
Ni2+-binding protein (NikA) nor the
-0.4
-2
2+
-0.6
transmembrane components of the Ni
-0.6
transporter (NikB and NikC) for a Ni2+
-4
-0.9
0
3
6
0
3
6
0
3
6
molar ratio
molar ratio
molar ratio
response. Then we performed ITC
Tsr
/ NiSO4
/ NiSO
Tarof/ CoSO
measurements of Ni2+ binding to the
FigureTar
1: ITC
measurements
of binding
Ni2+ to the
fragment
4
4 Tar periplasmic
periplasmic fragment of Tar. The
addition of Ni2+ to the Tar fragment caused heat release, indicating the direct binding of Ni2+ (Figure 1).
Furthermore, we examined the properties of chimeric proteins Tar and Tsr to determine the region of
Tar responsible for Ni2+ sensing.
Indole is known as an autoinducer, and increases
the drug resistance and inhibits the biofilm formation of E.
coli. It is known that repellent response to indole is mediated
by Tsr. However, we found that cells expressing Tar as a
sole chemoreceptor attracted to high concentration of indole
(2.5 mM) (Figure 2). The analysis using chimeric receptors
and pin-head Tar indicated that periplasmic domain of Tar is
dispensable for indole sensing.
Here, we will show the current progress of analysis
of the repellent-sensing mechanisms.
Figure 2: tethered cell assay with indole
Lab: Toshio Fukuda
____________
67
BLAST XII
_______________ Poster #17
IDENTIFICATION FOUR NEW GGDEF/EAL DOMAIN PROTEINS THAT REGULATE MOTILITY IN
SALMONELLA, ONE OF WHICH LOCALIZES TO THE MID-CELL AND INHIBITS CELL DIVISION
Hyo Kyung Kim, Vincent Nieto and Rasika M. Harshey
Section of Molecular Genetics and Microbiology, University of Texas at Austin, Austin, TX 78712
Cyclic-di-GMP (c-di-GMP) levels control the transition between motility and sessility. This
molecule is produced by diguanylate cyclases (DGCs) having a catalytic GGDEF domain and broken
down by phosphodiesterases (PDEs) containing an EAL domain. Our model organism Salmonella
enteric has 5 GGDEF domain proteins, 8 EAL domain proteins and 7 proteins having both GGDEF and
EAL domains. Thus far, absence of only the specific phosphodiesterase YhjH impairs motility in this
bacterium. Using the yhjH mutant as the starting strain, we have identified 4 additional GGDEF/EAL
domain proteins which affect motility. Most of these proteins have an N-terminal receiver domain,
coupling environmental signals to c-di-GMP levels in the cell. To test whether these proteins provide a
spatially compartmentalized c-di-GMP pool in the cell, we initially localized them by using fluorescent
fusion proteins. 4 of the 5 GGDEF/EAL proteins showed polar localization, suggesting that this location
was either non-specific or that they were associating with the chemoreceptor complex at the poles.
Interestingly, 1 GGDEF protein showed a striking spiral pattern all along the length of the cell that was
most obvious at the mid-cell position. This pattern is remarkably similar to that of FtsZ tubulin spirals,
thought to be early intermediates in formation of the division ring. Accordingly, mutant cells of this
protein were shorter than wild-type cells, and increased protein expression (native protein or GFP
fusion protein) produced longer cells. The localization of a DGC at division sites suggests a role for cdi-GMP signaling in cell division.
Lab: Rasika Harshey
_______________
68
BLAST XII
_______________ Poster #18
SIGNALLING SYSTEMS CONTROLLING DEVELOPMENTAL CELL FATE SEGREGATION IN
MYXOCOCCUS xanthus
Andreas Schramm, Vidhi Grover, and Penelope I. Higgs
Max Planck Institute for Terrestrial Microbiology, Dept. of Ecophysiology, 35043, Marburg, Germany
Myxococcus xanthus is a model organism for regulation of complex behavior. Under nutrient
limiting conditions, these bacteria enter a multicellular developmental program wherein cells follow
different fates: aggregation into mounds (fruiting bodies) followed by differentiation into environmentally
resistant spores; differentiation into a persister-like state termed peripheral rods; programmed cell
death; or cell clusters, a recently identified population whose role is not well understood. At least four
distinct histidine-aspartate phosphorelay (aka “two-component”) signaling systems are necessary for
appropriate cell fate segregation. Our results suggest that each of these signaling systems
independently regulates accumulation of an important transcriptional regulator, MrpC. Consistently, we
have demonstrated that MrpC accumulates heterogeneously in the developing cell population; rapid
MrpC accumulation is observed in cells induced to migrate into fruiting bodies, while little accumulation
is observed in cell clusters or in cells destined to become peripheral rods.
Our data indicate that one of these signaling systems, Esp, regulates proteolytic turnover of
MrpC. The Esp signaling system consists of two hybrid histidine kinases, EspA and EspC.
Phosphorylation of the receiver domains from both EspA and EspC is necessary to stimulate MrpC
turnover, and this occurs via a novel inter- and intra-hybrid kinase signaling mechanism which will be
presented.
Interestingly, the EspA:EspC ratio varies between developmental subpopulations
suggesting that the relative per cell signaling efficiency is controlled not just by signals perceived by
EspA and EspC , but also by the respective levels of the signaling components themselves. A model
for the role of Esp and the remaining signaling systems which control production of MrpC and cell fate
segregation will be presented.
Lab: Penelope Higgs
___________
69
BLAST XII
_______________ Poster #19
ATTEMPT TO INVESTIGATE THE INTERACTION BETWEEN THE ROTOR AND THE STATOR BY
USING SOLUTION NMR
Mizuki Gohara1, Rei Abe-Yoshizumi1, Shiori Kobayashi1, Yohei Miyanoiri2, Yoshikazu Hattori3, Chojiro
Kojima3, Masatsune Kainosho2,4, Michio Homma1
1
Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya,
Aichi, Japan 464-8602; 2Structural Biology Research Center, Graduate School of Science, Nagoya
University, Chikusa-ku, Nagoya, Aichi, Japan 464-8602; 3Institute for Protein Research, Osaka
University, 3-2 Yamadaoka, Suita, Osaka, Japan 565-0871; 4Center for Priority Areas, Tokyo
Metropolitan University, 1-1 Minamiosawa, Hachioji, Toyko, Japan 192-0397;
In the bacterial flagellar motor, torque is generated by the interactions between rotor and stator.
In the rotor, a component FliG, especially at its C-terminal domain, is mainly participated in torque
generation. In the H+-driven motor of Escherichia coli, mutational analyses suggested that the
interaction is mediated with charged residues between the FliG C-terminal domain and the MotA
cytoplasmic loop region. However, in the Na+-driven motor of Vibrio, the effects of mutations in the
charged residues on motility were very weak. So the important residues for torque generation in the
Na+-driven motor may be different from the H+-driven motor. We want to observe the interaction
between FliG and PomA (MotA ortholog) in the Na+-driven motor and decided to use the solution NMR.
In the previous meeting, we reported that N-terminally truncated FliG variants G214-FliG
(FliGC) gave much better NMR spectra by 1H-15N TROSY-HSQC. Here, we would like to present the
results of the measurement of the 3D-NMR(1H-13C-15N) and the on going assignments of the signals
to amino acids. Furthermore, we purified intact PomA, the PomA/B complex, and the PomA fragments
and currently we are trying to measure NMR spectra of FliGC in the presence and absence of the
purified proteins to investigate residues involved in the rotor-stator interaction.
Lab: Michio Homma
____________
70
BLAST XII
_______________ Poster #20
IMPORTANCE OF CHARGED RESIDUES OF POMA AND FLIG FOR ROTOR-STATOR
INTERACTION IN THE SODIUM-DRIVEN FLAGELLAR MOTOR OF VIBRIO ALGINOLYTICUS
Norihiro Takekawa, Seiji Kojima and Michio Homma
Division of Biological Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku,
Nagoya 464-8602, Japan
Bacterial flagellar motor is a reversible rotary motor located at the base of the flagellum. The
flagellar motor is consisted of two parts, rotor and stator. Essential components for torque generation in
the rotor is located at the C-ring composed of FliG, FliM, and FliN, and the stator is composed of the
membrane proteins MotA and MotB in H+-driven motor and PomA and PomB in Na+-driven motor that
form ion channel structure. In the H+-driven motor of E. coli, it was suggested that the interactions
between conserved charged residues of FliG and MotA were important for the torque generation of the
motor. However, the presence of such interactions had been unclear in the Na+-driven motor of V.
alginolyticus. In this study, we systematically made mutants whose charged residues of FliG and PomA
were replaced to uncharged or charge-reversed ones, and examined motility of those Vibrio mutants.
We found that some fliG/pomA double mutations gave the synergetic effects or suppression on the
motor rotation. Our results suggested that interactions between some charged residues of FliG and
those of PomA were important for force generation of Na+-driven flagellar motor in V. alginolyticus. In
the H+-driven motor, smaller number of electrostatic interactions between charged residues at the
rotor-stator interface are good enough for motor function, whereas in the Na+-driven motor of Vibrio
requires more charged residues at the rotor-stator interface. Such larger number of interactions may
cause robustness against the mutations at the rotor-stator interface in the Vibrio motor, and its
remarkably high-speed rotation.
Lab: Michio Homma
____________
71
BLAST XII
_______________ Poster #21
VIBRIO ALGINOLYTICUS REGULATION OF THE FLAGELLAR NUMBER IN: ROLE OF ATP
BINDING MOTIF OF FlhG AND THE NOVEL PROTEIN SflA
Seiji Kojima, Takehiko Nishigaki, Hiroki Ono and Michio Homma
Division of Biological Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku,
Nagoya 464-8602, Japan
Bacterial flagella are expressed in a wide variety of locations and numbers. Marine bacterium
Vibrio alginolyticus has a single polar flagellum whose number is regulated positively by putative
GTPase FlhF and negatively by putative ATPase FlhG. FlhF is also involved in its polar positioning in
flagellar formation. Our mutational and biochemical analyses showed that FlhG interacts with FlhF to
prevent FlhF from localization at cell pole. Here we investigated the role of ATP binding motif of FlhG.
Since FlhG is the homolog of the cell division inhibitor MinD, we mutated four residues in the ATP
binding motif of FlhG, whose corresponding mutations in MinD disrupted its ATPase activity. Two of
them (K31A and K36Q) could not complement the flhG defective mutant on motility. Investigation of
subcellular localization and binding to FlhF of these FlhG mutants are now ongoing. We also isolated a
suppressor mutant from the ∆flhFG strain that only occasionally confers peritrichous flagella in a small
number of cells. The responsible mutation was mapped to the non-flagella-related gene. This novel
gene, named sflA, is specific for Vibrio and is speculated to encode a transmembrane protein with a
DnaJ domain. Expression of the C-terminal cytosolic region of SflA containing DnaJ domain is sufficient
to suppress the flagellar generation. SflA is detected in the membrane fraction by immunoblot using an
antibody raised against the N-terminal region of SflA. Further analyses using the C-terminal GFP-fusion
variant are now ongoing and SflA function will be discussed.
Lab: Michio Homma
____________
72
BLAST XII
_______________ Poster #22
EFFECT OF CODON CONTEXT ON TRANSLATION SPEED IN VIVO
Kelly T. Hughes, Chakib Mouslim and Valentina Rosu
Department of Biology, University of Utah, Salt Lake City, UT 84112
Codon context plays a significant role in determining the speed at which the ribosome translates
through codon pairs. We have isolated a translation-slow “silent” mutant in the flgM gene of Salmonella
at amino acid codon 8. This results in inhibition of flgM translation and a restart of flgM translation at a
methionine codon near the 3’end of the 97 amino acid coding sequence. We developed a regulatory
system that provides a simple, yet elegant mechanism to determine the relative, in vivo speed of the
ribosome as it translates through codons and codon pairs in a manner that is independent of protein
and mRNA stability. The system utilizes the leader sequence of the Salmonella histidine biosynthetic
operon. Surprisingly, the rate at which the ribosome translates through single codon pairs in the his
leader RNA shows significant variation even for different codons used by a single tRNA species. Our
system allows us to rank codon pairs according to in vivo translational speed, and readily identifies the
fastest and slowest among the 3,721 coding pairs. Choice of slow codon pairs might be important to
allow proper protein folding, or differential expression levels in the products of polycistronic transcripts.
Of all possible amino acid-coding codon pairs, approximately 50, including all 16 Pro-Pro pairs result in
ribosome stalling in the His leader peptide equivalent to what is observed with a stop codon.
Implications for gene expression and evolution are discussed.
Lab: Kelly Hughes
______________
73
BLAST XII
_______________ Poster #23
SALMONELLA fliC mRNA TRANSLATION
Fabienne F.V. Chevance, Soazig Le Guyon and Kelly T. Hughes
Department of Biology, University of Utah, Salt Lake City, UT 84112
Production of the filament subunit protein FliC is regulated by multiple mechanisms. The fliC
gene is transcribed from a σ28-dependent flagellar class 3 promoter, which is subject to FlgM inhibitory
activity coupled to hook-basal body formation. Production of FliC is also regulated at the posttranscriptional. The FljA protein binds sequences in the 5’-untranslated region of fliC mRNA to inhibit
translation. A mutant duplicated for FljA binding domain in the fliC 5’UTR is actually dependent on FljA
for fliC mRNA translation. We have uncovered a second translational control mechanism in the 5’UTR
of fliC mRNA that is independent of FljA. A predicted stem-loop region near the 5’-end of the transcript,
SL2, was discovered as an inhibitory element for fliC mRNA translation. A single base change that is
predicted to destabilize SL2 formation results in the inhibition of fliC mRNA translation. An “opened”
SL2 region is believed to act to inhibit fliC mRNA translation because complete deletion of the SL2
sequence results in an increase in FliC production over wild-type. Furthermore, an 12 base sequence
coding for amino acids 12 through 15 of FliC (12-15 coding) is complementary in sequence to SL2.
Mutations that are predicted to increase 12-15 coding sequence interaction with SL2 sequence result in
decreased FliC production. Mutations that are predicted to decrease 12-15 coding sequence interaction
with SL2 sequence result in increased FliC production. A transposon mutagenesis experiment was
developed to identify genes whose products are required for fliC mRNA translation, but not for fliC
transcription. This was done using either wild-type fliC sequence, mutants that have enhanced SL2
interaction with the fliC amino acid 12-15 coding region (translation-down) and mutants that have
decreased interaction between SL2 and the fliC amino acid 12-15 coding region (translation-up).
Insertions mutants isolated included those affecting multi-drug efflux pumps, tolA and acrB, and a
number of genes affecting outer membrane/cell wall assembly, surA, rfaC, rfaQ, rfbA and rfbH, in
addition to known genes required for general mRNA translation rne and prfA. The role of outermembrane/cell wall integrity in fliC mRNA translation will be discussed.
Lab: Kelly Hughes
______________
74
BLAST XII
_______________ Poster #24
EFFECT OF A SINGLE motP MUTATION FOR MOTILITY AT NEUTRAL pH OF THE Na+-DRIVEN
FLAGELLAR MOTOR OF BACILLUS PSEUDOFIRMUS OF4
Yuka Takahashi, Yukina Noguchi, Masahiro Ito
Graduate School of Life Sciences, Toyo University
Many bacteria swim by rotating helical flagellar filaments that act as screw propellers. There is a
motor that consists of a rotor and some stators at the base of the flagellar. Stators such as the MotAB
complex work as an ion channel. The flagellar motor is driven by the electrochemical potential of a
coupling ion, H+ or Na+. Direct interactions between the rotor protein FliG and the stator protein MotA
are thought to generate the rotational torque. Escherichia coli and Salmonella typhimurium have a
MotAB complex as a stator and the flagellum is driven by a proton-motive force. On the other hand,
alkaliphilic Bacillus and marine bacteria, such as Vibrio species, have a MotPS or PomAB complex as a
stator and the flagellum is driven by a Na+-motive force.
Alkaliphilic Bacillus pseudofirmus OF4 has a MotPS complex as a stator and the flagellum is
driven by a sodium-motive force. Previous studies showed that B. pseudofirmus OF4 swimming is
dependent on the Na+ concentration at pH 8-10, but showed poor motility at neutral pH even in the
presence of a high Na+ concentration. It was hypothesized that there could be competitive inhibition by
H+ of the Na+ translocation by the stator-force generator MotPS (1). On the other hand, B. subtilis has a
MotPS complex similar to B. pseudofirmus OF4. However, it was reported that B. subtilis swimming is
dependent on the Na+ concentration at neutral pH (2).
Here, we made a mutant strain of motPS from B. pseudofirmus OF4 that was expressed in the
stator deletion strain of B. subtilis (ΔmotABΔmotPS), and we investigated whether the mutant strain can
complement motility on a soft agar plate. In addition we observed whether the mutant strain exhibited
motility at neutral pH.
In this study, we identified that an amino acid residue in MotP is involved in competitive
inhibition by H+ in the Na+-driven flagellar motor of B. pseudofirmus OF4.
1) Fujinami, S. et al. (2007) Arch. Microbiol. 187: 239-247
2) Ito, M.et al. (2005) J. Mol. Biol., 352: 396-408
Lab: Masahiro Ito
______________
75
BLAST XII
TOWARDS A MECHANISM
CRESCENTUS
_______________ Poster #25
FOR
SURFACE
MECHANO-SENSATION
IN
CAULOBACTER
Isabelle Hug1, Siddharth Deshpande2, Thomas Pfohl2, Urs Jenal1
1
Biozentrum, 2Department of Chemistry, University of Basel, Switzerland
The bimodal reproductive program of C. crescentus upon division generates two distinct
daughter cells, a sessile stalked and a flagellated swarmer cell. While the newborn stalked cell
reinitiates growth, replication and division immediately, the swarmer progeny remains in G1 for a
defined time before differentiating into a sessile stalked cell. During this process it looses its polar
flagellum and pili and assembles an adhesive organelle, the holdfast, at the same pole. The
progression through the cell cycle is controlled by the interplay between cyclic di-GMP signaling and
phosphorylation networks, following the dogma that high intracellular cyclic di-GMP levels favor
settlement and surface attachment, whereas low levels correlate with planktonic behavior. Recently it
was shown that swarmer cells can produce holdfast and attach immediately when encountering surface
(Li G. et al., Mol Microbiol, 2012). This suggested that the cell cycle program that directs the motilesessile transition can be overridden and accelerated when cells mechanically sense solid surface. To
decipher the molecular mechanisms responsible for mechano-sensation and to explore a potential role
of cyclic di-GMP regulation we used microfluidic devices to observe single dividing C. crescentus cells
and their offspring when challenged with surface. Using this assay we could confirm rapid surfacemediated attachment of newborn swarmer cells and the strict dependence of this process on pili, a
rotating flagellar motor and an intact holdfast machinery. Furthermore, surface induced attachment was
also dependent on the diguanylate cyclase DgcB, indicating a role for cyclic di-GMP signaling in this
process. Interestingly, the rapid surface-mediated transition to sessility was not accompanied by cell
cycle progression into S-phase, arguing that two c-di-GMP dependent processes in C. crescentus,
surface adherence and S-phase entry, are not interconnected. These findings suggest that C.
crescentus can effectively and rapidly uncouple the tight interplay between cell cycle progression and
pole differentiation and that mechano-sensing processes override the cell cycle program when cells
encounter surface.
