AN ABSTRACT OF THE DISSERTATION OF
Marion L. Brodhagen for the degree of Doctor of Philosophy in Molecular and
Cellular Biology presented on June 30, 2003.
Title: Pyoluteorin as a Signaling Molecule Regulating Secondary Metabolite
Production and Transport Genes in Pseudomonas fluorescens Pf-5.
Redacted for privacy
Abstract Approved:
Joyce E. Loper
A major factor in the ability of Pseudomonasfluorescens Pf-5 to act as a
biological control agent is its production of antibiotics, including pyoluteorin (PLT),
2,4-diacetylphloroglucinol (2,4-DAPG) and pyrrolnitrin (PRN). The data provided in
this thesis demonstrate that the presence of any of these antibiotics in the extracellular
milieu affects production of that same antibiotic, as well as others, by Pf-5. Amending
the growth medium with antibiotics had multiple effects on secondary metabolism in
Pf-5. i) PLT positively regulated its own production. ii) 2,4-DAPG positively
regulated its own production. iii) PLT suppressed 2,4DAPG production. iv) 2,4DAPG inhibited PLT production. v) PLT suppressed transcription of a heterologous
ferric-pyoverdine uptake gene. vi) PRN exerted a slight inhibitory effect on PLT gene
transcription and production.
PLT autoinduction by Pf-5 was extensively characterized, and was shown to
require concentrations of exogenous PLT in the nanomolar range. These low
concentrations are comparable to those of many molecules proposed to function in
signaling roles. PLT served as a signal between distinct populations of cells within the
rhizosphere, where it prompted autoinduction by those cells. Aside from effects of Pf5 antibiotics on one another, I also described the positive effect of exogenous PLT on
expression of a set of transport genes flanking the PLT biosynthetic gene cluster.
Sequence data and experimental evidence suggests that these genes encode a transport
apparatus for PLT. The deduced amino acid sequences for four adjacent open reading
frames together resemble Type I secretion apparatuses, which typically function in
transport of proteins rather than secondary metabolites. The intact transporter genes
are necessary for optimal PLT production.
Taken together, the data from the studies described herein demonstrate that i)
the production of PLT by Pf-5 can affect the production of PLT by neighboring cells,
and ii) PLT and other exogenous secondary metabolites have both autoregulatory and
cross-regulatory effects in culture. Because Pf-5 derivatives engaged in PLT crossfeeding in the rhizosphere, it is likely that cross-feeding occurs for other secondary
metabolites as well. Thus, production of an antibiotic by one cell can profoundly affect
secondary metabolism in neighboring cells occupying natural habitats.
©Copyright by Marion L. Brodhagen
June 30, 2003
All Rights Reserved
Pyoluteorin as a Signaling Molecule Regulating Secondary Metabolite Production and
Transport Genes in Pseudomonasfluorescens Pf-5.
by
Marion L. Brodhagen
A DISSERTATION
submitted to
Oregon State University
in partial fulfillment of
the requirements for the
degree of
Doctor of Philosophy
Presented June 30, 2003
Commencement June, 2004
Doctor of Philosophy dissertation of Marion L. Brodhagen presented on June 30,
2003.
APPROVED:
Redacted for privacy
Major Ptofessor, representing Molecular and Cellular Biology
Redacted for privacy
Director Molecular and Cellular Biology Program
Redacted for privacy
Dean of the graduate School
I understand that my dissertation will become part of the permanent collection of
Oregon State University libraries. My signature below authorizes release of my
dissertation to any reader upon request.
Redacted for privacy
Marion L. Bro'hagen, Author
ACKNOWLEDGEMENTS
I would like to express my gratitude to members of the Loper Lab past and
present who have made my tenure as a PhD student a truly wonderful time. Thank
you for the guidance, the ideas, and most of all, the friendship that you shared.
Thanks especially to Nancy Chaney, Marcella Henkels, Dr. Caroline Press, Brenda
Shaffer, and Dr. Virginia Stockwell for technical suggestions at crucial times, for
conversations that helped me understand the larger context of my research, for
mentoring me at the bench and in my development as a scientist, and for sharing your
broad perspectives - the knowledge you shared helped to make my time productive
and rewarding, and the warmth and humor you shared made it fun.
Thanks to Nathan Corbell, Cheryl Whistler, and Todd Temple for collegial
interactions that have been so helpful to me, but also for camaraderie on late nights in
the lab and elsewhere! I also am grateful to Meadow Anderson, Chelsea Meltzer, and
Kevin Hockett for all the things they did to make life easy for me in the lab - and for
the sparkle they bring to our lab! Special thanks go to Cara Taormina, Brie Sawtelle,
and Amy Davis who have made research contributions to this thesis and who were
each very much fun to collaborate with! Finally, the USDA Horticultural Crops
Research Laboratory has been a very special place to spend my time as a PhD student,
and I can't begin to list all the people that have befriended me here. I cannot imagine a
more genial group! The same is true for my friends, colleagues, and mentors within
the Botany and Plant Pathology department, the Molecular and Cellular Biology
Program, and all over the OSU campus. From backyard bonfires to weekend hikes to
much-needed coffee breaks, I could never have made it though this degree without the
friendship and support of countless friends.
Dr. Linda Thomashow generously provided me the opportunity to do research
in her lab; I am very grateful for the wonderful month that I spent at Washington State
University. Dr. Dmitri Mavrodi went out of his way again and again to make the time
spent there a success, as did everyone else in the lab. I have many fond memories of
my time there. Dr. Dan Arp also welcomed me into his lab, and Luis Sayavedra-Soto
and Alisa Vangnai spent many days mentoring me there. I am grateful for the chance
to have worked with so many warm people, doing exciting science and having fun!
My committee, Dr. Dan Arp, Dr. Lynda Ciuffetti, Dr. Walt Ream, and Dr.
Oscar Riera-Lizarazu, have guided me through this process and provided advice and
encouragement at every step. My sincere thanks for all they have done to help me
develop as a scientist. Finally, Dr. Joyce Loper has been a true mentor in every sense
of the word, and a scientific and personal role model of the highest caliber. She has
given me tremendous freedom, along with constant encouragement and support in all
my endeavors. I am very grateful for all the opportunities that she created for me both
inside and outside of her laboratory.
Lastly, I am grateful for financial support provided by a fellowship from the
Environmental Protection Agency Science to Achieve Results program.
CONTRIBUTION OF AUTHORS
Dr. Joyce Loper advised all of the work reported herein. Marcella Henkels was
involved in the design and implementation of experiments to demonstrate pyoluteorin
cross-feeding in the rhizosphere. This work is discussed in Chapter Two. Dr. Ian
Paulsen provided privileged access to information about sequence quality from the
region flanking pkJ in the unfinished Pf-5 genome being sequenced at The Institute
for Genomic Research. This work is discussed in Chapter Four.
TABLE OF CONTENTS
Chapter 1.
1.1.
Introduction. The Role of Secondary Metabolites in Biological
Control and in the Physiology of R1iizosphere Pseudomonads ................. 1
Biological control of plant pathogens by microorganisms........................ 1
1.1.1.
1.1.2.
1.2.
Antibiosis and its importance in biological control by
Pseudomonasspp ...................................................................................... 3
1.2.1.
1.2.2.
1.2.3.
1.2.4.
1.2.5.
1.3.
Limitations to biological control........................................................ 1
Mechanisms of biological control...................................................... 3
Evidence that antibiosis contributes significantly to biocontrol
in the soil environment ....................................................................... 5
Regulation of antibiotic production in Pseudomonas spp .................. 9
Transport of secondary metabolites from cells................................ 14
Regulation of pyoluteorin production by P. fluorescens Pf-5,
a model biocontrol organism ............................................................ 14
Biosynthesis of pyoluteorin.............................................................. 18
Secondary metabolites as signal molecules ............................................. 21
1.3.1.
1.3.2.
Role of secondary metabolites in Pseudomonas spp ....................... 22
Existing support for the hypothesis that secondary metabolites
function as signal molecules in Pseudomonas spp ........................... 23
1.3.3.
Signaling properties of antibiotics in non-Pseudomonas species....24
1.3.4. Regulation of antibiotic production by other secondary
metabolites ............................................................ 25
1.4. Statement of research objectives....................................................................25
1.5. References...................................................................................................... 28
TABLE OF CONTENTS (Continued)
Page
Chapter 2.
Positive Autoregulation of the Antibiotic Pyoluteorin in the Biological
Control Organism Pseudomonasfluorescens Pf-5 .................................. 43
2.1. Abstract....................................................................................................44
2.2 Introduction.............................................................................................45
2.3. Methods ................................................................................................... 47
2.3.1.
2.3.2.
Bacterial strains, plasmids, and culture conditions.......................... 47
Influence of exogenous antibiotics on antibiotic production and
PLT gene expression by Pf5 ............................................................. 50
2.3.3. Antibiotic quantification ..................................................................51
2.3.4. Transcriptional activity from PLT biosynthetic genes..................... 51
2.3.5. Assessment of PLT cross-feeding on roots ....................................... 52
2.3.6. Data analysis ....................................................................................53
2.4. Results ..................................................................................................... 53
2.4.1.
2.4.2.
2.4.3.
2.4.4.
2.4.5.
PLT production is autoregulated...................................................... 53
PLT and 2,4-DAPG exhibit mutual inhibition................................. 58
Effects of PLT and 2,4-DAPG on PLT production occur at the
transcriptional level .......................................................................... 62
PLT autoinduction is superseded by genetically and
environmentally repressive backgrounds ......................................... 64
PLT autoinduction occurs in the rhizosphere .................................. 64
2.5. Discussion................................................................................................ 67
2.6. Acknowledgements ................................................................................. 71
2.7.
References ............................................................................................... 72
TABLE OF CONTENTS (Continued)
Chapter 3.
Preliminary Evidence for Effects of Pyrrolnitrin,
2,4-Diacetyiphioroglucinol, and Pyoluteorin on Secondary Metabolism
in Pseudomonasfluorescens Pf-5 ............................................................ 79
3.1.
Introduction ............................................................................................. 79
3.2.
Methods ................................................................................................... 80
3.3.
Results and Discussion ............................................................................. 83
3.3.1.
3.3.2.
3.33.
3.3.4.
Lack of influence by exogenous PLT on PRN................................. 83
Minimal influence by exogenous PRN on PLT production ............. 85
Autoinduction by 2,4-DAPG in P. fluorescens Pf-5 ........................ 87
Exogenous PLT represses expression of pyoverdine biosynthesis
genes ................................................................................................. 89
3.4.
Conclusions ............................................................................................. 92
3.5.
References...............................................................................................94
Chapter 4.
Membrane Transport Genes Play a Role in Pyoluteorin Production
by Pseudomonasfluorescens Pf-5 ........................................................... 97
4.1.
Introduction............................................................................................. 97
4.2. Methods .................................................................................................101
4.2.1.
4.2.2.
4.2.3.
4.2.4.
4.2.5.
4.2.6.
4.2.7.
4.2.8.
4.2.9.
Bacterial strains, plasmids, and culture conditions........................ 101
DNA manipulations....................................................................... 105
RT-PCR.......................................................................................... 196
Nucleotide sequencing of pltH,pltI, and plt.J ................................. 107
Derivation of chromosomal p/tI: :inaZ and plt.J::inaZ
transcriptional fusions by marker exchange mutagenesis .............. 108
Derivation of chromosomalpltF:: aacCl mutant by marker
exchange mutagenesis.................................................................... 113
Antibiotic quantification................................................................ 114
Transcriptional activity from PLT biosynthetic genes................... 114
Statistical analysis .......................................................................... 115
TABLE OF CONTENTS (Continued)
4.3.
Results ................................................................................................... 115
4.3.1.
4.3.2.
Nucleotide sequence analysis ........................................................ 115
Phenotypic characterization of piti, p/tJ mutants ........................... 123
4.4. Discussion .............................................................................................. 138
4.4.1.
4.4.2.
4.4.3.
4.4.4.
4.4.5.
PLT production requires functionality of transport genes flanking
the PLT biosynthetic gene cluster .................................................. 138
Exogenous PLT induces expression of p/tI and p/tJ...................... 139
pitH ................................................................................................. 141
p/tI .................................................................................................. 143
pltJ .................................................................................................. 145
4.5.
References ............................................................................................. 147
Chapter 5.
Concluding Remarks............................................................................. 156
5.1.
Repercussions of PLT production for the rhizosphere community....... 156
5.2.
Functionality of bioactive compounds in a soil environment............... 158
5.3.
Secondary metabolite production by Pf-5 is affected by the profile of
metabolites already present ................................................................... 158
5.4.
Site of perception of the PLT signal may be cytoplasmic or
extracytoplasmic .................................................................................... 159
5.5
PLT accumulation requires a set of flanking transporter genes,
which are in turn regulated by PLT ....................................................... 160
5.6.
Significance ........................................................................................... 160
5.7 References..................................................................................................... 162
Bibliography ................................................................................................................ 164
Appendices .................................................................................................................. 187
LIST OF FIGURES
Figure
ig
1.1.
Antibiotics produced by P. fluorescens Pf-5 that have been
implicated in biological control......................................................................... 15
1.2.
Factors known to influence pyoluteorin biosynthetic gene transcription
in P. fluorescens Pf-5 ........................................................................................ 17
1.3.
Proposed pyoluteorin biosynthetic pathway..................................................... 20
2.1.
Structures of pyoluteorin (a) and 2,4-diacetylphloroglucinol (b) ..................... 46
2.2.
Influence of exogenous PLT on PLT and 2,4-DAPG production
byderivatives of Pf-5 ........................................................................................ 55
2.3.
Minimum effective concentration of PLT for autoinduction in Pf-5 ................ 57
2.4.
Influence of exogenous 2,4-DAPG on PLT production by derivatives
ofPf-5 ...............................................................................................................
2.5.
Minimum effective concentration of 2,4-DAPG for repression of
PLT production in Pf-5 ..................................................................................... 61
2.6.
Positive and negative influences by PLT and 2,4-DAPG, respectively,
on transcriptional activity within the PLT biosynthetic gene cluster................ 63
2.7.
Autoinduction of PLT as a result of cross-feeding in the rhizosphere ..............66
3.1.
Influence of exogenous PLT and PRN on PRN produced by Pf-5 iceC ......... 84
3.2.
Positive and negative influences by PLT and PRN, respectively,
on transcriptional activity of the PLT biosynthetic gene, pltB .......................... 86
3.3.
Influence of exogenous 2,4-DAPG on production of 2,4-DAPG by
derivativesof Pf-5 ............................................................................................. 88
3.4.
Negative influence by PLT on transcriptional activity of a
ferric-pyoverdine uptake gene ........................................................................... 91
4.1.
Map of PLT biosynthetic gene cluster and relationship to subclones
used as templates for sequencing templates ...................................................... 99
4.2.
Cloning strategy used to construct pJEL6300, which was used in
marker-exchange mutagenesis to derive Pf-5 pltI::inaZ ................................. 110
4.3.
Map and features of open reading frames flanking one end of the
PLT biosynthetic gene region ......................................................................... 116
LIST OF FIGURES (Continued)
Figure
4.4.
Alignment of predicted amino acid sequence of PItH with that of the
Ph1H sequence from P.fluorescens CHA0 ..................................................... 118
4.5.
Alignment of predicted amino acid sequence of PitT with HIyD
consensus domain
..................................................................................... 120
4.6.
Alignment of predicted amino acid sequence of PI1J with the consensus
ABC-binding cassette of ABC transporter proteins........................................ 122
4.7.
PLT production by Pf-5 and bypltl::inaZ,pltJ::inaZ, and
pltB::inaZ derivatives of Pf-5 ........................................................................ 124
4.9.
Influence of exogenous PLT on PLT production by Pf-5, and
by Pf-5 pltJ::inaZ and Pf-5 pltB::inaZ...........................................................128
4.10.
Possible P1tR binding sites from the pltR-pltL and pitH-p/tI intergenic
regions ............................................................................................................. 129
4.11.
INA activity from pltB::inaZ (a), pitI::inaZ (b), and
pltJ::inaZ (c) transcriptional fusions in wild-type (circles),
pltF.:aacCl (triangles) orpitR::aacCl (squares) backgrounds ..................... 131
4.12.
Positive influence by PLT on transcriptional activity from pltB::inaZ
in a wild type Pf-5 background (a); pltJ::inaZ in a wild type Pf-5
background (b);pltJ::inaZin apltF background (c), and pit.J::inaZ
in apltR background (d) .................................................................................. 134
4.13.
Positive influence by PLT on transcriptional activity from pltB::inaZ
in a wild type Pf-5 background (a); p/tI: :inaZ in a wild type Pf-5
background (b); p/tI: :inaZ in a pltF background (c), and pltI::inaZ
in apltR background (d) .................................................................................. 136
LIST OF TABLES
Table
Page
1.1.
Antibiotics produced by Pseudomonas spp. and implicated in biological
control................................................................................................................. 4
2.1:
Bacterial strains and plasmids used in this study.............................................. 49
3.1.
Bacterial strains and plasmids used in this study .............................................. 82
3.2.
Influence of exogenous PLT on PRN production by derivatives of Pf-5 ......... 83
4.1.
Bacterial strains and plasmids used in this study............................................ 102
4.2.
PCR primers used in this study....................................................................... 106
LIST OF APPENDICES
Page
Appendix 1. Overexpression of PItR in Escherichia
coli........_......................... 188
Al.l.
Introduction ................................................................................... 188
Al .2.
Characterization of P1tR ................................................................. 190
Al.2.l. A transcriptional reporter for pltR........................................... 191
A1.2.2. Overexpression of P1tR in E. coli ........................................... 193
A1.3.
Future recommendations................................................................ 200
Al .4.
Acknowledgements ........................................................................ 201
Al.5.
References ...................................................................................... 202
Appendix 2. Nucleotide and Amino Acid Sequence for pitH, piti, pltJ, orJK,
orJP, orfQ, orfU, orJX, and orf)'..................................................... 204
A2. 1.
Predicted amino acid sequences and positions of open reading
frames in nucleotide sequence ........................................................ 204
A2.2.
Relevant nucleotide sequence ........................................................ 207
LIST OF APPENDIX FIGURES
Figure
Al .1. Proposal for PItR/PLT interaction at PLT promoters ..................................... 189
Al .2.
Proposal for dual binding by PItR of PLT (co-activator) and 2,4-DAPG
(co-repressor).................................................................................................. 190
LIST OF APPENDIX TABLES
Table
Page
Al .1. Expression of P1tR from pTYB 1 in E. coli B ER2566 cells, as visualized
byimmunoblotting .......................................................................................... 198
Al.2. Expression of PItR in various hostivector combinations ................................ 199
1642) - (1564 Galilei Galileo
them.
discover to is point the discovered; are they once understand to easy are truths All
Pyoluteorin as a Signaling Molecule Regulating Secondary Metabolite
Production and Transport Genes in Pseudomonasfluorescens Pf-5.
Chapter 1: The Role of Secondary Metabolites in Biological Control and in the
Physiology of Rliizosphere Pseudomonads
The feeding of our burgeoning human population places ever-greater
demands on agriculture for increased crop yields. Although the past century has
certainly seen revolutionary advances in the efficiency of agricultural crop production,
some advances have come at the expense of ecological sustainability or environmental
health. For example, affordable, organic pesticides introduced in the 1940's were
powerfully effective at stemming crop yield losses due to plant diseases. However,
the drawbacks of widespread use of such toxins were soon realized, as demonstrated
by the prompt enactment of the Federal Insecticide, Fungicide, and Rodenticide Act
(FIFRA) in the United States in 1947. The struggle for effective plant disease control
without riskato environmental or human health continues: the FIFRA has been
recently updated by the Food Quality and Protection Act (FQPA) of 1996, which
requires the US Environmental Protection Agency to reevaluate the toxicity of
pesticide residues to human health based on cumulative exposure from multiple
sources and to groups of pesticides with similar mechanisms for toxicity
(http://www.epa.gov/opppspsl /fqpa/). Concerns about environmental health also
develop along with our understanding of the impact of chemical usage: the Montreal
Protocol of 1991 requires that usage of methyl bromide, a broadly effective and
widely used pesticide, be completely discontinued in developed nations by 2005
because of its adverse effects on the Earth's ozone layer. As a result, alternate control
measures must be found to replace methyl bromide use in over 100 crops, for control
of plant pathogens, nematodes, weeds, and insects
(http://w-ww.ars.usda.gov/is/mb/mebrweb.htm). The status of synthetic pesticides in
agriculture is suffering from such increasingly restrictive legislation governing
pesticide use (10, 36, 54,121), and from concerns of growers and consumers alike
about effects of agrochemicals on the environment and on human health (25, 54). Tn
addition, the use of synthetic pesticides can lead to unwanted development of resistant
populations (114,121) and can have adverse effects on non-target organisms (e.g. 38,
40). Moreover, despite modem agricultural practices and technologies, Agrios
estimated in 1988 (3) that approximately 12% of the potential yield for a given
agricultural crop is lost to plant disease before harvest, and up to another 20% may be
lost after harvest. Thus, there is room for improvement in both the efficacy (see also
reference 9) and safety of plant disease control.
1.1. Biological control of plant pathogens by microorganisms.
For the past two or three decades, biological control organisms have been a
focus of academic study and have increasingly gained commercial favor as viable
alternatives to synthetic pesticides in combatting plant disease (24, 43, 53, 83, 89,102,
135). Biological control, as defined by Cook (23), is the control of one organism by
another and implies the reduction in either pest populations or the severity of plant
disease. Biological control organisms have the following advantages over synthetic
pesticides, in the control of plant pathogens: 1) greater safety to human health; 2)
minimal potential for known harmful environmental effect; 3) low likelihood of
inducing resistance by target organisms; and 4) possibility for combination with
conventional (chemical) controls, if necessary for greater efficacy (9, 23, 54).
1.1.1. Limitations to biolo2ical control.
As with chemical control, biological control has its share of shortcomings.
In a market sense, these include a brief shelf life (122), the necessity for a lag time for
establishment (and thus a less immediate effect than that of chemical controls),
incomplete control of pathogen populations, and low profit margins for producers and
users alike (9). There is also a persistent problem of variable performance in the field
(9, 101,130,134) which may stem from any one, or a combination of, a number of
biological factors. These factors can include poor or patchy colonization, loss of
ecological competence of the biocontrol strain, susceptibility to plant and soil
influences, failure to express biocontrol traits, phytotoxicity of secreted metabolites,
tolerance of target organisms to secreted metabolites, and influx of non-target
pathogens due to alteration of the environment by the biocontrol agent (9, 50).
Understanding the mechanisms by which microorganisms exert biological control, and
the environmental constraints on the success of those mechanisms, can lead to
adoption of strains and application strategies with improved rates of biocontrol
success.
1.1.2. Mechanisms of biological control.
Biological control may be exerted by biological control agents directly or
indirectly. Direct effects result from i) competition for space, or for nutrients such as
C, N, or Fe; ii) antagonism of the pathogen by parasitism or predation, and/or iii)
antibiosis. Biological control may also be the result of an indirect interaction with the
pathogen, via the plant. In this case, physiological activities of the biological control
agent trigger plant host defense responses, which thwart the progress of the pathogen
(e.g. reviewed in 52, 130, and 135).
1.2. Antibiosis and its importance in biological control by Pseudomonas
spp.
Of the mechanisms described above, the work described in this dissertation
is focused solely on antibiosis. Antibiotic production is important to the success of
many biological control agents. Examples include the production of zwitteromycin A
and kanosamine production by Bacillus cereus (123,124), oligomycin A production by
Streptomyces libani (74), xanthobaccins from Stentrophomonas spp. (93), and
herbicolins 0 and I from Pantoea agglomerans (69). Among fluorescent
Pseudoinonas spp., possibly the largest, best characterized, and most promising group
of biocontrol bacteria (101,130), the importance of antibiotic production has been
especially well highlighted. Pseudomonas spp. produce an array of secondary
4
metabolites, many of which have been studied in the context of their utility as
antibiotics that inhibit microorganisms pathogenic to agricultural crops (Table 1.1).
For brevity, I will limit my discussion to four well-characterized Pseudomonas
antibiotics - phenazine, 2,4-diacetylphloroglucinol, pyoluteorin, and pyrrolnitrin.
Table 1.1. Antibiotics produced by Pseudomonas spp. and implicated in
biological control.
Antibiotic
Producing
Target Pathogen
Host
Plant
species
Selected
Reference
Anthranhlate
P. aeruginosa
Fusarium oxysporum f sp. ciceris
chickpea
5
2,4-
P. fluorescens
Gaeumannomyces grainini var.
tritici
tobacco
71
P. fluorescens
Thielaviopsis basicola
tobacco
71
P. fluorescens
Pythium ultimum
sugar beet
41
P. aurantiaca
Fusarium oxysporum
wheat
106
Hydrogen cyanide
P. fluorescens
Thielaviopsis basicola
tobacco
133
Oomycin A
P. fluorescens
Pythium ultimum
cotton
68
Phenazine-1-carhoxamide
P. chloraphis
Fusarium oxysporum f. sp. radicislycopersici
tomato
22
Phenazine-1-carboxylic
acid
P.fiuorescens
Gauemannomyces gramini var
tritici
wheat
128
P. aureofaciens
Gauemannomyces gramini var
tritici
wheat
108
Pyocyanin
P. aeruginosa
Seploria tritici
wheat
42
Pyoluteorin
P.fluorescens
Pythium ultimum
cotton
67
P. fluorescens
Pythium ultimum
cress
84
P. fluorescens
Rhizoctonia solani
cotton
67
P. cepacia
Aphanoinyces cochliodes
sugar beet
64
P. fluorescens
Pyrenophora tritici-repentis
wheat
105
P.fluorescens
Scierotinia homoeocarpa
bluegrass
112
P. fluorescens
Pythium ultimum
sugar beet
131
diacetylphloroglucinol
Pyrroluitrin
Viscosinainide
From Dowling and O'Gara, 1994; Camacho, 2001; Hans and Keel, 2003.
1.2.1. Evidence that antibiosis contributes siviificantly to biocontrol in the soil
environment.
The production of antibiotics by soilborne bacteria has long been postulated
to have evolved as a defense mechanism, similar to allelopathy in plants. That
antibiosis truly plays a significant ecological role in the soil environment has been
supported by multiple lines of evidence, as described below.
1.2.1.1. Inhibition of a pathogen by a pure antibiotic.
In most cases, the establishment of an antibioticts contribution to biological
control originates with evidence that a biologically active constituent is produced in
vitro, has been isolated from a producing bacterium, and has been chemically
characterized. Inhibition by the compound is then established on agar plates or in
rhizosphere assays during which the pathogen is presented with the compound in its
purified form. In this manner, phenazine derivatives produced by P. fluorescens and
P. chioraphis have been shown to suppress Gaeumannomyces graminis var. tritici,
which causes take-all disease of wheat (48), and Fusarium oxysporum f. sp. radicislycopersici, the causal agent of root rots for greenhouse plants such as tomato (22).