Lab: Urs Jenal
_________________
76
BLAST XII
_______________ Poster #26
SURFACE ACCESSIBILITY STUDY IDENTIFIES DOMAIN INTERACTIONS IN THE AEROTAXIS
RECEPTOR Aer
Darysbel Perez, Kylie J. Watts, Mark S. Johnson, and Barry L. Taylor
Division of Microbiology and Molecular Genetics, Loma Linda University, Loma Linda, CA, 92350
The Escherichia coli aerotaxis receptor, Aer, monitors cellular redox changes via FAD, which is
non-covalently bound to a PAS sensing domain. Changes in the redox state of FAD propagate from the
PAS domain to downstream HAMP and proximal signaling domains. Our recent disulfide crosslinking
studies demonstrate that the PAS β-scaffold can collide with the HAMP domain, and suggest that the
PAS β-scaffold is a site of PAS-HAMP interaction. If so, one would predict that surface residues in the
PAS β-scaffold would be partially hidden by the HAMP domain and would be less accessible to a
reactive hydrophilic probe.
To map PAS surface regions that are shielded in Aer, we created cysteine replacements for 57
PAS residues predicted to be surface accessible in an Aer-PAS homology model. Two additional
substitutions predicted to face the PAS interior served as inaccessible controls. The accessibility of the
substitutions was tested in vivo using the probe methoxypolyethylene glycol-maleimide 5000 (PEGmal). All mutants were tested for function as a proxy for native folding; only Aer-W94C abolished
aerotaxis. Residues were classified as having low (0-30% PEGylation), intermediate (31-50%
PEGylation), or high (≥51% PEGylation) accessibility according to the extent that the residue bound
PEG-mal in vivo.
Residues in the N-terminus of the PAS domain (the N-cap) were highly accessible (70-113%
PEGylated), suggesting that the N-cap is flexible and/or does not stably interact with other domains in
Aer. In contrast, a large area in the PAS β-scaffold (1,497 Å2) was inaccessible (0-26% PEGylated,
similar to inaccessible controls), and overlapped with a cluster of previously identified PAS signal-on
lesions, and with the proposed PAS-HAMP interaction site. Of the remaining residues tested, 9 had
low, 13 had intermediate, and 13 had high accessibility. Notably, nine of the residues with intermediate
accessibility bordered the hidden β-scaffold region and may delineate the boundary of the PAS-HAMP
contact surface.
To highlight potential PAS-PAS interaction sites, we tested whether the PAS cysteine
replacements could form disulfides in vivo in response to the oxidant copper phenanthroline.
We found that several of these cysteine replacements could form disulfides. Some of them occurred at
scattered positions around the PAS domain, but most of the PAS-PAS disulfides were in the flexible, Nterminal region, and were likely between (not within) dimers. Importantly, none of the cysteine
replacements in the inaccessible part of the PAS β-scaffold formed PAS-PAS disulfides.
To further map the PAS-HAMP interacting region, we chose 10 cysteine replacements in the
hidden region of the PAS domain and monitored disulfide formation between these and HAMP-Q248C.
Two of these cysteine replacements formed disulfides (V100C-Q248C and M112C-Q248C) (11-16%
crosslinking) and were located in the inaccessible region on the PAS β-scaffold. The remaining PASCys mutants surrounded this region but failed to crosslink, giving a clearer understanding of the PASHAMP interaction site. Together these data provide a putative contour map of PAS contact surfaces,
and provide novel insights into the PAS-HAMP signaling mechanisms used by Aer-type
chemoreceptors.
Lab: Barry Taylor
______________
77
BLAST XII
_______________ Poster #27
ROLE OF ENERGY SENSOR TlpD OF HELICOBACTER PYLORI IN GERBIL COLONIZATION AND
WHOLE GENOME ANALYSES AFTER ADAPTATION IN THE GERBIL
Wiebke Behrens1, Tobias Schweinitzer1, Sarah Klose1, Friederike Kops1, Birgit Brenneke1, Martina
Dorsch2, André Bleich2, Sebastian Suerbaum1, Peter Loewen3, Jonathan McMurry4, Christine
Josenhans1
1
Department for Medical Microbiology and Hospital Epidemiology,
Medical School Hannover, Carl-Neuberg-Straße 1, 30625 Hannover, Germany
2
Central Animal Laboratory, Medical School Hannover, Hannover, Germany
3
Department of Microbiology, University of Manitoba, Winnipeg, Canada
4
Department of Chemistry and Biochemistry, Kennesaw State University, Kennesaw, USA
Helicobacter pylori maintains colonization in its human host using a limited set of taxis sensors.
One sensor which appears dominant under environmental conditions of low bacterial energy yield is
TlpD, a proposed energy taxis sensor of H. pylori (Schweinitzer et al., 2008). Previous in vivo
colonization data of a H. pylori TlpD mutant in a mouse model (Williams et al., 2007) had indicated that
TlpD may not be essential in vivo. In addition, the mechanism of TlpD function remained unclear. One
important open question is the impact of H. pylori TlpD in vivo which was explored here using a gerbil
infection model, which closely mimicks the gastric physiology of humans. This approach included whole
genome analyses and comparisons of gerbil-adapted H. pylori strains.
A gerbil-adapted H. pylori strain, HP87 P7, was able to perform TlpD-dependent energy
behavior, while its isogenic mutant in tlpD had lost it. A complemented strain regained the ability. Gerbil
infections demonstrated that TlpD was essential for the initial colonization in the antrum as well as for
persistence. A long-term effect of tlpD-deficiency in reducing H. pylori colonization was observed both
in the antrum and corpus region of the gerbil stomach. The complete genome sequence of the gerbiladapted strain HP87 P7 was elucidated and compared with HP87 pre-gerbil, with a HP87 P7 tlpD clone
pre-gerbil and with a reisolate pool of HP87 P7 tlpD which persisted during the gerbil infection. Genetic
differences of the strains after gerbil adaptation were observed but no major losses or gains of genes or
gene functions. The tlpD mutant gerbil reisolate also had acquired several SNPs in comparison to the
gerbil-adapted wild type strain and the HP87 P7 tlpD input strain. The occurrence of potential
adaptations to the animal host or mutations compensating for the loss of TlpD in the gerbil model will be
discussed.
Schweinitzer T, Mizote T, Ishikawa N, Dudnik A, Inatsu S, Schreiber S, Suerbaum S, Aizawa S,
Josenhans C, 2008. J. Bacteriol. 190:3244-55.
Williams SM, Chen YT, Andermann TM, Carter JE, McGee DJ, Ottemann KM, 2007. Infect Immun.
75:3747-57.
Lab: Christine Josenhans
_______
78
BLAST XII
INTERCONNECTIVITY
ESCHERICHIA COLI
_______________ Poster #28
BETWEEN
TWO
LytTR-LIKE
TWO-COMPONENT
SYSTEMS
IN
Stefan Behr, Luitpold Fried, and Kirsten Jung
Center for Integrated Protein Science Munich (CiPSM) at the Ludwig-Maximilians-University Munich,
Microbiology, Großhaderner Str. 2-4, 82152 Martinsried, Germany
Bacteria use two-component systems (TCSs) to encounter fluctuating environmental conditions.
A membrane-bound histidine kinase (HK) senses a stimulus and transduces it into a cellular signal via
phosphorylation. The transfer of this phosphoryl group to a response regulator (RR) with DNA-binding
properties mediates the inert reaction, generally an alteration in gene expression (1). Based on the
limited number of TCSs in Escherichia coli (30/32 HK/RR) it is necessary to coordinate cellular
adaptions in order to respond to a multitude of environmental signals. To this end many so called
auxiliary proteins have been described recently (2). These proteins can be involved in sensing,
scaffolding or connecting TCSs and evolved to an emerging field of bacterial signal transduction.
Both LytS/LytR-like TCSs of E. coli, the YehU/YehT TCS (3) and YpdA/YpdB TCS are studied in
our laboratory. Based on bioinformatical data these two TCSs share an amino acid identity of more
than 30%. They are found in many γ-proteobacteria (4). The characterization of the YehU/YehT and the
YpdA/YpdB systems revealed reversed transcriptional effects on target genes.
Using the bacterial adenylate cyclase-based two-hybrid system YehS was uncovered as hub
connecting the two TCSs via protein-protein interactions. Surface plasmon resonance spectroscopy
with purified YehS and the RRs confirmed the interactions and suggest an interconnectivity between
YehU/YehT and YpdA/YpdB.
References:
1) Stock et al. (2000): Two-component signal transduction. Annu Rev Biochem 69:183-215
2) Jung et al. (2011): Histidine kinases and response regulators in networks. Curr. Opin. Microbiol. 2:118-24
3) Kraxenberger et al. (2012): First insights into the unexplored two-component system YehU/YehT in
Escherichia coli. J. Bacteriol. 194(16):4272-84
4) Szklarczyk et al. (2011): The STRING database in 2011: functional interaction networks of proteins, globally
integrated and scored. Nucleic Acids Res. 39:561-568
Lab: Kirsten Jung
______________
79
BLAST XII
_______________ Poster #29
LOCALIZATION CONTROL OF CHEMOTAXIS-RELATED SIGNALING COMPONENTS OF V.
CHOLERAE
Geetha Hiremath1, Hiroki Okabe2, So-ichiro Nishiyama2, Ikuro Kawagishi3
1
Research Center for Micro-Nano Technology, and 2Dept. of Frontier Bioscience, Hosei University,
Japan.
The genome sequence of V. cholerae has revealed that this bacterium possesses three sets of
proteins with similarity to conventional chemotaxis-signaling proteins (Che proteins). Each set
constitutes a distinct system, and only System II is directly involved in chemotaxis. To investigate the
roles of Systems I and III and also to understand how parallel (closely related) signaling systems can
perform distinct functions in V. cholerae, subcellular localization of their components were observed
using GFP fusions. Whereas System II components localized constitutively to a cell pole, the histidine
kinases (CheA1and CheA3) and adaptor proteins (Putative CheW, CheW2 and CheW3) of Systems I
and III showed clustering at a cell pole and lateral regions of the membrane in cells cultured with
standing (microaerobic) but not in cells cultured with shaking (aerobic), implying that Systems I and III
functions under oxygen-limiting conditions. Polar localization of the histidine kinases of Systems I and
III was induced by the addition of NaN 3 , but not that of chloramphenicol, to shaking cultures. Thus, it is
neither mechanical stimuli, oxgen limitation nor growth retardation per se, but changes in energy
metabolism that triggers the localization of these components. We further examined the effect of NaN 3
under various conditions. Increasing concentrations of NaN 3 at 30˚C enhanced localization of CheA1
and CheA3 to a cell pole and lateral membrane regions. Changes in temperature from 30˚C to 37˚C
showed that CheA1 localized to the pole in the presence of lower concentrations of NaN 3 , and CheA3
localized to the pole in the absence of NaN 3 . CheA2, which always localized to a cell pole at 30˚C even
in the presence of NaN 3 , altered its localization to cell pole and lateral membrane regions at 37˚C with
0.2% NaN 3 . Immunoblotting analyses demonstrated that these changes in localization of the CheA
proteins are not due to those in their expression levels. These results suggest that components of
Systems I and III of V. cholera may be assembled into active signaling complexes under energylimitating conditions.
Lab: Ikuro Kawagishi
___________
80
BLAST XII
_______________ Poster #30
THE ROLE OF FlgM IN REGULATING FLAGELLAR ASSEMBLY IN BACILLUS SUBTILIS
R. A. Calvo and D. B. Kearns
Indiana University, Bloomington, IN, USA
Flagella are composed of over thirty different proteins, and transit both the membrane and
peptidoglycan to provide motility to diverse bacterial species. The flagellum is composed of three parts:
a membrane-embedded anchor called the basal body, a universal joint called the hook, and an
extracellular helical propeller called the filament. Coordinated assembly of the flagellum requires
bacteria to couple gene expression with specific stages in assembly. Early flagellar genes are
transcribed by the housekeeping sigma factor σA. The early genes encode components of the hookbasal body (HBB) and a type-three secretion apparatus. The HBB serves as channel through which
extracellular components of the flagellum are secreted by the type-three secretion apparatus. Late
flagellar genes, such as the filament protein, are transcribed by the alternative sigma factor σD.
σD activity is inhibited by the anti-sigma factor FlgM prior to HBB completion. Once HBB
assembly is complete, FlgM is antagonized and inhibition of σD is relieved. Relief of σD inhibition allows
transcription of the filament gene and leads to the assembly of a functional flagellum. FlgM plays a
crucial role in flagellar morphogenesis in that it couples gene expression to assembly state. In
Salmonella enterica, FlgM is antagonized by secretion through the HBB. This regulatory paradigm has
only been observed in the γ-proteobacteria, and the mechanism by which the HBB antagonizes FlgM in
B. subtilis is not known.
By precipitating proteins from culture supernatant, we found that FlgM is secreted through the
HBB in B. subtilis. Furthermore, we determined that FlgM is degraded extracellularly by a specific
subset of the seven proteases secreted by B. subtilis. Although FlgM is secreted, secretion may not be
sufficient to regulate the anti-σD activity of FlgM. Mutations in the HBB abolish FlgM secretion and
σD activity is inhibited. However, certain HBB mutants that fail to secrete FlgM have active σD. Currently,
we are screening for regulation blind alleles of FlgM in an effort to understand the role of FlgM in
regulating σD activity in response to the assembly state of the flagellum.
Lab: Daniel Kearns
_____________
81
BLAST XII
_______________ Poster #31
ROLE OF THE PSEUDOMONAS AERUGINOSA FLAGELLAR MOTOR IN SWIMMING MOTILITY
AND CHEMOTAXIS
Chui Ching Wong, Chen Qian and Keng-Hwee Chiam
Mechanobiology Institute, National University of Singapore, Singapore
Flagellar-driven swimming motility is well-established in some bacterial model organisms, and it
is best described in the case of Escherichia coli. However, increasing genetic and structural data show
that diversity in flagellar motors exists across the bacterial kingdom, where new paradigms of swimming
motility may be discovered. In this report, we describe the flagellar motor function of monotrichous P.
aeruginosa, and show that unlike E. coli, it is a motor that rotates in both CCW and CW directions
giving rise to a ‘run-and-reverse’ trajectory. Additionally, the flagellar motor exhibits two-speeds in CCW
and CW direction. Using a microfluidic-based assay, we show that in the presence of a chemoattractant
(serine), the cells alter their run-length, switching frequency and motor speeds in order to move toward
favourable environments. Therefore, in chemotaxis, apart from varying the switch frequency, the P.
aeruginosa flagellar motor has an additional mechanism that allows it to favour the higher rotation
speed state. These findings are validated in a computational model of P. aeruginosa swimming and
chemotaxis.
Lab: Chiam Keng Hwee
_________
82
BLAST XII
_______________ Poster #32
BINDING AFFINITY PROVIDES KINETIC PREFERENCE, SPECIFICITY AND SIGNALING FIDELITY
FOR TWO-COMPONENT SYSTEMS REGULATING DEVELOPMENT IN MYXOCOCCUS XANTHUS
Jonathan W. Willett1, Nitija Tiwari2, Katherine Hummels1, Ernesto Fuentes2, John R. Kirby1
1
University of Iowa, Department of Microbiology
2
University of Iowa, Department of Biochemistry
Two-component signal transduction systems, composed of histidine kinases and their cognate
response regulators provide bacteria with the ability to sense and respond to a wide variety of signals.
Upon activation the histidine kinase, can both phosphosphorylate and subsequently dephosphorylate
the cognate response regulate. These reactions are highly specific and take place via a transiently
formed histidine kinase and response regulator complex. A highly tractable model system for studying
signaling specificity, fidelity and HK-RR interactions can be found in the organism Myxococcus xanthus.
Here we report a systems-level analysis on a highly homologous family of two-component systems.
The genome of M. xanthus encodes for over 125 two-component systems and as such has one of the
largest number of signal transduction proteins in any bacterium. Bioinformatic analyses led us to
conclude that M. xanthus possesses 27 NtrC homologs, which display high degrees of sequence
similarity. 21 of these NtrC homologs are encoded next to predicted cognate kinases. All of these TCS
have homology to the prototypical E. coli TCS system NtrB and NtrC. Using in vitro phosphotransfer
and phosphatase profiling methods we show that each tested HK has kinetic preference for its
predicted cognate regulator. Using isothermal titration calorimetry we demonstrate that cognate HK-RR
pairs display simmilar dissociation constants (K d ) of approximately 1 μM while non-cognate pairs have
no measureable binding affinity in this assay. Lastly, we generated a chimeric HK to demonstrate that
previously known residues conferring phosphotransfer specificity also impart binding preference.
These data suggest that phosphotransfer and phosphatase activities are predicated by discrete and
measurable protein-protein interactions.
Lab: John Kirby
________________
83
BLAST XII
_______________ Poster #33
SECRETION AND GLIDING OF BACTERODETES PHYLUM
Keiko Sato, Daisuke Nakane, Mark J. McBride and Koji Nakayama
Department of Molecular Microbiology and Immunology, Graduate
Sciences, Nagasaki University, 852-8588 Nagasaki, Japan
School
of
Biomedical
Flavobacterium johnsoniae is a Gram-negative bacterium, which moves rapidly over surfaces at
a speed of 1-3 um/s in gliding motility. In F. johnsoniae, the ahesion complexes containing SprB
adhesin and two proteins that are involved in the bacteriodete protein translocation systems (referred to
as the Por secretion system (PorSS)) are essential for gliding motility. The disruption of the PorSS
proteins resulted in defects in translocation and assembly of SprB to cell surfaces and therefore in
motility in F. johnsoniae. Many members of the phylum Bacteroidetes, including gliding bacteria such as
F. johnsoniae and Cytophaga hutchinsonii, and nomotile bacteria such as Porphyromonas gingivalis,
encode the genes of related to PorSS. Not only F. johnsoniae but also P. gingivalis secrete many
proteins ossessed the C-terminal domains (CTDs), which have been suggested to form the CTD
protein family including pathogen cysteine proteinase and hemagglutinin by PorSS.
Lab: Koji Nakayama
____________
84
BLAST XII
_______________ Poster #34
THE RESPONSE REGULATOR RRP1 REGULATES CHITOBIOSE UTILIZATION AND VIRULENCE
OF THE LYME DISEASE SPIROCHETE BORRELIA BURGDORFERI
Ching Wooen Sze1, Alexis Smith3, Young Hee Choi2, Xiuli Yang3, Utpal Pal3, Aiming Yu2, and Chunhao
Li1*
Department of Oral Biology, The State University of New York at Buffalo, Buffalo, New York1,
Department of Pharmaceutical Sciences, The State University of New York at Buffalo, Buffalo, New
York2,
Department of Veterinary Medicine, University of Maryland and Virginia-Maryland Regional College of
Veterinary Medicine, College Park, Maryland3
Life cycle alternating between arthropod and mammals forces the Lyme disease spirochete,
Borrelia burgdorferi, to adapt to different host milieus by utilizing diverse carbohydrates. B. burgdorferi
lacks the de novo biosynthesis pathway of N-acetylglucosamine (GlcNAc) but encodes a set of
chitobiose metabolic genes that allows conversion of chitobiose to GlcNAc. The chbC gene, a key
chitobiose transporter, is dually regulated by RpoD and RpoS. Rrp1 is a response regulator that
synthesizes cyclic diguanylate (c-di-GMP) in B. burgdorferi. In this report, we found that the rrp1 mutant
had growth defects and formed membrane blebs when GlcNAc was replaced by chitobiose in growth
medium. The expression level of chbC was significantly repressed in the mutant and constitutive
expression of chbC successfully reversed the phenotype. Immunoblotting and transcriptional studies
revealed that Rrp1 is required for the activation of BosR and its impact on chbC is likely mediated by
the BosR-RpoS signaling pathway. Although the rrp1 mutant was unable to be transmitted via tick bite,
exogenous supplementation of GlcNAc into unfed ticks partially rescued the transmission. Based on
these results, we propose that Rrp1 governs the chitobiose metabolism of B. burgdorferi via a BosRRpoS-ChbC regulatory network essential for the transmission of the spirochete.