Similarly, pure pyrrolnitrin (a compound produced by P. fluorescens and related
bacteria) added to cotton seeds prior to planting inhibited the cotton seedling
pathogens Rhizoctonia solani, Thielaviopsis basicola, Alternaria sp., and Verticillium
dahliae (66). Agar plate assays were used to demonstrate that 2,4diacetyiphioroglucinol, a compound produced by P. fluorescens and P. aureofaciens,
is an important factor in antagonism of T. basicola, which causes black root rot of
tobacco, (70, 71), Pythium ultimum, which causes damping-off of sugar beet seedlings
(71, 86), and the wheat take-all pathogen, G. g. tritici (55, 71). Pyoluteorin, a
compound produced by P. fluorescens, inhibited P. ultimum on agar plates (86) and on
cotton and cress seedlings (67, 84). Bacteria are thought to inhibit plant pathogens by
a number of mechanisms employed simultaneously, as described above. Thus, while
these studies demonstrate that antibiotics can suppress both pathogens and plant
disease, they do not prove a requirement for antibiosis during biocontrol in the
rhizosphere, when other mechanisms may also contribute in varying degrees to
pathogen suppression.
1.2.1.2. Comparison ofbiocontrol efficacy between antibiotic-deficient mutants and
wild type strains
In a number of systems, genetic studies have extended the evidence
described above to support an integral role for antibiosis in biological control.
Pathogen inhibition by strains bearing genes necessary for antibiotic biosynthesis has
been compared with pathogen inhibition by strains deficient in such genes. For
example, a phenazine-1-carboxylate producing P. fluorescens 2-79 suppresses G. g.
var. tritici, while a Tn5 mutant derivative deficient in phenazine-1-carbooxylate
production does not suppress the pathogen (129). P. chioraphis PL1391, which
produces the derivative phenazine- 1 -carboxamide, inhibited F. oxysporum in the
tomato rhizosphere, but a non-producing mutant did not (22). Similarly, Hill et al.
(61) and Pfender et al. (105) demonstrated that disruption of genes necessary for
pyrrolnitrin biosynthesis diminished biological control of R. solani and Pyrenophora
tritici-repentis, respectively, by P. fluorescens strains. Likewise, genes for 2,4diacetylphloroglucinol biosynthesis were required for optimal suppression by P.
fluorescens strains of the pathogens 7'. basicola, P. ultimum, and G. g. var. tritici, and
F. oxysporum (41, 70, 71, 86).
Genetic analysis has also proven useful in determining whether in vitro
observations about pathogen inhibition could be extrapolated to the rhizosphere
environment. P. fluorescens 2-79 inhibited F. oxysporum in agar plate assays, but the
in vitro effect was not observed in the tomato rhizosphere, presumably because strain
2-79 does not produce phenazine-1-carboxamide, the phenazine derivative most
effective at suppression ofF. oxysporum (22). In the case of pyoluteorin, antibioticproducing strains suppressed P. ultimum in agar plate assays (75) and protected cress
7
against P. ultimum while pyoluteorin-deficient mutants did not (75,84). However, the
pyoluteorin-deficient mutants were not compromised in their ability to inhibit P.
ultimum infection on cucumber seedlings (75, 84). Later, it was shown that when P.
fluorescens colonized cucumber seeds, pyoluteorin biosynthesis genes were expressed
late, after Pythium infection was expected to have already occurred(76). Thus, while
the presence or absence of antibiotic genes allows correlation of antibiotic production
with biological control, tracking the nuances of expression of those genes in the
rhizosphere may reveal sources of inconsistencies in the contribution of antibiosis to
biological control.
1.2.1.3. Expression of antibiotic genes in the natural environment.
Expression of antibiotic genes in the rhizosphere is predicted if they indeed
play a role in antagonism of plant pathogens on the root surface. Reporter gene
systems have been used to test this hypothesis in various antibiotic-producing
biological control agents. For example, genes for phenazine (22, 140), 2,4diacetylphloroglucinol (96, 97), and pyoluteorin (16, 76) biosynthesis are expressed in
the rhizosphere. Further, environmental factors such as plant species and fungal
exudates have been shown to modify antibiotic gene expression (e.g. 75, 97).
1.2.1.4. Isolation ofantibiotics from soil.
Because of the nature of soil and the tendency of hydrophobic secondary
metabolites to become immobilized by soil constituents such as clay particles and
humic substances, extraction of antibiotics from soil is a daunting feat. Yet, antibiotic
extractions from the rhizosphere provide the best direct evidence that antibiotics are
produced (and, by extrapolation, function antagonistically) in their natural habitat.
1.2.1.4.1. Phenazine.
Phenazine was extracted from soil surrounding wheat roots inoculated with
P. fluorescens 2-79 and P. aureofaciens 3 0-84, strains suppressive to take-all decline
of wheat. The antibiotic was extracted from natural soil in growth chamber
experiments and from field soil. A positive correlation was observed between
phenazine recovery and suppression of take-all disease in the presence of G. g. tritici
(128). h a separate study, P. putida WCS358r, or derivatives modified to contain the
genes for phenazine biosynthesis, were inoculated onto wheat seeds that were planted
into a field plot for two consecutive years. Soil surrounding roots of mature plants
was extracted and found to contain phenazine, and the authors suggest that varying
amounts of phenazine extracted from each strain might have accounted for different
influences of each strain on the surrounding fungal microflora (44).
1 .2.1 .4.2. 2,4-diacetylphloroA'lucjnoL
The polyketide 2,4-diacetylphloroglucinol has been extracted from
gnotobiotic soil (clay, sand, and quartz powder) in which wheat seedlings inoculated
with P. fluorescens CHAO were grown. Similar amounts of the compound were
recovered in the presence or absence of the take-all pathogen of wheat, G.g. tritici
(71). In later experiments, methods were developed to extract 2,4diacetylphloroglucinol from raw, virgin soils in which wheat plants colonized with P.
fluorescens Q2-87 were grown (14). Finally, it was demonstrated that 2,4diacetylphloroglucinol was produced by naturally occurring fluorescent
pseudomonads colonizing wheat roots in their native, take-all suppressive soils, but
the compound was not produced by the same isolates when they colonized wheat roots
grown in take-all conducive soils (110). Suppressive soils, which naturally develop
resistance to a pathogen after repeated seasons of planting with the same crop, have
been a source of inspiration for biological control upon which many research dollars
and hours have been expended. By extrapolation, this work of Raaijmakers et al.
(110) demonstrated that production of a pathogen-inhibiting antibiotic is one factor
that delineates a suppressive from a non-suppressive soil.
1.2. 1. 4. 3. Pyolu(eorin and pyrrolnilrjn.
Pyotuteorin and 2,4-diacetyiphioroglucinol together have been extracted
from soil surrounding the roots of wheat seedlings inoculated with P. fluorescens
CHAO in a gnotobiotic system. The presence of the damping-off pathogen P. ultimum
appeared to increase levels of both compounds recovered from the wheat rhizosphere,
although the reason for this is unknown. It is hypothesized that pathogen attack of
wheat roots might release nutrients conducive to antibiotic production (85).
Pyrrolnitrin has also been extracted from soil surrounding Pseudomonas-colonized
roots of cotton and sugar beet seedlings, and from barley seeds (130, and references
therein).
1.2.2. Regulation of antibiotic production in Pseudomonas spp.
A complex circuitry governs production of Pseudomonas antibiotics, and
includes factors ranging from sigma factors to noncoding RNAs. Examples are
described below.
1.2.2.1. GacS/GacA.
Two-component regulatory systems are protein pairs in which a histidine
protein kinase located in the cell exterior or the cytoplasm senses signals, and uses a
phosphorylation cascade to relay signal information to a response regulator that
activates transcription from promoters of genes encoding appropriate physiological
responses. The GacS/GacA two-component regulatory system is widespread among
gram-negative bacteria and frequently controls the production of exoproducts. These
exoproducts are often important for interactions with a eukaryotic host; therefore,
10
gacA or gacS mutations result in phenotypes associated with the host such as reduced
biocontrol ability and reduced virulence. Excellent reviews of the GacS/GacA
regulatory cascade and molecular mechanisms are available (50, 56). Of the
Pseudomonas antibiotics important for biocontrol listed in Table 1.1, 2,4diacetyiphioroglucinol, pyrrolnitrin, pyoluteorin, phenazine, and hydrogen cyanide are
known to be under the positive control of GacS/GacA. There is strong evidence that
the expression of GacA-regulated exoproduct genes is post-transcriptional. First,
promoter elements preceding GacA-regulated exoproduct genes that bind GacA have
not been identified (26, 50), demonstrating that control of expression of these genes by
the GacA transcriptional regulator is indirect. Second, in the case of genes for
biosynthesis of HCN in
P. fluorescens
CHAO, GacS/GacA regulation of hcnA
expression does not require the hcnA promoter. Rather, GacA control requires a
region surrounding the hcnA nbosome binding site (11, 13, and 56), implying that the
GacA effect is posttranscriptional. Whether GacA posttranscriptionally regulates all
the exoproduct genes under its jurisdiction remains to be determined.
1.2.2.2. Regulatory RNAs.
Untranslated RNAs have important functions in both eukaryotes (e.g. 28)
and bacteria (e.g. see reference 95). The functionality of these small RNAs has been
gaining so much recognition that databases of protein families have been joined by
databases for non-coding RNA families (e.g. http://rfam.wustl.edu; http://www.imb-
jena.deIRNA.html). In bacteria, untranslated RNAs can perform various functions by
binding to mRNAs (e.g. 4, 78, 79), to phospholipid bilayers (73), and to proteins (e.g.
141). For example, the binding of untranslated RNA to a translational repressor,
RsmA, is part of a regulatory cascade governing exoproduct formation (e.g. 20, 30, 31,
80, and 92) among the Enterobacteriaceae (29) and pseudomonads (12, 104). The
RsmAIRsmB system has been well characterized in Erwinia carotovora subsp.
carotovora. In. E. carotovora, the small protein RsinA inhibits the production of
various extracellular products by stimulating transcript degradation, presumably
11
because RsmA binds exoproduct mRNAs in a destabilizing manner (29). However,
RsmA inhibition is opposed by a small untranslated RNA, RsmB, which entangles
RsmA in an inactive nucleoprotein complex (81).
Homologs of RsmA and RsmB have been found in P.fluorescens; while
RsmA sequences are highly conserved among strains, the nucleotide sequences of the
RsmB homologs are not. However, the function of RsmA binding appears to remain
constant. In P. fluorescens, the RsmB homolog has been called either RsmZ (57) or
PrrB (1). Like their counterparts in Erwinia spp., RsmA and RsmZ control
exoproduct formation. The RsmA homolog is a translational repressor in P.
fluorescens
(12) and RsmZ appears to function by sequestering RsmA or a
homologous protein away from mRNA. This is accomplished by RNA secondary
structures containing multiple stem-loops bearing ribosome-binding site-like
AGG(G)A motifs, which presumably occupy RNA-binding sites on RsmA homologs.
RsmZ expression is under the control of GacA, and although GacA-controlled
exoproduct genes do not require GacA for transcription, the rsmZ gene from P.
fluorescens
CHAQ, which encodes an untranslated regulatory RNA, is dependent on
GacA for transcription. Expression of RsmZ requires an element upstream of the
rsmZ transcriptional start, implying that GacA must exert direct or indirect
transcriptional control over RsmZ expression (57). Another untranslated RNA,
RsmY, has recently been discovered in P. fluorescens CHAO; RsmY displays a similar
secondary structure to RsmZ and is under GacA control (50).
1.2.2.3. Sigma factors.
Sigma factors constitute a pooi of subunits for RNA polymerase that, when
bound to the core polymerase, complete the holoenzyme. The identity of the bound
sigma factor confers a primary level of transcriptional regulation, by directing the
polymerase to bind to a specific subset of promoters in the cell. In P. fluorescens, the
stationary phase and stress response sigma factor RpoS (c?) represses the production
12
of pyoluteorin, 2,4-diacetyiphioroglucinol and HCN, but is required for pyrrolnitrin
production (116, 137). In contrast, the housekeeping sigma factor RpoD (a70)
positively affects pyoluteorin and 2,4-diacetylphloroglucinol production (118). c? is
best described in E. co/i, where it confers transcriptional specificity for a subset of
genes expressed predominantly during stationary phase or during nutrient, osmotic,
oxidative, or other stresses (37, 58, 59, 94, and 126). In Pf-5, & is similarly
implicated in tolerance to osmotic and oxidative stresses (116), and is partially under
the control of the histidine protein kinase/response regulator pair GacS/GacA (137).
1.2.2.4. Pathway-linked regulatory genes.
Often, genes for biosynthesis of secondary metabolites are clustered, and
associated with these clusters are genes for regulation of expression of the same genes.
Among the compounds listed in Table 1.1, pathway-linked regulators have been
especially well characterized for the phenazine and 2,4-diacetylphioroglucinol gene
clusters. The phenazine biosynthetic genes are flanked by phzl and phzR, which
encode LuxIILuxR homologs involved in the synthesis and perception, respectively, of
an AHL signal (108). Although the phenazine biosynthetic pathway is responsive to
this quorum-sensing system, PhzI/PhzR do not appear to influence production of other
secondary metabolites (50) in phenazine-producing strains. The 2,4diacetyiphioroglucinol gene cluster contains two TetR-type transcriptional regulators,
ph/F and phlH. A function for the latter has not been reported, but ph/F has been
characterized.
In P. fluorescens,
Ph1F binds repressively to the ph/A CBD promoter in
the absence of 2,4-diacetyiphioroglucinol, but dissociates from its target sequence in
the presence of 2,4-diacetylphloroglucinol (2). The pyoluteorin gene cluster contains
the linked gene pltR, encoding a LysR-type transcriptional regulator. The pltR gene is
necessary for transcription of genes within the cluster (100). A DNA hybridization
array showed that effects of PItR on gene expression are not limited to the pyoluteorin
biosynthetic genes (Caroline Press, unpublished data), although it is unclear whether
these pleiotropic effects are direct or indirect. Another gene within the pyoluteorin
13
biosynthetic gene cluster, pitH, encodes a member of the TetR transcriptional
regulator family (Chapter 4). No pathway-linked regulator has been found for the
pyrrolnitrin gene cluster, but the sequence of the genome of P. fluorescens Pf-5 may
reveal candidate regulatory genes for this and other previously less-characterized
pathways.
1.2.2.5. Quorum sensing.
Extracellular concentrations of AHLs appear to govern production of
various antibiotics byP. aeruginosa, P.fiuorescens, and P. aureofaciens (e.g. 39, 107,
139, and 140). Although AHLs have not been implicated in production of the
majority of antibiotics listed in Table 1.1, phenazine production is under the control of
quorum sensing in P. aureofaciens and P. chiororaphis (21, 140). Additionally,
AHLs can mediate cross-talk among bacterial populations in the rhizosphere
environment, orchestrating the production of phenazines (107).
1.2.2.6. Autoregulation of antibiotic and secondary metabolite production.
Another layer in the regulation of Pseudomonas secondary metabolites is
autoregulation. For example, 2,4-diacetylphloroglucinol, which is produced by many
pseudomonads (e.g. 72) including Pf-5 and CHAO (71), is positively autoregulated in
CHAO (120; discussed further in Chapter 2). Oomycin A is produced byP.
fluorescens Hv37aR2 and has been shown to suppress Pythium on cotton and other
crops. A lacZ transcriptional fusion to afuE, a gene required for oomycin A
biosynthesis, was highly expressed in a wild type background while little or no
expression could be detected when the fusion was introduced into an oomycin A-
deficient mutant (49, 68). The phenazine operon experiences positive feedback
through PhzJlR autoregulation (51). Other examples of positive autoregulation by
extracellular metabolites in Pseudomonas include those of the siderophores pyochelin
(111) and pyoverdine (77) in P. aeruginosa. In the latter case, binding of extracellular
14
pyoverdine to the ferripyoverdine receptor protein FpvA in the outer membrane
initiates a regulatory cascade resulting in increased production of pyoverdine itself,
and of two other secreted virulence factors, exotoxin A and an endoprotease (PrpL).
1.2.3. Transport of secondary metabolites from cells.
Although many of the compounds described in Table 1.1 are known to be
extracellular, the mode of escape from the cell is known for few. The 2,4diacetyiphioroglucinol gene cluster in P. fluorescens Q2-87 contains phiE, a gene
encoding a putative integral membrane permease (7). The phiE gene resembles sugar
transporters; its role in 2,4-diacetyiphioroglucinol transport has not been reported, but
intact phlE is required for optimal 2,4-diacetylphloroglucinol production (6).
1.2.4. Regulation of pyoluteorin production by P.
biocontrol organism.
P. fluorescens
Pf-5 is a
fluorescens
Pf5g a model
model biocontrol organism. It has been a focus of
study since its isolation from the rhizosphere of cotton seedlings over two decades ago
(66). It produces a suite of secondary metabolites, including HCN, pyrrolnitrin, 2,4diacetyiphioroglucinol, and pyoluteorin, that contribute to biocontrol of
phytopathogens. The associations of pyrrolnitrin and pyoluteorin with disease
suppression represent two of the earliest assignments of biochemical mechanisms to
biocontrol (66, 67). The sequencing of the genome of Pf-5
is
currently nearing
completion at The Institute for Genomic Research (http://www.tigr.org/tdb/ufmgl).
Because Pf-5 shares biochemical traits with other biocontrol pseudomonads, valuable
comparisons can be made and insights gained about their genetic underpinnings. One
such trait is the production of antibiotics, which is especially well characterized in Pf5
and the similar strain P. fluorescens CHAO. The studies described in this
dissertation further our understanding of factors regulating antibiotic production in Pf5, with a specific focus on pyoluteorin.
15
Hydrogen cyanide
HCEN
OH
Pyoluteorin
EEIII;L11JiII>ct
OH
o
H
OH
2,4-diacetyiphloroglucinol
CI
Pyrrolnitrin
Fig. 1.1. Antibiotics produced by P. fluorescens Pf-5 that have been implicated in
biological control.
In Pf-5, pyoluteorin production is affected by external and intracellular
conditions. Susceptibility of pyoluteorin production to environmental influences
contributes to variability in the overall efficacy of biocontrol. Pyoluteorin production
in Pf-5 is affected by nutrient source (76, 99; Brodhagen and Loper, unpublished
data). Pyoluteorin production is also affected by osmotic potential (115), temperature
(98), and cell density (32). The links between extracellular conditions and
intracellular regulatory pathways are not yet well understood. However, a complex
picture is emerging of the intracellular regulatory circuit in Pf-5 that governs
production of pyoluteorin and other secreted products, and links the production of
these exoproducts to the physiological status and stress response of the cell (Fig. 1.1).
For example, the sigma factors RpoS (&) and RpoD
(a70)
influence pyoluteorin
production as described above. The repression of pyoluteorin production when the
stationary phase/stress response/starvation sigma factor as is present implies that the
antibiotic is not made under conditions of limited nutrient supply. Indeed, pyoluteorin
production is low in minimal media when compared to production in complex and rich
16
media (unpublished data). The serine protease Lon, which accumulates during heat
shock and degrades unstable, nonfunctional, or denatured proteins (46), represses
pyoluteorin production. Heat shock induces Lon accumulation in Pf-5. Moreover, the
ion gene is preceded by heat shock sigma factor (ar') recognition sequences, and
like & and
a70,
H
plays a role, albeit indirect, in pyoluteorin biosynthetic gene
expression (138). PtsP, a paralog of Enzyme I of the sugar phosphotransferase
system, also represses pyoluteorin production (136). The GacS/GacA pair positively
regulates production of antibiotics in Pf-5, including pyoluteorin, 2,4-
diacetyiphioroglucinol, pyrrolnitrin, hydrogen cyanide (27, 75). In addition to the
global regulatory proteins that affect transcription of pyoluteorin biosynthetic genes,
more localized regulatory elements also exist. A cluster of ten genes is necessary for
pyoluteorin biosynthesis, and transcriptional activity from all promoters within the
cluster requires the linked gene pltR, as described above. In the biochemically similar
strain P. fluorescens CHAO, pyoluteorin is also produced and is subject to regulation
by additional intracellular and extracellular factors. These factors include negative
regulation by pyrrolquinoline quinone (119), a cofactor necessary for activity of
glucose and alcohol dehydrogenases, and both positive and negative influences on
pyoluteorin production by a number of mineral salts (34). These regulatory
mechanisms are thought to function similarly in Pf-5, although that assumption has
not been verified.
The regulatory mechanisms described above could arguably provide
mechanisms to Pf-5 for "considering" the physiological status of the cell for regulation
of pyoluteorin production. Thus, in a broad sense, pyoluteorin, and perhaps other
antibiotics, may be thought of as carrying information about the physiological status of
a cell to the outside environment, and perhaps ultimately, to neighboring cells.
17
Starvation
Unknown signal
jr
Heat shock
I
Carbon
Source
Temperature
Fig. 1.2. Factors known to influence pyoluteorin biosynthetic gene transcription
in P. fluorescens
Pf-5.
18
1.2.5. Biosynthesis of pyoluteorin.
A cluster of genes required for pyoluteorin biosynthesis has been identified,
and gene functions have been predicted by sequence analysis, although these
predictions remain to be verified biochemically (100).
1.2.5.1. Organization ofgenes within the pyoluteorin biosynthetic gene cluster.
The pyoluteorin biosynthetic genes (Fig. 1.3) are contained on at least three
separate operons. The divergent transcription of pltL and pltR implies the existence of
separate promoters between these genes. There is evidence thatpltR andp/tMdo not
form a single operon, as transcription of pltM is not correlated with that of pltR
(Chapter 4). At least two promoters drive transcription of the p1tLABCDEFG gene
cluster. One of these has been shown experimentally to reside upstream of pltB (76),
presumably in the pltR-pltL intergenic region; another has been shown, via activation
in trans of a promoterless reporter gene, to precede eitherpltD orpltE (76).
1.2.5.2. Predicted functions ofgenes within the pyoluteorin biosynthetic gene
cluster.
The predicted functions of eight structural genes in the pyoluteonn
biosynthetic gene cluster are sufficient to account for pyoluteorin biosynthesis (100).
Briefly, pltB and pltC encode type I polyketide synthases (PKSs) and are predicted to
be involved in synthesis of the resorcinol (phenolic) moiety of pyoluteorin. The
precursor for the growing polyketide chain is likely to be proline, and the acyl-CoA
synthetase P1tF is predicted to activate this molecule. Neither PltB nor PItC contain a
thioesterase domain, which is normally necessary for cleavage of the growing
polyketide chain from the PKS. However, pltG is predicted to encode this function.
The pyrrole (proline-derived) moiety of pyoluteorin is activated by PltF as described
above, and modified by PItE. The pilE gene encodes an acyl-CoA dehydrogenase
19
thought to perform oxidation of the pyrrole ring prior to its incorporation as a
precursor for the polyketide ring. By analogy with homologous gene products, PltE
activity probably results in double bond formation adjacent to the ketone linkage of
the final pyoluteorin product. The pyrrole ring in pyoluteorin is aromatic; it is thought
that the second oxidation may occur spontaneously.
Finally, pitA, pltD, and pltM
encode halogenase enzymes. Because pyoluteorin exhibits only two chlorination sites,
the existence of three chlorinating enzymes is enigmatic. However, PltD lacks a
predicted NADH cofactor-binding site, and may therefore lack catalytic activity.
Nevertheless, a mutation inpltD resulted in loss of pyoluteorin production. A role for
pitL has not been predicted.
20
a
jItB/G
H
HQJ7 !!!*
FItB/C/
PItE
.
OH
OH
çç_ç
HO 0
PItAITj
H
a
(&X0
HO
0
H
Pyoiuteorb
çc
OH
HO 0 H
p1 tM
pltL
1
p1W
1kb
Gene
pliM
pliR
pilL
p/IA
puB
p/IC
p/ID
pilE
p/IF
puG
Putative protein function
halogenase
transcriptional regulator
unknown
halogenase
polyketide synthase
polyketide synthase
halogenase
acyl-00A dehydrogenase
adenylating enzyme
thioesterase
Fig. 1.3. Proposed pyoluteorin biosynthetic pathway. From Nowak-Thompson et
al. (100).
21
1.3. Secondary metabolites as signal molecules.
Primary metabolites are, by definition, necessary for survival of the
producing cells and therefore understood to play vital roles in the cell's physiology. In
many cases, these roles are quite well studied; it is also becoming clear that many
metabolites are not limited to a single role within the cell but play parts in various
reactions. On the other hand, secondary metabolites do not appear to be necessary for
survival to bacterial cells, and the impetus behind the production of these energetically
expensive and frequently toxic molecules remains, in many instances, enigmatic. As
described above, the role of antibiotic has been assigned to certain secondary
metabolites based on their demonstrated ability to suppress the growth of organisms
that might potentially co-inhabit their environment, and compete for niches or
nutrients. Indeed, phenazine antibiotics have been shown to contribute to ecological
fitness of Pf-5 in natural soils, but not in pasteurized soils in which resident microfiora
have already been destroyed (87). However, not all Pseudomonas antibiotics have
been shown to contribute to rhizosphere competence. A short term (12 day)
experiment suggested a lack of a role for oomycin A in competence of P. fluorescens
in cotton rhizospheres (68), but a longer colonization period may be necessary to see
differences, as demonstrated for phenazine (87). Rhizosphere populations of wild type
and 2,4-diacetylphloroglucinol-deficient strains of P. fluorescens Fl 13 remained
similar in natural soils containing sugarbeets over a period of 270 days (18). Thus, a
contribution to ecological fitness does not explain the selection pressure to retain 2,4-
diacetylphioroglucinol biosynthesis genes. Still, these genes are highly conserved
among Pseudomonas spp. collected from locations all around the world (72; L. de la
Fuente, personal communication).
Are there additional roles for antibiotics beside that of competition with
rhizosphere neighbors? Many other roles for secondary metabolites have been
suggested. Secondary metabolites have been postulated to serve as metal transporters,
22
metabolic waste or detoxification products, byproducts of primary metabolism, ancient
or extant effectors of catalytic RNA reactions, reactants with cellular macromolecules
(e.g. DNA and cell wall components), and providers of a pool of "biochemical
diversity" for the cell (to name a few). Secondary metabolites have also been
suggested to serve as signal molecules. Signaling roles are expected to include those
essential to eukaryote-bacterium interactions, as well as those involved in
communication between neighboring bacterial cells (19).
1.3.1. Role of secondary metabolites in Pseudomonas spp.
Based on the discussion above, it is possible that metabolites such as those
listed in Table 1.1 may have roles additional to that of extracellular toxins that
contribute to the overall physiology of the bacteria. It is apparent that production of
these compounds is highly regulated (e.g. see discussion above) and dependent on
specific growth phases, stress response, and metabolic and physiological status of the
cell. Moreover, at least two antibiotics, 2,4-diacetyiphioroglucinol and pyoluteorin,
affect the metabolism of the cell when applied exogenously (16, 120). Thus, I propose
a second role for secondary metabolites of pseudomonads. I hypothesize that some, or
possibly all, secondary metabolites of Pseudomonas spp. may function as signal
molecules. In this way, production of secondary metabolites would represent not
"dead ends" of the branches of regulatory cascades that govern their production, but
rather, intermediate points in cellular regulatory processes at which a new signal
molecule (the metabolite) is generated. According to this hypothesis, the generation
of a secondary metabolite would represent a node in the cell's regulatory circuitry,
and, much like the phosphorylation of a regulatory protein, could be expected to
generate multiple phenotypic changes.
Hypothesis: Secondary metabolites of Pseudomonas spp. that function as
extracellular toxins and are important for biological control may additionally function
as signal molecules.
23
1.3.2. ExiStjn2 support for the hypothesis that secondary inetabolites function as
siEnal molecules in Pseudomonas spp.
One well-documented example of secondary metabolites that act as signal
molecules in pseudomonads as well as other gram-negative bacteria is that of N-acyl
homoserine lactones (AHLs). These diffusible molecules mediate cell-densitydependent regulation (quorum sensing). Their re-uptake across cell membranes
presumably serves as an indicator of either cell density or confinement, and initiates
upregulation of their own biosynthetic genes as well as others in regulatory cascades,
resulting in profound changes in the cellular phenotype. For example, two AHL
systems, las and rhi, are together estimated to govern expression of 1-4% of all genes
in P. aeruginosa (139). However, some strains, like P. fluorescens Pf-5 and P.
fluorescens CHAO, produce no detectable AHLs (57; J. E. Loper, unpublished data).
It is unlikely that these rhizosphere bacteria lack extracellular signaling molecules.
Rather, it is more likely that AHLs are only one class of signal molecule of which
microbiologists have become acutely aware in the past two decades; the possibility
that others exist is quite logical. Indeed, secondary metabolites of other structural
classes have been shown to function as signal molecules in Pseudomonas. For
instance, a quinolone antibiotic from P. aeruginosa, PQS, was found to be an integral
part of the las/rhi regulatory network, which governs the production of virulence
factors and secondary metabolites (88, 103). Pyoverdine, a siderophore that chelates
ferric iron and returns it to the cell, was shown to have signaling properties with
repercussions for the synthesis of three secreted virulence factors, including itself, in
P. aeruginosa (8, 77). N-acyl homoserine lactones typically bind intracellular
receptors from the LuxR family; recently, it was found that a family of cyclic
dipeptides called diketopiperazines, or DKPs, from P. aeruginosa can induce LuxR-
regulated functions. This observation led to the hypothesis that diketopiperazines
function as signal molecules, and serve as modulators of quorum sensing pathways
(63). Similarly active diketopiperazines were isolated from other gram-negative
bacteria as well (63), and at least one of the active DKPs is also produced by the
fungal pathogen Alternaria alternata, and confers phytotoxicity to spotted knapweed
24
(Centaurea maculosa) (125). This latter observation invites the tempting speculation
that diketopiperazines could play a role in Pseudomonas-plant interactions, although
this proposal has not been experimentally tested.
1.3.3. Si2nalin properties of antibiotics in non-Pseudomonas species.
In non-Pseudomonas systems, other examples of secondary metabolites
with signaling properties also exist. An exhaustive listing of these is beyond the scope
of this chapter. However, it is worth noting that subinhibitory concentrations of
antibiotics commonly used in medicinal practice can induce non-lethal physiological
responses in their targets. Recently, it was shown that exposure to subinhibitory
concentrations of erythromycin and rifampicin resulted in altered transcription of as
many as 5% of genes in Salmonella typhimurium (45). Subinhibitory concentrations
of antibiotics have been observed to have non-toxic effects on other bacterial species
as well: clindamycin affects exoproduct expression in Staphylococcus aureus (60),
and quinolone antibiotics affect flmbrial expression and adherence in Escherichia coli
(15). Low concentrations of quinolone antibiotics also block intercellular signaling
and the production of virulence factors in P. aeruginosa (47, 127, and 132). These
observations lend credence to the ofi-touted view that antibiotics and other microbial
toxins may have intracellular significance in addition to their extracellular roles as
toxins. It seems intuitive to question the effect of bacterially produced antibiotics on
the cells that produce them. For example, erythromycin, which affected Salmonella
gene expression, is produced by Streptomyces erythreus. Tetracycline, streptomycin,
cycloheximide, neomycin, and kanamycin are other familiar antibiotics produced by
Streptomyces spp., while polymyxin B and bacitracin are produced by Bacillus spp.
However, I am unaware of studies that explore the effect of these antibiotics on their
producing organisms.
1.3.4. Re2ulation of antibiotic production by other secondary metabolites.
Production of Pseuc/omonas antibiotics is itself modulated by secondary
metabolites from both producing cells, and from other organisms. AHLs are a wellstudied example of such metabolite - metabolite regulation, and are discussed in a
separate section. However, other classes of secondary metabolites regulate antibiotic
production as well. 2,4-diacetylphioroglucinol production in P. fluorescens CHAO is
repressed by at least three secondary metabolites: salicylic acid, fusaric acid, and
pyoluteorin (35, 97, and 120). Salicylic acid and pyoluteorin are produced by CHAO,
but fusaric acid is secreted by the pathogenic fungus F. oxysporum, a potential
rhizosphere coinhabitant. Moreover, salicylic acid is produced by many organisms,
including plants, where it serves as a signaling molecule and is integral in mounting
induced systemic resistance against pathogens. "Foreign" secondary metabolite
influences do not appear to be unique. For example, production of the secondary
metabolite syringomycin, a phytotoxin produced by P. syringae pv. syringae, is
induced by various phenolic compounds produced by the host plant (90, 91, and 109).
1.4. Statement of research objectives.
As described above, the genes responsible for biosynthesis of pyoluteorin
have been sequenced, and several intra- and extracellular factors affecting its
production levels have been elucidated. This knowledge distinguishes pyoluteorin
production in Pf-5 as an enviable model system for the study of both intracellular and
environmental effects on regulation of antibiotic biosynthesis. I employed Pf-5 to test
the hypothesis, stated above, of signaling roles for Pseudomonas antibiotics, with
specific regard to pyoluteorin. Pyoluteorin, pyrrolnitrin, and 2,4diacetylphioroglucinol share facets of global regulation in Pf-5. Therefore, possible
shared or interrelated signaling roles for pyoluteorin, pyrrolnitrin and 2,4-
diacetylphlorglucinol were also explored. A recurring theme in antibiotic regulation
by biocontrol pseudomonads is autoregulation, as described above (51). Thus, the first
26
test for a signaling role for pyoluteorin was one to determine the influence of
exogenous pyoluteorin on its own production. Early results indicated that pyoluteorin,
like 2,4-diacetylphloroglucinol, was positively autoregulated, and my larger goal
became the characterization of the pyoluteorin autoinduction phenomenon. I also
dissected the involvement of pyrrolnitrin and 2,4-diacetyiphioroglucinol in pyoluteorin
production, and vice versa.
The phenomenon of pyoluteorin autoinduction implies that exogenous
pyoluteorin is either i) perceived at the cell surface, or ii) transported from the
extracellular milieu back into the cytoplasm, where it is perceived by an intracellular
pyoluteorin receptor. Pathway-linked LysR- and TetR-type transcriptional regulators
are often activated by cofactor binding, and cofactors frequently are, in turn, products
of the biosynthetic pathway under regulation (e.g. 62, 115). By regulating
transcription from their own promoters, thus enhancing or repressing cellular levels of
the cofactor receptor, LysR and TetR-type regulators can in some instances facilitate
positive or negative autoregulation by products of the pathways that they regulate.
Two such linked regulatory genes, pltR and pitH, occur in the pyoluteorin pathway
and encode a LysR- and TetR-type regulator, respectively. In the related stain CHAO,
autoinduction of 2,4-diacetylphloroglucinol is mediated by the pathway-linked TetR-
type transcriptional regulator, PhlF. These observations added appeal to the
hypothesis of an intracellular pyoluteorin receptor. Because an intracellular
pyoluteorin receptor would necessitate the existence of a pyoluteorin uptake
mechanism, a search was initiated for potential pyoluteorin transporter genes. Genes
encoding enzymatic functions for biosynthesis of secondary metabolites, like
pyoluteorin, are often flanked by genes for immunity to, or transport of, the metabolite
(reviewed by 82). Examples of such clustering are given in Chapter 4. Therefore, the
pyoluteorin biosynthetic gene cluster was further delineated. The early discovery of
both regulatory and transport genes on one end of the antibiotic gene cluster prompted
a shift in focus from sequencing to characterization of those genes. In light of its
potential as a pyoluteorin receptor, attempts were also made to biochemically
characterize the pathway-linked regulator PltR.
27
The results presented herein do not completely delineate the pyoluteorin
biosynthetic pathway, nor do they provide a comprehensive understanding of the
intracellular roles for Pf-5 antibiotics. However, they lay solid groundwork for fuzther
studies which are already ongoing to better understand the function of pyoluteorinregulated transport genes that flank the pyoluteorin biosynthetic gene cluster, and to
elucidate on a more global scale the physiological responses of Pf-5 to its antibiotic,
pyoluteorin.
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43
Chapter 2
Positive Autoregulation of the Antibiotic Pyoluteorin in the Biological Control
Organism Pseudomonasfluorescens Pf-5
Marion Brodhagen, Marcella D. Henkels, and Joyce E. Loper
Submitted to
Applied and Environmental Microbiology
American Society for Microbiology
2.1. Abstract
Pseudomonasfluorescens Pf-5, a rhizosphere bacterium, produces a suite of
secondary metabolites that are toxic to seed- and root-rotting plant patbogens. Among
these are the polyketide compounds pyoluteorin (PLT) and 2,4-diacetyiphioroglucinol
(2,4-DAPG). We provide evidence that PLT production is influenced by positive
autoregulation. Addition of PLT to liquid cultures of Pf-5 enhanced PLT production.
In addition, PLT and 2,4-DAPG mutually inhibit one another's production in Pf-5. For
PLT, both positive autoregulation and negative influences on production by 2,4DAPG were demonstrated at the transcriptional level, by measuring activity from
transcriptional fusions of an ice nucleation reporter gene to three separate PLT
biosynthetic genes. The occurrence of PLT autoregulation in the rhizosphere was
assessed on cucumber seedlings in pasteurized soil, using cross-feeding experiments.
In the rhizosphere, a Plt derivative of Pf-5 carrying a reporter gene fusion to a PLT
biosynthetic gene responded positively to PLT produced by coinoculated cells of Pf-5
or a derivative. These data establish for the first time that the polyketide PLT is an
autoregulatory compound and functions as a signal molecule influencing the spectrum
of secondary metabolites produced by the bacterial cell.
2.2 Introduction
Microbial biological control agents such as the rhizosphere bacterium
Pseudomonasfluorescens Pf-5 represent alternatives to synthetic chemicals for
combating plant disease in agriculture. An important aspect of plant disease
suppression by rhizosphere bacteria is the production of low molecular weight
metabolites with antibiotic properties against certain plant pathogens (reviewed in 8,
12, 17, 18, 36). P. fluorescens Pf-5 produces an array of secondary metabolites that
inhibit plant pathogens, including pyoluteorin (PLT), pyrrolnitrin, 2,4-
diacetyiphioroglucinol (2,4-DAPG) and hydrogen cyanide (23,24, 26, 29, 43, 47).
PLT and 2,4-DAPG (Fig. 2.1) are synthesized by polyketide synthase complexes (4,
42, 44) and are secreted from cells of Pf-5. The focus of this research is PLT, a
chlorinated polyketide compound (3) that suppresses the Oomycete Pythium ultimum,
a seed- and root-rotting pathogen (24, 37). PLT production is conferred by a
biosynthetic gene cluster encompassing nine structural genes whose predicted
functions are sufficient for the biochemical transformations required for PLT
biosynthesis from acetate and proline precursors (11, 42, 44). Also within the PLT
biosynthetic gene cluster is pltR, which encodes a LysR transcriptional regulator and is
required for transcription of PLT biosynthesis genes and resultant PLT production
(42). PLT production by Pf-5 is affected by nutrient source (30, 43), temperature (41),
and cell density (11). Although the links between these extracellular factors and the
intracellular regulatory pathways controlling PLT production are not yet understood, a
complex picture is emerging that links regulation of production of PLT and other
exoproducts to the physiological status of the cell.
46
0
0
ççrc'
OH
a
Figure 2.1. Structures of pyoluteorin (a) and 2,4-diacetylphioroglucinol (b).
Several global regulatory genes control PLT biosynthesis in Pf-5 and in P.
fluorescens CHAO, a biochemically similar strain in which regulation of PLT
production also has been extensively characterized. The production of many
antibiotics and exoproducts by Pseudomonas spp. and other Gram-negative bacteria
requires the histidine protein kinase/response regulator pair GacS/GacA (e.g. see 21);
the signal(s) that activates GacS is not known. In Pf-5, the GacS/GacA pair positively
regulates production of several secreted products, including PLT, 2,4-DAPG,
pyrrolnitrin, hydrogen cyanide and an extracellular protease (10, 29). At least two
common components of stress response systems, the stationary-phase and stressresponse sigma factor RpoS (aS) and Lon protease, are implicated in regulation of
antibiotic production in Pf-5. Relative levels of as and the housekeeping sigma factor
RpoD (&) influence PLT, 2,4-DAPG, and pyrrolnitrin production. Abundant as is
required for the production of pyrrolnitrin, but is repressive to PLT and 2,4-DAPG
production (50, 52). Lon, a serine protease induced by heat shock, also is repressive to
PLT production (58). The importance of nutrient status to PLT production is
corroborated by the observation that pyrrolquinoline quinone, a cofactor required by
glucose and alcohol dehydrogenases, represses PLT production (53). Thus, PLT
production in Pf-5 is linked to the physiological status of the cell.
In this manuscript, we present evidence that the regulatory circuit governing
PLT production includes positive autoregulation. The most familiar example of
positive autoregulation is that of the N-acyl homoserine lactones (AHLs). In Gramnegative bacteria, AULs are synthesized and perceived, respectively, by Luxl/LuxR
47
homologs, and are postulated to function as signals of cell density and/or confinement.
AHL production, along with that of other cellular products, is upregulated as a
consequence of AHL accumulation in the extracellular milieu and re-entry into
producer cells. AHLs have been demonstrated to function as intercellular signal
molecules in the rhizosphere, where their production by rhizosphere inhabitants can
induce phenazine antibiotic production by Pseudomonas auerofaciens (48, 59). NonAHL extracellular metabolites produced by Pseudomonas spp. also undergo positive
autoregulation, including the antibiotics 2,4-DAPG (54) and oomycin A (16,25), and
the siderophores pyochelin (49) and pyoverdine (31). This report adds PLT to the
growing list of non-AHL autoinducers known in Pseudomonas spp. Because Pf-5
produces several antibiotics, we investigated the potential for regulation of PLT
production by a heterologous antibiotic. We demonstrate that PLT production and the
transcription of PLT biosynthesis genes are influenced by the presence of another
antibiotic produced by Pf-5, 2,4-DAPG. Finally, we present evidence that PLT
produced by Pf-5 can induce the expression of PLT biosynthesis genes by neighboring
bacterial cells in the rhizosphere.
2.3. Methods
2.3.1. Bacterial strains plasmids, and culture conditions.
Bacterial strains and plasmids used in this study are listed in Table 2.1. Pf-5
and derivative strains were cultured routinely on King's Medium B agar (KMB; 28)
containing streptomycin (100 pg/ml). KMB was supplemented with other antibiotics
(kanamycin, 50 jig/mI; gentamycin, 40 j.tg/ml) when appropriate. Inoculum of Pf-5
and derivatives was grown overnight with shaking (200 rpm) at 27°C in either KMB
broth or KMBGIu, modified from KMB broth by substituting the glycerol with 1%
(w/v) glucose to repress PLT production. For experiments evaluating antibiotic
production or PLT biosynthetic gene transcription, cultures were grown at 20°C with
shaking (200 rpm) in nutrient broth (pH 6.8; Difco Laboratories, Detroit, MI) without
48
selection and supplemented with either 2% (w/v) glycerol for NBG1y, a medium
conducive to PLT production, or with 2% (w/v) glucose for NBGIu, a medium
repressive to PLT production by Pf-5 (30, 43). For experiments evaluating antibiotic
production or PLT biosynthetic gene transcription in defined medium, bacterial
cultures were grown in OS medium (45) amended with 0.5% (w/v) glycerol (OSG1y)
at 20°C with shaking (200 rpm).
Table 2.1: Bacterial strains and plasmids used in this study
Strain
Strain
Number
Relevant
JL4474
Pseudomonasfluorescens Pf-5
Reference
or Source
characteristicsa
P. fluorescens
Pf-5
23
rhizosphere isolate; Pitt, Ina
Pf-5 iceC
JL4303
Pf-5 pltE::inaZ
JL4365
Pf-5 (pJELI 703); Kmr Plt, Ina
34
pltE::Tn3 -nice derivative of Pf-5, P1t,
30
Ina, Kmr
Pf-5pItB::inaZ
JL4389
pItB::Tn3-n ice derivative of Pf-5,
P1t,
30
Ina, Kmr
Pf-5pltF::inaZ
JL4390
pltF::Tn3 -nice derivative of Pf-5, PIf,
Ina, KThr
30
Pf-5 rpoS::lacZ
JL4489
rpoS::lacZ derivative of Pf-5, Plt, Ina
57
Pf-5 gacA:.aphl
JL4577
gacA ::aphl derivative of Pf-5; Pif, Ina,
9
Pf-5 pltR::aacCl JL4563
pltR::aacCl derivative of Pf-5; Pif, Ina
42
GmT
Plasmids
pJELI 703
iceC from
P. syringae cloned into
pVSP61 (nucleotide sequences of iceC
and inaZ are nearly identical)
34
pVSP61
Stable plasmid vector containing pVS1
replicon and mobilization region and
p1 5A replicon; Mob,
W. Tucker,
DNA Plant
Technolog
KThr
yCorp.,
Oakland,
Calif.
aplt+ produces pyoluteorin; Plf, does not produce detectable pyoluteorin; P1t,
overproduces pyoluteorin relative to the parental strain; 1na, expresses ice nucleation
activity at -5°C; In&, does not express ice nucleation activity at -5°C; KmT and GmT,
resistant to kanamycin and gentamycin, respectively.
50
2.3.2. Influence of exogenous antibiotics on antibiotic production and PLT gene
expression by Pf-5.
Cultures of experimental strains were grown overnight in KBGIu, and cells
were harvested by centrifugation (2,500 x g, 5 mm), washed twice in sterile KMBGIu
to eliminate traces of exported metabolites, and resuspended in 5 ml of NBG1y to
achieve an OD6 of 0.08. Purified PLT and synthetic 2,4-DAPG standards dissolved
in methanol were added to NBG1y immediately prior to inoculation with Pf-5 or
derivative strains. An equivalent volume of methanol was added to negative controls.
Except where noted, antibiotics were added to the medium at 4
jig/mi
PLT and 12.5
,.tg/ml 2,4-DAPG. These represent the low end of a range of concentrations typically
detected in supernatants of Pf-5 cultures grown under conditions optimizing antibiotic
production (10, 43, 57), and were selected to mimic extracellular antibiotic
concentrations that Pf-5 cells would encounter in a stationary-phase culture.
For experiments assessing transcriptional activity of PLT genes using the inaZ
reporter gene, samples were harvested periodically for 24 h and assessed for ice
nucleation activity (NA) as described below. At 18 Ii after inoculation, both 2,4DAPG and PLT were present at measurable concentrations in the supernatant of Pf-5
cultures grown in NBG1y. Therefore, for all experiments that were not harvested at
multiple timepoints, PLT and 2,4-DAPG concentrations were determined from
cultures harvested 18 h after inoculation. Bacterial population densities were
determined by spreading serial dilutions of cultures onto KMB and counting CFU on
the plates after 48 h incubation at 27°C. In each experiment, triplicate 5 ml cultures
were grown and evaluated; each experiment was performed at least twice; and the
results from a representative experiment are presented.
51
2.33. Antibiotic quantification.
Cultures were centrifuged (5,000 x g, 5 mm) to pellet cells, and supernatants
were extracted for recovery of PLT and 2,4-DAPG. Extractions were performed by a
modification of the methods of Sarniguet et al. (50). Culture supernatants were
acidified to pH
2.0 with 1 M HC1 and extracted twice with 0.4 volume of ethyl
acetate. Ethyl acetate was removed from extracts under vacuum, and the resultant
residues were resuspended in 100 p.! methanol, from which 10 p.! samples were
evaluated by high performance liquid chromatography (HPLC). Samples were
separated over a Nova-Pak C18 reversed-phase column (Waters Corporation, Milford,
MA) eluted (1 mI/mm) with 90% water + 0.1% acetic acid:10% acetonitrile + 0.1%
acetic acid (v/v) for two mm, followed by a linear gradient to 100% acetonitrile +
0.1% acetic acid over 18 mm. Antibiotics were detected with a photodiode array
detector (PLT, X = 310 nm and retention time (tr) = 13.1 mm; 2,4-DAPQ,A = 270 nm
and tr =15.6 miii). Quantification was done by comparison to a standard curve
generated from authentic compounds. The detection limit was 0.02 jig/mI for PLT
and 0.01 p.g/ml for 2,4-DAPG. Recovery of compounds using the extraction
procedure described above averaged 70% for PLT and 45% for 2,4-DAPG.
2.3.4. Transcriptional activity from PLT biosynthetic genes
Transcriptional fusions of a promoterless inaZ gene to three PLT biosynthetic
genes of Pf-5 were constructed previously (30). Transcription of the inaZ reporter
gene from an upstream promoter results in expression of the membrane-bound InaZ
protein, which forms aggregates that nucleate water molecules to form ice crystals at
temperatures slightly below 0°C (32). Expression of INA from inaZ transcriptional
fusions was quantified using a droplet freezing assay at -5°C as described previously
(35) and reported as logio(JNA), where INA = ice nuclei/CFU.
52
2.3.5. Assessment of PLT cross-feeding on roots
Assays were performed in pasteurized Newberg fine sandy loam, wetted 24b
in advance of planting to water holding capacity. Cucumber seeds (Cucumis sativis L.
cv. Marketmore 86) were surface disinfested by immersion in 95% ethanol for 1 mm,
followed by a 20% (v/v) sodium hypochiorite solution for 15 mm. Seeds were rinsed
thoroughly in sterile distilled water and placed on sterile, wetted filter paper within
Petri plates to germinate, and incubated at 27°C in darkness for two days before
seedlings were removed and inoculated with bacterial suspensions. Inoculum of
inducing strains Pf-5, Pf-5 pltR::aacCl, and Pf-5 rpoS::lacZ was produced in KMB
broth to encourage PLT production. Inoculum of reporter strain Pf-5 pltB: :inaZ was
produced in KMBG1u to discourage pltB induction. Cultures were grown with
shaking (200 rpm) overnight at 27°C. Cells were then harvested by centrifugation
(2,500 x g, 5 mm) and resuspended in 20 mM potassium phosphate buffer (pH 7.0).
Suspensions of three inducing strains, Pf-5, Pf-5 pltR::aacC] , and Pf-5 rpoS::lacZ
were adjusted to an OD6 of 1.0 while a suspension of Pf-5 pltB::inaZ was adjusted to
an 0D600 of 0.1. The suspension of reporter strain Pf-5 pltB::inaZ was mixed 1:1 with
suspensions of each of the other three strains, providing three inoculum mixtures in
which the reporter and inducing strains were present at final cell density ratios of 1:10,
respectively. Cucumber seedlings were inoculated by soaking them in the bacterial
suspensions for 10-20 mm. Inoculated seedlings were planted individually into wells
of plastic punch trays (35 mm diameter; 25 ml of soil per well). Treatments within
trays were arranged by a complete randomized design. Punch trays were placed in
closed plastic containers, and incubated in darkness at 20°C and 100% humidity. At 0,
10, 24, and 48 h, seedlings were retrieved from ten replicate wells, placed in culture
tubes containing 20 mM potassium phosphate buffer (pH 7.0), and sonicated for 5 mm
in a bath-style sonicator, to remove bacterial cells from roots of the seedlings. Serial
ten-fold dilutions were made in 20 mM potassium phosphate buffer (pH 7.0) for
determination of INA and CFU. Total CFU per sample were determined by spreading
diluted samples onto KMB agar containing streptomycin, while CFU of the reporter
53
strain Pf-5 pltB::inaZ were enumerated separately on KMB agar amended with
streptomycin and kanamycin.
2.3.6. Data analysis
Treatment means were compared using a Student's t-test following an analysis
of variance. Two-tailed P values are reported. Statistical analyses were performed
using JMP, version 3 (SAS Institute, Cary, NC).
2.4. Results
2.4.1. PLT production is autoreuIated
To evaluate the possibility that PLT induces its own production in Pf-5, PLT
was added to NBG1y prior to inoculation with either Pf-5 pltB.:inaZ or Pf-5 iceC. In
preliminary experiments, Pf-5 and Pf-5 iceC were similar in production of PLT and
2,4-DAPG (data not shown), so Pf-5 iceC' filled a dual role as an experimental Plt
strain for evaluation of antibiotic production and a positive control for NA. Similarly,
Pf-5 pltB: :inaZ provided a negative control for PLT production while serving as a
reporter of PLT biosynthetic gene transcription. The concentrations of PLT in PLTamended cultures of Pf-5 pltB::inaZ, a Plt strain, initially reflected the amount
detectable in supernatant extracts as a result of PLT amendment, but PLT
concentrations declined over time in these cultures, presumably because the compound
was degraded (Fig. 2.2a). Amendment of NBG1y with PLT resulted in earlier,
enhanced PLT production by Pf-5 iceC' (Fig 2.2a). At 48 h, PLT was detectable in
both non-amended and PLT-amended cultures of Pf-5 iceC, but PLT concentrations
were 4.5 times greater in amended cultures of Pf-5 iceC than in non-amended
cultures, after subtracting from the amount of PLT detected in amended Pf-5 iceC
cultures the amount detected in amended cultures of Pf-5 pltB::inaZ. As expected, no
PLT was detected in Pf-5 pltB::inaZ cultures grown in non-amended medium.