Lab: Chunhao Li
_______________
85
BLAST XII
_______________ Poster #35
CRYO-ELECTRON TOMOGRAPHY OF ESCHERICHIA COLI MINICELLS REVEALS MOLECULAR
ARCHITECTURE OF INTACT FLAGELLAR MOTOR
Bo Hu1, Xiaowei Zhao1, William Margolin2 and Jun Liu1
1. Department of Pathology and Laboratory Medicine, University of Texas Medical School at Houston,
Houston, TX 77030, USA
2. Department of Microbiology & Molecular Genetics, University of Texas Medical School at Houston,
Houston, TX 77030, USA
Cryo-electron tomography (cryo-ET) has emerged as a powerful technology to study intact
flagellar motor structure in living cells. However, the power of cryo-ET is significantly compromised in
Escherichia coli because of their larger diameters, resulting in limited structural information. To
circumvent this limitation, we constructed a mreB mutant of E. coli that produces cells with smaller
diameters. Additional inactivation of clpX and min and overexpression of flhD/flhC genes resulted in
highly motile and tiny minicells, which are about 0.3 micrometers in diameter and also contain one to
three flagella. Cryo-ET and image analysis of several thousands of the wild-type cells yielded a more
detailed three-dimensional (3-D) structure of the intact E. coli flagellar motor. Our comparative analysis
of the flagellar motor structures from the wild-type cells, non-motile motA/B deletion mutants and
purified flagellar motors provides new insights into the dynamic of the major components of the motor:
the stator, C ring and L/P rings. Our results suggest that the flagellar motor is not a rigid, but a highly
dynamic and flexible structure, which could undergo large elastic deformation in cells.
Lab: Jun Liu
___________________
86
BLAST XII
_______________ Poster #36
THE TORQUE GENERATOR OF BACTERIAL FLAGELLAR MOTOR REVEALED IN BORRELIA
BURGDORFERI
Xiaowei Zhao1, Kihwan Moon2, Tristan Boquoi2, Steven, J. Norris1, Md A. Motaleb2, Jun Liu1
Department of Pathology and Laboratory Medicine, University of Texas Medical School at Houston,
Houston, TX 77030
2
Department of Microbiology and Immunology, Brody School of Medicine, East Carolina University,
Greenville, NC 27834
1
The bacterial flagellum is the core organelle of motility in bacteria, and is often important for the
virulence of many bacterial pathogens including Lyme disease spirochete Borrelia burgdorferi. Powered
by the membrane ion-motive force, the flagellar motor converts electrochemical energy into torque
through an interaction between the rotor and its surrounding stator. The stator is the torque-generating
unit composed of the membrane protein complex MotA/MotB. The mechanism of how stator interacts
with the rotor remains elusive due to the lack of structural information of these components. Here we
present the three-dimensional (3-D) structures of B. burgdorferi periplasmic flagellar motor in wild-type
(WT), motA, and motB mutants by utilizing high-throughput cryo-electron tomography (cryo-ET) and
sub-volume analysis. Comparative analysis of those motor structures revealed the 3-D in situ structure
of the torque generating unit and its interaction with the rotor. Sixteen stator units are assembled
peripherally around the rotor. The cytoplasmic domain of the stator is located adjacent to the C-terminal
domain of the rotor protein FliG. This stator-rotor interaction induces an unexpected conformational
change of FliG. We also mapped the association of stator-rotor in a nonmotile motBD24N mutant and a
less motile motBD24E mutant, in which the proton translocation is blocked or limited, respectively. The
results show that the incorporation of stators into the motor is closely associated with the proton flux.
Collectively, our results provide novel structural insights into the rotor-stator interaction, which is
fundamentally important for understanding the mechanism of flagellar rotation and bacterial motility.
Lab: Jun Liu
___________________
87
BLAST XII
_______________ Poster #37
THE CYTOPLASMIC AROMATIC ANCHOR OF TM2 REGULATES TRANSMEMBRANE SIGNAL
TRANSDUCTION BY THE Tar CHEMORECEPTOR
Christopher Adase, Roger Draheim, Raj Desai and Michael D. Manson
Texas A&M University, BSBE Rm 303, MS3258, College Station, TX 77843
The chemoreceptor Tar of Escherichia coli (Tar Ec ) interacts with two of its principal attractant
ligands very differently. L-aspartate binds directly to the receptor; whereas maltose interacts with the
receptor indirectly though the maltose-binding protein. The repellent nickel also appears to interact
directly with the periplasmic domain, although the exact binind site is currently not known. Thus, Tar
must communicate transmembrane signals in response to at least three modes of ligand binding.
Our specific interest is in how particular combinations of aromatic residues at the cytoplasmic
(C-terminal) end of transmembrane helix 2 (TM2) contribute to baseline signal output and to ligand
sensitivity. Each of the four MCPs of E.coli has a unique aromatic anchor composition, and Tar Ec has
been subjected to evolutionary selection for the ability to respond to divergent ligands. This process is
occurs for each transmembrane sensor leading to the variety of aromatic anchor residue combinations
and number of aromatic anchors found in various transmembrane sensors of two component systems.
We report here studies in which both the position and residue composition of the aromatic
anchor of Tar Ec have been altered. Our results indicate which parameters of the aromatic anchor are
most critical to its function. They also demonstrate the importance of Gly-211, which immediately
follows Trp-209/Tyr-210 of the aromatic anchor of wild-type Tar Ec anchor, in the response to ligands.
Together with previously published information on the TM2-HAMP connector cable of Tsr (Kitanovic et
al., 2011), our data can be used to generate a model for tunable signal transduction by transmembrane
receptors.
Lab: Michael Manson
___________
88
BLAST XII
_______________ Poster #38
COMPARATIVE ANALYSIS OF FLAVOBACTERIUM JOHNSONIAE
ALGICOLA GLIDING MOTILITY AND PROTEIN SECRETION
AND
CELLULOPHAGA
Yongtao Zhu and Mark J. McBride
Department of Biological Sciences, University of WI-Milwaukee
Many members of the phylum Bacteroidetes glide rapidly over surfaces. Bacteroidete gliding
has been well studied in Flavobacterium johnsoniae. Gliding is powered by proton motive force and
involves the rapid movement of adhesins such as SprB along the cell surface. Nineteen gld and spr
genes are required for efficient gliding. The distantly related bacteroidete Cellulophaga algicola has
orthologs for most of these but lacks gldA, gldF, and gldG. F. johnsoniae GldA, GldF and GldG are
similar in sequence to ATP-binding-cassette (ABC) transporter components. The absence of these
proteins in C. algicola suggests that they do not have a central or irreplaceable role in gliding.
To address the possibility of the involvement of another transporter in gliding we developed
techniques for HimarEm1 transposon mutagenesis, site-directed mutagenesis, and complementation
analyses for C. algicola. Sixty-one HimarEm1-induced motility mutants that formed nonspreading
colonies were isolated and characterized. Mutants containing transposon insertions in gldB, gldI, gldJ,
gldK, gldL, gldM, gldN, sprA, sprB, sprC, sprE, sprF, and sprT, which correspond to F. johnsoniae
gliding motility genes, were identified. No ABC transporter genes were identified and no novel genes
required for C. algicola gliding were found. The results suggest that an ABC transporter is not required
for C. algicola gliding motility. They also reinforce the importance of the other Gld and Spr proteins for
gliding. gldD and gldH are required for F. johnsoniae gliding and orthologs are found in C. algicola but
no insertions were identified in these genes. gldD and gldH are small, which probably explains the
absence of transposon insertions. We disrupted gldD and gldH by insertion mutagenesis and
demonstrated that each mutant had severe motility defects. Surprisingly, unlike F. johnsoniae gldD
mutants the C. algicola gldD mutants exhibited some motility. The results suggest that GldD may be
less critical than some of the other components of the motility machinery.
Some of the motility proteins (GldK, L, M, N, SprA, E, T) are components of a novel
Bacteroidetes-specific protein secretion system, the Por secretion system, also referred to as the Type
IX secretion system (T9SS). The F. johnsoniae T9SS is needed for secretion of SprB to the cell surface
and for secretion of an extracellular chitinase. The C. algicola T9SS is probably involved in secretion of
SprB, but it also appears to be required for secretion of one or more proteases. Mutations in gldK, L, M,
N, sprA and sprT resulted in inability to digest casein. Surprisingly, sprE mutant cells digested casein
as well as the wild type. This suggests that SprE may have a less central role in protein secretion than
the other components. These studies of C. algicola complement those performed on F. johnsoniae and
help to identify the most critical components of the gliding motility and T9SSs of members of the phylum
Bacteroidetes.
Lab: Mark McBride
_____________
89
BLAST XII
_______________ Poster #39
GLIDING OF MYCOPLASMA MOBILE CAN BE EXPLAINED BY DIRECTED BINDING
Akihiro Tanaka, Daisuke Nakane, Takayuki Nishizaka and Makoto Miyata
3-3-138 Sugimoto Sumiyoshi-ku Osaka, Japan
Mycoplasma mobile, a fish pathogen, forms a membrane protrusion, namely the gliding
machinery, and glides on solid surface smoothly up to 4.5 μm/s and 27 pN in the direction of protrusion.
The “directed smooth gliding” has been suggested to occur through repeated catch-pull-release of
sialylated oligosaccharide existing on animal cells by hundreds of 50 nm flexible “legs” sticking out from
the gliding machinery. However, the directed displacement is difficult to explain, because the legs
responsible for gliding is very flexible under electron microscopy and fast scan AFM (atomic force
microscopy). In the present study, we measured the force to detach the cell laterally from glass surface
by using laser trap. A single cell binding to sialylated oligosaccharide fixed on glass was pulled forward
and backward by manipulating the plastic bead attached to front or tail of the cell. The forces required
to detach the cell were 22.5 ± 13.9 pN and 48.4 ± 28.0 pN for forward and backward, respectively.
These results suggest that M. mobile binds to solid surface in a direction dependent manner, and this
feature may cause the directed gliding.
Lab: Makoto Miyata
_____________
90
BLAST XII
_______________ Poster #40
GLIDING MACHINERY OF MYCOPLASMA MOBILE OBSERVED BY ELECTRON MICROSCOPY
Hiroki Yamamoto, Makoto Miyata
3-3-138 Sugimoto Sumiyoshi-ku Osaka Japan
Mycoplasma, a fish pathogen, forms a membrane protrusion at a cell pole and glides in the
direction of protrusion, by a unique mechanism. The structural information on the gliding machinery is
critical to elucidate this unique mechanism. The gliding machinery is composed of mainly three huge
proteins, Gli521, Gli349, and Gli123, weighing 521, 349, and 123 kDa, respectively. To date, the “leg”
structure on the surface of the gliding machinery and the molecular shapes of isolated component
proteins have been clarified, by freeze-fracture and rotary-shadowing electron microscopy (EM),
respectively. However, the whole structure of the machinery or the assignment of component proteins
in the machinery remains unclear. We examined negative staining and rotary-shadowing EM under
reduced backgrounds, and then achieved the better image of gliding machinery featured with two types
of fibers. To assign filamentous proteins, Gli349 and Gli521, involved in the gliding mechanism, onto
two types of fibers, we examined the subcellular localization and amounts of gliding proteins by
immunofluorescence microscopy, for the wild type and mutant strains, and compared these results with
their EM images. The results suggested that Gli349 sticks out from the membrane and Gli521 lines
along the membrane.
Lab: Makoto Miyata
_____________
91
BLAST XII
_______________ Poster #41
GENE MANIPULATION OF GLIDING BACTERIUM, MYCOPLASMA MOBILE
Isil Tulum, Atsuko Uenoyama, and Makoto Miyata
Graduate School of Science, Osaka City University
Mycoplasma mobile, a fish pathogen, is the fastest-gliding mycoplasma. It glides on solid
surfaces at an average speed of 2.0 to 4.5 μm/s, exerting a force up to 27 pN. M. mobile exhibits
gliding motility in the direction of the protrusion, which is essential for mycoplasma infection. This
motility is caused by a unique mechanism composed of at least three huge surface proteins and an
ATPase, and probably more than ten internal proteins. To elucidate this mechanism, genetic
manipulation should be a powerful tool. However, this organism has not been transformed so far by
standard procedures used for other Mycoplasma species. In this study, we examined and improved
each step of transformation, and found and solved the possible problems. (i) The electroporation
conditions have been adjusted to fit to the M. mobile cells which are much tougher than the other
species. (ii) The recovery time after electroporation was elongated from 2 to 12 h. (iii) The growth
medium was modified to get clearer colony shapes. Then, we achieved the transformation of M. mobile
with efficiencies ranging 10-5 to 10-7 per competent cells by using 10 μg of oriC plasmids.
Transformation was confirmed by colony PCR, and labeling of cells by EYFP. In order to improve the
transformation efficiencies, we have developed vectors containing different marker genes under the
control of different M. mobile specific promoters. The results suggested the best combination for
transformation.
Lab: Makoto Miyata
_____________
92
BLAST XII
IS MOTILITY OR POSSESSION OF
PATHOGENESIS OF LYME DISEASE?
_______________ Poster #42
PERIPLASMIC
FLAGELLA
CRUCIAL
FOR
THE
S. Z. Sultan1, A. Manne1, X. Zhao2, Y. Chien2, Jun Liu2, P. Sekar3, R. M. Wooten3 and M. A. Motaleb1.
1
Dept. of Microbiology and Immunology, East Carolina University; 2Dept. of Pathology and Lab
Medicine, University of Texas Medical School at Houston; 3Dept. of Medical Microbiology and
Immunology, University of Toledo College of Medicine, Toledo, OH.
Motility and chemotaxis are known to be important for host tissue colonization and disease
production by many bacteria, including the spirochetes. Rotation of flagellar motors is required for
propulsion of bacteria. However, flagellin molecules are recognized by Toll-like receptor 5, which
activates host inflammatory responses. Additionally, bacterial flagella are potent immunogens and elicit
adaptive immune response, and the immunogenicity of flagellin has been the basis for several vaccine
strategies. However, the role of Borrelia burgdorferi motility in the pathogenesis of Lyme disease has
not been rigorously studied. The genome of B. burgdorferi contains more than fifty genes encoding the
motility and chemotaxis proteins. We have recently inactivated the flaB gene, which encodes the major
periplasmic flagellar filament. The flaB mutants were rod shaped and non-motile, whereas the wild-type
cells are motile and flat-wave. The flaB mutants were unable to infect experimental mice by needle or
ticks, suggesting that either motility and/or the presence of the periplasmic flagella is crucial for
infection. To verify if the mere presence of structural periplasmic flagella without their motor function
can establish an infection, we created a motB-specific mutant. B. burgdorferi ΔmotB retained their
periplasmic flagella, but were paralyzed. ΔmotB display a flat-wave morphology similar to wild-type
cells, however, they possess a rod-shaped section in the middle of the cell body. Using cryo-electron
tomography, we revealed that ΔmotB periplasmic flagella form a ribbon in the flat-wave region, while
the ribbon is not well-organized in the rod-shaped area. Mouse and tick-mouse infection studies using
ΔmotB determined that motility, and not just the possession of periplasmic flagella, is important for the
pathogenesis of Lyme disease. Further study of ΔmotB and ΔflaB may discriminate the effect of these
immune agonists during the disease process.
Lab: Md Motaleb
_______________
93
BLAST XII
_______________ Poster #43
Fe(II) SENSING IN PSEUDOMONAS AERUGINOSA
Naomi Kreamer, Maureen Coleman, James Boedicker, Dianne Newman
California Institute of Technology 1200 E. California Blvd. Pasadena, CA 91125
Pseudomonas aeruginosa is a ubiquitous Gram-negative bacterium best known as the
predominant opportunistic pathogen infecting the lungs of cystic fibrosis patients. In this context, it is
thought to form biofilms, within which locally reducing and acidic conditions can develop that favor the
stability of ferrous iron [Fe(II)]. Because iron is a signal that stimulates biofilm formation within a
particular concentration range, we performed a microarray study to determine whether P. aeruginosa
exhibits a specific transcriptional response to extracellular Fe(II). We identified a novel two-component
sensor histidine kinase (BqsS) – response regulator system (BqsR) that responds specifically to Fe(II).
BqsS distinguishes between Fe(II) and Fe(III) and other dipositive ions, responding solely to Fe(II). The
periplasmic domain of the sensor contains a motif similar to a known Fe-binding motif: RExxE. Sitedirected mutants in this putative Fe(II)-binding motif abolish the Fe(II) shock transcriptional response.
Using bioinformatics, we identified a putative DNA sequence that BqsR binds based on identifying
conserved patterns in regions upstream of the genes most highly upregulated in an Fe(II) shock
microarray experiment. We are in the process of testing this prediction biochemically using gel-shift
assays with purified BqsR. In parallel, we are attempting an in vivo assay to verify the specificity of this
motif by employing a LacZ-reporter construct to measure transcription from engineered promoter sites.
Lab: Dianne Newman
___________
94
BLAST XII
_______________ Poster #44
DIRECT OBSERVATION OF UNITARY STEP OF GLIDING MACHINERY IN MYCOPLASMA MOBILE
Yoshiaki Kinosita1, Daisuke Nakane2, Tomoko Masaike1, Kana Mizutani1, Makoto Miyata2, Takayuki
Nishizaka1
1
Dept. Phys., Gakushuin Univ.,2Dept. Biol., Osaka City Univ.
Mycoplasma mobile (M.mobile) is a fish pathogenic flask-shaped bacterium of about 0.8 μm
long and glides on the substrate at speed of 2.5-4.0 μm/sec. The mechanism of the gliding motility has
been unique because the gene of M. mobile contains no homologs related to known bacterial motility or
conventional motor proteins such as kinesin and myosin. With a series of recent contributions, it was
revealed that M. mobile has four novel proteins, all of which are involved in the gliding motion, Gli123,
Gli349, Gli521 and P42 (1). To establish the molecular basis of the gliding mechanism, we here
develop a method to detect the unitary step of the gliding machinery comprised of these proteins under
the changing ATP concentrations in the medium. Three strategies were adopted: 1) The cell membrane
was stained with a fluorescent dye, Cy3, so that we could observe the motility of single bacterium under
fluorescent microscope at high temporal resolution (2 msec). 2) The addition of Triton X-100
permeabilized the cells, called them gliding ghosts (2). M. mobile gliding is supplied by ATP hydrolysis
and so we could control its gliding speed by changing ATP concentrations in the medium 3) To reduce
the number of legs contributing to the motility, free sialylated oligosaccharide, binding target for the
gliding motility, were added. Through these improvements we successfully observed repetitive stepwise
motions with the size of 70 nm. The speed during each stepping coincides with the maximum speed,
suggesting that the continuous gliding motion comprises assemblies of the unitary step we detected.
The 70 nm step size is the largest one ever reported amongst all motor proteins and possibly
contributes to the characteristic fast gliding of M. mobile.
(1) Miyata, M. Annu Rev Microbiol 64, 519-37 (2010).
(2) Uenoyama A, Miyata M. Proc. Natl. Acad. Sci. USA 102:12754–58 (2005).