54
Although some variation occurred in the timing and levels of PLT production across
multiple time course experiments that we performed, PLT amendments at 4 jig/mI to
initial culture medium always hastened PLT production by Pf-5 or Pf-5 icecF, and
enhanced it between 4.5- and 20-fold by late stationary phase.
55
10
a.
14
12
0
10
.
E
0
10
0
8
3
6
0
0
0
I.
0
.0
0.
Ce
Ce
.
2
'0
1
0
11
10
U
9
0
8
p Pf-Splt&:inaZ
-- Pf-5plt&:inaZ+PLT
7
6
12
18
24
30
36
42
48
Time (hours)
Figure 2.2. Influence of exogenous PLT on PLT and 2,4-DAPG production by
derivatives of Pf-5. Cultures were grown in nonamended NBGIy or in NBG1y
amended with 4 .Lg/ml PLT. At the indicated sampling times following inoculation,
PLT concentrations (a), 2,4-DAPG concentrations (b), and population densities of
cultures (c) were assessed. Population sizes did not differ significantly between PLTamended and nonamended treatments at any time point. All three panels represent
data from the same experiment. Error bars denote one standard error.
56
To estimate the minimum concentration of PLT needed to induce PLT
autoregulation, Pf-5 was grown in NBG1y amended with PLT at concentrations
ranging from 0.004 to 4,000 ng/ml (Fig. 2.3a). Amendment of NBGIy with PLT at
concentrations greater than or equal to 0.4 ng/ml (1.47 nM) enhanced PLT production
by Pf-5 (P = 0.002), with PLT production increasing with increasing concentrations of
PLT up to 400 ngfml in the initial growth medium.
Addition of 4 j.tg!mI PLT to the growth medium did not significantly alter the
population sizes of Pf-5 iceC or Pf-5 pltB::inaZ (all P> 0.05; Fig. 2.2c); lower
concentrations similarly had no effect on bacterial populations (P
0.53; Fig. 2.3b),
indicating that the influence of exogenous PLT on PLT production was not achieved
through indirect effects on bacterial growth.
57
16
14
a.
.
e
PLT in Pf-5 due to biosynthesis
'
d
0 Pf-5 pltB::inaZ
12
......
10
C
8
-=0
6
4
2
0
10x10
b.
8x10'
6x109-
4x10'
2x109
0
0.004
0.04
0.4
4
40
400
4000
ng/mI PLT initially present in medium
Figure 2.3. Minimum effective concentration of PLT for autoinduction in Pf-5. PLT
concentrations (a) and CFU (b) were measured in Pf-5 and Pf-5 pltB::inaZ cultured
for 1 8h in NBGIy that was amended with the concentrations of PLT indicated on the
abscissa. Black symbols connected by a solid line represent actual concentrations of
PLT recovered from Pf-5 and black symbols connected by a dotted line represent PLT
that resulted from biosynthesis by Pf-5, approximated by subtracting concentrations of
PLT recovered from Pf-5 pltB::inaZ cultures (P11) amended with identical
concentrations of PLT. PLT amendments did not significantly alter population sizes.
Differing lefters above symbols denote treatments significantly different from one
another (P 0.05; two-sample t-test). Error bars denote one standard error.
2.4.2. PLT and 2.4-DAPG exhibit mutual inhibition
Pit- mutants produced more 2,4-DAPG than did
Pitt
strains, as exemplified by
comparing 2,4-DAPG production in cultures of Pf-S iceC and Pf-5 pltB::inaZ (Fig.
2.2b). This observation is consistent with the hypothesis that PLT represses 2,4DAPG production in Pf-5. To test this hypothesis, the influence of PLT on 2,4-DAPG
production was determined for Pf-5
iceC
and Pf-5 pltB. :inaZ grown inNBGIy, a
medium conducive to both PLT and 2,4-DAPG production. Exogenous PLT (4
jig/mi) added to NBG1y repressed 2,4-DAPG production by both strains. At 12 h,
when 2,4-DAPG was at peak levels, PLT repressed 2,4-DAPG production in Pf-5
iceC
and Pf-5 pltB.:inaZ by a factor of 3.8 and 13.6, respectively (Fig. 2.2b). The
reverse scenario was also tested: we examined whether 2,4-DAPG repressed PLT
production by Pf-5. Prior to inoculation with Pf-5 iceC and Pf-5 pltB::inaZ, NBGIy
was amended with 12.5 jig/mi 2,4-DAPG. Amendment of the medium with 2,4DAPG reduced PLT production by Pf-5
iceC
at 18 h after inoculation, when PLT
production was first detected, and at each time point thereafter (Fig. 24a). As
expected, Pf-5 pltB::inaZ did not produce detectable PLT. Again, despite some
variation in timing and levels of PLT production across multiple time course
experiments, 2,4-DAPG amendments at 12.5 jig/mI to initial culture medium always
slowed PLT production in Pf-5 or Pf-5
late stationary phase.
iceC
and repressed it between 2- and 5-fold by
16
a.
14
12
10
©
8
4
2
0
11
b.
10
U
9
0
7
0
6
12
18
24
30
36
42
48
Culture age (hours)
Pf5 iceC
0 Pf5 iceC + 2,4-DAPG
V Pf-5pltB::inaZ
-v-- Pf-5 pltB::inaZ
+ 2,4-DAPG
Figure 2.4. Influence of exogenous 2,4-DAPG on PLT production by derivatives of
Pf-5. Cultures were grown in nonamended NBG1y or in NBG1y amended with
12.5 jig/mi 2,4-DAPG. At the indicated sampling times following inoculation, PLT
concentrations (a) and population densities of cultures (b) were assessed. Population
sizes did not differ significantly between 2,4-DAPG-amended and nonamended
treatments at any time point. Error bars denote one standard error. Symbols denoting
PLT concentrations for unamended Pf-5 pltB::inaZ cultures are occluded by those
denoting 2,4-DAPG-amended Pf-5 pltB::inaZ cultures.
To estimate the minimum concentration of 2,4-DAPG needed to inhibit PLT
production, Pf-5 was grown in NBG1y amended with 2,4-DAPG concentrations
ranging from 0.0 125 to 12,500 ng/ml (Fig. 2.5a). Amendment of NBG1y with 2,4-
DAPG at concentrations greater than or equal to 125 ng/ml (0.595 jiM) inhibited PLT
production by Pf-5, with PLT production decreasing as the initial concentrations of
2,4-DAPG in the growth medium exceeded that level (P
0.04). In contrast, when
cultures were amended with very low concentrations (between 0.0125 ng/ml and 0.125
ng/ml, or 59.5 pM to 0.595 nM) of 2,4-DAPG, PLT production was reproducibly
enhanced over that in non-amended cultures (P = 0.04).
Addition of 12.5 jig/mi 2,4-DAPG to the growth medium did not significantly
alter the population sizes of Pf-5 iceC or Pf-5 pltB : : inaZ (P 0.09; Fig. 2.4b); lower
concentrations similarly had no effect on growth of Pf-5 or Pf-5 pltB::inaZ (P
0.55;
Fig. 2.5b), indicating that differences in PLT production could not be attributed to an
impact of 2,4-DAPG amendments on growth.
61
5
a.
4
b
b
ac
e
f
I
0
I
I
I
I
1Z.
I2
10x109
b.
8x109
6x10'
14
4x109
2x109
U
U.UIL
U.1
I.Z
1,h3U
Jh,W
ng/mI 2,4-DAPG initaily present in medium
Figure 2.5. Minimum effective concentration of 2,4-DAPG for repression of PLT
production in Pf-5. PLT concentrations (a) and CFU (b) were measured in Pf-5 after
culturing for 18 h in NBG1y that was amended with the concentrations of 2,4-DAPG
indicated on the abscissa. 2,4-DAPG amendments did not significantly alter
population sizes. Differing letters above symbols denote treatments significantly
different from one another (P 0.05; two-sample t-test). Error bars denote one
standard error.
62
2.4.3. Effects of PLT and 2.4-DAPG on PLT production occur at the
transcriDtional level
The effects of exogenous PLT and 2,4-DAPG on PLT production were
accompanied by corresponding effects on transcription from PLT biosynthetic gene
promoters, as measured by activity of the ice nucleation transcriptional reporter gene
(inaZ) fused to three separate chromosomal PLT biosynthesis genes, pltB, pitE, and
pltF. Because preliminary experiments indicated that inaZ expression fromplt
promoters reached a maximum by 24 h after inoculation, JNA was measured
periodically from 0 to 24 h after inoculation (Fig. 2.6). INA was enhanced in all three
reporter strains when grown in NBGIy amended with 4 .tg/ml PLT. At 24 h after
inoculation, INA expressed by Pf-5 derivatives containing pltB::inaZ,pltE::inaZ, and
pltF: :inaZ was enhanced by one to two orders of magnitude by the addition of PLT.
Conversely, transcription of PLT biosynthetic genes was reduced when NBGIy was
amended with 2,4-DAPG (12.5 pg!ml) prior to inoculation. At 24 h after inoculation,
2,4-DAPG amendment of the culture medium reduced INA expressed by Pf-5
derivatives containing pItB: : inaZ, pitE: :inaZ, and pltF: :inaZ by three to twelve-fold,
respectively. These data provide evidence that autoinduction of PLT and repression of
PLT production by 2,4-DAPG both occur, at least in part, at the level of transcription
from PLT biosynthetic gene promoters. Addition of 4 tg/ml PLT or 12.5 tg!ml 2,4DAPG to the growth medium did not significantly alter the population sizes of any
strain at any timepoints sampled (all P> 0.05; Fig. 2.6d).
2
a. plL&:inaZ
b. pltE:inaZ
*
*
0
*
I:
-8
-10
0
6 12182430
I
c. pIth:inaZ
0 612182430
0
6 12182430
0
6 12 18 24 30
Cuiture age (hours)
I
pltB::inaZ
pli&:inaZ+PLT
4 jiL&uuzZ+2,4-DAPG
A
pfr:inaZ
...A.... pItE:inaZ+PLT
pfrE:ina2+2,4-DAPG
plLF.:inaZ
p1tF:inaZ+ILT
4-- p11F:inuZ+ 2,4-DAPG
Figure 2.6. Positive and negative influences by PLT and 2,4-DAPG, respectively, on
transcriptional activity within the PLT biosynthetic gene cluster. Transcriptional
fusions were previously made of the ice nucleation reporter gene (inaZ) to three PLT
biosynthetic genes. Panels a-c depict the response of individual reporter strains to
exogenous PLT and 2,4-DAPG. Pf-5 pltB: :inaZ (a, square symbols); Pf-5 pitE: :inaZ
(b, triangular symbols); and Pf-5 pltF::inaZ (c, round symbols); were grown in
unamended NBG1y (solid lines), or in NBG1y amended with 4 j.ig/ml PLT (dotted
lines) or 12.5 jig/mI 2,4-DAPG (dashed lines). Population densities (d) are shown for
all strains, and did not differ significantly among treatments at any time point.
Asterisks above symbols denote treatment means which differ significantly from the
mean of the unamended treatment at the identical timepoint (P 0.05; Student's ttest). Error bars denote one standard error.
64
2.4.4. PLT autoinduction is superseded by eneticaIly and environmentally
repressive backgrounds
In NBG1u, a medium that is not conducive to PLT production by Pf-5 (30, 43),
the addition of 4 j.tg/ml PLT did not result in detectable PLT production by the
bacterium (data not shown). Thus, PLT amendments in NBGIy and NBG1u had
different effects on PLT production by Pf-5, suggesting that the PLT autoinduction
phenomenon was overridden by glucose repression. Similarly, Pf-5 pltR: :aacCl and
Pf-5 gacA::aphl derivatives grown in NBG1y failed to produce detectable
concentrations of PLT, even in the presence of exogenous PLT (data not shown),
suggesting that autoinduction does not supersede control of PLT production by the
LysR transcriptional regulator encoded bypltR or the by the GacA/GacS histidine
kinas c/response regulator pair. Other workers observed that autoinduction of 2,4DAPG is not entirely subordinate to gacA in CHAO (54), indicating that the
mechanisms of autoinduction may differ between the two systems.
2.4.5. PLT autoinduction occurs in the rhizosphere
To determine whether PLT could influence PLT biosynthetic gene expression
by bacterial populations in the rhizosphere as well as in culture, we performed a PLT
cross-feeding assay. The PIf strain Pf-5 pItB::inaZ was used as a reporter strain to
detect the presence of PLT produced by other bacteria in the rhizosphere. Cucumber
seedlings were coinoculated, at a 1:10 ratio, with Pf-5 pltB::inaZ and one of three
other Pf-5 derivatives that differ in PLT production: Pf-5, which produces PLT; Pf-5
rpoS::lacZ, which overproduces PLT; or pltR::aacCl , which does not produce
detectable PLT (Fig. 2.7). When coinoculated with pltR::aacCl in the rhizosphere of
cucumber, Pf-5 pltB::inaZ produced a mean of -4.0 logio(ice nuclei/CFU) from 10 to
48 h after inoculation, which represented a basal level of pltB::inaZ expression in the
absence of PLT cross-feeding. When coinoculated with the PLT-producing strain Pf-
65
5, rhizosphere populations of Pf-5 pltB::inaZ expressed significantly greater INA [-2.5
logio(ice nucleiICFU)}. The greatest level of INA expressed by Pf-5 pltB::inaZ in the
rhizosphere, averaging -.1.1 logio(ice nuclei/CFU) from 10 to 48 h after inoculation,
occurred when Pf-5 pltB::inaZ was coinoculated with the PLT-overproducer Pf-5
rpoS::lacZ. Thus, PLT produced by P1-5 and the overproducing derivative Pf-5
rpoS::lacZ was perceived by coinoculated cells of Pf-5 pltB::inaZ in the rhizosphere,
as evidenced by enhanced transcription of pltB in those coinoculated cells. Population
sizes (mean log CFU/seedling) of the inducing strains averaged 6.5 at the time of
inoculation, and increased to 6.8 over 48 hours. Log CFU/seedling for the reporter
strain coinoculated with each inducer averaged 5.3 at the time of inoculation and
increased to 5.9 over 48 hours. While slight differences in the rhizosphere population
sizes of Pf-5 pltB::inaZ were observed among treatments at two time points, these
differences were not found to significantly affect NA determinations.
0
-1
z -4
-5
-6
-7
0
10
24
48
Hours post-inoculation and planting
Pf-5 pltR::aacCl; P1t
V
Pf-5;Plt
Pf-5 rpoS:.iacZ, Plt
Figure 2.7. Autoinduction of PLT as a result of cross-feeding in the rhizosphere. A
Pif reporter strain (Pf-5 p/tB:: inaZ) was coinoculated onto cucumber seedlings at a
cell density ratio of 1:10 with each of the following inducing strains: Pf-5 (Plt), Pf-5
pltR: :aacCl (Pit) and Pf-5 rpoS::IacZ (Plt). After seedlings were grown in
pasteurized soil for the durations indicated, bacteria were harvested and evaluated for
INA conferred by Pf-5 pftB::inaZ in the presence of each coinoculant. Values
represent the mean of ten replicate seedlings and error bars indicate one standard error,
which are obscured by the symbols for some timepoints.
67
2.5. Discussion
The data presented in this paper provide strong evidence that PLT serves as an
autoregulator, positively influencing its own production in P.fiuorescens Pf-5. Three
lines of evidence from experiments with Pf-5 and derivative strains support this
conclusion: 1) exogenous PLT enhanced PLT production, ii) exogenous PLT
enhanced transcriptional activity of three PLT biosynthetic genes, and iii)
coinoculation with Plt cells induced expression of PLT biosynthetic gene
transcription by a Pit- reporter strain in the rhizosphere. Furthermore, PLT functioned
as an inducer of its own production when present in culture medium at an extremely
low concentration (1.47 nM). In this respect, PLT is similar to established signaling
molecules, such as the A-factor of Streptomyces griseus (19, 22, 27), or AHLs of
Vibriofischeri and Serratia liquefaciens (13, 14), which influence cell physiology and
gene expression when extracellular concentrations are in the nanomolar range. In
addition to its autoregulatory role, PLT influenced the production of at least one other
secondary metabolite, 2,4-DAPG. Exogenous PLT repressed 2,4-DAPG production
by Pf-5, as was previously reported in the related strain, P.Jluorescens CHAO (54).
Thus, in addition to its established extracellular role as an antibiotic, PLT contributes
to regulation of at least two metabolic pathways within the bacterial cell.
The roles of antibiotics in the ecology and physiology of microorganisms have
been debated for decades, and evidence is accruing to support intracellular roles for
bacterial antibiotics. For example, P. aeruginosa produces a 4-quinolone compound
(PQS, for Pseudomonas guinolone igna1) that, when present in micromolar
concentrations, plays an integral role in signal transduction of producing bacterial cells
(38, 46). Although toxicity has not been demonstrated for PQS itself (46), related
molecules in the 4-quinolone class have antibacterial activity and are used medicinally
(55). More recently, it has been shown that exposure to subinhibitory concentrations
of several commonly used antibiotics results in altered transcription of as many as 5%
of genes in Salmonella typhimurium (15); by extension, one might predict that these
antibiotics also have physiological relevance in the cells in which they are produced.
The finding that nanomolar concentrations of PLT can enhance PLT production and
biosynthetic gene expression by Pf-5 provides another example of a regulatory role for
antibiotics.
PLT biosynthesis genes were transcribed in early to mid-log phase, but PLT
did not accumulate to detectable levels until cells were entering or in stationary phase.
The lag between induction of gene expression and accumulation of detectable levels of
PLT might be explained by slow rates of PLT biosynthesis, or by posttranscriptional
control mechanisms like those affecting the antibiotics 2,4-DAPG and hydrogen
cyanide in the related P. fluorescens strains CHAO and Fl 13 (1, 5, 6, 20). Transcripts
of genes for biosynthesis of hydrogen cyanide and 2,4-DAPG are sequestered by a
small RNA-binding protein (RsmA), but a noncoding RNA, PrrB (RsmZ), whose
expression is regulated by GacA/GacS, can in turn bind RsmA, and thus relieve RsmA
suppression in these strains. An alternative hypothesis is that low levels of PLT,
below our detection limits, may gradually accumulate in cultures until a threshold rate
of PLT re-entry into producer cells is achieved, whereupon PLT begins autoinduction.
Such a mechanism is reminiscent of the prototypic LuxlILuxR autoinduction systems,
which require accumulation of an autoinducer in the extracellular milieu for induction
of gene expression (reviewed in 39).
Established positive autoregulation systems, such as LuxIILuxR, rely upon
diffusion or secretion and re-uptake of the signal molecule across the membrane, and
include an intracellular signal receptor(s) that mediates signal transduction within the
cell. Neither a PLT receptor nor a mechanism for PLT transport across the bacterial
membrane is known in Pf-5, but candidates for both are found within the PLT
biosynthetic gene cluster. The nucleotide sequence of the p/tHu cluster, which is
directly adjacent to the PLT biosynthesis genes p/tLABCDEFG, includes genes
encoding members of the ABC transporter family (7). Transcription of p/ti and pltf is
enhanced by exogenous PLT (7), indicating that regulation of the genes is coordinated
with PLT production, as would be expected if the region encodes proteins that
function in transport of PLT into or out of the cell. The possibility also remains that
the small hydrophobic PLT molecule could diffuse across the bacterial cell membrane.
A candidate PLT receptor(s) is PItR, a LysR transcriptional regulator required for
transcriptional activity of pltB,pltE, and pith' and for the accumulation of detectable
levels of PLT in culture supernatants (42). LysR. family members generally require a
cofactor for activity (e.g. reviewed in 51).
The findings of this study demonstrate that regulation of 2,4-DAPG and PLT
production is interrelated in Pf-5; both compounds were repressive of the other's
production. In P. fluorescens strain CHAO, 2,4DAPG autoinduction and repression
by PLT, salicylate and a flingal metabolite, fusaric acid, are mediated by PhIF (54),
whose repressive binding to the ph1ACBD promoter is directly stabilized by salicylate
but alleviated by 2,4-DAPG (2). Whether the mechanism of 2,4-DAPG autoregulation
and repression is similar in Pf-5 remains to be determined; we have not ruled out the
possibility that precursor competition (e.g. for malonyl-CoA needed to extend the
growing polyketide chains) contributes to PLT repression of 2,4-DAPG in Pf-5.
The concentration of 2,4-DAPG (595 nM) required for repression of PLT
production in Pf-5 was much higher than the concentration of PLT (1.47 nM) required
for autoinduction. Furthermore, the bimodal response of PLT production to increasing
concentrations of 2,4-DAPG suggests a concentration-dependent role reversal of 2,4-
DAPG from activator to inhibitor. The reason for this shift is unknown. However,
similar concentration-dependent role reversals have been observed in other systems.
For example, in Vibriofischeri MJ-1, AHL concentrations between 0.01 and 5 jig/mi
enhanced luciferase activity, but higher concentrations were inhibitory (13).
Similarly, in Bacillus subtilis, a small peptide (CSF for competence and porulation
factor) controls gene expression in a cell-density dependent fashion. The response of
srfA, a CSF-regulated gene for surfactin biosynthesis, was upregulated at low (1-5
nM) CSF concentrations, but inhibited at higher (>20 nM) concentrations of the signal
peptide (56).
70
To test the level of PLT autoinduction in the regulatory hierarchy surrounding
antibiotic production in Pf-5, exogenous PLT was added to Pf-5 in an environmental
condition and in a genetic background repressive to PLT production. The positive
effect of exogenous PLT does not supersede the negative effect of a gacA mutation on
PLT production, nor can it override repression of PLT production in the presence of
glucose. Because low concentrations of PLT are still detectable in Pf-5 cultures
grown in the presence of glucose, lack of PLT does not explain the inability for
autoinduction. Rather, this result implies that PLT autoinduction requires a factor(s)
which is lacking during glucose repression, or is inhibited by a factor(s) produced in
the presence of glucose. For the gacA mutant, which produces no detectable
pyoluteorin, this observation implies that factors necessary for PLT production or
autoinduction are lacking.
This study established that PLT functions as signal molecule triggering the
transcription of a PLT biosynthesis gene in bacterial cells in the rhizosphere, as well as
in culture. When Pf-5 pltB::inaZ was co-inoculated with PLT-producing strains,
expression of p/tB, monitored with the inaZ reporter, was enhanced. Thus, PLT
served as an intercellular signal between distinct populations of bacterial cells co-
inhabiting the rhizosphere. This observation implies that, in the rhizosphere, PLT
biosynthetic genes are upregulated by exogenous PLT, and PLT autoinduction
contributes significantly to PLT regulation by Pf-5. To our knowledge, this is the
first report demonstrating that an antibiotic functions as a signal molecule in a natural
environment.
PLT is an antibiotic toxic to Pythium ultimum and other plant pathogenic
Oomycetes, and has been shown to be an important factor in biological control of such
pathogens (24, 37). The finding that PLT serves as signal in the rhizosphere,
influencing the expression of a gene required for its biosynthesis by neighboring
bacterial cells, has important implications for biological control of plant disease. PLT
71
can now be added to the growing list of microbial metabolites shown to function as
signal molecules influencing the expression of biocontrol traits by Pseudomonas spp.
in the rhizosphere. Other examples include AHLs (48, 59), which influence the
expression of phenazine biosynthesis genes by P. aureofaciens; fusaric acid (40),
which represses the expression of 2,4-DAPG biosynthesis genes by P.fiuorescens
CHAO; and siderophores (33), which influence the expression of pyoverdine
biosynthesis genes by P. putida. When taken together, these examples provide
convincing evidence that the expression of biocontrol traits by Pseudomonas spp. is
influenced profoundly by signal molecules produced by their coinhabitants in the
rhizosphere. It seems likely that secreted metabolites with various functions in the
extracellular environment, retrieved or intercepted by producing or neighboring cells,
supply information about the ecological and physiological status of the surrounding
community. Therefore, the composition and physiological status of the microbial
community is likely to be an important factor influencing the expression of biocontrol
traits by bacterial antagonists in the rhizosphere, and consequently, their success as
biological control agents.
2.6. Acknowledgements
We thank Brian Nowak-Thompson and Regina Notz for providing pure PLT
and 2,4-DAPG, respectively. We are grateful to Meadow Anderson, Walter Mahaffee,
Dmitri Mavrodi, Caroline Press, and Brenda Shaffer and Virginia Stockwell for
critical reviews of the manuscript. This research was supported in part by a fellowship
to M.B. from the U.S. Environmental Protection Agency Science to Achieve Results
program (U-91582801-0).
72
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1995. Amplification of the housekeeping sigma factor in Pseudomonas
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abilities. J. Bacteriol. 177:5387-5392.
53.
Schnider, U., C. Keel, C. Voisard, G. Defago, and D. Haas. 1995. Tn5directed cloning of pqq genes from Pseudomonasfluorescens CHAO:
mutational inactivation of the genes results in overproduction of the
antibiotic pyoluteorin. App!. Environ. Microbiol. 61:3856-3864.
54.
Schnider-Keel, U., A. Seematter, M. Maurhofer, C. Blumer, B. Duffy,
C. Gigot-Gonnefoy, C. Reimmann, R. Notz, G. Défago, D. Haas, and C.
Keel. 2000. Autoinduction of 2,4- diacetylphioroglucinol biosynthesis in
the biocontrol agent Pseudomonasfiuorescens CHAO and repression by the
bacterial metabolites salicy!ate and pyoluteorin. J. Bacteriol. 182:12151225.
55.
Smith, J. T., and H. -J. Zeiler. 1998. History and introduction, p. 1-11.
In J. Kuhlmann, A. Dalhoff, and H.-J. Zeiler (ed.), Quinolone antibacterials.
Springer, Berlin, Germany.
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1998. The two-component regulators GacS and GacA influence
accumulation of the stationary-phase sigma factor a' and the stress response
in Pseudomonasfluorescens Pf-5. J. Bacteriol. 180:6635-6641.
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Whistler, C. A., V. 0. Stockwell, and J. E. Loper. 2000. Lon protease
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rhizosphere. J. Bacteriol. 179:7663-7670.
79
Chapter 3. Preliminary evidence for effects of pyrrolnitrin, 2,4diacetyiphioroglucinol, and pyoluteorin on secondary metabolism in Pf-5.
3.1. Introduction
Because pyoluteonn (PLT) affects its own production as well as that of another
polyketide antibiotic, 2,4-diacetyiphioroglucinol (2,4-DAPG) (Chapter 2), I
hypothesized that PLT influences levels of other antibiotics produced by Pf-5. PLT is
synthesized by a polyketide synthase complex (14,16) and is secreted from cells of Pf-
5. PRN is remarkably similar in structure to PLT (Fig. 1.1), but PRN is derived from
tryptophan (8), and is largely retained within the bacterial cell. Because of the
similarity in structures, it was postulated that PLT and PRN might compete for the
active site on the hypothetical PLT receptor protein that mediates PLT autoinduction.
Experiments were performed to identify potential effects of pyrrolnitrin (PRN) and
PLT on one another's production in culture.
Pyoverdine is a siderophore compound: it is secreted into the extracellular
environment where it chelates ferric iron, and subsequently imported back into the cell
to release its bound iron. In P. aeruginosa, pyoverdine uptake is also a signal
activating the expression of several virulence factors (10). Because of the pyoverdine-
virulence factor connection in P. aeruginosa, I tested whether pyoverdine production
might be affected by exogenous PLT.
Finally, 2,4-DAPG autoinduces its own production in P. fluorescens CHAO.
The results presented below demonstrate that the same is true for 2,4-DAPG
production in Pf-5. Together, the results presented herein provide evidence that the
secondary metabolites secreted by Pf-5 influence their own and one another's
production as a rule, rather than as an exception.
3.2. Methods
For all experiments evaluating the effects of PLT, PRN, or 2,4-DAPG
amendments on antibiotic production, culture conditions and experimental setup was
exactly as described in Chapter 2. Bacterial strains and plasmids used in this study are
listed in Table 3.1. Pf-5 and derivative strains were cultured routinely on King's
Medium B agar (7) containing streptomycin (100 tg/ml). KMB was supplemented
with other antibiotics (kanamycin, 50 .tgIml; gentamycin, 40 pg/ml) when culturing
strains with antibiotic resistance. For experiments evaluating antibiotic production or
PLT biosynthetic gene transcription, cultures were grown at 20°C with shaking (200
rpm) in nutrient broth (pH 6.8; Difco Laboratories, Detroit, Mich.) without selection
and supplemented with 0.5 % (w/v) glycerol for NBG1y, a medium conducive to PLT
production (unpublished data). For experiments evaluating antibiotic production or
PLT biosynthetic gene transcription in defined medium, bacterial cultures were grown
in OS medium (17) amended with 0.5% (w/v) glycerol (OSGIy) at 20°C with shaking
(200 rpm). Pure antibiotics dissolved in methanol were added to NBGIy immediately
prior to inoculation with experimental cultures; where indicated, an equivalent volume
of methanol was added to controls. Amendments were made at the following rates: 4
jtg/ml PLT, 12.5 jtg/ml 2,4-DAPU, and 0.7 .tg/ml PRN. PRN amendments were
based on the concentrations of PRN found in stationary phase supernatants, which are
similar to concentrations extracted from cells. In non-timecourse experiments, PRN
was measured at 48h after inoculation, because it reaches maximum concentrations in
late stationary phase cultures of Pf-5. Inoculum of Pf-5 and derivatives was obtained
from overnight cultures grown with shaking at 200 rpm at 27°C in KMBGIu, modified
from KMB broth by substituting the glycerol with 1% (w/v) glucose to repress PLT
production. Cells from twice-washed overnight seed cultures grown in KMBG1u were
suspended in NBG1y or OSG1y to an OD6 of 0.1. In all experiments, triplicate 5 ml
E31
cultures were grown and evaluated; each experiment was performed at least twice; and
the results from a representative experiment are presented. For experiments assessing
transcriptional activity of PLT genes using the inaZ reporter gene, samples were
harvested periodically and assessed for ice nucleation activity (INA) as described in
Chapter 2.
Antibiotic extractions and HPLC analyses were performed as described in
Chapter 2. Cultures were centrifuged (5,000 X g, 5 mm) to pellet cells, and
supematants were extracted as described for recovery of PLT and 2,4-DAPG, which
are secreted into the culture medium. Because PRN is found both intracellularly and
in the supematant, cell pellets were also extracted for PRN by suspending in 5 ml
acetone, vortexing for 10 sec, placing in a sonicating bath for 30 sec, and subsequently
centrifuging (10,000 X g, 10 mm) to separate the cell debris from the organic extract.
Acetone was removed from extracts under reduced pressure, and the resultant residues
were resuspended in 100
tl
methanol, from which 10
HPLC as described. PRN was detected at ?.
ILl
samples were evaluated by
254 nm and t1 = 17.9 mm. The
detection limit for PRN was 0.01 j.tglml, and the efficiency of recovery for PRN was
77%. Values for other antibiotics are listed in Chapter 2.
82
Table 3.1. Bacterial strains and plasmids used in this study
Strain
Strain
Number
Relevant characteristics"
Reference
or Source
Pf-5
JL4474
Pseudomonasfluorescens Pf-5 rhizosphere
isolate; Plt, Ina
5
Pf-5 iceC
JL4303
Pf-5 (pJEL1 703); jr Plt, Ina
12
Pf-5 pvd-inaZ
JL4304
Pf-5 (pJEL17O1); Rif Kmr Plt, 1na
12
Pf-5
JL4389
P. fluorescens
pltB..inaZ
Pf-5
pltR.:aacCl
pltB:Tn3 -nice derivative of Pf-5, PIt, Ina,
9
KmT
JL4563
pltR.:aacCl derivative of Pf-5; P1t, Gm',
14
mi
Plasmids
pJEL1 696
4 kb fragment containing inaZ cloned into
pVSP61, in opposite orientation to lac
promoter
P. Mukeiji
pJEL17O1
8 kb fragment containingpvd genes from
P. syringae 31R1 cloned into pJEL1 696,
upstream of the promoterless inaZ
12
pJEL1 703
iceC from P. syringae cloned into pVSP61
(nucleotide sequences of iceC and inaZ are
nearly identical)
12
pVSP61
Stable plasmid vector containing pVS1
replicon and mobilization region and pl5A
replicon; Mob, KmT
W. Tucker
"Plt, produces pyoluteorin; Pif, does not produce detectable pyoluteorin; 1na,
expresses ice nucleation activity at -5°C; maT, does not express ice nucleation activity
at -5°C; KmT and GmT, resistant to kanamycin and gentamycin, respectively.
83
33. Results and Discussion
3.3.1. Lack of mfluence by exogenous PLT on PRN
In multiple PRN amendment experiments, I failed to see an effect of
exogenous PLT on the production of PRN. Similar concentrations of PRN were
produced in NBG1y by Pf-5, Pf-5 p/tB ::inaZ, and Pf-5 pltR::aacCl , whether or not the
culture medium was amended with 4 pg/ml PLT (Table 3.2). PLT amendment also
had no reproducible, statistically significant effect on PRN production by Pf-5 iceC
grown in defined OSGly medium (P O.14) (Fig. 3.1).
Table 3.2. Influence of exogenous PLT on PRN production by derivatives of Pf-5.
Amendment of culture medium
PRN concentrations
No amendment
+ 4j.tg/ml PLT
Pf-5
1.85±0.12
2.07± 0.21
Pf-5 pltB::inaZ
2.02 ± 0.21
2.24 ± 0.05
Pf-5pltR::aacCl
1.81 ± 0.27
2.27 ± 0.06
(.tg/m1 ± SE)
84
1.6
a.
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
0
12
24
36
48
60
-Ju.0
'
-.
9.5 lb.
9.0
0
pf_5 iceCF
S
L)
8.0
Pf-5 keCE + PLT
7.5
s Pf-5 iceC + PRN
70
0
12
24
36
48
60
Culture age at harvest (hours)
Fig. 3.1. Influence of exogenous PLT and PRN on PRN produced by Pf-5 iceC.
Cultures were grown in nonamended NBG1y, in NBGIy amended with 4 .tg/ml PLT,
or in NBG1y amended with 0.7 .tg/ml PRN. At the indicated sampling times
following inoculation, PRN concentrations (a), and population sizes of cultures (b)
were assessed. Error bars denote one standard error.
85
3.3.2. Minimal influence by exogenous PRN on PLT production
The opposite scenario was also tested by evaluating the influence of exogenous
PRN (0.7 p.g/ml) on PLT production by Pf-5 iceC grown in OSGIy. In contrast to the
lack of an effect of PLT on PRN production, amendment of OSG1y with PRN
repressed PLT production by Pf-5 iceC slightly in one trial (14.2% reduction; P =
0.01) although the repressive effect was not significant in another experiment (data not
shown). Amendment of OSG1y with PRN also reduced INA in Pf-5 pltB::inaZ
between 10 and 20% at all timepoints after 24 h in two separate experiments, but the
repressive effect was only significant at 22 h (12.7%) in one experiment, and at 44 h
(13.9%) in another experiment (both P 0.04) (Fig. 3.2). PRN amendments also
affected the production of PRN itself. PRN amendments caused a reproducible
increase in PRN production by late stationary phase (67 hours) (P = 0.013). It remains
to be determined whether this positive feedback regulation occurred at the level of
transcription. The mutually repressive impacts of 2,4-DAIPG and PLT on one
another's production occurs, at least in part, at the level of transcription (Chapter 2;
Press and Loper, unpublished data). The slight inhibitory effect of PRN on PLT gene
transcription and production cannot be explained by precursor competition, as it is
unlikely that these compounds share precursors (8,14).
1
0
0
-1
-2
-3
-4
-5
v
-6
-7
0
6
12
18
Ff-5pltB::inaz
Pf-5 pltB::inaz + PLT
Pf-5pltB::inaz + PRN
24
30
36
42
48
Culture age (hours)
Fig. 3.2. Positive and negative influences by PLT and PRN, respectively, on
transcriptional activity of the PLT biosynthetic gene, p/tB. A transcriptional fusion
was previously made of the ice nucleation reporter gene (inaZ) to the chromosomal
p/tB gene, to create Pf-5 pltB::inaZ. The transcriptional reporter strain was grown in
unamended NBGIy (round symbols), in NBGIy amended with 4 tg/ml PLT (triangular
symbols) or in NBG1y amended with 0.7 j.tg/ml PRN (square symbols). Error bars
denote one standard error, and are occluded by symbols at many points.
3.3.3. Autoinduction by 2,4-DAPG in P. fluorescens Pf-5
In P. fluorescens
CHAO, 2,4-DAPG autoinduces its own production. The
results of this study demonstrate that the same is true for 2,4-DAPG production in Pf-
5. 12.5 ig/ml 2,4-DAPG was added to NBG1y prior to inoculation; this concentration
represents the low end of concentrations of 2,4-DAPG normally detected in NBG1u,
the medium typically used for 2,4-DAPG production (4, 15, 22). 2,4-DAPG
concentrations were enhanced in the 2,4-DAPG amended cultures, although the timing
and magnitude of 2,4-DAPG production and autoinduction by Pf-5 were variable
among experiments under the culture conditions used in these studies. Two selected
experiments are shown (Fig. 3.3). In Fig. 3.3a, at 12 hours, amended cultures
contained 3 to 29-fold more 2,4-DAPG than nonamended cultures, even after
accounting for the original 2,4-DAPG amendment (by subtracting 2,4-DAPG
concentrations detectable at the earliest timepoint). In contrast, in Fig. 3.3c, amended
cultures contained 2 to 8-fold more 2,4-DAPG than nonamended cultures, after
accounting for the original 2,4-DAPG amendment. The average recovery for 2,4DAPG using the extraction method described in Chapter 2 is 45%. Thus,
approximately 5.6 jig/mI was expected from extractions of 2,4-DAPU in amended
cultures at the earliest timepoint. However, 2,4-DAPG concentrations in amended
cultures of the experiment in Fig. 3.3.a were less than 2 tg/m1, suggesting that 2,4-
DAPG recovery was lower than normal. Thus, inconsistent extraction efficiencies
may explain some of the experiment-to-experiment variation in 2,4-DAPG
autoinduction. As noted by members of our laboratory as well as those of other
laboratories (L. Thomashow and G. Phillips, personal communication), detection of
2,4-DAPG from culture supernatants is quite variable, and 2,4-DAPG productionmay
even appear to cease altogether for certain laboratory strains.
ilo
:
=
0
10
8
0
U
6
0
P9.4
0.
4
U
'
2
I
0
0
11
10
.9
6
12
18
24
30
36
42
48
7,-==
8
Pf5keC
0 Pf5 weC+ 2A-DAPG
,-- Pf.5plzB::inaZ
v-- Pf-5pltB::inaZ+ 2,4-DAPG
6
12
26
-- Pf 5 icecF
0-- P15 iceC+ 24-DAPG
-- Pf5pftB::iaZ
v
Pf.5pltB::inaZ+2,4-DAPG
Fig. 3.3. Influence of exogenous 2,4-DAPG on production of 2,4-DAPG by
derivatives of Pf-5. Pf-5 iceC (round symbols) is a Plt strain, and Pf-5 pltB::inaZ
(triangular symbols) is a Plf strain. Cultures were grown in nonamended NBGIy
(filled symbols) or in NBGIy amended with 12.5 tg/ml 2,4-DAPG (open
symbols). At the indicated sampling times following inoculation, 2,4-DAPG
concentrations (a,c) and population densities of cultures (b,d) were assessed.
Population sizes did not differ significantly between 2,4-DAPG-amended and
nonamended treatments at any time point. Two separate experiments (a,b and c,d) are
shown to demonstrate the variation in observations. Error bars denote one standard
error, and are occluded symbols at some time points.
Confirming that 2,4-DAPG exerts positive autoregulation in Pf-5 as it does in
CHA0 was expected, because of the genetic (e.g. 13, 19) and biochemical similarity of
these strains. However, a regulatory role for a particular compound may not
necessarily be extrapolated to every strain in which that compound is produced. For
instance, pyoverdine exhibits evidence of positive autoregulation in P. fluorescens
Ml 14, P. putida WCS358, and Pseudomonas strain BlO (1,3, 21), but does appear to
do so in P. aeruginosa PAO1, based on studies in BlO and PAO1 comparing the
effects of pyoverdine amendments upon activity from structurally similar promoters
preceding genes that encode functionally interchangeable gene products (1).
3.3.4. Exogenous PLT represses expression of pyoverdine biosynthesis genes
Although it is not known whether pyoverdine autoregulates its production in
Pf-5, a serendipitous observation suggests that it, too, may be under regulation by
another secondary metabolite. Strain Pf-5 pvd-inaZ is a reporter strain for inaZ
expression from the promoter precedingfpvA, a gene encoding the outer membrane
ferric-pyoverdine receptor from P. syringae. Pf-5 pvd-inaZ is used as an iron reporter
strain. I discovered that adding PLT at 4 tg/ml to NBG1y prior to inoculation with Pf5 pvd-inaZ reduced transcription offpvA. INA from Pf-5 pvd-inaZ remained similar
between 0 and 48 hours in the presence of exogenous PLT, whereas expression
increased approximately 150-fold over 48 hours in unamended cultures, in two
separate experiments (Fig. 3.4). Because these results are from an unexpected
observation rather than a planned experiment, several conditions throw doubt on the
results. First, thepvd genes used to construct the pvd-inaZ reporter are from another
species, P. syringae. Secondly, the pvd-inaZ reporter gene is plasmid-bome and
therefore present in multiple copies. Thus, the expression of INA from the pvd::inaZ
fusion is likely to be several-fold higher than expression from the chromosomalpvd
genes. Third, the medium used for this experiment was NBGIy, an undefined medium
that is not an optimal choice for measuring the expression of iron-responsive genes.
Fourth, PLT autoinduction occurred as a result of the PLT addition (data not shown).
It is possible that the excessive amount of PLT produced by these cultures placed a
metabolic load on Pf-5 (pvd::inaZ) so that the response was a decrease in the synthesis
of other energetically expensive metabolites, such as pyoverdine. Fifth, it is unclear
whether the effect of PLT on pyoverdine gene expression is due to direct effects of
one compound upon the other's production, or whether PLT might alter the iron
availability to Pf-5 cells and thus indirectly affect pvd gene expression. A follow-up
experiment to adthess these issues and test the verity of the results presented here
might include the following parameters:
1)
The real-time RT-PCR, rather than the Pf-5 pvd-inaZ reporter strain, to
assess the effect of PLT on the fpvA homolog in Pf-5, as well as
pyoverdine biosynthesis genes
2)
The use of a defined medium, such as RSM, with or without a source of
iron, such as ferric citrate (e.g. as in reference 11)
3)
The use of a Pf-5 pvd::inaZ strain that was also P11, to circumvent the
issue of metabolic load by PLT autoinduction
Recent findings have linked ferric-pyoverdine uptake by FpvA to regulation of
multiple virulence factors in P. aeruginosa. The binding of ferric-pyoverdine to FpvA
generates a signal which is passed to the antisigma factor FpvR, and then to either
FpvI or PvdS (both xtraçytopIasmic family, or ECF, sigma factors). Binding of FpvI
and PvdS to the RNA polymerase results in transcription of thefpvA gene (FpvI) or of
genes for biosynthesis of at least three exoproducts: pyoverdine, exotoxin A, and
PrpL endoprotease (PvdS) (2, 10, 18, 20). Therefore, follow-up of a possible
analogous link between FpvA and exoproducts of Pf-5 could be very exciting.
91
0.0
Exp.1
-0.5
-1.0
-1.5
-2.0
0.
-2.5
-3.0
0
I
I
10
20
Pf-5 (pvd-inaZ)
Pf-5 (pvd-inaZ)
30
PLT
40
0
-a
I::
-2.
-3,
Culture age (hours)
Fig. 3.4. Negative influence by PLT on transcriptional activity of a ferric-pyoverdine
uptake gene. A transcriptional fusion of a pyoverdine biosynthesis and uptake region
to the promoterless ice nucleation reporter gene (inaz) was made previously. Pf-5
pvd- inaZ contains the transcriptional fusion on the stable plasmid pVSP6 1. Pf-5 pvdinaZ was grown in unamended NBG1y (solid symbols), or in NBG1y amended with
4 pg/ml PLT (open symbols). Error bars denote one standard error.
3.4. Conclusions
Results from Chapter 2 demonstrated that exogenous PLT suppresses 2,4-
DAPG production. The results from this chapter expand those results to suggest the
involvement of two more secondary metabolites (PRN and pyoverdine) of Pf-5 in
cross-regulation of one another's production. Although the repression of the
pyoverdine reporter by exogenous PLT was observed under non-optimal experimental
conditions, this result was recently confirmed in the parental strain, P. fluorescens Pf-5
using both DNA hybridization analysis (microarrays) and real-time RT-PCR. Recent
experiments (Caroline Press, personal communication) showed that expression of
pvsA, a pyoverdine synthetase gene, is repressed in Pf-5 in the presence of exogenous
PLT. Notably, I was unable to demonstrate a significant effect of exogenous PLT
upon a structurally similar compound, PRN. Thus, although PLT affects the
production of at least three (PLT, 2,4-DAPG, and pyoverdine) secreted metabolites of
Pf-5 under the conditions tested, its influence is not so global that it influenced all
secondary metabolites that I tested in Pf-5.
In conclusion, I provide evidence for positive feedback of three secondary
metabolites produced by Pf-5: PLT (Chapter 2), 2,4-DAPG, and PRN. Each of these
compounds is important for biological control because of its antibiotic properties.
Pyoverdine exhibits autoinduction in several related strains; it is not known whether
this siderophore also undergoes autoinduction in Pf-5. From examples in the
literature, it appears that negative feedback control for secondary metabolites is a more
common phenomenon than positive feedback control. The most familiar example of
the latter is positive autoregulation by N-acyl-homoserine lactones (AHLs), as
described in Chapter 1. The rationale generally given for autoinduction of AHLs is
that they provide a mechanism for rapidly ramping up a response to a stimulus. In
many cases, the response involves the production of exoproducts secreted into the
extracellular environment. Could the antibiotics of Pf-5 be signals for the ramping up
93
of such a response - a response that includes the production of more of the same
compound that acted as a signal? What is the significance of the repression, by certain
secondary metabolites, of the production of others? Recent DNA hybridization
experiments, using an array comprising a limited set of Pf-5 genes important to
biological control, have shown that exogenous PLT affects the transcription of a
number of these genes. Thus, during PLT autoinduction, increased extracellular PLT
concentrations will be expected to wreak profound effects on the metabolism of the
cell. The imminent availability of the entire genome sequence of Pf-5, and the
possibility for an unabridged Pf-5 microarray, will permit further exploration of the
transcriptional responses of Pf-5 genes to autoinducmg antibiotics, and will build a
better understanding of the interrelatedness of these transcriptional responses.
94
3.5. References
Ambrosi, C., L. Leoni, and P. Visca. 2002. Different responses of
pyoverdine genes to autoinduction in Pseudomonas aeruginosa and the
group Pseudoinonasfiuorescens-Pseuclomonas putida. App!. Environ.
Microbiol. 68(8): 4122-4126.
2.
Beare, P. A., R. J. For, L. W. Martin, and I. L. Lamont. 2003.
Siderophore-mediated cell signalling in Pseudomonas aeruginosa:
divergent pathways regulate virulence factor production and siderophore
receptor synthesis. Mo!. Microbiol. 47:195-207.
3.
Callanan, M., N.. Sexton, D. N. Dowling, and F. O'Gara. 1996.
Regulation of the iron uptake genes in Pseudomonasfluorescens Ml 14 by
pseudobactin Ml 14: the pbrA sigma factor does not mediate the
siderophore regulatory response. FEMS Microbiol. Left. 144: 61-66.
4.
Corbell, N., and J. E. Loper. 1995. A global regulator of secondary
metabolite production in Pseudomonasfluorescens Pf-5. J. Bacteriol.
177:6230-6236.
5.
Howell, C. N.., and R. D. Stipanovic. 1979. Control of Rhizoctonia solani
on cotton seedlings with Pseudomonasfluorescens and with an antibiotic
produced by the bacterium. Phytopathology 69:480-482.
6.
Keel, C., D. Weller, A. Natsch, G. Défago, R. J. Cook, and L. S.
Thomashow. 1996. Conservation of the 2,4-diacetylphloroglucinol
biosynthesis locus among fluorescent Pseudomonas strains from diverse
geographic locations. App!. Environ. Microbiol. 62: 552-563.
7.
King, E. 0., M. K. Ward, and D. E. Raney. 1954. Two simple media for
the demonstration of pyocyanin and fluorescein. J. Lab. Clin. Med. 44:
301-307.
8.
Kirner, S., P. E. Hammer, B. S. Hill, A. Altmann, I. Fischer, L. J.
Weislo, M. Lanahan, K.-H. van Pee, and J. M. Ligon. 1998. Functions
encoded by pyrrolnitrin biosynthetic genes from Pseudomonasfluorescens.
J. Bact. 180:1939-1943.
9.
Kraus, J., and J. E. Loper. 1995. Characterization of a genomic region
required for the production of the antibiotic pyoluteorin by the biological
control agent Pseudoinonasfluorescens Pf-5. Appi. Environ. Microbiol.
61(3): 849-854.
95
10.
Lamont, I. L., P. A. Beare, U. Ochsner, A. I. Vasil, and M. L. Vasil.
2002. Siderophore-mediated signaling regulates virulence factor production
in Pseudomonas aeruginosa. Proc. Nati. Acad. Sci. USA 99:7072-7077.
11.
Loper, J. E., and M. D. Henkels. 1999. Utilization of heterologous
siderophores enhances levels of iron available to Pseudomonas putida in the
rhizosphere. AppI. Environ. Microbiol. 65:5357-5363.
12.
Loper, J. E., and S. E. Lindow. 1994. A biological sensor for iron
available to bacteria in their habitats on plant surfaces. AppI. Environ.
Microbiol. 60:1934-1941.
13.
McSpadden Gardener, B. B., K. L. Schroeder, S. E. Kalloger, J. M.
Raaijmakers, L. S. Thomashow, and D. M. Weller. 2000. Genotypic
and phenotypic diversity ofphlD-containing Pseudomonas strains isolated
from the rhizosphere of wheat. Appi. Environ. Microbiol. 66: 1939-1946.
14.
Nowak-Thompson, B., N. Chaney, S. J. Gould, and J. E. Loper. 1999.
Characterization of the pyoluteorin biosynthetic gene cluster of
Pseudomonasfluorescens Pf-5. J. Bacteriol. 181:2166-1274.
15.
Nowak-Thompson, B., S. J. Gould, and J. E. Loper. 1997. Identification
and sequence analysis of the genes encoding a polyketide synthase required
for pyoluteorin biosynthesis in Pseudomonasfluorescens Pf-5. Gene
204:17-24.
16.
Nowak-Thompson, B., S. J. Gould, J. Kraus, and J. E. Loper. 1994.
Production of 2,4-diacetylphloroglucinol by the biocontrol agent
Pseudomonasfluorescens Pf-5. Can. J. Microbiol. 40:1064-1066.
17.
Ornston, L.N. and R. Y. Stanier. 1966. The conversion of catechol and
protocatechuate to -ketoadipate by Pseudomonas putida. J. Biol. Chem.
241: 3776-3786.
18.
Redly, G. A., and K. Poole. 2003. Pyoverdine-mediated regulation of
FpvA synthesis in Pseudomonas aeruginosa: involvement of a probable
extracytoplasmic-function sigma factor, FpvI. J. Bacteriol. 185:1261-1265.
19.
Sharifi-Tehrani, A., M. Zala, A. Natsch, Y. Moënne-.Loccoz, and G.
Defago. 1998. Biocontrol of soilbome fungal plant diseases by 2,4diacetylphloroglucinol-producing fluorescent pseudomonads with different
restriction profiles of amplified 16S rDNA. Eur. J. Plant Pathol. 104: 631643.
20.
Shen, J., A. Meidrum, and K. Poole. 2002. FpvA receptor involvement
in pyoverdine biosynthesis in Pseudomonas aeruginosa. J. Bacteriol.
184:3268-3275.
21.
Venturi, V., Ottewanger, C., Bracke, M., and P. J. Weisbeek. 1995.
Iron regulation of siderophore biosynthesis and transport in Pseudomonas
putida WCS358: involvement of a transcriptional activator and of the Fur
protein. Mo!. Microbiol. 17: 603-610.
22.
Whistler, C. A., N. A. Corbell, A. Sarniguet, W. Ream, and J. E. Loper.
1998. The two-component regulators GacS and GacA influence
accumulation of the stationary-phase sigma factor c? and the stress response
in Pseudomonasfluorescens Pf-5. J. Bacteriol. 180:6635-6641.
97
Chapter 4. Membrane transport genes play a role in pyoluteorin production by
Pseudomonasfluorescens Pf-5
4.1. Introduction
Pyoluteorin (PLT), a chlorinated polyketide antibiotic secreted by
Pseudomonasfluorescens strain Pf-5 suppresses the plant pathogen Pythium ultimum.