Lab: Takayuki Nishizaka
________
95
BLAST XII
_______________ Poster #45
THE HELICOBACTER PYLORI TlpD CHEMORECEPTOR IS CRITICAL FOR MULTIPLE STAGES OF
NORMAL STOMACH COLONIZATION AND MEDIATES A RESPONSE TO IRON AND OTHER
REACTIVE OXYGEN SPECIES-GENERATING CONDITIONS
Susan M. Williams, Tessa M. Andermann, Lisa Sanders, Sarina Porcella, Kieran Collins, and Karen M.
Ottemann
Department of Microbiology and Environmental Toxicology, UC Santa Cruz, Santa Cruz, CA 96054,
USA
Helicobacter pylori is an epsilon proteobacterium that uses chemotaxis to infect the human
stomach and promote inflammation in tissues. The H. pylori chemotaxis system contains four
chemoreceptors, called TlpA, TlpB, TlpC and TlpD. TlpD is a soluble chemoreceptor that has been
implicated in an energy taxis response (J Bacteriol, 2008 190: 3244-55). We have explored TlpD’s role
in stomach colonization and conditions that it responds to. Toward this inquiry, we generated a tlpD
mutant in strains SS1 and mG27. tlpD mutants have a substantial initial colonization defect, and also
show defects after two weeks of colonization, suggesting tlpD is needed for both initial and sustained
colonization. The tlpD-associated defect could be complemented by expression of tlpD from a
heterologous locus, confirming that TlpD itself is required for mouse colonization. To determine what
TlpD responds to, we employed a variety of chemotaxis assays. Initially, we observed that tlpD mutants
fail to form a bacterial band in response to iron chloride in a modified agarose plug bridge assay. The
wild type band formed at a slight distance from the plug that could represent either an attractant or
repellent response. We thus analyzed bacterial temporal swimming behavior, and found that iron
caused a tlpD-dependent repellent response in a strain mG27 isolate, but an attractant response in
strain SS1. We furthermore used the temporal swimming assay to determine that H. pylori uses TlpD to
detect hydrogen peroxide as a repellent. These studies indicate that wild-type sensing of iron and
hydrogen peroxide require TlpD, suggesting that TlpD is part of H. pylori’s ability to sense intracellular
stress, particularly oxidative stress.
Lab: Karen Ottemann
___________
96
BLAST XII
_______________ Poster #46
THE ROLE OF RECEPTOR-KINASE INTERACTIONS IN ARRAY SIGNALING
Germán Piñas and John S. Parkinson
Biology Department, University of Utah, Salt Lake City, Utah 84112
Recent models of chemoreceptor array organization (Briegel et. al, 2012; Liu et al., 2012)
predict a contact between the tip of the receptor dimer and the P5 domain of the CheA dimer that might
be important to signaling. The CheA kinase has five domains with different signaling roles: P1, the
phosphorylation site domain; P2, a phosphotransfer target acquisition domain; P3, the dimerization
domain; P4, the ATP-binding domain; and P5, a domain that resembles the CheW protein required for
coupling CheA to chemoreceptor control. P5 appears to mediate binding interactions with CheW and
with receptors, but only the P5-CheW interaction is known to be critical for receptor coupling control.
To explore the signaling role of the predicted P5-receptor interaction, we constructed a series of amino
acid replacements in E. coli CheA at P5 residues proposed, by biochemical and structural studies from
various groups, to lie at the receptor-binding surface: L528, V531, S534, I581, L599. The mutant CheA
proteins were encoded on a regulatable plasmid that also expressed CheW, to maintain normal
stoichiometry of these interacting components. The mutant plasmids were transferred to various host
strains to test their behaviors in soft agar chemotaxis assays and with in vivo FRET kinase assays. We
found that most amino acid replacements at V531 and S534 had little effect on chemotaxis in plate
assays, whereas roughly one-half of the amino acid replacements at L528, I581, and L599 impaired
chemotactic ability on plates. The most deleterious amino acids at all three positions were ones with
charged sidechains (D, E, K, R) or with very small (G) or very large (W) sidechains. These findings
indicated that putative P5-receptor contact residues were important for operation of the signaling array.
These P5 mutants evinced a variety of signaling defects, often rather subtle ones, in FRET
kinase assays with wild-type Tsr partners in the QEQE modification state. Replacements at L528
typically increased serine sensitivity; those at I581 mainly reduced cooperativity; and those at L599
usually exhibited changes in both parameters. To ask whether these P5 lesions might have additive
effects on signaling proficiency, we constructed and characterized a series of triple mutants with G, S,
or W replacements at all three residues (L528/I581/L599). The S and W triple mutants still formed
ternary complexes in vivo and showed chemotactic ability on soft agar plates, but had threshold and
cooperativity shifts in FRET assays. The triple G mutant could not support ternary complex formation
or chemotaxis, and showed no serine responses in FRET tests.
These results show that a P5-receptor interaction is important for chemotaxis, but that the P5
residues that comprise the interaction surface are not individually critical for array signaling. The shifts
in serine response threshold and cooperativity observed in the CheA-P5 mutants imply that the multiple
connections between CheA, CheW and receptors in the array probably shift, break, and reform during a
serine-induced signaling cycle. We are currently studying the effect of these P5 mutations on sensory
adaptation.
Lab: John Parkinson
___________
97
BLAST XII
TRANSMEMBRANE
SIGNALING
IN THE SERINE CHEMORECEPTOR TSR
_______________ Poster #47
VIA
SYNTHETIC
CONTROL
CABLES
Smiljka Kitanovic and John S. Parkinson
Biology Department, University of Utah, Salt Lake City, Utah 84112
Ligand binding in the periplasmic domain of the E. coli serine receptor promotes inward piston
displacement of one TM2 helix in the Tsr homodimer. Piston motions in turn influence the structure or
stability of the cytoplasmic HAMP domain to control receptor output signals to the flagellar motors. A
five-residue control cable connects TM2 to the AS1 helix of the HAMP bundle and transmits
conformational signals between them. To assess the role of control cable secondary structure in Tsr
signaling, we constructed 32 synthetic control cable mutants that had all possible combinations of
alanines (A) and glycines (G) at the five wild-type control cable residues (G213, I214, K215, A216,
S217). We assumed that alanines would favor helical secondary structure of the control cable,
whereas glycines would destabilize control cable helicity. We characterized the synthetic control cable
mutants for serine chemotaxis on soft agar plates, for pre- and post-stimulus flagellar rotation pattern,
for ability to undergo adaptational modifications, and for serine responses in FRET-based in vivo kinase
control assays.
The Tsr synthetic control cable mutants fell into several distinct groups, based on their serine
response thresholds. In hosts lacking the CheR and CheB adaptation enzymes, some Tsr mutants,
e.g., AAAAA, failed to respond to any level of serine. In contrast, the GGGGG mutant exhibited the
most sensitive serine response. Surprisingly, in hosts containing the CheR and CheB adaptation
enzymes, every mutant receptor except the one with an all-glycine control cable (GGGGG) supported
chemotaxis at some serine concentration in soft agar assays.
In vivo FRET kinase assays revealed that the AAAAA and other control cable mutants with high
serine thresholds in adaptation-deficient hosts had greatly reduced serine thresholds and underwent
sensory adaptation in adaptation-proficient hosts. In contrast, the GGGGG mutant had low serine
response thresholds, but failed to undergo sensory adaptation. The adaptation defect of the GGGGG
mutant most likely accounts for its inability to mediate serine chemotaxis in soft agar assays.
These results suggest a trade-off between two receptor functions: stimulus response and
adaptational modification. The AAAAA control cable is defective in responding to a serine stimulus, but
its output is still subject to sensory adaptation control. The GGGGG control cable, on the other hand,
mediates very sensitive responses to serine, but is not subject to sensory adaptation control. These
behaviors imply that attractant stimuli may initiate HAMP signaling by relaxing control cable helicity.
The GGGGG control cable is less helical than the AAAAA control cable and, therefore, more easily
driven to the attractant-stimulated signaling state. Additionally, a glycine in the first position of the
control cable appears to play a prominent role in lowering the serine response threshold, possibly
functioning as a structural swivel. Adaptational modifications, however, may depend on a more stable
helical control cable.
Lab: John Parkinson
___________
98
BLAST XII
_______________ Poster #48
TSR-E502: AN UNORTHODOX CHEMORECEPTOR SENSORY ADAPTATION SITE
Xue-sheng Han and John S. Parkinson
Biology Department, University of Utah, Salt Lake City, Utah 84112, USA
Methyl-accepting chemotaxis proteins (MCPs) mediate diverse chemotactic responses in motile
microbes. These chemoreceptors sense temporal changes in chemoeffector concentrations by
comparing current ligand occupancies with a memory of the recent chemical past, recorded in the form
of reversible methyl-group modifications of glutamate residues in the receptor’s cytoplasmic signaling
domain. MCP methylation state is regulated by the stimulus-modulated interplay of two MCP-specific
enzymes, CheR, a methyltransferase, and CheB, a methylesterase. The best-studied MCPs, Tar
(aspartate/maltose sensor) and Tsr (serine sensor) of E. coli, have four canonical methylation sites in
each subunit, located near the dimer interface. Methylation at these sites should enhance inter-subunit
packing of the four-helix methylation (MH) bundle. Tsr has a fifth methylation site, E502, located on a
different helical face, that would most likely modulate intra-subunit packing interactions in the MH
bundle. Does E502 play a different signaling role than the four conventional modification sites in Tsr?
Is E502 essential for Tsr function? Is it sufficient for Tsr function? We took three approaches to
answer these questions.
We first constructed and characterized Tsr receptors with all possible amino acid replacements
at E502. Nearly all of the mutant receptors mediated robust serine chemotaxis on tryptone soft agar
plates, indicating that E502 is not critical to Tsr function. Only two mutant receptors, E502P and E502I,
lacked function in soft agar assays. Flagellar rotation tests and in vivo FRET kinase assays
demonstrated that these mutant receptors had aberrant signal outputs. Tsr-E502P was shifted toward
the attractant-induced kinase-off state; Tsr-E502I was shifted toward a kinase-on state. Both mutant
receptors mediated responses to serine stimuli, but with higher (E502P) or lower (E502I) detection
sensitivity than wild-type Tsr.
To determine whether E502 underwentboth methylation and demethylation reactions, we used
combinations of D and N replacements to approximate the wild-type E and Q residues found at the
other four Tsr methylation sites. D produced signaling effects similar toE, but proved to be an
ineffective substrate for CheR-dependent methylation; N produced signaling effects similar to Q, but
was refractory to CheB-dependent deamidation. Tsr variants with only the E502 site intact underwent
methylation and demethylation, but could not support chemotaxis in soft agar assays, demonstrating
that E502alone is not sufficient for Tsr signaling and sensory adaptation.
To compare the signaling effects of modifications at E502 with those at the canonical
methylation sites, we analyzed, with in vivo FRET kinase assays, the serine thresholds and response
cooperativities of receptors with different mutationally adjusted modification states. All five sites
influenced Tsr signaling in the same manner, implying that overall MH bundle packing
stabilitydetermines receptor signaling characteristics, rather than a particular intra- or inter-subunit
packing conformation.
In sum, our study of Tsr-E502supports predictions of the dynamic bundle model of HAMP
signaling, which proposes that stimulus responses and sensory adaptation occur through shifts in the
relativepacking stabilities of the oppositionally coupled HAMP and MH bundles. The especially strong
effects of E502 modifications on signaling behavior probably reflect the facts that this site lies closest to
the adjoin HAMP domain and that it uniquely influences intra-subunit helix packing in the receptor’s
methylation bundle.
Lab: John Parkinson
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99
BLAST XII
_______________ Poster #49
CHARACTERIZATION OF A CHEMOTAXIS LOCUS IN LEPTOSPIRA INVOLVED IN MOTILITY AND
VIRULENCE
Ambroise Lambert1,2, Jérôme Ng Wong3, Mathieu Picardeau1
1
Institut Pasteur, Unité de Biologie des Spirochètes, Paris, France
2
Université Paris Diderot, Sorbonne Paris Cité, Cellule Pasteur, Paris, France
3
Institut Pasteur, Unité de Physique des Systèmes Biologiques
Leptospira belong to the spirochete family of bacteria existing as both saprophytes and
pathogens. Pathogenic species are the causative agents of leptospirosis, which is currently recognized
by the WHO as an emerging zoonotic disease. Leptospires express flagella that are localized in the
periplasm and attached at each polar end of the cell. Leptospira coordinate their flagellar motor
together asymetrically to allow bacterial motility. Although chemotaxis and motility might have an
important role in the infection process (Lambert et al., 2012), the regulation of the flagellar-based
motility in relation to chemotaxis remains unexplored in these atypical bacteria.
We generated a library of random transposon mutants in the pathogen L. interrogans which
included insertions in genes located in the same chromosomal locus. This operon is constituted by
genes encoding homologues of chemotaxis proteins CheA, CheW, CheD, CheB, CheY and MCP.
Swarm plate assays suggested that the disrupted chemotaxis genes were involved in
chemotaxis. Microscopy of Leptospira in liquid medium demonstrated loss of motility of one of the
mutants and this mutant was attenuated in the gerbil model of infection. Analysis via videomicroscopy
combined with determination of bacterial trajectories has allowed us to show significant differences in
the motility of the mutants compared to the wild-type. This is the first study demonstrating that a
chemotaxis regulatory system is required for both motility and virulence of Leptospira.
Lambert A, Takahashi N, Charon N, Picardeau M. Chemotactic behavior of pathogenic and nonpathogenic Leptospira species. Appl Environ Microbiol. 2012 Sep 21.
Lambert A, Picardeau M, Haake DA, Sermswan RW, Srikram A, Adler B, Murray GA. FlaA proteins in
Leptospira interrogans are essential for motility and virulence but are not required for formation of the
flagellum sheath. Infect Immun. 2012 Jun;80(6):2019-25. Epub 2012 Mar 26.
Lab: Mathieu Picardeau
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100
BLAST XII
_______________ Poster #50
STIMULATORY INTERACTIONS BETWEEN HYBRID HISTIDINE PROTEIN KINASES IN THE
VIRULENCE SIGNALLING NETWORK OF PSEUDOMONAS AERUGINOSA
Vanessa I. Francis and Steven L. Porter
Biosciences, College of Life and Environmental Sciences, Geoffrey Pope Building, Stocker Road,
University of Exeter, Exeter, Devon, EX4 4QD, United Kingdom
The GacS signalling network of P. aeruginosa plays a key role in regulating the transition between
acute and chronic modes of infection. This network comprises multiple histidine protein kinases (HPKs)
that have the remarkable ability to directly affect one another’s signalling through physical interactions.
The HPK, RetS has previously been reported to inhibit the signalling of the HPK, GacS (1). In this
study, we have identified stimulatory interactions between several of the HPKs comprising this network.
In particular, we find that the phosphorylation of the HPKs, RetS and PA1611, can be stimulated by
other HPKs in the GacS network. PA1611-P levels are increased by over 15-fold of that seen from
autophosphorylation alone, while RetS, which is unable to autophosphorylate, becomes highly
phosphorylated upon coincubation with the activating HPKs. We discuss the potential implications of
this HPK to HPK signalling for signal integration and downstream signalling.
1. Goodman, A. L., M. Merighi, M. Hyodo, I. Ventre, A. Filloux, and S. Lory. 2009. Direct interaction
between sensor kinase proteins mediates acute and chronic disease phenotypes in a bacterial
pathogen. Genes Dev. 23:249-259.
Lab: Steven Porter
_____________
101
BLAST XII
_______________ Poster #51
TAKING CONTROL OF THE BACTERIAL FLAGELLAR MOTOR
Guillaume Paradis, Ismaël Duchesne and Simon Rainville
Department of Physics, Engineering Physics and Optics and Centre of Optics, Photonics and Lasers,
Laval University, Québec, Québec, CANADA
The bacterial flagellar motor is a fairly complex machine embedded in the multiple layers of the
bacterial membrane. The focus of our laboratory for the past years has been the development of a
unique in vitro assay to study the bacterial flagellar motor. Our setup consists of a filamentous
Escherichia coli bacterium partly introduced inside a micropipette. Femtosecond laser pulses (60 fs and
~ 15 nJ/pulse) are then tightly-focused on the part of the bacterium that is located inside the
micropipette. This vaporizes a small portion of the membrane, leaving an essentially permanent hole in
the wall of the bacterium. Using a patch-clamp amplifier, we then apply an external voltage between the
inside and the outside of the micropipette. That voltage then directly contributes to the proton-motive
force (pmf) that powers the flagellar motor. As we change the applied potential, variations in the motor's
rotation speed are observed.
In addition to granting us full access to the inside of the cell, this in vitro assay gives us direct
control over both components of the proton-motive force. Indeed, we can apply an electric potential
across the membrane, control the pH both inside and outside of the cell and expose the motor to
various concentrations of proteins. That system therefore opens a world of new possibilities.
Interestingly, we recently used this novel system to expose a ∆FliL mutant of Salmonella enterica (from
R. Harshey) to very high pmf in an attempt to explain why these mutants lose their filaments when
swarming. Our system allowed us to expose these cells to pmf up to -320mV and invalidate the
hypothesis that the high pmf caused the filament to fall off.
The rotation speed is measured using 2 different techniques: high-speed video microscopy of
fluorescently labeled filaments and gold nanoparticules using an avalanche photodiode. Image
sequences from a fast EMCCD camera are analyzed with custom MatLab code while signal from the
photodiode is analyzed with LabView code. Using the strong link between gold and the thiol group
genetically added to the end of the fliC protein we can label the filaments with gold nanoparticules. This
technique gives the opportunity to have a high ratio of filament labeled. It should also be possible to
shine a new light on the switching mechanism as each event can be recorded and analyzed in an
easier way using those probes.
Finally, the fabrication of the micropipettes was recently automated. Using image analysis in
real time, the system allows a much more precise measurement of the constriction in the micropipette
and hence a more flexible and robust fabrication process.
Lab: Simon Rainville
____________
102
BLAST XII
_______________ Poster #52
THE MOTILITY OF BACTERIA IN AN ANISOTROPIC LIQUID ENVIRONMENT
Ismaël Duchesne1, Anil Kumar2, Guillaume Paradis1, Tigran Galstian1 and Simon Rainville1
1
Department of Physics, Engineering Physics and Optics and Centre of Optics, Photonics and Lasers,
Laval University, Québec, Québec, Canada
2
Department of Chemical Engineering, Indian Institute of Technology Delhi, Hauz Khas, New Delhi,
India
While the influence on bacterial motility of many genetic and biochemical factors has been
extensively studied, there have been limited studies of the impact of physical parameters. Indeed,
despite the fact that natural environments are often asymmetric (such as stretched supramolecular
structures), the majority of behavioural experiments with bacteria have been done in isotropic liquid
solutions. In the present work, we show that the behaviour of living microorganisms is dramatically
different in media that are asymmetric. The example of E. coli bacteria swimming in a bulk uniaxial
liquid environment is used to demonstrate this phenomenon. The results of our study shine light on the
behaviour of bacteria in conditions that they may encounter in real environments and open new
avenues for the control of their movements.