A region of the Pf-5 chromosome required for PLT production was identified (34) and
sequenced (49, 51) and found to comprise nine biosynthetic genes and a
transcriptional regulator. The predicted functions of these genes are sufficient to
account for PLT biosynthesis (49). Also within the PLT biosynthetic gene cluster is
pltR, which encodes a LysR-type transcriptional regulator (49). ApltR derivative of
Pf-5 does not exhibit transcriptional activity from promoters within the PLT
biosynthetic region, and produces no detectable PLT. Additional loci found to affect
PLT production in Pf-5 or the related strain P. fluorescens CHAO contain global
regulatory genes or genes encoding proteins that are responsive to the physiological
status of the cell. These include the histidine protein kinase/response regulator pair
GacS/GacA (15), the protease Lon (82), sigma factors
and aD (62, 64), and
pyrrolquinoline quinone, a cofactor required by glucose and alcohol dehydrogenases
(65).
The pit biosynthetic genes (Fig. 4.1) are contained on at least three separate
operons. The divergent transcription of pltL and pltR implies the existence of separate
promoters between these genes. Although the proximity ofp/tR and pltM suggests
that these genes may form a single operon, RT-PCR experiments repeatedly failed to
demonstrate a correlation between the presence of apitR transcript and that of the
pltM transcript (data not shown). At least two promoters drive transcription of the
pltLABCDEFG gene cluster. One of these has been shown experimentally to reside
upstream of pltB (34), presumably in the pltR-pltL intergenic region; another has been
shown, via activation in trans of a promoterless reporter gene, to precede either p/tD
orpltE (43).
98
Genes encoding enzymatic functions for biosynthesis of secondary
metabolites, like PLT, are often clustered with genes for immunity to, or transport of
the metabolite (reviewed by 39). Moreover, regulatory genes are frequently included
in such regions, whose products coordinate expression of genes within the cluster. For
example, the gene cluster encoding 2,4-diacetyiphioroglucinol (DAPG) biosythesis in
P. fluorescens contains phiE, a gene encoding a putative integral membrane permease
(6) with similarity to genes encoding transporters of sugars and other substrates.
Intact phlE is required for optimal DAPG production (7). Also within the DAPG gene
cluster isphlF, which encodes a transcriptional repressor of DAPG biosynthetic genes,
and whose repression is lifted by binding of its cofactor, DAPG itself (6, 1). In P.
syringae pv. glycinea PG4 180, the coronatine biosynthetic gene cluster contains genes
encoding regulators of coronatine production (77), additionally, a possible membrane
permease is encoded by the linked gene cmaU although its function remains unknown
(76). The carbapenem biosynthetic gene cluster from Erwinia carotovora subspecies
carotovora also includes linked genes encoding regulatory and resistance functions
(41,42). In P. syringae pv. syringae, the syringomycin biosynthetic region contains a
gene for regulation of syringomycin production (85) and an ABC transporter predicted
to be involved in syringomycin secretion (57). ABC transporters, which are ATPpowered, multi-subunit substrate transporters, frequently afford antibiotic producing
organisms (prokaryotic and eukaryotic) a mechanism by which to resist selfintoxication (44).
Figure 4.1. Map of PLT biosynthetic gene cluster and relationship to subclones used
as templates for sequencing templates. All elements in the region are drawn to size.
Lettered abbreviations designate the following restriction sites: H, HindUl; B, EcoRI;
B, BamHI; P, PstI; S. Smal.
643
E
ortR
E
E
E
p2tM pltR
E
0
pitA
E
pitB
E
H
0
pitC
0
E
0
PS
BE
0B
p.ItD pitE pitF0pItG
BEP
H
rtI p1.tJ
pitH
ortI<
1kb
6120
Hi
5569
PP
P
limit of sequence hifomiafion
6247
H
6130
E
H
6246
BP
P
6198
S
H
6274
Fig. 1
C
C
101
Like the examples above, the PLT biosynthetic gene cluster in Pf-5 contains a
linked transcriptional regulator necessary for PLT gene expression, pltR (49). I
hypothesized that the PLT region might contain additional, unknown regulatory
functions. PLT is broadly toxic to Gram negative and Gram positive bacteria (5) but
similar concentrations have no significant effect on growth of Pf-5 (11), therefore, I
hypothesized that self-resistance or transport functions might be encoded by additional
genes flanking the known PLT biosynthetic genes. This report provides evidence for
new regulatory and transport functions encoded within the PLT biosynthetic gene
region. Adjacent topltG is a region containing pitH, a gene encoding a putative
transcriptional regulator, and pill and pltJ, two genes encoding a membrane fusion
protein and a subunit of an ABC transporter, respectively. I demonstrate
transcriptional regulation of p/tI and p/ti by exogenous PLT. I also provide evidence
that p/tI, p/ti, and/or adjacent genes encoding transport functions are necessary for
PLT production. The results herein provide the basis for the testable hypothesis that
p/tI and plt.J are involved in PLT transport and PLT autoinduction.
4.2. Methods
4.2.1. Bacterial strains, plasmids, and culture conditions
Bacterial strains and plasmids used in this study are listed in Table 4.1. E. co/i
strains were cultured routinely in Luria-Bertani (LB) medium (61) and maintained on
LB agar. Pf-5 and derivative strains were cultivated routinely on Kings Medium B
agar (KMB; 33). The following antibiotics were included in growth media as
required: spectinomycin, 50 jig/mi; streptomycin, 100 jig/mi; kanamycin, 50 jig/mi;
gentamycin, 40 jig/mI (P. fluorescens) or 15 jig/mi (E. co/i); tetracycline, 200 jig/mi
(P. fluorescens)
or 20 jig/mi (E. coli).
102
Table 4.1. Bacterial strains and plasmids used in this study
Strain
Strain
Number
Reference
Descriptiona
P. fluorescens
Pf-5
Pf-5pitB::inaZ
JL4474
JL4389
Pf-5 pitR..aacCl
JL4563
Pf-5 pItF::aacCl
JL4629
Rhizosphere isolate.
pitB::Tn3 -nice derivative of Pf5, PIf, that, Kmr
pltR:: aacCl derivative of Pf-5,
Pit-, Gm'
pltF..- aacCl derivative of Pf-5,
31
34
49
This study
Pit-, G'
Pf-5 pit.!: .inaZ
JL4669
Pf-5 p/ti: .inaZ
JL4671
pltJ::inaZ derivative of Pf-5,
Ina, Kmt.
pltJ:.inaZ derivative of Pf-5,
This study
This study
Ina, Kmr
Pf-5 pltI..inaZ
JL4673
pltI..inaZ derivative of Pf-5,
This study
Jna, Kmr.
Pf-5 pitR:: aacCl,
pltJ::inaZ
JL4675
Pf-5pltR.: aacCl,
JL4677
pitR:: aacCl, pltJ:.inaZ
This study
derivative of Pf-5, Ina, Gm',
plt.J::inaZ
pltR:. aacCl,pitJ:.inaZ
denvative of Pf-5, ma+ Gm,
This study
pltR:. aacCl pltI:.inaZ
This study
r
-
,
Pf-5 pltR:: aacCl,
pltI::inaZ
JL4679
Pf-5 pitF:. aacCl,
pltJ::inaZ
JL468 1
,
+
denvative of Pf-5, ma Gm,
I.
,
pitF:. aacCl pit. J: .inaZ
This study
,
derivative of Pf-5, 1na, Gm',
Kmr
Pf-5 p/tF.. aacCl,
pltJ::znaZ
JL4683
pitF:: aacCl pltJ:.inaZ
+
denvative of Pf-5, ma Gm,
,
This study
I.
,
KmT
Pf-5pltF:: aacCl,
pitI:.inaZ
JL4685
pltF:: aacCl,pitI::inaZ
This study
derivative of Pf-5, Jna, Gmr,
KmT
E. coli
S 17-1
DH5cx
Res, Mod, recA, Tra
V endAl hsdRl7 (rKmK)
supE44 thi-1 recAl gyrA96
re/Al 48OdiacZA Ml 5
-.
69
61
103
Table 4.2, continued
Plasmids
pRK415
pRK2O13
JncPl replicon, polylinker of
pUC19, Mob, Tc'.
Mobilizing plasmid, Tra, Kmr
pUC19
pMGm
ColE 1 replicon, Apr.
ColE 1 replicon, source of
pJEL6O33
32
21
61
47
aacCl (Gm') cassette on a 2.0kb Smal fragment, GmT, Tc'.
pJEL1938
pJELI939
pTn3 -nice
pJEL55O8
pJEL5569
29kbofPf-5DNAthat
complements a Plt mutant
cloned into pLAFR3, Tcr
Ca. 24 kb of Pf-5 DNA that
complements a Pit- mutant
cloned into pLAFR3, Tc'
pTn3-nice; pMB8 replicon,
contains Tn3 -nice, Ap', Kmr
12 kb HindIlI fragment from
pJEL1938, cloned into pUC18,
34
34
34
S. Carnegie
Apr
pJEL6 198
pJEL6O27
pJEL6I 19
pJEL6 120
pJIEL6 199
pJEL6200
1.7 kb PstI fragment from
pJEL1 938 containing pltF,
cloned into pUC19, Apr
8.1 kb HindIII-BamHI fragment
from pJEL1 939 containing
orJN, cloned into pUC19, Ap'
5.8 kb EcoRil fragment from
pJEL1 939 containing orfN,
cloned into pUC19, Apr
1.0 kb EcoRI fragment from
pJEL1 939 containing pltN,
cloned into pUC19, Apr
3.7 kb PstI fragment containing
p/IF disrupted by aacCl
cassette at an internal BsmI site,
contained in pUC19, ApT, Gmr
3.7 kb KpnI - Hindlil fragment
containing pltF disrupted by
aacCl cassette at an internal
BsmI site, cloned into pRK4l5,
TCr. GmT. Mob
This study
N. Chaney
This study
This study
This study
This study
104
Table 4.2, continued
Plasmids
pJEL6274
pJEL6277
pJEL6279
3.4 kb Smal - HindIH fragment
from pJEL1938 encompassing
pitI and pltJ, cloned into
pUC19, Apr
4.6 kb BamHI - PstI fragment
spanning inaZ gene from pTn3nice, cloned into pUC19, Apr,
lila
1.2 kb PstI fragment containing
nptl gene from pJEL5SO8,
cloned into PstI site of
This study
This study
This
study
pJEL6277, Apr, [r Jna
pJEL6283
6.2 kb BamHI - AfihII fragment
containing inaZ and nptl from
pJEL6279, inserted at the NruI
site internal to pltJ in
This study
6.2 kb BamHI - AflhII fragment
containing inaZ and nptl from
pJEL6279, inserted at the SphI
site internal to pltJ in
r Apr
pEJL6274,
6.2 kb BamHI - AJ1lII fragment
containing inaZ and nptl from
pJEL6279, inserted at the
BsaAI site internal to pitI in
pJEL6274, Kmr Ap'
pJEL6283 linearized at NdeI
site and fused to pRK415
linearized at EcoRI site, Kmr,
Tcr, Mob
pJEL6285 linearized at NdeI
site and fused to pRK4l5
linearized at EcoRI site, Kmr,
Tc', Mob
pJEL6287 linearized at NdeI
site and fused to pRK4l5
This study
pJEL6274, jr Apr
pJEL6285
pJEL6287
pJEL6298
pJEL6299
pJEL6300
This study
This study
This
study
This study
linearized at EcoRI site, Kf
Tc', Mob
aApr Gmr, Kr and Tcr indicate resistance to ampicillin, gentamycin, kanamycin, and
tetracycline, respectively. Pit indicates lack of pyoluteorin production. Ina indicates
ice nucleation activity at -5°C.
105
For experiments evaluating antibiotic production or gene transcription, cultures
were grown at 20°C with shaking (200 rpm) in nutrient broth (pH 6.8; Difco
Laboratories, Detroit, Mich.) without selection and supplemented with either 0.5%
(wlv) glycerol for NBGIy, a medium conducive to PLT production, or with 2% (w/v)
glucose for NBGIu, a medium repressive to PLT production by Pf-5 (34, 50).
Jnoculum for experimental cultures of Pf-5 and derivative strains was derived from
overnight cultures grown with shaking at 200 rpm at 27°C in KMB broth, modified by
omitting glycerol and substituting 1% (w/v) glucose (KBG1u) to repress PLT
production before inoculation of experimental media. Inoculum was washed twice in
sterile KBG1u prior to inoculation, and resuspended in 5 mis of NBGIy or NBGIu to
0D600
of 0.1. Where indicated in the Results, pure PLT dissolved in methanol was
added to the culture medium immediately prior to inoculation with Pf-5 or derivative
strains. An equivalent volume of methanol was added to negative controls. Bacterial
population densities were determined by spreading serial dilutions of cultures onto
KMB and counting CFUs on the plates after 48 h incubation at 27°C. In each
experiment, triplicate 5 ml cultures were grown and evaluated; each experiment was
performed at least twice; and the results from a representative experiment are
presented.
4.2.2. DNA manipulations
Restriction digests, phenol chloroform extraction and ethanol precipitation of
DNA, blunting of DNA fragment ends with the Kienow fragment or T4 polymerase,
dephosphorylating of DNA fragment ends with calf intestinal alkaline phosphatase,
and agarose gel electrophoresis were performed according to standard procedures (4).
Plasmids were isolated from E. coli DHSa using alkaline lysis (4) or the PerfectPrep
Plasmid Mini kit (Eppendorf AG, Hamburg). DNA fragments were extracted from
agarose gels using the QlAquick gel extraction kit (QIAGEN Inc., USA). E. co/i
DH5a and E. coli S 17-1 cells were made chemically competent by treatment with
CaCl2 prior
to transformation (4). Southern analysis was performed using DNA
probes labeled by random priming with either digoxigenin- 11 -dUTP using the Genius
kit (Roche Applied Science, Indianapolis, IN) or biotin-14-dCTP using the BioPrime
DNA Labeling System (Jnvitrogen, Carlsbad, CA). Hybridizations were performed
according to the manufacturers instructions. Custom oligonucleotides for sequencing
or for PCR were synthesized by Operon (Alameda, CA), Macromolecular Resources
(Colorado State University, Fort Collins, CO) or the Central Services Laboratory
(Oregon State University, Corvallis, OR). PCRs were performed with Taq polymerase
(Promega, Madison, WI), using the primers listed in Table 4.2. Reaction mixtures
were heated for 3 mm at 94°c, and then subjected to 35 cycles of 45 sec at 94°C, 45
sec at the indicated annealing temperature (Table 4.2), and 1.5 to 2.25 mm at 72°C
(depending on the product size), prior to a final elongation for 10 mm at 72°C.
Table 4.2. PCR primers used in this study
Target
gene
pitA
Forward primer
(5'-3')
cgagaaagccaacttccc
pltG
ctgttcacctttccctatgcc
pitH
atctatcaccgccagcatc
pitI
gtgtucctggtttctcc
plt.J
gggaactgtggagcatcatcg
orjK
atgcgtgacaagagcaacc
odP
attgctgttcggctacgg
orfQ
ctggtggcaggtgtttgg
oijU
cgaaatgctcggctacacc
oijX
gcccacacctattccaacc
orfY
ccaacgcagacaaccttcc
pltI::inaZ gtgtttcctggtttctcc
pitJ::inaZ
Reverse primer
Product
(5'-3')
size (l'p
951
ttcaggcggtaatagagcg
668
agcactgttttgacccgc
517
acgcagaaccagcagaag
943
aggtcttccgtttccacc
1039
atcgtcgtgccgctttgc
866
aagaggaaccccgagagc
857
gcagcggatcgagatagg
1083
cgctggtattcgttgtgg
aaaacagcggtccagaaacc 401
1000
tccctgctcgacgtactgc
ggcgtectcatacatcactagc 795
ccaacgccttgtcgagattcat 691
2139
Annealing
Tm(°C)
55
51
51
51
51
51
51
51
51
51
51
48
48
4.2.3. RT-PCR
For RT-PCR experiments, RNA was extracted from Pf-5 and derivative strains
during mid-log phase. RNA was extracted from cultures in two different experiments
107
using the RNAqueous kit from Ambion (Austin, TX). RNA templates were LiCl2
precipitated and then treated twice with DNaseI according to the manufacturer's
recommendations for the DNA-free kit (Ambion, Austin, TX). Samples from each
experiment were subjected to reverse transcription (RT) to derive cDNA, using
SuperScript 11 RNaseH reverse transcriptase (Invitrogen, Carlsbad, CA) according to
the manufacturer's recommendations. Amplification from 100
.tg
of eDNA template
was performed on a Robocycler Gradient 96 Temperature Cycler (Stratagene, La Jolla,
CA), using conditions described above. PCRs were also performed on genomic DNA
templates for positive controls, and on RNA templates not treated with reverse
transcriptase, as controls to indicate contamination of RNA and subsequent cDNA
samples with genomic DNA. RNA and RT-PCR products were stored at 80°C.
4.2.4. Nucleotide sequencing of pitH, pitl, and pit.!
Two regions flanking the PLT biosynthetic genes in Pf-5 were represented by
pJEL6 130, pJLE6246, and pJEL6247, and by pJEL6O27, pJEL61 19, and pJEL6I2O,
respectively (Fig. 4.1). Sequencing of the regions was initiated using primers
complementary to the pUC19 multiple cloning site, and was then continued by primer
walking: information from each consecutive sequencing run in a particular direction
was used to design a the sequencing primer to begin the next sequencing run, until
there were runs spanning the entire insert in the desired direction. Nucleotide
sequencing reactions were carried out at the Central Services Laboratory (Oregon
State University, Corvallis, OR), using ABI Prism BigDye Terminator Cycle
Sequencing chemistry with Amplilaq DNA polymerase, and separated on ABI model
377 and 373A sequencers (Perkin-Elmer, Applied Biosciences Division). Sequence
information for the region was considered reliable when a minimum of 3X coverage,
with sequencing runs in both directions, was available for the entire region.
Assembly, contig joining, and contig editing were performed using the Staden package
(71). Finished consensus sequences were analyzed using the algorithms of the
University of Wisconsin Genetics Computer Group (version 10). Predicted amino
ID1
acid sequences of each gene were searched for patterns, profiles, and motifs listed in
the Pfam, ProSite, and InterPro databases (8, 68, 84) for predictions about
functionality. Transmembrane regions were predicted by the Dense Alignment
Surface method (17) and the "positive inside" rule (14).
4.2.5. Derivation of chromosomal pltI::inaZ and ylt.J::inaZ transcriptional
fusions by marker exchanie mutaenesis
Strains bearing chromosomal transcriptional fusions of the promoterless ice
nucleation reporter gene, inaZ (38) within pltI and p1LJwere constructed as follows
(also see Fig. 4.2 for an example of the construction of pJEL6300, used to create Pf-5
pltI::inaZ via marker-exchange mutagenesis). The plasmid pJEL5 569 is pUC 18
containing a 12 kb HindIll fragment from pJEL 1938 and spanning the right end (as
depicted in Fig. 4.1) of the PLT biosynthetic gene cluster. The 3.4 kb SmaI-HindIII
fragment from pJEL5569 was excised and cloned into pUCI9 linearized with SmaI
and Hindlil. The resultant plasmid, pJEL6274, was checked for directionality of
inserts by restriction digest mapping. A 4.6 kb BainHI-PstI fragment from pTn3-nice
that contains inaZ was excised and cloned directionally into pUC 19 linearized with
BamHI and PstI, to yield pJEL6277. The 1.2 kb PstI fragment containing the nptl
gene originally derived from Tn903 was also excised from pTn3 -nice, and cloned into
pJEL6277 that had been linearized with Pstl. The resultant plasmid, pJEL6279, was
checked for directionality of the nptl insert by restriction mapping. Before
proceeding, pJEL6279 was also tested for ice nucleation activity (INA) conferred by
inaZ fused to the lacZ promoter. A 6.2 kb BamHI - AfthII fragment from pJEL6279
encompassing the inaZ and nptl genes was excised, and cloned via blunt-ended
ligation into the BsaAl site of pill in pJEL6274 and into the SphI site of pltJ in
pJEL6274. Resultant plasmids (pJEL6287 and pJEL6285, respectively) were checked
for directionality of the insert by restriction digest mapping and by PCR using a
primer complementary to the first 22 nt of the inaZ ORF in combination with a primer
comprising nt 53-70 of the phI gene (Table 4.2). The pltI::inaZ and pltJ::inaZ
transcriptional fusions were introduced into the chromosome of P1-5 by marker
109
exchange mutagenesis. For this purpose, constructs were moved into pRK41 5, which
is unstable in Pf-5. Briefly, plasmids pJEL6285 and pJEL6287 were linearized by
digesting a unique Ndel site. The resultant fragments were blunted with T4 DNA
polymerase, and ligated to EcoRI-linearized, T4 DNA polymerase-blunted, and
dephosphorylated pRK415. The resultant plasmids (pJEL6299 and pJEL6300) were
transformed into chemically competent E. co/i Si 7-1 cells. Putative transformants
were verified by restriction digest mapping of the re-extracted plasmids. pJEL6299
and pJEL6300 were mobilized from E. co/i S 17-1 into Pf-5, Pf-5 pltR::aacCl and Pf-
5 pltF::aacCl. Putative transconjugants were screened by plating on KMB containing
streptomycin (to which Pf-5, but not the E. coli donor, is naturally resistant) and
kanamycin and tetracycline (to select for plasmid transfer to Pf-5 derivatives).
Verification that putative transconjugants were Pf-5 derivatives and notE. co/i strains
was achieved by patching putative transconjugants onto KMB containing
spectinomycin, to which Pf-5 is naturally resistant. Single colonies were then
inoculated into KMB broth with streptomycin and kanamycin, but no tetracycline, to
maintain selection for the nptl insertion but not for the pRK415 derivative plasmid.
After three days, serial dilutions were plated onto KMB containing streptomycin and
kanamycin; single colonies from these platings were patched onto KMB containing
streptomycin and kanamycin, and onto replica plates containing streptomycin,
kanamycin, and tetracycline. Genomic DNA was extracted from strains that were
resistant to streptomycin and kanamycin but sensitive to tetracycline, and insertions
were further verified via PCR using the p111-F and inaZ-R primers described above
(Table 4.2), and by Southern analysis using PCR-generated (Table 4.2) probes
complimentary to p/tI and pltJ, labeled as described above.
110
Figure 4.2. Cloning strategy used to construct pJEL6300, which was used in markerexchange mutagenesis to derive Pf-5 pltI::inaZ. Numbers in parentheses indicate
distance of restriction sites from 5' end of insert (in pJEL5569) or from bp 1 ofpUCl9
or a pUC19 derivative, when known. Block arrows or thickened lines represent open
reading frames and denote directionality, when known. Checkered spaces indicate
noncoding regions. Elements within each plasmid are drawn to size with respect to
that individual plasmid, but plasmids are not drawn to size with respect to other
plasmids.
$aml-H (4099)
saAI (4224)
SmaI (3034)
BamHl (578)
PstJ (2369)
EcoRl (32)
$amHI (4)
Smal (4cI)
SphI (74) \\\
i
coRI (4311)
(4845)
\
rul (4869)
$amHI (4981)
PstI (224 )
SmaI)
,PstI (5310)
/ SphI (5620) HiudIII (6470)
I
I
[[
ii
4
S
pUCl
9
EcoRJ (3)
ma I (4i)
amHI (418)
\\PstI (440)
\$phl (446)
HindIIL (448)
region flanking PLT gene cluster
(3' end of inse in pJEL5569)
i/HindIii digest of pUCI9
ligation
SmaiIHindUi digest of pJEL5569
TSmaI(415)
I
pJEL6274 JBamHI (1480)
BsaAJ (1605)
6089 bp
EcoRI (1692)
oriksp
(2226)
Hu1III (3851)
NruI (2250)
BamHI (2362)
PstI (2691)
Sh 1(3001)
Fig. 2
Continued on next page
NdeI
BsaAl digest of pJEL6274
On
Two fragments from pJEL55O8 cloned into pUCI9:
I
4.6 kb
and
BamHlIPstI fragment containing inaZ
Pstl fragment containing npt
F'Iac
ligation
1.2 kb
pJEL6287
12.2kb
,BamHI
Digest pJEL6279 with BamHl, AfIlIl to
yield 6.2 kb cassette containing inaZ and nptl.
Blunt ends.
tIüxlHI
Ori/'''_
ti
PstI
8.4 kb
IIlkLac
HindIH
ligation
digest to linearize.
Blunt ends.
Tr
N
1415
10.5kb
/ I \
IlindIH PstI BamHI EcoRI
Fig. 2, continued
113
4.2.6. Derivation of chromosomal pltF:: aacCl mutant by marker exchan2e
mutaenesis
The constructs described above for assessing transcriptional activity of p/tI and
plt.J include an nptl gene that confers kanamycin resistance. To assess the activity of
these transcriptional reporter genes in a Pf-5 derivative bearing a mutation in aplt
structural gene, it was necessary to disrupt the desired pit structural gene with a
cassette other than one conferring kanamycin resistance. The pltF gene, which is
predicted to encode an acyl-CoA synthetase necessary for precursor activation during
induction of PLT biosynthesis (49), was mutagenized by insertion of a gentamycin
resistance-conferring gene, aacCl. A 1.7 kb PstI fragment containing the pltF gene
and flanking sequences was cloned into pUC19 to yield pJEL6 198, and the aacCl
gene was transferred on a 2.0 kb SmaI fragment from pMGm (47) into a blunted BsmI
site located between nt 937 - 942 of the pltF gene, to yield pJEL6 199. The insert
disrupts the predicted sequence of PIItF five amino acid residues beyond the conserved
core D sequence shared by adenylate-forming enzymes (49). The integrity of the
constructs was verified by restriction digest mapping and by nucleotide sequence
analysis. A 3.7 kb HindIII KpnI fragment containing the resultant pltF::aacCl was
cloned into pRK4I5 to make pJEL6200. pJEL6200 was mobilized from E. co/i DH5a
into Pf-5 via triparental mating, using pRK2Ol3 as a helper plasmid, for marker-
exchange mutagenesis of the chromosomal pltF in Pf-5. Transconjugants were grown
on KMB containing gentamycin (to select for pJIEL6200 or for the subsequent
incorporation ofpltF:: aacCl into the chromosome) and streptomycin (to select
against the E. co/i strains) and replica plated on KMB containing tetracycline (200
.tg/ml) to test for loss of the pJEL6200. Chromosomal mutations of gentamycin
resistant, tetracycline sensitive strains were confirmed by Southern analysis as
described below, and evaluated for PLT production.