Lab: Simon Rainville
____________
103
BLAST XII
_______________ Poster #53
PHENOTYPIC VARIATION AND BISTABILITY WITHIN FLAGELLAR GENE NETWORK IN
SALMONELLA ENTERICA SEROVAR TYPHIMURIUM
Santosh Koirala, Phillip Aldridge, Christopher V. Rao
University of Illinois at Urbana Champaign, Newcastle University
Availability of nutrients in the cellular environment plays a key role in switching between motile
or sessile phenotypes. However, the response to nutritional cues could be very different even in closely
related species. For example, E. coli up regulates flagellar synthesis whereas Salmonella enterica
serovar Typhimurium downregulates flagellar synthesis under low-nutrient condition. YdiV is
responsible for the repression of flagellar synthesis in Salmonella enterica serovar Typhimurium which
acts as an anti-FlhD 4 C 2 factor. Moreover, FliZ-dependent activation of P class2 promoters is more
pronounced in low nutrient condition and is achieved by repression of the ydiV gene by FliZ. YdiV
expression is enhanced in poor media and greatly reduced in rich media. Thus, FliZ and YdiV, in poor
media, form an overall positive feedback loop which results in a bistable motility phenotype. This study
aims to characterize the extent of bistability within the flagellar network in Salmonella enterica serovar
Typhimurium. Our results suggest that two distinct phenotypes, motile and sessile, coexist in an
isogenic cell population under limited nutrient condition and the heterogeneity is enabled by FliZ-YdiV
feedback loop.
Lab: Christopher Rao
___________
104
BLAST XII
_______________ Poster #54
IDENTITY AND FUNCTION OF A LARGE GENE NETWORK UNDERLYING MUTAGENIC REPAIR
OF DNA BREAKS UNDER STRESS
Abu Amar M Al Mamun1, Mary-Jane Lombardo1*, Chandan Shee1, Andreas M Lisewski1, Caleb
Gonzalez1‡, Dongxu Lin1†, Ralf B Nehring1, Claude Saint-Ruf2, Janet L Gibson, Ryan L Frisch, Olivier
Lichtarge1,3, P. J. Hastings1, Susan M Rosenberg1,,3,4,5
1
Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030-3411
USA; 2INSERM, France; 3Department of Biochemistry and Molecular Biology, 4Department of Molecular
Virology and Microbiology, and 5The Dan L Duncan Cancer Center, Baylor College of Medicine,
Houston, TX 77030.
Mechanisms of DNA repair and mutagenesis are defined based on relatively few proteins acting
on DNA, yet the identities and functions of all proteins required are unknown. Here, we identify the
network that underlies mutagenic repair of DNA breaks in stressed Escherichia coli and define
functions for much of it. Using a comprehensive screen, we identified 77 new, and a total network of
≥93 genes that function in mutation. Most operate upstream of activation of three required stress
responses (RpoS, RpoE and SOS), key network hubs, apparently sensing stress. An autophagy-like
starvation-signaling pathway is identified. The results identify how a network integrates mutagenic
repair into the biology of the cell, reveal specific pathways of environmental sensing, demonstrate the
centrality of stress responses and imply their attractiveness as potential drug targets for blocking
evolution of pathogens.
Lab: Susan Rosenberg
__________
105
BLAST XII
IS BACTERIAL TWITCHING
SYMBIOSIS?
_______________ Poster #55
MOTILITY REQUIRED TO
ESTABLISH THE SQUID/VIBRIO
Aschtgen, MS., Brennan, C.A., Schaefer, A.L.*, Ruby, E.G.
Department of Medical Microbiology and Immunology, University of Wisconsin-Madison, Madison, WI
53705, * Department of Microbiology, University of Washington, Seattle, WA 98195
The symbiotic infection of the light organ of the squid Euprymna scolopes by the luminous
bacterium Vibrio fischeri has been studied experimentally to define the bacterial basis for the
biochemical and molecular events that characterize the colonization of animal epithelial tissue. The light
organ consists of a specific superficial ciliated epithelium and a series of deeply invaginated epitheliumlined crypt spaces, the site where symbiont colonization can occur. Within the first 2-3 hours after
inoculation, V. fischeri cells form external aggregates, migrate to and through surface pores leading into
ducts, and finally arrive in the crypt spaces. It has been previously shown that flagellar swimming
motility is not required for the migration from the aggregate to the pores, suggesting that another kind of
motility could be involved. For pathogenic associations, twitching motility is an essential virulence factor
allowing bacteria to contact specific cells and disseminate within a host.
In that context, we aim to (i) determine whether twitching motility is also essential for
establishing a symbiotic association, and (ii) characterize the underlying molecular mechanism. In a
preliminary study, we made a pilT/U mutant to describe the importance of this locus in the ability of V.
fischeri to move by twitching on 1% agar plates. We next showed that twitching motility is an important
determinant for host colonization. We also determined that the loss of twitching motility results in a
decrease in biofilm formation in V. fischeri. This last observation and the fact that the colonization
efficiency for a twitching motility mutant is dose and time dependent indicate, for the first time, the
importance of this particular form of motility in the first steps of colonization in this model symbiosis.
Lab: Edward Ruby
______________
106
BLAST XII
_______________ Poster #56
THE PSEUDOMONAS AERUGINOSA TYPE THREE SECRETION SYSTEM TRANSCRIPTIONAL
ACTIVATOR ExsA INTERACTS WITH THE ANTI-ACTIVATOR PROTEIN ExsD IN A TEMPERATUREDEPENDENT MANNER
Robert C. Bernhards, Anne E. Marsden, Shannon K.
Schubot
Virginia Polytechnic Institute & State University
Esher, Timothy L.
Yahr and Florian D.
The opportunistic pathogen Pseudomonas aeruginosa ranks among leading causes of
nosocomial infections. The type III secretion system (T3SS) aids acute P. aeruginosa infections by
injecting potent cytotoxins into host cells to suppress the host's innate immune response. Expression
of all T3SS-related genes is strictly dependent upon the transcription factor ExsA. Consequently, ExsA
and the biological processes that regulate ExsA function are of great biomedical interest. The
presented work focuses on the ExsA-ExsC-ExsD-ExsE signaling cascade that ties host cell contact to
the up-regulation of T3SS gene expression. Prior to induction, the anti-activator protein ExsD binds to
ExsA and blocks ExsA-dependent transcription by interfering with ExsA dimerization and promoter
interactions. Upon host cell contact, ExsD is sequestered by the T3SS chaperone ExsC resulting in the
release of ExsA and an up-regulation of the T3SS. Previous studies have shown that the ExsD-ExsA
interactions are not freely reversible. Because independently folded ExsD and ExsA were not found to
interact, it has been hypothesized that folding intermediates of the two proteins form the complex. Here
we demonstrate for the first time that ExsD alone is sufficient to inhibit ExsA-dependent transcription
and that no other cellular factors are required. More significantly, we show that independently folded
ExsD and ExsA are capable of an interaction, but only at 37°C and not at 30°C. Guided by the crystal
structure of ExsD, we designed a monomeric variant of the protein and demonstrate that ExsD
trimerization prevents ExsD from inhibiting ExsA-dependent transcription at 30°C. We propose that this
unique mechanism plays an important role in T3SS regulation.
Lab: Florian Schubot
___________
107
BLAST XII
_______________ Poster #57
A SURFACE-REGULATED ORPHAN ParA-LIKE PROTEIN HAS AN IMPACT ON GLIDING MOTILITY
IN BDELLOVIBRIO BACTERIOVORUS
David S. Milner, Emma Saxon and Liz Sockett
The Centre for Genetics and Genomics, University of Nottingham, NG7 2UH, United Kingdom
Bdellovibrio bacteriovorus is a small, liquid- and surface- motile, predatory bacterium which
invades other Gram-negative bacteria to replicate. Inside the periplasm of the host bacterium,
Bdellovibrio do not replicate by binary fission, but by synchronous septation of a multiploid filament.
Bdellovibrio has two genes encoding orphan ParA proteins which have no partner ParB, in
addition to the usual parAB gene pair coexpressed in an operon. Whilst ParA ATPases (usually with
ParB partners) are involved in chromosome segregation in some other bacteria; orphan ParAs have a
wide variety of functions, including aiding localisation of the Z ring and positioning of large chemotaxis
clusters.
It was hypothesised that one of the orphan ParA proteins would assist in division site selection,
due to the complex division strategy of Bdellovibrio. Instead we discovered that one of these
Bdellovibrio orphan ParA proteins (ParA2) is regulated in a surface-responsive manner. We show that
although transcription of parA2 was unaltered upon surface exposure, fluorescent tagging of the protein
revealed localisation is affected when Bdellovibrio cells are applied to a solid surface. The incidence of
ParA2 foci alters, and the sites where these foci reside correlates with the surface-related gliding
motility of that cell. Directed gene deletion demonstrated that although not essential for predation, loss
of the parA2 gene alters surface gliding motility behaviour by Bdellovibrio.
Lab: Liz Sockett
_______________
108
BLAST XII
_______________ Poster #58
THE ROLE OF POLYPHOSPHATE RELATED GENES IN THE PREDATORY BACTERIA
BDELLOVIBRIO BACTERIOVORUS
Sarah M Basford, Andrew Gilbert, Liz Sockett.
Centre for Genetics and Genomics, School of Biology, Medical School, QMC, University of Nottingham,
Nottingham, NG7 2UH UK.
Bdellovibrio bacteriovorus is a small predatory Gram-negative bacterium which invades other
Gram-negative bacteria. Once inside prey, the Bdellovibrio grow and divide before causing lysis. This
lifestyle means that Bdellovibrio has potential as a “living antibiotic” as it attacks and kills pathogens.
They small numbers of the cells also have the ability to grow host independently in rich media.
Survival in the time between prey invasions and the active processes of invasion itself are
energetically costly. This is especially important as Bdellovibrio don’t “feed” extensively outside prey to
produce the ATP required. Like many other bacteria, Bdellovibrio are reported to have phosphate-rich
granules, hundreds of nanometers in diameter. They also have genes encoding enzymes with
homology to exopolyphosphatases (Ppx), which could break down polyphosphate or the secondary
messenger pppGpp. Many roles have been assigned to polyphosphate in other bacterial species,
including uses as an intermediate-energy or phosphate store for metabolism, and (p)ppGpp is
important in the stringent response during amino acid starvation. This project is investigating how the
exopolyphosphatase homologues are used in the unique Bdellovibrio system.
Directed mutagenesis was used to test the role of polyphosphate degradation during
Bdellovibrio development and survival, in order to assess the ideal storage conditions to preserve
Bdellovibrio actively for medical application.
One ppx gene is involved in development as its deletion causes cells to be shorter and wider
than wild type cells and slows their development inside prey, whereas deletion of a different ppx gene
homolog does not cause such a phenotype. The Ppx gene product of this second homolog is involved
in allowing Bdellovibrio to grow host independently, as its removal decreases the number of cells which
are able to convert from host dependent growth to host independent growth.
Lab: Liz Sockett
_______________
109
BLAST XII
_______________ Poster #59
IDENTIFICATION OF A NOVEL CELL-ENVELOPE SUBCOMPLEX INVOLVED IN GLIDING
MOTILITY IN MYXOCOCCUS XANTHUS
Beata Jakobczak, Daniela Keilberg, Lotte Søgaard-Andersen
Max Planck Institute for Terrestrial Microbiology, Marburg, 35043 Germany
Myxococcus xanthus is a rod-shaped, Gram-negative bacterium that has two different motility
systems: the A- and S-motility system. A-motility, also referred to as gliding motility, allows movement
of single cells, while S-motility is cell-cell contact-dependent and is similar to twitching motility. If genes
of one of the motility systems are deleted, cells remain motile by the means of the remaining
system. Mutations which abolish both systems lead to non-motile cells. While S-motility depends on
extension and retraction of type-IV-pili, the A-motility is powered by the pH gradient across the
cytoplasmic membrane through the AglQRS and/or the AglXV complex. The exact mechanism of Amotility is not known; however, numerous proteins essential for A-motility have been described, four of
them (AglQ, AglZ, mxan_4868/GltF and AgmU/GltD) have been shown to localize to focal adhesion
complexes (FACs). FACs are distributed along the cell body and are stationary with respect to the
substratum in moving cells.
We identified two genes, agmO and mxan_2541, by a transposon mutagenesis screen to be
required for A-motility. Further experiments gave evidence that the two genes between agmO and
mxan_2541 (mxan_2539, mxan_2540) are also required for A-motility. agmO encodes a protein with a
lipoprotein signal peptide. Furthermore, AgmO belongs to the group of contact dependent gliding
motility proteins and is likely localized to the outer membrane. In contrast, mxan_2539, mxan_2540 and
mxan_2541 encode proteins with a signal peptide I. Moreover mxan_2539 and mxan_2540 are
predicted to contain a domain similar to the the outer membrane domain of OmpA, while mxan_2541 is
predicted to contain TPR domain. Bioinformatic analyses showed conservation of these genes in
Myxococcales genomes suggesting that the four proteins interact. Interestingly, the three genes
mxan_2539, mxan_2540 and mxan_2541 were often found in the genetic context of homologs of
known A-motility genes, such as agmU and aglT. Our data demonstrate that these four proteins localize
to the cell envelope as predicted from the bioinformatic analyses and are dependent on each other for
stability. Furthermore, interaction studies strongly indicate that these four A-motility proteins form a
complex in the cell envelope. We are currently determining the localization of the four proteins as well
as their putative interaction with known FAC components using bioinformatic, biochemical and
localization studies.
Lab: Lotte Søgaard-Andersen
____
110
BLAST XII
_______________ Poster #60
DECIPHERING THE ASSEMBLY PATHWAY OF TYPE IV PILI IN MYXOCOCCUS XANTHUS
Carmen Friedrich, Iryna Bulyha & Lotte Søgaard-Andersen
Max Planck Institute for Terrestrial Microbiology, Marburg, 35043 Germany
Myxococcus xanthus moves by gliding. For this purpose, M. xanthus uses two independent
motility engines. The Social motility system is powered by type IV pili (T4P). T4P are built at the leading
cell pole and generate movement by undergoing cycles of extension, attachment to the surface of other
cells and retraction, which provides the force to pull a cell forward. When cells reverse, T4P switch
poles. T4P are composed of thousands of copies of the type IV pilin. The pilin subunits are incorporated
into the base of the growing pilus from a reservoir of pilins in the inner membrane. Similarly, when pili
retract, pilin subunits are removed from the base of the pilus and incorporated back into the inner
membrane. The channel in the outer membrane through which the pilus is extruded is built by the
secretin PilQ. The energy required for T4P extension and retraction is provided by two cytoplasmic
ATPases PilB and PilT, respectively. Furthermore, the inner membrane platform protein PilC is required
for T4P biogenesis. While some of the T4P-proteins, i.e. PilC, PilM and PilQ, localize to both poles and
remain stationary during a reversal, PilB and PilT localize mainly to one pole and switch poles upon a
reversal. These data suggest that some T4P-proteins build a preassembled complex at both poles and
are complemented by the dynamic proteins PilB or PilT to build a functional pilus extension and
retraction complex. The proteins PilM, PilN, PilO and PilP are also important for T4P-dependent
motility, but their function remains unclear.
Approximately 10 pil-genes are conserved and essential for T4aP assembly and function. We
have as a working hypothesis that these proteins interact to form a trans-envelope macromolecular
complex supporting T4P assembly and dynamics. However, it is currently unknown how the T4Pproteins interact to make a functional T4P machinery. To analyze in which order and dependence these
proteins work together in the T4P assembly machinery, we determined the stability of every T4P protein
in the absence of each individual T4P protein. Similarly, we determined the localization of every T4P
protein and how correct localization of each T4P protein depends on other T4P proteins. Our protein
stability and localization data suggest that PilM/N/O/P/Q interact and that PilM/N/O/P depend on the
outer membrane secretin PilQ and the outer membrane lipoprotein Tgl for correct bipolar localization.
To verify the interconnection between single components of the protein complex, we are currently
analyzing direct interactions between PilM, PilN, PilO PilP and PilQ via co-purification experiments or
site-directed mutagenesis.
Further localization experiments revealed that the inner membrane platform protein PilC
depends on PilN, PilQ and Tgl for correct localization. In contrast, the two cytoplasmic ATPases PilB
and PilT, which switch poles during a reversal, localize independently from the presence of
PilM/N/O/P/Q/Tgl. This suggests that the assembly of the T4P machinery does not follow a hierarchical
pathway, but involves at least two independent pathways.
Lab: Lotte Søgaard-Andersen
____
111
BLAST XII
_______________ Poster #61
DIVERSITY OF SMALL Ras-LIKE G-PROTEINS IN PROKARYOTES
Kristin Wuichet, Stuart Huntley, and Lotte Søgaard-Andersen
Max Plank Institute for Terrestrial Microbiology, Marburg, 35043 Germany
Members of the Ras superfamily of small G-proteins are ubiquitous in eukaryotes and function
as nucleotide-dependent molecular switches that cycle between an active GTP-bound form and an
inactive GDP-bound form. The inherent GTPase activity of these proteins is low and requires the
assistance of a GTPase activating protein (GAP). Additionally, they have a high affinity for both GTP
and GDP and utilize guanine nucleotide exchange factors (GEFs) to exchange GDP for GTP. Ras-like
G-proteins were initially identified in and considered to be exclusive to eukaryotes. Experimental
studies demonstrated that Ras-like G-proteins have important functions in e.g. signal transduction, cell
polarity and motility. Experimental studies and sequence characterization revealed MglA, the master
motility regulator of Myxococcus xanthus, to be a Ras-like small G-protein and its partner protein, MglB,
which is a member of the superfamily of Roadblock proteins, functions as a GAP. Currently, no GEF
has been identified as part of this system. Interestingly, the MglA/MglB module functions to regulate cell
polarity and motility.
Here, we performed a computational analysis of 1611 prokaryotic genomes to tally the
distribution of small Ras-like G-proteins in prokaryotes. Using sequence similarity and genome context
methods, we expanded on previous studies, identifying and computationally characterizing 630
prokaryotic Ras-like G-proteins in 277 genomes from diverse lineages including Acidobacteria,
Actinobacteria, Aquificales, Bacteriodetes, Chlorobi, Chloroflexi, Cyanobacteria, Deferribacteres,
Deinococcus/Thermus,
Dictyoglomi,
Fibrobacteres,
Gemmatimonadetes,
Proteobacteria,
Verrucomicrobia as well as archaeal species. MglA-like sequences make up the largest family of
prokaryotic Ras-like G-proteins and co-evolve with MglB-like sequences. MglA-like proteins can be
divided into five subfamilies. Our previous structural analysis of a close homolog of the canonical MglA
from M. xanthus revealed a novel catalytic mechanism. Interestingly, sequence diversity among
signature motifs of the other four subfamilies suggests additional G-protein catalytic mechanisms that
have yet to be revealed. While MglA-like proteins only maintain conservation of four of the five Ras
signature motifs that were established in eukaryotic studies, a few families of prokaryotic Ras-like Gproteins maintain conservation of all five Ras signature motifs. Genome context analyses hint at coevolving proteins that may function as GAPs or GEFs for these family members; however, none of
these G-proteins or neighboring sequences have been characterized experimentally. The abundance
and wide distribution of small Ras-like G-proteins suggest that they may have critical functions in
prokaryotic physiology most of which have yet to be uncovered. Our analysis provides valuable
predictions that can be tested experimentally.
Lab: Lotte Søgaard-Andersen
____
112
BLAST XII
_______________ Poster #62
AN ENGINEERED SERINE CHEMORECEPTOR WITH SYMMETRIC INSERTIONS MIMICS
NATURAL TRANSDUCERS OF THE 40H CLASS
Herrera Seitz, M.K, Massazza, D.A. and Studdert, C.A
Instituto de Investigaciones Biológicas, Universidad Nacional de Mar del Plata, Buenos Aires, Argentina
Bacterial chemoreceptors usually detect extracellular signals through a periplasmic sensing
domain and transmit them to a highly conserved intracellular domain. The signal then reaches the
flagellar motors to control swimming behavior.
The cytoplasmic signaling domain consists of a long alpha-helical hairpin that forms, in the
dimer, a coiled-coil four-helix bundle. The huge variety of chemoreceptors identified from genomic
analysis in Bacteria and Archaea can be classified into a small number of classes according to the
length of their cytoplasmic signaling domain. Differences in length are due to the presence of pairs of
insertions or deletions of seven-residue stretches (heptads), located symmetrically with respect to the
hairpin turn. The size and location of the indels highlight the importance of the coiled-coil structure and
suggest the existence of specific interactions between the two arms of the hairpin, that are needed to
preserve proper signal transmission.
To understand the structural requirements of signal transmission that led to this peculiar
evolution pattern, in our lab we are engaged in the construction and characterization of derivatives of
the serine chemoreceptor of E. coli, Tsr (36H-class because it possesses 36 heptads in its cytoplasmic
domain), to mimic natural transducers of different length classes.
In this work, we built a 40H-class derivative of Tsr through the introduction of symmetric 14-residue
insertions, whose sequence and position were chosen based on the alignment of Tsr with PctApp, a
40H-class receptor from Pseudomonas putida.
The 40H-class derivative, TsrH18, was able to activate the CheA kinase at a high level.
However, it was insensitive to serine and unable to mediate serine taxis in soft agar plates. Random
mutagenesis applied to the construct allowed the isolation of two functional derivatives (TsrH18*) that
carry single point mutations, indicating that subtle changes restored signaling abilities to the enlarged
hairpin. TsrH18* derivatives were able to control CheA activity in response to serine. They also
localized to the poles of the cell, as assessed with the fluorescent reporters YFP-CheZ or YFP-CheR.
In contrast, the original TsrH18 construct failed to mediate YFP-CheZ localization, suggesting its
inability to form properly assembled ternary complexes.
Unlike the derivative belonging to the 34H class that he had characterized in previous work, this
40H-class derivative does not interfere with the chemotactic function of native Tar (36H class), and it
does not form mixed trimers of dimers with Tar, probably due to the larger length difference.
The availability of functional derivatives that mimic receptors of different classes represent
useful tools to study interactions between different receptors in the same cell and the requirements for
co-operation within the same chemoreceptor cluster.
Lab: Claudia Studdert
___________
113
BLAST XII
_______________ Poster #63
FUNCTIONAL AND STRUCTURAL ANALYSIS OF HOMOLOGOUS CONSERVED RESIDUES CheWR62 AND CheA-R555 WITHIN THE CHEMOTAXIS TERNARY COMPLEX
Pedetta A.1, Parkinson J.S.2, Studdert C.A.1
1
Instituto de Investigaciones Biológicas, Universidad Nacional de Mar del Plata, Mar del Plata,
Argentina
2
Biology Department, University of Utah, Salt Lake City, USA
The small protein CheW, structurally homologous to the P5 regulatory domain of CheA, couples
the receptors with the kinase, playing an essential role in CheA control. Various studies in recent years
indicate that the role of CheW may be more complex than that of a simple bridge between the two
proteins.
We focused our study on the conserved residues R62 in CheW and R555 in CheA. These
arginine residues lie in equivalent positions in CheW and the homologous P5 domain in CheA, and
replacements in either of them cause severe impairment of chemotactic behavior. However, the mutant
proteins mediate the formation of ternary complexes with normal kinase activity when studied in vitro,
using chemoreceptor-containing membranes.
To assess the effect of these replacements on the signaling ability of the complex in living cells we
performed a FRET-based assay. The assay, based on CheY-CheZ interaction as a read-out of kinase
activity, allowed us to compare the sensitivity and cooperativity of the response in cells expressing the
wild-type versions of CheA and CheW with those of cells expressing the mutant variants. Whereas in
CheW-R62 mutants the sensitivity and cooperativity were slightly reduced, in CheA-R555 mutants both
parameters were more significantly affected.
In order to contribute to the understanding of the arrangement of CheW within the signaling
complex, CheW R62C was co-expressed with Tsr variants carrying single cysteine replacements, and
cells were subjected to oxidizing conditions through diamide treatment. The co-expression of CheW
R62C with Tsr V398C (but not Tsr carrying other cysteine replacements) at physiological levels led to
the formation of a disulfide-bonded product between the two proteins, indicating that the crosslinking
reflects a specific interaction. Moreover, the presence of Tsr V398C corrects the chemotaxis defect of
cells expressing CheW R62C, indicating that this protein pair is part of a fully functional complex.
The extent of crosslinking is somehow increased in cells that lack the CheA kinase. This fact
could be explained if there exists some competition between CheW and CheA (P5 domain) for
interaction with Tsr. However, we could not detect any disulfide bond formation between CheA R555C
and Tsr V398C.
Our results suggest that small changes in the conformation of the ternary complexes may lead
to reduced sensitivity and/or cooperativity of the system, with consequent alterations in signal
amplification and in the chemotactic response.
Lab: Claudia Studdert
___________
114
BLAST XII
_______________ Poster #64
EFFECT OF A CHEMOEFFECTOR ON THE STABILITY OF CHEMORECEPTORS CLUSTERING
STUDIED BY PHOTOACTIVATED LOCALIZATION MICROSCOPY
F. Anquez and T.S. Shimizu
FOM Institute AMOLF, Amsterdam
Bacterial chemoreceptors cluster into partially ordered arrays which provides cooperative signal
amplification and their sensitivity can be tuned via covalent modification, allowing adaptation.
A number of studies have addressed the in vivo stability of the large polar clusters upon addition
of chemoattractants with conflicting results : while some studies [1-3] yielded data indicating that polar
clusters are disrupted upon attractant stimulation, others found that similar stimuli do not appear to
significantly affect the appearance of clusters [4,5]. Notably, all of these studies have employed
diffraction-limited fluorescence microscopy, the resolution of which is limited to ~250 nm.
Here, we employ photoactivation localization microscopy (PALM) which can achieve resolution
down to 10nm [6,7], to probe the distribution of Tar-receptor cluster sizes (a) in the absence of
chemoeffector stimuli, (b) shortly after exposure to the attractant methylaspartate (MeAsp), and (c) after
adaptation to sustained MeAsp stimulation. Preliminary results suggest that exposure to this chemoeffector can disrupt polar clusters shortly after MeAsp addition, but the cluster size distribution is
restored to its pre-stimulus state upon prolongated stimulation.
[1] Lamanna et al., Mol. Microbiol. 57 (3) pp. 774-785 (2005)
[2] Borrock et al., ACS Chem. Biol. 3 (2) pp. 101-109 (2008)
[3] Wu et al., J. Biol. Chem. 286 pp. 2587-2595 (2011)
[4] Homma et al., PNAS 101 (10) pp. 3462-3467(2004)
[5] Liberman et al. J. Bacteriol. 186 (19) pp. 6643-6646 (2004)
[6] Betzig et al. Science 313 (5793) pp.1642-1645 (2006)
[7] Greenfield et al. Plos Biol.7 (6) pp. e1000137 (2009)
Lab: Tom Shimizu
______________
115
BLAST XII
_______________ Poster #65
THE FLIP OF A COIN: ACETYLATION OR PHOSPHORYLATION BY ACETYL PHOSPHATE IN
BIOFILMS
Abouelfetouh, Alaa A. Y.1,2, Zemaitaitis, Bozena1 and Wolfe, Alan J.1
1
Department of Microbiology and Immunology, Loyola University Chicago, Stritch School of Medicine,
2160 S. First Ave. Bldg. 105, Maywood, IL 60153.
2
Department of Pharmaceutical Microbiology, Alexandria University, Faculty of Pharmacy, 1 Khartoum
Sq., Alexandria, Egypt 21521.
The high-energy intermediate acetyl phosphate (acetyl-P) plays a pivotal role in metabolism. It
accumulates during growth in the presence of large amounts of carbon. As the flux of carbon through
glycolysis puts pressure on the limited supply of coenzyme A, the acetyl group from acetyl-CoA is
transferred to inorganic phosphate (by the enzyme phosphotransacetylase, Pta). Because acetyl-P
retains the high-energy status of acetyl-CoA, it can donate its phosphoryl group to ADP to generate
ATP (by the enzyme acetate kinase, AckA). Acetyl-P can also donate its phosphoryl group to certain
two-component response regulators (e.g. RcsB and CpxR). We recently discovered a third function:
acetyl-P donates its acetyl group to a large set of proteins that function in diverse cellular processes,
including many critical to the development of biofilms. We previously reported that acetyl-P influences
biofilm development by E. coli K-12 (Wolfe et al., 2003) and that this effect acts in part through the
response regulator RcsB (Fredericks et al., 2006). Since acetyl-P can donate its phosphoryl group to
response regulators and its acetyl groups to diverse proteins (including some response regulators), it is
clear that acetyl-P could influence biofilm development by many mechanisms. This abstract describes
early studies designed to dissect those mechanisms.
One strategy is to identify genes whose deletion either enhances or suppresses phenotypes of
an ackA mutant, including reduced motility, increased mucoidy, defective biofilm development and
increased protein acetylation. We therefore have generated and screened double mutants of the type
ackA yfg. We have begun by constructing double mutants with genes that encode proteins involved in
acetyl transfer, e.g. the protein deacetylase CobB, one known protein acetyltransferase (YfiQ/Pat) and
several predicted GCN5-like acetyltransferases. At present, we can conclude that acetyl-P and CobB
affect protein acetylation by independent pathways: the double mutant is more hyper-acetylated and
more defective for biofilm development than either single mutant. On the basis of the behaviors of the
acetyltransferase mutants, this inverse relationship may be general.
A complex relationship is emerging between the deacetylase CobB and the response regulator
RcsB. Based on the assessment of either biofilm development or protein acetylation, we come to the
conclusion that CobB and RcsB function in the same pathway. The order of the proteins, however,
appears to be reversed depending on which phenotype we analyze. Most understandable is the
prediction that CobB inhibits the activity of RcsB, which regulates protein acetylation. This fits with other
evidence that our laboratory has obtained: RcsB becomes acetylated in vivo and some of that
acetylation is sensitive to CobB (Hu et al., submitted).
Fredericks C. E. et al (2006): Acetyl phosphate-sensitive regulation of flagellar biogenesis and capsular
biosynthesis depends on the Rcs phosphorelay. Molecular Microbiology. 61(3), 734–747.
Wolfe A. J. et al. (2003): Evidence that acetyl phosphate functions as a global signal during biofilm development.
Molecular Microbiology. 48(4), 977–988.
Lab: Alan Wolfe
________________
116
BLAST XII
_______________ Poster #66
ACETYL PHOSPHATE IS A POTENT REGULATOR OF BACTERIAL PROTEIN ACETYLATION
Alan J. Wolfe1*, Birgit Schilling2, Bozena Zemaitaitis1, Linda Hu1, Bruno Lima1, Bradford Gibson2
1
Loyola University Chicago, Maywood, IL 60153
2
Buck Institute for Research on Aging, Novato, CA 94702
Reversible Nε-lysine (protein) acetylation of bacterial proteins is a previously unrecognized
regulatory mechanism. Since the study of bacterial protein acetylation is in its infancy, many questions
remain unanswered. The mechanisms responsible for most protein acetylations remain obscure, their
effect on protein function remains unclear, and their global impact remains unquantified.
Earlier studies provided evidence for this post-translational modification on about two hundred
proteins in E. coli and S. enterica. We now report that that the acetylated proteome (‘acetylome’) is
much larger than previously reported. Our preliminary experiments using a novel label-free quantitative
mass spectrometry analysis identified 1515 unique acetylation sites on 541 E. coli proteins that function
in diverse cellular processes, including signal transduction, transcription, quorum sensing, biofilm
formation and pathogenesis. Some acetylated proteins include the response regulator RcsB, the global
transcription factor CRP, and RNA polymerase. If only a fraction of the detected acetylations exert a
significant impact on their protein’s function, then this post-translational modification would easily
surpass phosphorylation as the primary regulatory mechanism.
The Pta-AckA pathway links the central metabolite acetyl-CoA to ATP generation. The pathway
intermediate acetyl phosphate has been shown to donate its phosphoryl group to two-component
response regulators, e.g. CpxR and RcsB. We now propose that this pathway is a potent regulator of
bacterial protein acetylation. Anti-acetyllysine Western immunoblot and quantitative mass spectrometry
analyses showed hyperacetylation of mutants that lack AckA. In contrast, mutants that lack the entire
Pta-AckA pathway are less acetylated like the WT parent. Since these pta ackA mutants differ from the
ackA mutants by their retention of Pta and its ability to synthesize acP, these data argue that protein
acetylation is regulated by Pta and/or acP. More surprising is the observation that the Pta-AckA
pathway is a more potent regulator of protein acetylation than the known E. coli KAT and KDAC (YfiQ
and CobB, respectively), as similar analyses of cells that lack these enzymes show more minor effects.
Since similar effects are observed in other organisms including pathogens, we conclude that
acetylation is both universal and relevant to public health.
Lab: Alan Wolfe
________________
117
BLAST XII
_______________ Poster #67
A NOVEL CHEMORECEPTOR COUPLES DEGRADATION OF AROMATIC POLLUTANTS WITH
CHEMOTAXIS
Zhihong Xie, Jiangfeng Gong, Yongming Luo
CAS and Shandong Provincial Key Laboratory of Coastal Environmental Processes, Yantai Institute of
Coastal Zone Research, Chinese Academy of Sciences, 264003, Yantai Shandong, PR China
Pesticides and industrial wastes often contain aromatic constituents, and most of them are
toxic to living organisms. The use of plants and bacterial to clean up in soil has gained increasing
interest in past years. Rhizobia are motile alpha-proteobacteria that can establish a nitrogen-fixing
symbiosis within the roots of leguminous plants. These bacteria show chemotaxis to aromatic
constituents and soybean root exudates, such as organic acid, sugar, and amino acids, which is an
initial and important step for establishing the symbiotic relationship with legumes. But the relationship
between chemotaxis response and behavioral variability in nodule bacteria is not clear. In this study,
the functions of the methyl-accepting chemotaxis protein TLP5 in Azorhizobium caulinodans ORS571 is
determined, since this protein were predicted to be important in chemotaxis and microbe-host
interactions.
By generating a tlp5 deleted mutant strain, we analyzed its behavior in chemotaxis, nodulation
and degradation of 4-Hydroxybenzoate. We found that tlp5 was unaffected in its aerotaxis response but
was affected in its chemotactic ability. We have found that TLP5 modulate the motility swimming bias of
Azorhizobium cells and have demonstrated that TLP5 promotes competitive nodulation of the legumes
and degrades the 4-Hydroxybenzoate. We therefore hypothesize that tlp5 is an energy sensing
chemotaxis protein and represents a novel function of the chemoreceptor.This finding implies that the
environmental cue(s) triggering chemotaxis of Azorhizobium cells towards the roots of legumes and
facilitating degradation of pollutants in soil by root-nodule symbiosis.
The findings obtained from this study will help to investigate novel regulatory mechanisms and
catabolic activities that can be of great biotechnological interest for improving the microbial degradation
of aromatic environmental pollutants. Further studies will be performed for better understanding of the
mechanisms of Che-like pathways and their potential use in optimization of conditions for applications
of Rhizobium species in bioremediation.
Lab: Zhihong Xie
_______________
118
BLAST XII PARTICIPANT LIST
119
Alaa Abouelfetouh
Loyola University Chicago
Microbiology and Immunology
128 Washington Blvd
Oak Park, IL 60302
alaa.abouelfetouh@pharmacy.alexu.edu.eg
Christopher Adase
Texas A&M University
Biochemistry
3258 TAMU
BSBE Room 303
College Station, TX 77843
Phone: (979) 845-1249
xpage@neo.tamu.edu
Oshri Afanzar
Weizmann Institute of Science
Biological Chemistrey
Rehovot 76100
Israel
Phone: +972 8 934 3685
afanzar@gmail.com
Abu Amar Al Mamun
Baylor College of Medicine
Molecular and Human Genetics
One Baylor Plaza, MSC 225
Houston, TX 77030
Phone: (713) 798-6693
mamun@bcm.edu
James Allen
Oxford University
Biochemsitry
Parks Road
New Biochemistry
Oxford, Oxon OX1 3QU
United Kingdom
Phone: +44 1865 613 315
james.allen@wadh.ox.ac.uk
Francois Anquez
FOM Institute AMOLF
P.O. Box 41883
Science Park 102
Amsterdam 1009 DB
Netherlands
Phone: +31 20 754 7278
anquez@amolf.nl
Judith Armitage
Oxford
Biochemistry
Department of Biochemistry
Oxford OX1 3QU
United Kingdom
Phone: +44 1865 613 293
judith.armitage@bioch.ox.ac.uk
Marie-Stephanie Aschtgen
University of Wisconsin - Madison
Department of Medical Microbiology and
Immunology
Microbial Sciences Building Room 5245
1550 Linden Drive
Madison, WI 53706
Phone: (608) 262-5550
aschtgen@wisc.edu
Sonia Bardy
University of Wisconsin-Milwaukee
Biological Sciences
3209 N Maryland Ave
Milwaukee, WI 53211
Phone: (414) 229-6415
bardy@uwm.edu
Szilvia Baron
Weizmann Institute of Science
Biological Chemistry
Ulmann Building
Room 17
Rehovot 76100
Israel
Phone: +972 8 934 2702
szilvia.baron@weizmann.ac.il
120
Sarah Basford
University of Nottingham
Centre for Genetics and Genomics
C15, Centre for Genetics and Genomics
Medical School, QMC
Nottingham NG7 2UH United Kingdom
sbxsb@nottingham.ac.uk
Morgan Beeby
Imperial College London
Life Sciences
Division of Molecular Biosciences
South Kensington Campus
London SW7 2AZ United Kingdom
mbeeby@imperial.ac.uk
Stefan Behr
Ludwig-Maximilians-University
Grosshaderner Strasse 2-4
Martinsried - Munich, Bavaria D-82152
Germany
Phone: +49 89 2180 74535
Fax: +49 89 2180 74520
stefan.behr@campus.lmu.de
Wiebke Behrens
Hannover Medical School
Institute for Medical Microbiology
Carl-Neuberg-Strasse 1
Hannover 30625
Germany
Phone: +49 511 532 8028 ext. 8028
behrens.wiebke@mh-hannover.de
Robert Belas
University of Maryland Baltimore County
Marine Biotechnology
Institute of Marine & Environmental Technology
701 East Pratt Street
Baltimore, MD 21202
Phone: (410) 234-8876
belas@umbc.edu
Howard Berg
Harvard University
Molecular and Cellular Biology
Harvard Biological Laboratories
16 Divinity Avenue, Room 3063
Cambridge, MA 02138
Phone: (617) 495-0924
Fax: (617) 496-1114
hberg@mcb.harvard.edu
Indranil Biswas
University of Kansas Medical Center
3901 Rainbow Blvd
Kansas City, KS 66160
Phone: (913) 588-7019
Fax: (913) 588-7295
ibiswas@kumc.edu
Bob Bourret
University of North Carolina
Microbiology & Immunology
Chapel Hill, NC 27599-7290
Phone: (919) 966-2679
Fax: (919) 962-8103
bourret@med.unc.edu
Caitlin Brennan
University of Wisconsin
1550 Linden Drive, 5245 MSB
Madison, WI 53706
Phone: (608) 262-5550
cabrennan@wisc.edu
Ariane Briegel
California Institute of Technology
Biology
1200 E. California Blvd.
Mail code 114-96
Pasadena, CA 91125
Phone: (626) 395-8848
briegel@caltech.edu
121
Rebecca Calvo
Indiana University Bloomington
Biology
1001 E. Third St.
Bloomington, IN 47405
rcalvo@indiana.edu
Nyles Charon
West Virginia Uninversity
Microbiology, Immunology, and Cell Biology
Health Sciences Center North
Box 9177
Morgantown, WV 26506-9177
Phone: (304) 293-4170
Fax: (304) 293-7823
ncharon@hsc.wvu.edu
Remy Colin
Rowland Institute at Harvard
100 Edwin H. Land Bvd
Cambridge, MA 02142
Phone: (617) 497-4715
colin@rowland.harvard.edu
Kieran Collins
UC Santa Cruz
METX
1156 High Street
Santa Cruz, CA 95064
Phone: (415) 613-3316
kdcollin@ucsc.edu
Brian Crane
Cornell University
Chemistry and Chemical Biology
G75 Chemistry Research Building
Ithaca, NY 14853
Phone: (607) 254-8634
Fax: (607) 255-1248
bc69@cornell.edu
Rachel Creager-Allen
University of North Carolina
Campus Box 7290
Chapel Hill, NC 27599
Phone: (919) 966-2679
rcreager@email.unc.edu
Sean Crosson
University of Chicago
Biochemistry and Molecular Biology
929 E. 57th St.
GCIS-W138
Chicago, IL 60637
Phone: (773) 834-1926
scrosson@uchicago.edu
Rick Dahlquist
Department of Chemistry & Biochemistry
University of California Santa Barbara
Santa Barbara, CA 93106
Phone: (805) 893-5326
dahlquist@chem.ucsb.edu
Georges Dreyfus
Univ. Nacional Autónoma de México (UNAM)
Inst de Fisiología Celular
Ap. Postal 70243
Mexico, Mexico City 04510
Mexico
Phone: +52 55 5622 5618
Fax: +52 55 5622 5611
gdreyfus@ifc.unam.mx
Ismael Duchesne
Université Laval
Physique
618 rue Fraser
Quebec, QC G1S 1R7
Canada
Phone: (418) 656-2131 ext. 3788
ismael.duchesne.1@ulaval.ca
122
Michael Eisenbach
Weizmann Institute of Science
Biological Chemistry
PO Box 26
Rehovot 76100
Israel
m.eisenbach@weizmann.ac.il
Marc Erhardt
Helmholtz Centre for Infection Research
Infection Biologie of Salmonella
Inhoffenstraße 7
Braunschweig, Niedersachsen 38124
Germany
Phone: +49 531 6181 5700
marc.erhardt@helmholtz-hzi.de
Lewis Evans
University of Cambridge
Pathology
Tennis Court Road
Cambridge, Cambridgeshire CB2 1QP
United Kingdom
Phone: +44 1223 333 733
le227@cam.ac.uk
Joseph Falke
Univ. Colorado
Dept. of Chem. & Biochem.