114
4.2.7. Antibiotic guantification
Cultures were centrifuged (5,000 X g, 5 mm) to pellet cells, and supematants
were extracted for recovery of PLT and 2,4-DAPG while cell pellets were extracted
separately for pyrrolnitrin (PRN). Culture supematants were acidified to pH
2.0
with 1M HCI, extracted twice in 0.4 volume of ethyl acetate, and the extracts were
pooled. Cell pellets were suspended in 5ml acetone, vortexed for 10 sec, placed in a
sonicating bath for 30 sec, and subsequently centrifuged (10,000 X g, 10 mm) to
separate the cell debris from the organic extract. Ethyl acetate and acetone were both
removed from extracts under reduced pressure, and the resultant residues were
resuspended in 100 tL methanol, from which 10 .tL samples were evaluated by high
performance liquid chromatography (HPLC). Samples were separated over a Nova-
Pak C18 reversed-phase colunm (Waters Corporation, Milford, MA) eluted (1 mi/mm)
with 90% water + 0.1% acetic acid:10% acetonitrile + 0.1% acetic acid (v/v) for two
minutes, followed by a linear gradient that changed from 90% water + 0.1% acetic
acid:10% acetonitrile + 0.1% acetic acid to 100% acetonitrile + 0.1% acetic acid over
18 mm. Antibiotics were detected with a photodiode array detector (PLT, X = 310 nm
and retention time (ti) = 13.1 mm; 2,4-DAPG, ? = 270 mn and
t1
=15.6 mm; PRN, 2
254 nm and tr = 17.9 mm). Quantification was done by comparison to a standard
curve generated from authentic compounds. The detection limit was 0.02 p.g/ml for
PLT, 0.01 tg/ml for 2,4-DAPG, and 0.01 tg/ml for PRN. Recovery of compounds
using the extraction procedures described above averaged 70% for PLT, 45% for 2,4DAPG, and 77% for PRN.
4.2.8. Transcriptional activity from PLT biosynthetic genes
Transcriptional fusions of a promoterless inaZ gene to several PLT
biosynthetic genes of Pf-5 were constructed previously (34). 1NA activity from strains
carrying the inaZ reporter gene was assessed as described in Chapter 2 and in
reference 11.
115
4.2.9. Statistical analysis
Treatment means were compared using a Student's t-test following an analysis
of variance. Two-tailed P values are reported. Statistical analyses were performed
using JMP, version 3 (SAS Institute, Cary, NC).
4.3. Results
4.3.1. Nucleotide seguence analysis
Nucleotide sequence was determined for the region of the Pf-5 genome
spanned by pJEL6246, pJEL6247, and pJEL6 130, and by pJEL6 120 and portions of
pJEL6I 19 and pJEL6O27 (Fig. 4.1), yielding information extending 3776 bp beyond
the 3' end of pltG and 1618 bp beyond the 3' end of pltM. Codon preference analysis
(81) was used to identify three open reading frames (ORFs) designated pitH, pitI and,
pit..!. Fig. 4.3 describes salient characteristics of features of all three ORFs.
116
pltF
pitH
pitJ
orfP
)
pitG pltI
orfK
ortu
art Q
artY
H-4
orfl
ORF size (at)
Predicted
protein size (aa)
Putative protein function
pitH
636
211
TetR-tWe transcriptional regulator
pill
1014
337
HIyD homolog (membrane fission protein)
p11.1
1770
589
Cytoplasmic ATP-binding cassette of
ABC transporter
orfk
1149
382
Transmembrane subunit of ABC
transporter
oiJP
1119
372
Transmembrane subunit of ABC
transporter
oifQ
1497
498
Outer membrane protein or channel
orf U
606
201
LysE-type outer membrane channel
oiJX
1146
381
Zinc-binding oxidoreductase
orfY
879
292
Chloroperoxidase
Figure 4.3. Map and features of open reading frames flanking one end of the PLT
biosynthetic gene region. pltF and pUG are previously described PLT biosynthetic
genes (49).
117
4.3.1.1. orfN
I partially sequenced (1491 bp) an open reading frame downstream of pltM
that I have designated orJN. The partial sequence of orJN gene reveals a gene whose
translated product is highly similar (88% - 93% amino acid identity) to proteins from
Pseudomonas spp. and the closely related genera Azotobacter and Raistonia, that
belong to both the OsmC/Ohr family (accession number PF02566) and to COG! 944,
a family of conserved hypothetical proteins with unknown function. OsmC is an
osmotically inducible protein; Ohr is induced by organic hydroperoxides and
detoxifies these compounds (3, 46). Because no homologs with predicted protein
functions were found for orjN at the time of original analysis, the involvement of orJN
in PLT production has not been experimentally tested.
4.3.1.2. pitH.
Sequence analysis downstream of pltG revealed a convergently transcribed
ORF designated pitH. PSI-BLAST searching (2) revealed sequence similarity (35%
identity and 60% similarity at the amino acid level; Fig. 4.4a) to PhIH (GenBank
accession number AF207529), a putative TetR transcriptional regulator from the 2,4-
diacetlyiphioroglucinol gene cluster of P. fluorescens CHAO. The predicted amino
acid sequence contains an N-terminal DNA binding motif (Fig. 4.4b) that typifies the
TetR family of transcriptional regulators (Pfam accession number PFOO4O). A
potential transmembrane region between amino acid residues 174 and 185 (Fig. 4.4c)
suggests that PItH may be anchored within the cytoplasmic membrane.
118
a.
PitH: 3
62
T
Ph1H: 10
PitH: 63
Ph1H: 70
RT R DG T+ -*-ILE A +LFA+ GYP,NT SK ICE + A+ A+NYHF +++ LY
TAARTPRQDGQATRQQILETAGQLFAELGyANTTSKQI CENSRNASVNYHFENKDGLY 69
ICAVLIEGHKQLVSFEALSQLAQSEEPALIKLIJSFIDAIVTRVLDE-QSWQSKVCAREILA 121
+AVL H H LV H +
Kb + I ++ + ++ + W XV RE+L+
RAVLREAHDRLVRIETMISLSESQGSPEDKLRAI ITVLIEGLGNQREGWALKVLTRELLS 129
L+S
PitH: 122 PTVHFTSLVQEEVMPKFRLLHALISEITGFPIGDPALARCTISIIAPCLMLAVIDRQQPS 181
P+
++++E+ 91< +++ ++ +1
DP
R +S+ APCL L + +
Ph1H: 130 PSAVLPAILEEQAFPKAKIVRTILGQIMDLSPEDPVTLRSAVSVFAPCLFLLIAHQPLAR 189
PitH: 182 PLQAVLQHDANALKAHFKLFARSGLAAIAQ 211
+
L+ D AL H
+A +GL+A+++
Ph1H: 190 NVLPGLELDPPALIEHMMSYALAGLSAI4SR 219
b.
Consensus
ILdAAiebfaekGydat svrelAkrAGvskgaiYrhFksKeebl lal
IL+A++Lfa+Gy+t
I ++AG
+a+ +hFs e+L+ a+
31 ILEVAARLFAQHGYANTASKLICEEAGADLAAINYHFGSREALYKIW 77
PitH
C.
"DAS
n4-segnet uredictio
I-,
0
S
0)
0
0
ID
0
50
100
150
200
250
Query sequence
loose cutoff -- - -
strict cutoff
Figure 4.4. a. Alignment of predicted amino acid sequence of PitH with that of the
PhJH sequence from P.fluorescens CHAO. The conserved helix-turn-helix motif is
shown in bold characters. b. Alignment of PItH with the consensus DNA-binding
domain for TetR family proteins (8). Capital letters designate residues aligned to
match states in the profile hidden Markov model. c. Prediction of a PitH
transmembrane domain by the Dense Alignment Surface (DAS) method.
119
4.3.1.3.
LL
The ORF adjacent to and divergently transcribed from pitH is designated p/tI,
and shares similarity at the amino acid level (E= 0.0001) across its entire length with
the HIyD family of secretion proteins (Pfam accession number PF00529); Fig. 4.5a).
A single transmembrane domain is predicted for PitI, between amino acid residues 6
and 21 (Fig. 4.5b). The predicted PitI amino acid sequence is similar to numerous
hypothetical and confirmed membrane fusion proteins, which are components of
multidrug efflux pumps of the major facilitator superfamily. One of these is FusE, a
fusaric acid resistance protein from E. co/i (GenBank accession no. BAA1 5404.1; Evalue = 0.002).
120
a.
consensus
pitI
rskevqpqvsGiVieilvkeGdrvkaGdvLvrlflptpaeaala.
r+++
s ++
+ ++eGd+V GvL+1D ++++ + ++++
42
consensus
RQVSL.FDGSERIAALYEEGDLVQPGQVLAELDTRTLRLEINrska 88
raeaelalaqadaaqarlaaekldrlqalvlpaaiq. asaqeaaaarral
r a+ +++ +++
r +++ ++ +a++ +a++q 4--i-ag
++r
89 RIGAQEQALLRLKNGTRPQEV- -EQSKARFDAAQAQtnQLAQLHMQRLR-
p1 tI
consensus
134
realaasqrealaaskaqleaardnleaakatlpasiak ......... ig
r-i-a
+
+s+i-+1+-i-a -i--i-i-i- aka+++ ++
+ + +++
135 RIADDTQG- - - KGVSQQRLDQAAARLQVAKAQSQEQRESwt lakigprNE 181
p1 tI
consensus
risasqatlgaqltntllrklqaqglaakaqldeaylqlaetaaellrli
+i++++a Li-a ++
id + la+++
+
182 DIAQATADLQASRAD ---------------- LDLLEHYLARAQ ------ L 209
pitI
consensus
pitI
rapaqgervsleakersvqvgqevgsevgypLiugvvpfndvevdanl eea
+ap g+
++++r+-4-+g+ ++ + p-I-+++
+v a++ e
210 KPTQA ----- RIRTRLLEPGDMAS--PQRPVFALALTDPKWITRAYVNER 252
consensus
qlrlvrsgiraplsggvyvlvvhteGgvvgagei. . lmpivpvdrllpve
+ a++ + +++
G+v
i++
+p++ + ++
253 QLGHIRADQMMWYTDSFPD-QAIDGKVGY- --Is5VAEFTPKSVETED- 297
ql +-i-r
pitI
consensus
pitI
4
aqvavvnidkvvqglpVeikldpgafnqtttplvpGevviv
+++ +
++ ++ 1+-i- + V
+++++++
298 --LRTSL ------ VYEIRVLVK---DPDDRL.RLGMPATVYL
327
b.
TM-sequent ztrediction
DAS
0
Pa
Pt
0Pa
I-
I-.
S
0
Pt
S
0
50
100
150
200
250
300
350
Query sequence
loose cutoff
strict cutoff
-- -
Figure 4.5. a. Alignment of predicted amino acid sequence of PitT with HIyD
consensus domain (8). Capital letters designate residues aligned to match states in the
profile hidden Markov model. An arrow designates site of insertion of the inaZ
cassette in Pf-5 pltI::inaZ derivatives. b. Prediction of a PitI transmembrane domain
by the DAS method.
121
4.3.1.4.
itL
Adjacent to pill and transcribed from the same strand is pltL The pltJORF
overlaps the last four nucleotides of p/tI, and also lacks a consensus Shine-Dalgarno
sequence preceding the ATG start codon. Two separate consensus ABC transporter
ATP-binding motifs (Pfam accession number PF00005) reside inpltf (Fig. 4.6).
Expect (E) values for the consensus with the model were 1.44 e55 and 3.8 e55 for the
N-terminal and C-terminal ABC transporter motifs, respectively. The nucleotide
binding site comprises a Walker A box, a Walker B box, and a switch region (28, 48,
63, 79). Both ABC transporter motifs in pltJ include the y-phosphate-binding Walker
A box (accession number PD0000017) and magnesium-binding Walker B box, as
well as a switch region thought to be important for ATP hydrolysis. Also present in
each ABC transporter motif ofpltj are ABC transporter signature motifs (previously
termed linker peptides) next to the Walker B boxes. For ABC transporters involved
in import of substrates, the signature motif is proposed to be involved in interactions
with periplasmic substrate binding proteins (9). A single switch region (9) follows the
Walker B box in the C-terminal ABC transporter motif. There were no apparent
transmembrane domains, indicating thatpltjis likely to be cytoplasmic.
122
N-terminal
ATP-binding domain
consensus
pltJ
Gev1aIv3pNGaGKSrLLk1isG11ppteGt illdGardlsdlsklk
G++ alvfp GaGK+IrLL+iG11+ +eG +++ G d++ +
GKLSALVPDGAGK*LLR4IAGLLKADEGLLKVLG-LDVAADP- 79
37
Walker A
consensus
pltJ
erleslrknigvvfQdptlfpnpeltvreniafglrlslglskdeqddrl
++ + i++ +Q +1+
lt++en+ ++ 1
+ ++++
80 --QQVQDL-ISYMPQKFGLYE--DLTIQENLELYADL ------ HGVRAQQ 118
consensus
p1 tJ
kkagaee1Ler1g1gydd11drrptLsGGqkQRvafiARaL1tkpk.L1L
4+4+
IL i-+ 1+ +++dr ++LsGG+kQ44+A4 L++ p LlL
119 REERFSRLLQMMDLT- RFRDRLAdOLSGGMKpKL(ACTLVRSPQtLLL_166
Signature motif
consensus
p1 tJ
DEPTagLDasraq11e11re1rqq . ggTvllitHdldlldrlaDrilvl
DEPT g D sr++1+ ++ +1 q++ Tvl+t ++d+ ++ + ++vl
167 DEPTVGVD'LSRRELWSIIEQLIEQeNLTVLISTAYMDE-AQRCAQVFVL 215
Walker B
edG
pltJ
216 YQG
218
C-terminal ATP-bindiug domain
consensus
pltJ
Gevlal pNGaGKS LLklisGllppteGtilldGardlsdlsklk
Ge+ +1+ pNGaGK+ ++++Gllp G + + G
1++
GEIFGL PNG GK
FRMLCGLLPASSGYLEVAG-VNLRTA---- 408
Walker A
367
consensus
p1 tJ
erleslrknigvvfQdptlfpnpeltvreniafglrlslglskdeqddrl
++ r++ig+v+Q
1+
+1+++en++f++
++++r
gi
409 -RAAARRKIGYVSQKFALYA- -NLSALENLRFFGGA-YGLHGAKLKAR- 452
consensus
p1 tJ
453
kkagaeelLerlglgyddlldr
+ +Le+ 1+ + + r
-
-VALMLEQFDLD- -EHKHLRS
LSGGq QRV ARaL1t3cp4LI
L GG kQR+ A aLl++p L+L
LPGGYK R
VALLHEPQJ
Signature motif
496
consensus
p1 tJ
DEPTagLDasraq11e11re1rqqgTv1l itRdldlldrilaDrilvle
DEPT+g+E4 +r++++ + +1 q +T+++tH +4+ +.4Dri+ +
497 DEPTSGIDLARRAFWRRITALAQSTTIVITTHFMEE-AE'4CDRIVIQD 545
Walker B
Switch region
consensus
pltJ
dG
546 AG
547
Figure 4.6. Alignment of predicted amino acid sequence of P1tJ with the consensus
ABC-binding cassette of ABC transporter proteins (8). Capital letters designate
residues aligned to match states in the profile hidden Markov model. Regions
important for nucleotide binding are boxed and identified in bold typeface. The
conserved histidine residue in the switch region is boWed. An arrow designates site of
insertion of the inaZ cassette in Pf-5 pltJ::inaZ derivatives.
123
4.3.1.5. Analysis of adjoining sequence
Analysis of preliminary data from the unfinished genome of Pf-5
(http://www.tigr.org) revealed that four more ORFs reside downstream ofpltf and are
transcribed from the same strand (Fig. 4.3). These include, in order of increasing
distance from pltJ, two consecutive ABC transporter transmembrane proteins (orfK
and orfP), an outer membrane efflux protein (orfQ), and a LysE-type efflux protein,
orf U, with homology to cmaU (GenBank accession number U 14657) from the
coronatine biosynthesis pathway in P.
syringae
pv. glycinea. The closest homologs of
pitH, p/tI, pltj, orJK, and orJP (but not pltG or orfQ) in the extant databases reside in a
cluster of genes with identical synteny, from Azotobacter
vinelandii (GenBank
accession numbers ZP_00090839.1 through ZP 00090843.1). Expect (H) values for
similarity gene pairs in the Pf-5 cluster and the A.
vinelandii
cluster ranged from 3e-41
(pitH! ZP 00090839); 45% identical and 62% similar at the amino acid level, to 0.0
(pltJ/ ZP00090841.l); 65% identical and 76% similar at the amino acid level.
4.3.2. Phenotypic characterization of pill, pltJ mutants
Pf-5 derivatives bearing chromosomalpltl::inaZ orpitf::inaZ disruptions
accumulated approximately 4.7 times less PLT in their culture supernatants than did
wild-type Pf-5 at 48 hours (P = 0.011) (Fig. 4.7a). PLT concentrations in cell extracts
ofpltl and pkJ derivatives were, similarly, 5.2 times lower (P = 0.0085) than PLT
concentrations in cell extracts of Pf-5 (Fig. 4.7b). Population sizes (not shown) did
not differ significantly among strains at any time point (P
0.43).
124
2.0
PLT detected in supernatant extracts
13
1.0
0.5
0.0
0.16
lb. PLT detected in cell extracts
0.14
0.12
0.10
0.08
I-
0.06
0.04
0.02
0.00
:1
F
1'
Figure 4.7. PLT production by Pf-5 and bypltl::inaZ,plt.J::inaZ, and pltB:.inaZ
derivatives of Pf-5. Cultures were grown for 48 h in NBGIy before harvesting and
extracting supernatants (a) and cells (b) to determine PLT concentrations. Both panels
represent data from the same experiment. Error bars denote one standard error.
125
I examined whether the effects on PLT production could be attributable to
polar effects on transcription caused by the inaZ insertion mutations in p/ti and pltJ.
RT-PCR was utilized to test transcription of pltJ in apltl mutant and in wild-type Pf-5.
Amplification pkl cDNA occurred reproducibly in the wild type but not the pitI
mutant strain (Fig. 4.8). This result raises the possibility that the inaZ reporter gene
used to disrupt pill and pltJ has polar effects on transcription of downstream genes.
Although p/tI: :inaZ and plt.J::inaZ derivative strains have utility as transcriptional
reporter strains, the phenotypes of these strains cannot be attributed to the p/ti and plt.J
mutations without further analysis.
126
Pf-5
Pf-5 pltI::inaZ
I-
z
z
z
12,000
5,000
2,000
1,600
1,000
4
plt.J
850
650
500
Figure 4.8. RT-PCR products obtained from pltJ forward and
reverse primers (Table 4.2) and the templates indicated above each
lane. Templates were: lane 2, Pf-5 genomic DNA; lane 3, Pf-5
cDNA; lane 4, Pf-5 RNA; lane 5, none; lane 6, Pf-5 pltI::inaZ
genomic DNA; lane 7, Pf-5 pill: :inaZ cDNA; lane 8, Pf-5 p/tI: :inaZ
RNA. Lane 1 contains a 1 kb MW ladder, with band sizes indicated
on the left margin. The expected migration destination for the pltJ
PCR product is depicted by an arrow.
127
4.3.2.1. PLT auloinduction is inhibited in a pill derivative of P1-5
Recently, PLT has been shown to positively regulate its own production (11).
Although pitI and pltJ mutants produce PLT, they do so at a reduced level. Therefore,
I hypothesized that reduced PLT production inpltl and pltJmutants might be due to
defective PLT autoinduction. To test this hypothesis, I compared the capacity of Pf-5
plt.J::inaZ for PLT autoinduction with that of Pf-5 (Fig. 4.9). Pf-5 and Pf-5 pltJ::ina.Z
were grown in NBGIy without amendment and in NBG1y to which 4 .tg/ml PLT had
been added. The non-PLT producer Pf-5 pltB::inaZ
was
included in each treatment to
demonstrate the fraction of PLT from the original amendment detectable in culture
supernatants. In Pf-5, PLT production (calculated as {PLTJ in supematant minus
[PLT} detectable in supernatants of Pf-5 pltB::inaZ) in PLT-amended cultures were
2.8 times greater than those in non-amended cultures by 48 hours. However, no such
enhancing effect by PLT was observed on PLT production in apltJ mutant. Pf-5
pltJ::inaZ produced a small amount of PLT (0.53 .Lg/ml at 48 h), but PLT amendment
did not increase PLT production by Pf-5 pltJ::inaZ. Rather, PLT detected in culture
supernatants of PLT-amended Pf-5 plt.J::inaZ was less than or equal to that detected in
the P1t strain Pf-5 pltB::inaZ at all time points. There were no differences in
population sizes between PLT-amended or non-amended cultures at any time points.
128
10 a.
8
16
I-
2
0
le+1O
b.
le+9
E
U
v
U
0
v-- Pf-5 plt&:inaZ
le+8
le+7
0
6
12
18
24
30
36
Pf-SpltB::inaZ + PLT
Pf-5 pItJ.:inaZ
Pf-5pltJ::inaZ + PLT
42
48
Culture age (hours)
Figure 4.9. Influence of exogenous PLT on PLT production by Pf-5, and by Pf-5
pltJ::inaZ and Pf-5 pltB::inaZ. Cultures were grown in nonamended NBGIy or in
NIBG1y amended with 4 tg/ml PLT. At the indicated sampling times following
inoculation, PLT concentrations (a) and population densities of cultures (b) were
assessed. Population sizes did not differ significantly between PLT-amended and
nonamended treatments at any time point. Error bars denote one standard error.
129
4.3.2.2. Expression
ofpltl and
pltJ is inhibited in Pl( strains
Within the PLT biosynthetic gene cluster ispltR, a gene encoding a predicted
LysR-type transcriptional regulator that is necessary for PLT biosynthetic gene
transcription. A shared feature of the promoter targets for LysR-type transcriptional
regulators is an inverted repeat built around the core sequence T-N11-A. These dyad
sequences are most often located 50-60 bp upstream of the regulated gene (23). One
such dyad (Fig. 4.10) exists 32 bp upstream of pltR in the pltR-pltL intergenic region,
and has potential to form a stem-loop structure (G -2.9). Thep/tH-pltlintergenic
region is 111 bp in size and contains an inverted repeat sequence similar to one found
in the pltR-pltL intergenic region (Fig. 4.10), which was previously proposed to be a
P1tR-binding site (49). The potential PItR-binding site in the pitH-p itI intergenic
region resides 54 bp upstream of pitH and 40 bp upstream of the divergently
transcribed piti. The dyad has no predicted stem-loop potential surrounding the TN1 1-A core sequence, unlike that of its counterpart in the pltR-pltL intergenic region
(G = -2.9), but rather forms one side of a larger stem-loop (G -7.5). The
possibility of PltR binding to the pitH-pill intergenic region led me to test the effect of
pltR on transcription of p/ti and pltJ.
p1 tR -p1 tL:
TGTCTTATTTTTGTAAAGGCAAATTACAAATAGATCATGAC
p1 tH-pl tI:
TCTGTTATTT TCTAATTTAAATTCAAATTGAATTTTAATT
IIIIiIIlIIII
,I.11
II
I
I
Figure 4.10. Possible PItR binding sites from the pltR-pltL and pitH-p itI intergenic
regions. Dyad regions are depicted in bold, and the center of symmetry is in large
font. The T-N11-A core sequence is underlined. Each complete predicted stem-loop is
indicated by converging arrows. Identical nucleotides are indicated by vertical lines.
130
I evaluated chromosomal transcriptional reporter gene fusions to pill and pltJ
in wild-type (Plt) Pf-5, and inpltR and pltF backgrounds (both Plt)(Fig. 4.11).
After 18 h of growth, expression of 1NA from both pltl::inaZ and pltJ::inaZ was
approximately one order of magnitude higher in the Pf-5 wild type background than
INA from the same fusions in apltR orpltF background (P 0.025). The expression
of 1NA from Pf-5 pltB::inaZ was assayed simultaneously to compare activity from the
pltI::inaZ and pltJ::inaZ fusions with activity of a similar fusion to a PLT
biosynthetic gene. Expression of INA from pltB::inaZ was at least three orders of
magnitude greater than INA from eitherpltl::inaZ orpltJ::inaZ at all times. INA
expressed from pltI::inaZ was lower than that expressed from pltJ::inaZ, exceeding
background levels (P < 0.05) in only case (Pf-5 pltI.:inaZ at I 8h). At no time were
population sizes significantly different among strains; thus, growth differences among
strains do not account for differences in INA.
131
-L
-h
*-- Pf-5pltB::inaZ
-3
-4
-5
-6
-7
-8
-9
-In
C.?
1e4-1O
le-+-1O
Ie4
le+9
Ie+8
le+8
-I
le+7
Ie+7
J
Ie+1O
19
Ie+8
P111
d.
Ie-I-6
6
12
18
le+7
I
1e-1-6
-j
-1
pitt
Ie-I-6
0
I
24
0
6
12
18
pit.!
24
0
6
12
18
Culture age (hours)
Figure 4.11. NA activity from pltB::inaZ (a),pltI.:inaZ (b), and pltJ::inaZ (c)
transcriptional fusions in wild-type (circles), pltF::aacCl (triangles) orpltR::aacCl
(squares) backgrounds. The parental Pf-5 (Ina; open circles) was included as a
negative control (a). Cultures were grown in NBG1y and harvested at 1, 6, and 18
hours for assessment of NA (a-c) and population densities (d-e). Population sizes
did not differ significantly among strains at any time point. Error bars denote one
standard error.
24
132
To evaluate the possibility that the difference between INA expression of Pf-5
pltI::inaZ and Pf-5 pltJ::inaZ derivatives was due to positional effects of the inaZ
insertion, a second pltJ::inaZ fusion, bearing the inaZ insertion at the upstream Nrul
site ofpltf (Fig. 4.2), was tested. INA expressed by Pf-5 bearing the inaZ insertion at
the NruI site of pit..! lower than that expressed by Pf-5 bearing the inaZ insertion at the
SphI site ofplt.J and essentially identical to 1NA expressed from Pf-5 pltI::inaZ.
These results indicate that the difference in expression levels between Pf-5 pltI::inaZ
and Pf-5 plt.J::inaZ are likely to be artefactual, resulting from posttranscriptional
effects due to placement of the reporter construct within the transcript (see Section
4.4.5). Only results from pltJmutants bearing the inaZ insertion at the SphI site are
reported in all experiments, for brevity.