UCB 596
Univ. Colorado
Boulder, CO 80309-0596
Phone: (303) 492-3503
falke@colorado.edu
Milana Fraiberg
Weizmann Institute of Science
Biological chemistry
P.O. Box 26 Rehovot 76100 Israel
Rehovot 76100
Israel
Phone: +972 8 934 2710
Fax: +972 8 947 2722
milana.fraiberg@weizmann.ac.il
Carmen Friedrich
Max Planck Institute for Terrestrial Microbiology
Ecophysiology
Karl-von-Frisch-Straße 10
Marburg 35043
Germany
Phone: +49 6421 178 211
carmen.friedrich@mpi-marburg.mpg.de
Hajime Fukuoka
Tohoku University
IMRAM
Katahira 2-1-1
Aoba-Ku
Sendai, Miyagi 980-8577
Japan
Phone: +81 22 217 5804
Fax: +81 22 217 5804
f-hajime@tagen.tohoku.ac.jp
Michael Galperin
NCBI, NLM, National Institutes of Health
Computational Biology Branch
8600 Rockville Pike, MSC3830
Bldg. 38A, Rm. 5N507
Bethesda, MD 20894
Phone: (301) 435-5910
Fax: (301) 435-7793
galperin@ncbi.nlm.nih.gov
Susanne Gebhard
Ludwig-Maximilans-University Munich
Dept. Biology I, Microbiology
Grosshaderner Str. 2-4
Planegg-Martinsried, Bavaria 82152
Germany
Phone: +49 89 2180 74601
susanne.gebhard@bio.lmu.de
Mizuki Gohara
Nagoya Univercity
Furo-cho, Chikusa-ku
Nagoya, Aichi 4648602
Japan
Phone: +81 52 789 2991
gouhara.mizuki@c.mbox.nagoya-u.ac.jp
123
Lindsie Goss
Columbia University
Microbiology & Immunology
100 Haven Ave
T2-16B
New York, NY 10032
lag2147@columbia.edu
Xuesheng Han
University of Utah
Biology
Department of Biology
257 South 1400 East
Salt Lake City, UT 84112-0840
Phone: (801) 581-6307
xuesheng.han@utah.edu
Rasika Harshey
University of Texas at Austin
Mol. Gen. & Microbiology
1 University Station
A1000
Austin, TX 78712
Phone: (512) 471-6881
Fax: (512) 471-1218
rasika@uts.cc.utexas.edu
Gerald Hazelbauer
University of Missouri
Biochemistry
117 Schweitzer Hall
Columbia, MO 65211
Phone: (573) 882-4845
Fax: (573) 882-5635
hazelbauerg@missouri.edu
Penelope Higgs
Max-Planck-Institute for Terrestrial Microbiology
Ecophysiology
Karl-von-Frisch Strasse 10
Marburg, Hessen D35043
Germany
Phone: +49 6421 178 301
Fax: +49 6421 178 309
higgs@mpi-marburg.mpg.de
Geetha Hiremath-Mendez
Hosei University
Research Center for Micro-Nano Technology
Koganei-shi, Kajino-cho, 3-7-2
Higashi kan E-3001
Tokyo 184-8584
Japan
Phone: +42 387 7173
geetha.2225@gmail.com
Basarab Hosu
Harvard University
Molecular and Cellular Biology
16 Divinity Avenue
Bio Labs Buliding Room 3068
Cambridge, MA 02138
Phone: (617) 495-6127
Fax: (617) 496-1114
ghosu@mcb.harvard.edu
Bo Hu
IBM T.J. Watson Research Center
1101 Kitchawan Road, Route 134
Yorktown Heights, NY 10598
Phone: (914) 945-2857
bhu@us.ibm.com
Isabelle Hug
University of Basel
Biozentrum
Klingelbergstrasse 50/70
Basel CH-4056
Switzerland
Phone: +41 61 267 2117
isa.hug@unibas.ch
Kelly Hughes
Utah
Biology
257 South 1400 East
Salt Lake City, UT 84112
Phone: (801) 587-3367
hughes@biology.utah.edu
124
Akihiko Ishijima
Tohoku University
Katahira 2-1-1,Aoba-ku
Sendai 980-8577
Japan
ishijima@tagen.tohoku.ac.jp
Masahiro Ito
Toyo University
1-1-1 Izumino, Itakura-machi
Oura-gun, Gunma 3740193
Japan
Phone: +81 27 682 9202
masahiro.ito@toyo.jp
Beata Jakobczak
Max Planck Institute for terrestrial microbiology
Karl-von-Frisch-Straße 10
Marburg 35043
Germany
Phone: +49 6241 178 222
beata.jakobczak@mpi-marburg.mpg.de
Urs Jenal
Biozentrum, University of Basel
Infection Biology
Biozentrum der Universität Basel
Klingelbergstrasse 70
Basel, BS 4056
Switzerland
Phone: +41 61 267 2135
Fax: +41 61 267 2118
urs.jenal@unibas.ch
Grant Jensen
Caltech
Biology
1200 E California Blvd
MC 114-96
Pasadena, CA 91125
Phone: (626) 395-8827
Fax: (626) 395-5730
jensen@caltech.edu
Mark Johnson
Loma Linda University
Biochemistry and Microbiology
AHBS 120
Loma Linda, CA 92350
Phone: (909) 558-4480 ext. 42765
Fax: (909) 558-4035
mjohnson@llu.edu
Christopher Jones
University of Oxford
Biochemistry
St. Peter's College
New Inn Hall Street
Oxford OX1 2DL
United Kingdom
Phone: +44 1865 613 315
christopher.jones2@spc.ox.ac.uk
Christine Josenhans
Medical University Hannover
Institute for Medical Microbiology
Carl-Neuberg-Strasse 1
Hannover 30625
Germany
Phone: +49 511 532 4354 ext. 4354
Fax: +49 511 532 4354
josenhans.christine@mh-hannover.de
Kirsten Jung
Ludwig-Maximilians-Universitaet Muenchen
Microbiology
Biocenter
Grosshaderner Str. 2-4
Martinsried, Bavaria 82152
Germany
Phone: +49 89 2180 74500
jung@lmu.de
Dan Kearns
Indiana University
Biology
1001 E. 3rd St
Bloomington, IN 47405
Phone: (812) 856-2523
dbkearns@indiana.edu
125
Daniela Keilberg
Max-Planck-Institute for Terrestrial Microbiology
Karl-von-Frisch-Straße 10
Marburg, Hessen D-35043 Germany
daniela.keilberg@mpi-marburg.mpg.de
Shahid Khan
Molecular Biology Consortium
Cellular Biophysics
MS6-2100 1 Cyclotron Road
Berkeley, CA 94720
Phone: (312) 996-1216
khan@mbc-als.org
Hyo Kyung Kim
University of Texas, Austin
7630 Wood Hollow Dr. #272
Austin, TX 78731
Phone: (512) 963-3021
hkkim2009@gmail.com
Yoshiaki Kinosita
Gakushuin
Physics
Toshimaku Mejiro 1-5-1
Tokyo 1718588
Japan
Phone: +81 3712 0033
12144002@gakushuin.ac.jp
John Kirby
University of Iowa
Microbiology
51 Newton Road
Iowa City, IA 52242
Phone: (319) 335-7818
Fax: (319) 335-9006
john-kirby@uiowa.edu
Smiljka Kitanovic
University of Utah
Biology
257 South 1400 East
201 SB
Salt Lake City, UT 84112
Phone: (801) 581-3592
smiljka_kitanovic@yahoo.com
Anna Koganitsky
Weizmann Institute of Science
Biological Chemistry
234 Herzel St,
Rehovot 76100
Israel
Phone: +972 8 934 2701
anna.koganitsky@weizmann.ac.il
Santosh Koirala
University of Illinois
Chemical and Biomolecular Engineering
600 S Mathews
RAL210
Urbana, IL 61801
Phone: (217) 344-7528
koirala2@illinois.edu
Seiji Kojima
Nagoya University
Division of Biological Science, Graduate school of
Science
Furo-cho, Chikusa-ku
Nagoya, Aichi 464-8602
Japan
Phone: +81-52-789-2992
Fax: +81-52-789-3001
z47616a@cc.nagoya-u.ac.jp
Naomi Kreamer
California Institue of Technology
Biochemistry and Molecular BIophysics
1200 E California Blvd
Pasadena, CA 91125
Phone: (626) 395-4856
nkreamer@gmail.com
126
Tino Krell
Estación Experimental del Zaidín
Albareda 1
Granada 18008 Spain
tino.krell@eez.csic.es
Ambroise Lambert
Institut Pasteur-Paris 7 University
25-28 rue du Docteur Roux
Paris 75015 France
Phone: +33 1 40 61 34 06
alambert@pasteur.fr
Jürgen Lassak
Ludwig Maximillians University Munich
Biology I; Microbiology
Großhaderner Straße
2-4
Martinisried, Bavaria 82152
Germany
Phone: +49 89 2180 74508
Fax: +49 89 2180 74520
juergen.lassak@lmu.de
Milena Lazova
FOM Institute AMOLF
Systems Biology
Sciencepark 104
Amsterdam, Noord Holland 1098 XG
Netherlands
Phone: +31 20 754 7302
lazova@amolf.nl
Yi-Ying Lee
University of Maryland, Baltimore County
Department of Marine Biotechnology
701 E Pratt Street
Baltimore, MD 21202
Phone: (410) 234-8877
yyinglee@umbc.edu
Pushkar Lele
Harvard University
Molecular and Cellular Biology
Harvard Biological Laboratories
16 Divinity Avenue, Room 3063
Cambridge, MA 02138
Phone: (617) 495-4217
lele@fas.harvard.edu
Robert Levenson
UC Santa Barbara
Chemistry & Biochemistry
761 Birch Walk
Apt J
Goleta, CA 93117
Phone: (858) 342-5006
rlevenson@chem.ucsb.edu
Chunhao Li
SUNY at Buffalo
Oral Biology
3435 Main St.
Buffalo, NY 14214
Phone: (716) 829-6014
Fax: (716) 829-3942
cli9@buffalo.edu
Tao Lin
UT Medical School at Houston
Pathology and Laboratory Medicine
PO Box 20708, Room MSB 2.124
Houston, TX 77225-0708
Phone: (713) 500-5350
Fax: (713) 500-0730
tao.lin@uth.tmc.edu
Jun Liu
UT Houston Medical School
Department of Pathology
6431 Fannin, MSB 2.228
Houston, TX 77030
Phone: (713) 500-5342
Jun.liu.1@uth.tmc.edu
127
Michael Manson
Texas A&M University
Biology
3258 TAMU
College Station, TX 77840
mike@bio.tamu.edu
Ana Martinez-del Campo
Universidad Nacional Autonoma de Mexico
Circuito Exterior S/N Ciudad Universitaria
Coyoacan
Mexico, Distrito Federal 04510 Mexico
Phone: +52 55 5622 5618
acampo@email.ifc.unam.mx
Shonna McBride
Emory University
Microbiology and Immunology
1510 Clifton Rd
Rm 3022
Atlanta, GA 30322
Phone: (404) 727-6192
shonna.mcbride@emory.edu
Regina McGrane
Iowa State University
Plant Pathology
103 14TH ST
Ames, IA 50010
Phone: (641) 521-6759
rnickel@iastate.edu
Jonathan McMurry
Kennesaw State University
Chemistry & Biochemistry
1000 Chastain Rd. MB #1203
Kennesaw, GA 30144
Phone: (770) 499-3238
jmcmurr1@kennesaw.edu
Vishnu Menon
Weizmann Institute of Science
Ullman Building of Life Sciences
Rehovot 76100
Israel
Phone: +972 8 934 2702
menonvishnu@gmail.com
David Milner
The University of Nottingham
Centre for Genetics and Genomics
Queen's Medical Centre
Nottingham NG7 2UH
United Kingdom
Phone: +44 115 823 0317
plxdsm@nottingham.ac.uk
Makoto Miyata
Osaka City University
Department of Biology, Graduate School of Science
3-3-138 Sugimoto Sumiyoshi-ku
Osaka 5588585
Japan
Phone: +81 6 6605 3157
Fax: +81 6 6605 3158
miyata@sci.osaka-cu.ac.jp
Andreas Möglich
Humboldt-Universität zu Berlin
Institut für Biologie
Invalidenstrasse 42
Neubau Raum 304
Berlin 10115
Germany
Phone: +49 30 2093 8850
andreas.moeglich@hu-berlin.de
Michael Morse
Brown University
Physics
182 Hope St
Providence, RI 02912
Phone: (207) 838-5885
michael_morse@brown.edu
128
Md Motaleb
East Carolina University
Microbiology and Immunology
600 Moye Blvd
BT 116
Greenville, NC 27834
motalebm@ecu.edu
Chakib Mouslim
University of Utah
Biology
257 S 1400 E, Department of biology
Kelly Hughes lab
Salt Lake City, UT 84112
Phone: (801) 585-6950
ymouslim@gmail.com
Tarek Msadek
Institut Pasteur
Biology of Gram-Positive Pathogens, Department of
Microbiology, Biology of Gram-Positive Pathogens
25 Rue du Dr. Roux
Paris 75015
France
Phone: +33 1 45 68 88 09
Fax: +33 1 45 68 89 38
tmsadek@pasteur.fr
Sampriti Mukherjee
Indiana University Bloomington
1001 E 3rd St
Bloomington, IN 47405
Phone: (812) 856-2559
sampmukh@indiana.edu
Daisuke Nakane
Nagasaki University
Biomedical Sciences
1-7-1 Sakamoto
Nagasaki 852-8588
Japan
Phone: +81 95 819 7649
jj20110031@cc.nagasaki-u.ac.jp
Beiyan Nan
UC Berkeley
Molecular and Cell Biology
16 Barker Hall, UC Berkeley
University of California, Berkeley
Berkeley, CA 94720-3204
Phone: (510) 643-5457
nanbeiyan@gmail.com
Masayoshi Nishiyama
Kyoto University
The HAKUBI Center
Sakyo-ku
Kyoto 606-8302
Japan
Phone: +81 75 753 9828
mnishiyama@icems.kyoto-u.ac.jp
Catherine Oikonomou
Caltech
1200 E. California Blvd.
Pasadena, CA 91125
Phone: (626) 395-8848
coiko@caltech.edu
Manuel Osorio-Valeriano
Universidad Nacional Autónoma de México
Molecular Genetics
Circuito Exterior S/N Ciudad Universitaria
Coyoacán, 04510, México D.F.
Mexico City, Distrito Federal 04510
Mexico
Phone: +52 55 5622 5618
mosorio@email.ifc.unam.mx
Karen Ottemann
UC Santa Cruz
Microbiology/Env Toxicology
1156 High Street
METX
Santa Cruz, CA 95064
Phone: (831) 459-3482
ottemann@ucsc.edu
129
Stephani Page
University of North Carolina
Biochemistry & Biophysics
116 Manning Drive
CB# 7290
Durham, NC 27713
stephani_page@med.unc.edu
Guillaume Paradis
Laval University
Physics, Engineering Physics and Optics
2610 d'Oviedo
Quebec, QC G2B0G6 Canada
Phone: (418) 656-2131 ext. 3788
guillaume.paradis.5@ulaval.ca
John Parkinson
University of Utah
Biology
257 South 1400 East
Salt Lake City, UT 84112
Phone: (801) 581-7639
parkinson@biology.utah.edu
Jonathan Partridge
University of Texas at Austin
Molecular Genetics and Microbiology
2506 Speedway Stop A5000
Austin, TX 78712
Phone: (512) 471-6799
j.partridge@gmail.com
Darysbel Perez
Loma Linda University
Microbiology and Molecular Genetics
Lindsay Hall Box#284
Loma Linda, CA 92350
Phone: (909) 558-1000 ext. 42758
Fax: 909 558 4035
daperez@llu.edu
Kene Piasta
University of Colorado at Boulder
Chemistry and Biochemistry
Jennie Smoly Caruthers Biotechnology Building
596 UCB
Boulder, CO 80309
Phone: (303) 492-3597
kene.piasta@colorado.edu
German Pinas
University of Utah
Biology
257 S 1400 E 201 SB
Salt Lake City, UT 84112
Phone: (801) 581-6307
g.pinas@utah.edu
Steven Porter
University of Exeter
Biosciences
Biosciences, Geoffrey Pope Building
Stocker Road
Exeter, Devon EX4 4QD
United Kingdom
Phone: +44 1392 722 172
s.porter@exeter.ac.uk
Birgit Prüβ
North Dakota State University
Veterinary and Microbiological Sciences
1523 Centennial Blvd.
Fargo, ND 58108
Phone: (701) 231-7848
Birgit.Pruess@ndsu.edu
Simon Rainville
Laval University
Physics
Pavillon optique photonique
2375, rue de la Terrasse
Quebec, QC G1V 0A6
Canada
Phone: (418) 656-2131 ext. 12511
Fax: (418) 656-2623
simon.rainville@phy.ulaval.ca
130
Christopher Rao
University of Illinois
Chemical Engineering
211 Roger Adams Lab
600 S Mathews Ave
Urbana, IL 61801
Phone: (217) 244-2247
chris@scs.uiuc.edu
S. James Remington
University of Oregon
Molecular Biology
297 Klamath Hall
1229 University of Oregon
Eugene, OR 97403
remington@molbio.uoregon.edu
Anna Roujeinikova
Monash University
Microbiology
Building 76
Monash University
Clayton, N/A 3800 Australia
Phone: +613 9902 9194
Anna.Roujeinikova@monash.edu
Keiko Sato
Nagasaki University
1-7-1 Sakamoto
Nagasaki 852-8588
Japan
Phone: +81 95 819 7649
satou@nagasaki-u.ac.jp
Florian Schubot
Virginia Tech
Biological Sciences
Life Science I, Room 125
Washington Street
Blacksburg, VA 24061
Phone: (540) 231-2393
fschubot@vt.edu
Matthew Sears
Texas A&M University
Biology
3258 TAMU
College Station, TX 77843
Phone: (979) 845-1249
matthewrsears@gmail.com
Larry Shimkets
University of Georgia
Microbiology
527 Biological Sciences
Athens, GA 30602
Phone: (706) 542-2681
Fax: (706) 542-2674
shimkets@uga.edu
Joshua Shrout
University of Notre Dame
156 Fitzpatrick Hall
Notre Dame, IN 46556
Phone: (574) 631-1726
joshua.shrout@nd.edu
Ruth Silversmith
University of North Carolina
Microbiology and Immunology
Room 804 Mary Ellen Jones
Chapel Hill, NC 27599
Phone: (919) 966-2679
Fax: (919) 962-8103
silversr@med.unc.edu
Ria Sircar
Cornell University
Chemistry & Chemical Biology
Physical Sciences Building, Room # 360
Cornell University
Ithaca, NY 14853
Phone: (607) 255-0752
rs693@cornell.edu
131
Matthew Smith
University of Oxford
Biochemistry
OCISB New Biochemistry
South Parks Road
Oxford OX1 3QU
United Kingdom
Phone: +44 1865 613 317
matthew.smith@chem.ox.ac.uk
Lotte Søgaard-Andersen
Max Planck Institute for Terrestrial Microbiology
Karl-von-Frisch. Str.
Marburg 35043
Germany
sogaard@mpi-marburg.mpg.de
Claudia Studdert
Universidad Nacional de Mar del Plata
Instituto de Investigaciones Biológicas
Funes 3250
Mar del Plata, Buenos Aires 7600
Argentina
Phone: +54 223 475 3030 ext. 12
Fax: +54 223 475 3150
studdert@mdp.edu.ar
Syed Sultan
East Carolina University
Microbiology & Immunology
BT-115, 600 Moye Blvd
Greenville, NC 27834
Phone: (252) 744-3128
sultans@ecu.edu
Ching Wooen Sze
University at Buffalo
Oral Biology
316 Foster Hall 3435 Main Street
Buffalo, NY 14214
Phone: (716) 829-6329
chingsze@buffalo.edu
Hendrik Szurmant
The Scripps Research Institute
Molecular and Experimental Medicine
10550 N Torrey Pines Rd
MEM-116
La Jolla, CA 92037
Phone: (858) 784-7904
Fax: (858) 784-7966
szurmant@scripps.edu
Yuhei Tahara
Osaka City University
Biology
3-3-138 Sugimoto Sumiyoshi-ku
Osaka 558-8585
Japan
Phone: +81 6 6605 2575
Fax: +81 6 6605 3158
tahara@sci.osaka-cu.ac.jp
Hirotaka Tajima
Nagoya University
Chikusa-ku, Furo-cho
Nagoya, Aichi Prefecture 464-8603
Japan
Phone: +81 52 789 2717
tajima@mein.nagoya-u.ac.jp
Norihiro Takekawa
Nagoya University
Division of Biological Science
Furo-cho, Chikusa-ku
Nagoya, Aichi 464-8602
Japan
Phone: +81 52 789 3543
takekawa.norihiro@d.mbox.nagoya-u.ac.jp
Akihiro Tanaka
Osaka City University
Biology
3-3-138 Sugimoto Sumiyoshi-ku Osaka
Osaka 558-8585
Japan
Phone: +81 6 6605 2575
Fax: +81 6 6605 3158
atanaka@sci.osaka-cu.ac.jp
132
Lynmarie Thompson
University of Massachusetts
Chemistry
LGRT 122
Amherst, MA 01003
Phone: (413) 545-0827
thompson@chem.umass.edu
Yuhai Tu
IBM Research
1101 Kitchawan Rd./Rt. 134
Yorktown Heights, NY 10598
yuhai@us.ibm.com
Isil Tulum
Osaka City University
Grad.Sch.of Sci. Depart. of Bio. Cell Function Lab
Sugimoto, Sumiyoshi-ku
Osaka 558-8585
Japan
Phone: +81 6 6605 2575
Fax: +81 6 6605 3158
iciltlm@gmail.com
Ady Vaknin
Hebrew University
Givat Ram
Jerusalem 91904
Israel
Phone: +972 50 250 2873
avaknin@phys.huji.ac.il
Barry Wanner
Purdue University
BioSci
915 West State Street
BioSci Lilly Hall
West Lafayette, IN 47907
Phone: (765) 494-8034
blwanner@purdue.edu
Kylie Watts
Loma Linda University
Microbiology and Molecular Genetics
Div. of Microbiology and Molecular Genetics
AHBS 102
Loma Linda, CA 92350
Phone: (909) 558-1000 ext. 83394
Fax: 909 558 4035
kwatts@llu.edu
Ben Webb
Virginia Tech
Biological Sciences
Life Sciences 1 Washington St.
Blacksburg, VA 24060
Phone: (571) 426-9433
mercury1@vt.edu
Laurence Wilson
The Rowland Institute at Harvard
100 Edwin H Land Boulevard
Cambridge, MA 02142
Phone: (617) 497-4643
wilson@rowland.harvard.edu
Alan Wolfe
Loyola University Chicago
Microbiology and Immunology
2160 South First Avenue
Maywood, IL 60153
Phone: (708) 216-5814
awolfe@lumc.edu
Chui Ching Wong
National University of Singapore
Mechanobiology Institute
5A Engineering Drive 1
T-Lab Building, #09-01
Singapore 117411
Singapore
Phone: +65 8188 7475
mbiwong@nus.edu.sg
133
Liang Wu
Iowa State University
Department of Plant Pathology and Microbiology
207 Science I
Iowa State University
Ames, IA 50010
Phone: (515) 294-3198
liangwu@iastate.edu
Kristin Wuichet
Max Plank Institute for Terrestrial Microbiology
Karl von Frisch Str. 10
Marburg, Hesse 35043
Germany
kristin.wuichet@mpi-marburg.mpg.de
Elsio Wunder
Yale School of Public Health
EMD
60 College Street, LEPH 607
New Haven, CT 06510
Phone: (203) 737-6412
elsio.wunder@yale.edu
Zhihong Xie
Yantai Institute of Coastal Zone Research,Chinese
Academy of Sciences
Chinese Academy of Sciences
17 Chunhui Rd, Laishan District
Yantai, Shandong 264003
China
Phone: +86 535 210 9183
Fax: +86 535 210 9000
zhxie@yic.ac.cn
Hiroki Yamamoto
Osaka City University
Biology
3-3-138 Sugimoto Sumiyoshi-ku Osaka Japan
Osaka 558-8585
Japan
Phone: +81 6 6605 2575
Fax: +81 6 6605 3158
hiroki-y@sci.osaka-cu.ac.jp
Xiaowei Zhao
University of Texas Health Science Center at
Houston
Department of Pathology and Laboratory Medicine
6431 Fannin
Houston, TX 77054
Phone: (713) 500-5245
xiaowei.zhao@uth.tmc.edu
Shiwei Zhu
University of Wisconsin-Milwaukee
Division of Biological Science
3209 N. Maryland Ave.
Milwaukee, WI 53211
Phone: (414) 229-2910
zhu6@uwm.edu
Yongtao Zhu
Nagoya University
Furo-cho, Chikusa-ku
Nagoya, Aichi 464-8602
Japan
Phone: +81 52 789 2993
zhushiwei23@hotmail.com
David Zusman
University of California
Molecular and Cell Biology
16 Barker Hall #3204
Berkeley, CA 94720-3204
Phone: (510) 642-2293
Fax: (510) 642-7038
zusman@berkeley.edu
134
BLAST STAFF
Tarra Bollinger
Molecular Biology Consortium
835 S. Wolcott Ave. (M/C 790)
Chicago, IL 60612
Phone: (312) 996-1216
Fax: (312) 413-2952
tbolli1@uic.edu
Peggy O’Neill
Molecular Biology Consortium
835 S. Wolcott Ave. (M/C 790)
Chicago, IL 60612
Phone: (312) 996-1216
Fax: (312) 413-2952
oneill@uic.edu
135
INDEX
136
Participant’s Name, Abstract page(s), Contact Information Page – PI Lab
A
Abouelfetouh, Alaa, 116, 120 - Wolfe, Alan
Adase, Christopher, 88, 120 - Manson, Michael
Afanzar, Oshri, 120 - Eisenbach, Michael
Al Mamun, Abu Amar, 105, 120 - Rosenberg, Susan
Allen, James, 52, 120 - Armitage, Judith
Anquez, Francois, 115, 120 - Tom, Shimizu
Armitage, Judith, 120 - Armitage, Judith
Aschtgen, Marie-Stephanie, 106, 120 - Ruby, Edward
B
Bardy, Sonia, 54, 120 - Bardy, Sonia
Baron, Szilvia, 120 - Eisenbach, Michael
Basford, Sarah, 109, 121 - Sockett, Liz
Beeby, Morgan, 2, 121 - Beeby, Morgan
Behr, Stefan, 79, 121 - Jung, Kirsten
Behrens, Wiebke, 78, 121 - Josenhans, Christine
Belas, Robert, 56, 121 - Belas, Robert
Berg, Howard, vi, 121 - Berg, Howard
Biswas, Indranil, 121 - Biswas, Indranil
Bollinger, Tarra, ii, 135 - Matsumura, Philip
Bourret, Bob, ii, 58, 121 - Bourret, Robert
Brennan, Caitlin, 31, 121 - Ruby, Edward
Briegel, Ariane, 16, 121 - Jensen, Grant
C
Calvo, Rebecca, 81, 122 - Kearns, Dan
Charon, Nyles, 122 - Charon, Nyles
Colin, Remy, 122 - Wilson, Laurence
Collins, Kieran, 122 - Ottemann, Karen
Crane, Brian, ii, 122 - Crane, Brian
Creager-Allen, Rachel, 10, 122 - Bourret, Robert
Crosson, Sean, ii, 61, 122 - Crosson, Sean
D
Dahlquist, Rick, 122 - Dahlquist, Rick
Dreyfus, Georges, 122 - Dreyfus, Georges
Duchesne, Ismael, 103, 122 - Rainville, Simon
E
Eisenbach, Michael, 123 - Eisenbach, Michael
Erhardt, Marc, 65, 123 - Erhardt, Marc
Evans, Lewis, 3, 123 - Hughes, Colin
F
Falke, Joseph, ii, 66, 123 - Falke, Joseph
Fraiberg, Milana, 4, 123 - Eisenbach, Michael
Friedrich, Carmen, 111, 123 - Søgaard-Andersen, Lotte
Fukuoka, Hajime, 5, 123 - Ishijima, Akihiko
137
Participant’s Name, Abstract page(s), Contact Information Page – PI Lab
G
Galperin, Michael, 123 - Galperin, Michael
Gebhard, Susanne, 46, 123 - Gebhard, Susanne
Gohara, Mizuki, 70, 123 - Homma, Michio
Goss, Lindsie, 11, 124 - Dworkin, Jonathan
H
Han, Xuesheng, 99, 124 - Parkinson, John S.
Harshey, Rasika, ii, 124 - Harshey, Rasika
Hazelbauer, Gerald, 124 - Hazelbauer, Gerald
Higgs, Penelope, 69, 124 - Higgs, Penelope
Hiremath-Mendez, Geetha, 80, 124 - Kawagishi, Ikuro
Hosu, Basarab, 57, 124 - Berg, Howard
Hu, Bo, 39, 124 - Tu, Yuhai
Hug, Isabelle, 76, 124 - Jenal, Urs
Hughes, Kelly, viii, 73, 74, 124 - Hughes, Kelly
I
Ishijima, Akihiko, 125 - Ishijima, Akihiko
Ito, Masahiro, 75, 125 - Ito, Masahiro
J
Jakobczak, Beata, 110, 125 - Søgaard-Andersen, Lotte
Jenal, Urs, ii, 125 - Jenal, Urs
Jensen, Grant, 125 - Jensen, Grant
Johnson, Mark, 125 - Johnson, Mark
Jones, Christopher, 17, 125 - Armitage, Judith
Josenhans, Christine, ii, 125 - Josenhans, Christine
Jung, Kirsten, ii, 125 - Jung, Kirsten
K
Kearns, Dan, 125 - Kearns, Dan
Keilberg, Daniela, 12, 126 - Søgaard-Andersen, Lotte
Khan, Shahid, 41, 126 - Khan, Shahid
Kim, Hyo Kyung, 68, 126 - Harshey, Rasika
Kinosita, Yoshiaki, 95, 126 - Nishizaka, Takayuki
Kirby, John, ii, 83, 126 - Kirby, John
Kitanovic, Smiljka, 98, 126 - Parkinson, John S.
Koganitsky, Anna, 126 - Eisenbach, Michael
Koirala, Santosh, 104, 126 - Rao, Christopher
Kojima, Seiji, 72, 126 - Homma, Michio
Kreamer, Naomi, 94, 126 - Newman , Dianne
Krell, Tino, 30, 127 - Krell, Tino
L
Lambert, Ambroise, 100, 127 - Picardeau, Mathieu
Lassak, Jürgen, 47, 127 - Jung, Kirsten
Lazova, Milena, 18, 127 - Shimizu, Tom
Lee, Yi-Ying, 24, 127 - Belas, Robert
Lele, Pushkar, 6, 127 - Berg, Howard
138
Participant’s Name, Abstract page(s), Contact Information Page – PI Lab
Levenson, Robert, 62, 127 - Dahlquist, Frederick
Li, Chunhao, 85, 127 - Li, Chunhao
Lin, Tao, 43, 127 - Norris, Steven
Liu, Jun, 86, 87, 127 - Liu, Jun
M
Manson, Michael, ii, 128 - Manson, Michael
Martinez-del Campo, Ana, 63, 128 - Dreyfus, Georges
McBride, Shonna, 13, 128 - McBride, Shonna
McGrane, Regina, 55, 128 - Beattie, Gwyn
McMurry, Jonathan, 128 - McMurry, Jonathan
Menon, Vishnu, 128 - Eisenbach, Michael
Milner, David, 108, 128 - Sockett, Liz
Miyata, Makoto, 128 - Miyata, Makoto
Möglich, Andreas, 33, 128 - Möglich, Andreas
Morse, Michael, 34, 128 - Tang, Jay
Motaleb, Md, 129 - Motaleb, Md
Mouslim, Chakib, 48, 129 - Hughes, Kelly
Msadek, Tarek, viii, 14, 129 - Msadek, Tarek
Mukherjee, Sampriti, 25, 129 - Kearns, Daniel
N
Nakane, Daisuke, 26, 129 - Nakayama, Koji
Nan, Beiyan, 7, 129 - Zusman, David
Nishiyama, Masayoshi, 44, 129 - Nishiyama, Masayoshi
O
Oikonomou, Catherine, 129 - Jensen, Grant
O'Neill, Peggy, ii, 135 - Matsumura, Philip
Osorio-Valeriano, Manuel, 64, 129 - Dreyfus, Georges
Ottemann, Karen, ii, 96, 129 - Ottemann, Karen
P
Page, Stephani, 60, 130 - Bourret, Robert
Paradis, Guillaume, 102, 130 - Rainville, Simon
Parkinson, John S, ii, 19, 130 - Parkinson, John S.
Partridge, Jonathan, 8, 130 - Harshey, Rasika
Perez, Darysbel, 77, 130 - Taylor, Barry
Piasta, Kene, 20, 130 - Falke, Joseph
Piñas, Germán, 97, 130 - Parkinson, John S.
Porter, Steven, vi, 101, 130 - Porter, Steven
Prüβ, Birgit, viii, 49, 130 - Prüβ, Birgit
R
Rainville, Simon, 130 - Rainville, Simon
Rao, Christopher, ii, 35, 131 - Rao, Christopher
Remington, S. James, 131 - Remington, S. James
Roujeinikova, Anna, 40, 131 - Roujeinikova, Anna
S
Sato, Keiko, 84, 131 - Nakayama, Koji
139
Participant’s Name, Abstract page(s), Contact Information Page – PI Lab
Schubot, Florian, 107, 131 - Schubot, Florian
Sears, Matthew, 32, 131 - Manson, Michael
Shimkets, Larry, vii, 131 - Shimkets, Larry
Shrout, Joshua, 27, 131 - Shrout, Joshua
Silversmith, Ruth, 59, 131 - Bourret, Robert
Sircar, Ria, 42, 131 - Crane, Brian
Smith, Matthew, 53, 132 - Armitage, Judith
Søgaard-Andersen, Lotte, 132 - Søgaard-Andersen, Lotte
Studdert, Claudia, 113, 114, 132 - Studdert, Claudia
Sultan, Syed, 93, 132 - Motaleb, Md
Sze, Ching Wooen, 50, 132 - Li, Chunhao
Szurmant, Hendrik, 15, 132 - Szurmant, Hendrik
T
Tahara, Yuhei, 28, 132 - Miyata, Makoto
Tajima, Hirotaka, 67, 132 - Fukuda, Toshio
Takekawa, Norihiro, 71, 132 - Homma, Michio
Tanaka, Akihiro, 90, 132 - Miyata, Makoto
Thompson, Lynmarie, 21, 133 - Thompson, Lynmarie
Tu, Yuhai, vii, 133 - Tu, Yuhai
Tulum, Isil, 92, 133 - Miyata, Makoto
V
Vaknin, Ady, 22, 133 - Vaknin, Ady
W
Wanner, Barry, 133 - Wanner, Barry
Watts, Kylie, ii, 23, 133 - Watts, Kylie
Webb, Ben, 36, 133 - Scharf, Birgit
Wilson, Laurence, 37, 133 - Wilson, Laurence
Wolfe, Alan, ii, 117, 133 - Wolfe, Alan
Wong, Chui Ching, 82, 133 – Chiam, Keng-Hwee
Wu, Liang, 29, 134 - Beattie, Gwyn
Wuichet, Kristin, 112, 134 - Søgaard-Andersen, Lotte
Wunder, Elsio, 45, 134 - Ko, Albert
X
Xie, Zhihong, 118, 134 - Xie, Zhihong
Y
Yamamoto, Hiroki, 91, 134 - Miyata, Makoto
Z
Zhao, Xiaowei, 9, 134 - Liu, Jun
Zhu, Shiwei, 38, 134 - Homma, Michio
Zhu, Yongtao, 89, 134 - McBride, Mark
Zusman, David, 134 - Zusman, David
140