4.3.2.3. Expression ofpltl and pit..! in Pl( strains is restored by exogenous FL T
Because pltR and pltF mutants both exhibit a P11 phenotype, I hypothesized
that the effects of pltR and pltF mutations on pitI and pltJ transcription might be due
to a requirement for PLT. When cells were cultured in NBGIy to which 4 .tg/m1 PLT
had been added at the time of inoculation, INA from both plt.J::inaZ (Fig. 4.12) and
pltI::inaZ (Fig. 4.13) was enhanced in wild type, pltR, andp/tF mutant backgrounds.
1NA from pltJ::inaZ (Fig. 4.12) was enhanced by PLT amendment as early as two
hours after inoculation, by more than three orders of magnitude, in all backgrounds (P
0.000l). In contrast, Pf-5 pltB::inaZ showed no difference in 1NA expression at 2
hours (P
0.95) between PLT-amended and non-amended treatments. By 48 h, INA
expressed bypltf::inaZ derivative strains in PLT-amended medium was at least 3.6
log units higher (P 0.0001) than that expressed in non-amended medium, and 1NA
reached similar levels among strains in PLT-amended medium regardless of genetic
background (P = 0.96). In this and several other experiments, NA expressed by Pf-S
pltI::inaZ and Pf-5 plt.J::inaZ peaked at 12 hours, and was stable between 12 and 48
hours. Therefore, in subsequent experiments, NA expressed by Pf-5 pltI::inaZ and
derivatives was assessed at 0,2, and 48 hours to evaluate the induction of, and
133
maximum levels of, expression (Fig. 4.13). Like pltJ::inaZ derivatives,pltI::inaZ
derivatives grown in PLT-amended medium showed more than 10-fold enhanced INA
in all backgrounds, as early as two hours after inoculation (P O.0004). By 48 h,
INA expressed from pltI::inaZ was at least 3.4 log units higher (all P 0.004) in
PLT-amended cultures than in non-amended cultures, and did not differ (P = 0.78)
regardless of genetic background. Slight differences in population sizes were
observed between PLT-amended and non-amended cultures ofPf-5pltF::aacCl,
plt.J::inaZ at 12 hours, and of Pf-5 pltR::aacCl, pltI::inaZ at each timepoint. Because
1NA values are normalized for CFU, differences in population size did not account for
differences in INA.
134
Figure 4.12. Positive influence by PLT on transcriptional activity from pltB::inaZ in
a wild type Pf-5 background (a); plt.J::inaZ in a wild type Pf-5 background (b);
pltJ::inaZ in apltF background (c), and pltJ::inaZ in apltR background (d). The
parental Pf-5 (mi; triangles) was included as a negative control (a). Cultures were
grown in NBG1y with (open symbols) or without (filled symbols) an amendment of 4
pg/mI PLT added at the time of inoculation and were harvested at 0, 2, 12, 24, and 48
hours for assessment of INA (a-d) and population densities (e-h). Error bars denote
one standard error.
0
0
0
0
-2
-2
-2
-2
-4
-4
-4
I:
-6
-6
-6
-8
-8
-8
-8
-10
-10
le+10
-lO-
le+10
le+1O
le+8
7l1fl
/
.f
Pf-5 pltR::inaZ + PLT
le+8
.
I
I
I5p1:in
0
I
U
0 6 12 18 2430 3642 48
le-4-7
i
I
I
'plt:aa1,
pftJ:inaZ
le+8
0-- Pf-5 pltF::aacCl,
Pf-5 plt.J.:inaZ + PLT
V Pt$ + PLT
le-$-7
le+10
g.
e.
le+9
-10
ii.
le+9
1pIcCI,
pltJ.:inaZ
le+8
0 Pf-5 pIeR::aacCl,
pltJ::inaZ + PLT
le-1-7
I
0 6 12 18 24 30 3642 48
le-I-7
0
6 12 18 24 303642 48
pltJ::inaZ + PLT
I
I
I
0 6 12 18243036 42 48
Culture age (hours)
Fig. 4.12
(-'I
136
Figure 4.13. Positive influence by PLT on transcriptional activity from pltB::inaZ in
a wild type Pf-5 background (a); pltI::inaZ in a wild type Pf-5 background (b);
pltI::inaZ in apltF background (c), and pill: :inaZ in apltR background (d). The
parental Pf-5 (Ina; triangles) was included as a negative control (a). Cultures were
grown in NBG1y with (open symbols) or without (filled symbols) an amendment of 4
jtg/ml PLT added at the time of inoculation and were harvested at 0, 2, and 48 hours
for assessment of 1NA (a-d) and population densities (e-h). Population sizes did not
differ significantly between treatments at any time point. Error bars denote one
standard error.
E
U
U
U
U
-2
-2
-2
-2
-4
-4
-4
-4
-6
-6
-6
-8
-8
-8
-8
_1 (I
_1 ft
ft
-10
e
le+11
le+10
le+11
e.
le-f-11
1e+9
le+9
L? le+8
le+7
le+7
0
12
24
36
48
60
le+11
g.
J f.
19
19
le+8
le+8
le-I-7
0
12
24
36
48
60
0
Culture age (hours)
Fig. 4.13
12
I
I
24
36
-i
le-+-7
48
60
0
12
24
36
48
60
138
4.4. Discussion
4.4.1. PLT production requires functionality of transport genes flanking the
PLT biosynthetic gene cluster
This study provides evidence that genes with predicted transport functions are
necessary for PLT production. The P11 phenotype of Pf-5 pltJ::inaZ and Pf-5
pltJ::inaZ could result from polar effects of the inaZ insertions on any or all of four
downstream ORFs, each of which is predicted to encode a protein with a transport
function. Thus, regardless of the specific gene(s) responsible for the phenotype of
reduced PLT production, blockage of expression of a transport function is correlated
with this phenotype. The mechanism by which PitI, PItJ, and/or flanking gene
products affect PLT production remains to be explored. Low concentrations of PLT
produced by pitI and pltf derivatives of Pf-5 could be a consequence of enhanced PLT
degradation or reduced PLT production. This study hints that apklmutant degrades
PLT more than apltB mutant. In all experiments, Pf-5 pltf: :inaZ produced a small
amount of PLT (e.g. 0.53 tg/ml at 48 h; Fig. 4.9), whereas Pf-5 pltB::inaZ produced
none. However, PLT recovered from supematants of PLT-amended Pf-5 pltJ::inaZ
cultures was less than or equal to that recovered from cultures of the completely P1t
strain Pf-5 pltB::inaZ at all time points (Fig. 4.9). Although these results suggest PLT
degradation by Pf-5 pltJ: :inaZ, the rates are very small compared to the differences in
PLT production between Pf-5 and Pf-5 plt.J::inaZ. Thus, PLT degradation might
account for some, but not all, of the reduction in PLT accumulation bypltl orplt.J
derivatives (Fig. 4.7). Therefore, PLT degradation cannot fully account for the Plt
phenotypes of pitI and pit..! derivatives. Alternatively, it is possible that expression of
PLT biosynthetic genes is affected at the transcriptional or posttranscriptional level in
pitI and pit..! mutants. This hypothesis remains to be explored.
The influence of transport genes in regulation of PLT production suggests that
PLT production may be coordinated with transport of a substance to or from the cell.
139
The substrate for PitI and P1tJ is unknown, as is the directionality of transport. PLT
export is a possible function of PitI and P1tJ, but PLT does not appear to accumulate
in cells of pill and p11.1 strains. Rather, PLT concentrations in p/tI and p/U strains are
similarly reduced relative to the wild type, in both cell and supernatant extracts. PLT
may, however, be able to utilize an alternative mechanism for escape from cells even
if p/tI and pitJ represent its primary export apparatus.
PLT import is another potential function for PltI and PltJ. Support for the
notion of PLT import by PItI and PltJ comes from the observation thatp/tlandpld
derivatives of Pf-5 are unable to undergo PLT autoinduction, which is presumed to
require either an extracellular sensor of PLT or the transport of extracellular PLT into
cells of Pf-5 for perception by a cytoplasmic PLT sensor. Therefore, loss of PLT
transport to the inside of the cell could explain lack of autoinduction in p/tI and p/ti
mutant derivatives.
4.4.2. Exo2enOuS PLT induces expression of pitI and pltJ
This study demonstrates that pill and pltJ are transcriptionally regulated by
PLT. The rapid, positive effect of PLT on 1NA expressed by Pf-5 pltI::inaZ and Pf-5
pli.J::inaZ (Figs. 4.12 and 4.13) suggests that transcription of p/tI and p/tJ is tightly
regulated by the presence of PLT. Although PLT also positively affected
transcription of PLT biosynthetic genes (e.g. pltB; Chapter 2), the magnitude of the
response to exogenous PLT by Pf-5 pltB::inaZ was much greater than that of Pf-5
pltI::inaZ or Pf-5 plt.J::inaZ, suggesting that the mechanism by which PLT induced
transcription of pilB differed from the mechanism by which PLT induced
transcription of pill and pilJ. This difference could reflect separate proteins to
mediate the effect of exogenous PLT on promoters, or subtle differences in promoter
structures of pltB, pill, and p11.1 causing differential activation by a single
transcriptional regulator. In the absence of exogenous PLT, levels of p/tB expression
were greater than those of pill orplti (Figs. 4.12 and 4.13). Thus, it is also possible
140
that basal pltB expression is already near a threshold which pitI and pltJ approach
only in the presence of PLT. Regardless of the mechanism, it is clear that the effect
of PLT on transcription of p/tI and p/ti is qualitatively, but not quantitatively, similar
to the positive effect of PLT onpltB transcription.
The accumulation of PLT both influences, and is influenced by, Piti, PItJ,
and/or downstream gene products. PLT induced pltI and pltJ transcription, and
mutations in pill or pltf reciprocally repressed PLT accumulation in cells and culture
supematant. Precedents exist in the literature for regulation by substrate levels, and of
substrate metabolism, with regard to genes encoding transport machinery for the same
substrate. For example, transcription of genes involved in transport and genes for
assimilation of inorganic phosphate
(P1)
in Escizerichia
co/i
both require an intact
ABC transporter encoded bypstABC and the binding protein gene pstS. Mutations
that functionally disable the periplasmic binding protein PstS or the ATP-binding
component of the ABC transporter PstB simultaneously obviate P1 control of the Pho
regulon. Reciprocally, the Pst system itself is under
P1
of the Pho regulon, which is regulated by extracellular
control. The pst genes are part
P1
concentrations (reviewed in
80). Another example is that of MaIFGK2, an ABC maltose transporter in E. co/i and
part of the ma! regulon (see references 9 and 10 for reviews). A mutation in the ATPbinding component, MaIK, results in the loss of maltose transport and also in
constitutive expression of genes involved in maltose metabolism (30, 67); conversely,
MaIK overexpression has a repressive effect on ma! gene expression (59). The
influence of Ma1FGK2 on genes for maltose metabolism is mediated by a physical
interaction with the transcriptional regulator MalT (54). Again, reciprocal regulation
is evident: maIEFGK, encoding the maltose transporter, are among those genes under
MalT regulation. Examples also exist of non-ABC transporters which play roles in
substrate-regulated gene expression, and are themselves under substrate regulation.
Such "circular" regulation also appears to be common to the
phosphoenolpyrouvate:sugar phosphotransferase system (PTS) for sugar uptake in
bacteria. The glucose-specific permease of the phosphotransferase system, PtsG,
141
binds to the transcriptional regulator Mic, and thereby controls Mic-regulated
expression a number of glucose-regulated genes, including ptsG itself (36, 73).
Similarly, depending on presence of substrate, the -glucoside transporter BgIF from
E. coli directly alters the phosphorylation state of the transcriptional regulator BgIG,
which in turn regulates the expression of the f-glucoside transporter gene, blgF, and
other genes (13, 24). Bg1G is the prototype of a family of transcriptional regulators
that share this type of direct transporter/regulator interaction (72, 75). Transport of
compounds other than those involved in primary metabolism can also experience
transporter substrate-dependent regulation of gene expression for transport and
biosynthetic genes. The secondary metabolite pyoverdine, an iron-binding
siderophore of P. aeruginosa, regulates expression of genes for pyoverdine
biosynthesis (35) and transporter synthesis (22, 56, 58) in such a manner, via the
ferripyoverdine receptor and associated proteins (35, 58).
4.4.3. p1111
The influence of PLT on activity from the promoter(s) of pitI and pltJ suggests
the existence of a PLT receptor which directly or indirectly activates transcription
from the pltlandp/tfpromoter(s). One possible candidate is the TetR transcriptional
regulator encoded bypitH. TetR homologs are often involved in cofactor-dependent
regulation of genes, frequently linked, encoding biosynthesis or export functions for
hydrophobic compounds (e.g. 18, 29, 66, 83). Although the cofactor may be the very
compound whose biosynthesis or transport is under regulation (e.g. 18, 29, 66),
regulation of non-linked genes, whose product is not a cofactor for the TetR regulator,
also occurs (52, 53). In the 2,4-DAPG gene cluster of P. fluorescens CHAO, two
TetR family members, phiF and phlH, have been identified. In strain CHAO, PhlF is
required for 2,4-DAPG autoinduction of a 2,4-DAPG biosynthetic gene (66). Ph1F
binds repressively to the phiA CBD promoter in the absence of 2,4-DAPG, but
dissociates from its target sequence in the presence of 2,4-DAPG (1). The amino acid
sequence of PItH closely resembles that of PhIH in the 2,4-DAPG gene cluster of
142
strain CHAO. Pf-5 also produces the polyketide 2,4-DAPG, and a search of the
incomplete genomic sequence of Pf-5 from The Institute for Genomic Research
(http://www.tigr.org) revealed an ORF in Pf-5 with 99% similarity to phlH at the
nucleotide level. Thus,pltHandphlHare predicted to be paralogous genes in Pf-5.
The function of either pitH orphlH has yet to be revealed for either strain. A testable
hypothesis is that PItH, like other TetR homologs, acts as a transcriptional repressor.
The promoters of PLT biosynthetic genes, of the adjacent transport genes, and of pitH
itself are candidate targets for regulation. In keeping with this line of thought, PLT
might act as a cofactor. Cofactor binding in the case of the prototype TetR protein not
only derepresses transcription of tetA, a gene encoding a tetracycline efflux protein,
but also derepresses transcription of the tetR gene itself (60). The observation that
transcription ofpitH itself is upregulated in the presence of exogenous PLT (Caroline
Press, unpublished data), supports the hypothesis that PltH may be involved in
regulatory functions involving the perception of PLT. Support for the hypothesis of a
functional relationship between PltH and the products of pitl and plt.J lies in the
colinearity of pitH, piti, pit.J, orJK, and orfP with a homologous cluster from A.
vinelandii. The tight homology of the five genes in this cluster between two bacterial
species suggests evolutionary conservation of a mechanism for transport and/or
regulation. It is not known whether bacterial species other than A. vinelandii and Pf-5
regulate or transport exoproducts by this mechanism, or whether A. vinelandii
produces PLT.
PitH may be anchored in the cytoplasmic membrane, as suggested by the
prediction of a transmembrane domain using DAS analysis. Transmembrane domains
are typically 15-30 residues in length (17); the predicted PitH transmembrane domain
is only 12 residues in length. However, the ends of such domains are only
approximated by homology- and hydophilicity-based algorithms. The predicted
domain (TISIIAPCLMLA) can be extended by including one polar (C) and one
charged (R) residue that separate the predicted transmembrane domain from a stretch
of four additional hydrophobic residues (PALARCTISIIAPCLMLAVI). The
143
modified transmembrane domain prediction includes 20 amino acids, resulting in a
more typical length for a transmembrane domain. The validity of the transmembrane
prediction can only be determined experimentally; it is possible that this hydrophobic
region has an alternative function, such as substrate binding. Could PItH function as a
transcriptional regulator while membrane-bound? Two well-studied transcriptional
activators, TcpP and ToxR from Vibrio choierae, are anchored in the cytoplasmic
membrane but have N-terminal DNA-binding domains (27,45). Additionally, it has
been shown that association with the cytoplasmic membrane does not impede proteinnucleic acid interactions which take place at a discreet location in the cytoplasm:
BglG, a transcriptional antiterminator, acts on nascent mRNA which is indirectly
anchored to the DNA strand via RNA polymerase. BgIG functions effectively while
artificially tethered to the cytoplasmic membrane (25).
4.4.4. yltI
The occurrence of pill in a cluster of genes encoding putative ABC transporter
subunits is enigmatic. The piti gene bears homology with the membrane fusion
protein HJyD. H1yD homologs are found in ABC transporters of Gram negative
bacteria specifically associated with Type I secretion of protein toxins, proteases, or
lipases from the cell - such as the protein toxin hemolysin A in E. coil (reviewed in
86), or metalloprotease in Erwinia chrysanthemi (reviewed in 19). Membrane fusion
proteins are also associated with the major facilitator superfamily (MFS) class of
substrate transporters (20, 43), which are quite distinct from ABC transporters in form
and function. MFS transporters are single-polypeptide permeases that use protonmotive force to drive transport of small molecules across the membrane, while ABC
transporters are multi-subunit assemblies that use ATP hydrolysis to drive substrate
transport. In Gram positive bacteria, ABC transporters whose substrates are small
molecules (bacteriocins and peptides) utilize membrane fusion proteins as accessory
factors (26); however, I am aware ofno examples in which Gram negative bacteria
incorporate membrane fusion proteins into the ABC transporter machinery except for
144
the case of Type I secretion mentioned above. Still, a shared function for INtl and P1tJ
is suggested by the transcriptional activation ofbothpltlandpltfby exogenous PLT,
and the occurrence of pill in the cluster of colinear genes also found to reside in A.
vinelandjj. Because the substrate for PItI and PItJ is not known, it remains possible
that a protein or peptide substrate is transported by Type I apparatus including both
Pill and Pill, under the tight regulation of extracellular PLT. I also speculate that PLT
may be transported by an unusual transmembrane complex of which PitT and PIIJ are
both subunits.
The predicted amino acid sequence of PitT exhibits a predicted N-terminal
transmembrane domain conserved among H1yD homologs. N-terminal
transmembrane domains may sometimes be difficult to distinguish from signal
sequences that are cleaved upon passage of the nascent polypeptide through the cell
membrane (78). It remains possible that PitT is not an integral membrane protein, but
rather a periplasmic protein that uses the general secretory pathway (Type II secretion)
to insert into the membrane, and whose N-terminal signal sequence is subsequently
cleaved. Because periplasmic binding proteins and HlyD homologs both associate
with periplasmic portions of ABC transporters (9, 37, 74), it is not altogether unlikely
that certain motifs might be shared between these proteins. This rationale is seductive
because ABC transporters involved in substrate import require a periplasmic binding
protein to deposit substrate into the transport apparatus. Although it is possible that
pitI and p11.1 are involved in import rather than export, no such binding protein gene is
apparent in the region flanking the PLT gene cluster. Another viable hypothesis is that
a binding protein might be encoded by a gene within another regulon, perhaps one in
which the binding protein performs additional functions. Substrate-laden binding
proteins may interact with multiple proteins in the periplasmic space; for example,
ChvE from Agrobacterium tumefaciens is a monosaccharide-binding protein that,
when complexed with substrate, interacts with a monosaccharide transport system,
with chemosensory proteins necessary for chemotaxis, and with VirA, a histidine
kinase member of a two-component regulatory system governing virulence (12, 16).
145
By analogy, a hypothetical PLT-binding protein serving multiple functions might be
encoded by a regulon outside of the PLT biosynthetic gene cluster.
4.4.5. pltJ
The p/ti ORF lacks a typical ribosome binding site upstream from the start
codon. Consensus Shine-Dalgarno sites are not always found preceding the start
codon of prokaryotic genes. Indeed, Shine-Dalgamo site anchoring appears to be the
final step in a series of subtle interactions between the 30S subunit and the mRNA
strand (40). Thus, the ribosomal binding information inherent in the plt.J sequence
may simply elude detection. It is also possible that p/ti translation is coupled to that
of phi via ribosome frameshifting. Ribosomal frameshifting normally requires a
slippery region for slippage at the P site and an mRNA pseudoknot to stall the
progress of the ribosome on the mRNA strand (55). At least one niRNA pseudoknot
was found between nucleotides 8 and 502 ofplti(AG = -329.98; mfold program,
Zuker et al. [87}). The proximity of the pseudoknot to the start codon ofpltfmight
imply a role in ribosomal stalling; however, no consensus (XXXYYYZ) slippery
region (55) preceding the start codon was apparent although a near-consensus
sequence (GGGGCCA) begins at position -11 from the start codon. The consequence
of such a frameshifted translational product would be a fused polypeptide, comprising
PltI and PltJ. A protein expression system could be employed to determine the
molecular mass of the translated product of the p/ti gene alone, compared to that of
p/tI adjoining p/t.J.
The predicted P1tJ polyp eptide is homologous to the ATP-binding subunit of
ABC transporters; the single open reading frame contains two fused ATP-binding
domains. This arrangement is not uncommon for ABC transporters, whose subunits
occur in nearly every possible combination of fused and individual subunits (9). As
ABC transporters comprise paired cytoplasmic ABC binding domains and paired
transmembrane domains, the predicted PltJ polypeptide represents only half of a
FE!i
putative ABC transporter. Transmembrane domains, but no ATP-binding motifs, are
found in the adjacent genes orf K and orfP. Thus, a reasonable hypothesis is that orJK
and orfP together, or a dimer of either gene product, form(s) the transmembrane
portion of the ABC transporter of which PItJ is part. Because no other ORFs with
homology to ABC transporter genes appear adjacent to orJP, it is likely that p/tJ
orJK, and orjP encode subunits of the same ABC transporter.
In conclusion, I have provided preliminary evidence for the existence ofa new
mechanism for regulation of PLT production, which involves substrate transfer. The
substrate for the transporter(s) encoded by genes flanking the PLT region remains
unknown, but the transport machinery itself is necessary for optimal PLT production.
In turn, the expression of transport genes pitI and p11,1 is regulated by extracellular
PLT. Whether the substrate for PitI and P1tJ is PLT remains to be explored
experimentally. Iii at least one example (prodigiosin in Serratia sp. ATCC 39006),
there is genetic evidence that nutrient (P1) transport may regulate antibiotic production
(70). However, to my knowledge this is the first report of coordinate regulation of
antibiotic biosynthetic genes and a transport apparatus by the antibiotic itself.
Biochemical characterization of the mechanism for PLT regulation of p/tI and pltf,
and of the reciprocal influence of PitI, PltJ, and/or adjacent transport gene products on
PLT production, is likely to reveal a novel mode of regulation of antibiotic production
in Gram negative bacteria.
147
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Chapter 5. Concluding Remarks
A goal of this study was to describe autoinduction of the polyketide
pyoluteorin (PLT) in P. fluorescens Pf-5, and to explore possible cross-regulatory
relationships between PLT and two other secondary metabolites [2,4diacetyiphioroglucinol (2,4-DAPG) and pyrrolnitrin (PRN)J important in biological
control by Pf-5. I showed that concentrations of exogenous PLT as low as 1.5 nM
initiated autoinduction. These low concentrations are comparable to those of many
molecules proposed to function in signaling roles. Moreover, I demonstrated PLT
autoinduction as an ecologically relevant phenomenon. In rhizosphere soil,
derivatives of Pf-5 that carried a reporter gene fusion to a PLT biosynthetic gene, but
which were unable to produce PLT, expressed the reporter gene at higher rates when
co-inoculated as a mixed population with PLT-producing strains. This observation i)
demonstrates that PLT can serve as a signal between distinct populations of cells
within the rhizosphere, and ii) implies that PLT biosynthetic genes are upregulated in
the rhizosphere by exogenous PLT, and therefore, by extension, PLT autoinduction is
a significant component of in
situ
PLT regulation by Pf-5. This, and other new
information presented in this thesis, prompts the prediction of several repercussions of
PLT production by Pf-5 on fellow members of a rhizosphere community.
5.1. Repercussions of PLT production for the rhizosphere community
PLT produced and secreted by some Pf-5 cells within a community will
stimulate PLT production by others nearby, via autoinduction. This
autoinduction will result in an intensification of the other community
effects listed below.
2.
PLT will adversely affect Oomycete plant pathogens, inhibiting their
growth.
157
3.
The data from Chapter 3 demonstrate that PLT can affect expression
from the promoter offpvA, the ferric-pyoverdine receptor gene from P.
syringae. FpvA is highly conserved among Gram negative bacteria, so it is
likely that this result can be extrapolated tofivA of P.fluorescens Pf-5. In
support of this assumption, PLT also affects expression of pvsA, a gene for
pyoverdine biosynthesis, in Pf-5 (Caroline Press, personal communication).
These results together raise the question of whether secreted PLT in the
rhizosphere has potential to affect the iron status of conspecific cells, andlor
that of other species that utilize the pyoverdine system, provided that PLT is
taken up into, or sensed by, those cells.
4.
High concentrations of PLT in the microenvironment will inhibit production of
2,4-DAPG by cells of P. fluorescens within that environment. Therefore,
while Pf-5 can inhibit Pythium, it is not a good choice for biocontrol in a
situation dominated by pathogens better controlled by 2,4-DAPG (e.g.
Fusarium oxysporum, Gaeumannomyces gramini var. tritici, or Thielaviopsis
basicola. In such a situation, a 2,4-DAPG producer that doesn't produce PLT
may be a better biocontrol agent. As a matter of fact, this idea has
experimental support (9). Large collections exist of P.fluorescens 2,4DAPG-producers, collected from rhizospheres in suppressive soils worldwide
(e.g. 4, 8). Phylogenetic grouping of these strains by amplified spacer
ribosomal DNA restriction analysis (ARDRA) placed them into several
groups. ARDRA1 strains had the phenotype Plt, 2,4-DAPG, while
ARDRA2 strains were Pif, 2,4-DAPG. The latter group was better in
rhizosphere assays at inhibiting Fusarium oxysporum (9). One explanation
might be antibiotic antagonism, hindering the production of 2,4-DAPG by
ARDRA1 strains. On the other hand, ARDRA1 strains are closely related, and
therefore might share another characteristic that lowers biocontrol efficacy.
158
5.2. Functionality of bioactive compounds in a soil environment
The soil environment presents a challenging environment through which to
relay a biochemical signal. For example, Howell and Stipanovic (3) demonstrated the
inactivation or adsorption of PLT by passage through non-sterile soils, although pure
PLT applied to cotton seeds in a carrier of diatomaceous earth retained the ability to
protect seeds in non-sterile soil from Pythium ultimum. In truly natural field soils,
PLT might be carried by soil water away from producing cells, or be degraded or
utilized by heterologous bacteria, or compete with other substrates for binding to the
PLT receptor hypothesized to mediate autoinduction (Chapter 2; Appendix 1). In a
similar system, indirect evidence for receptor competition exists (6). In CHAO, 2,4DAPG autoinduction and repression of 2,4-DAPG by PLT, salicylate and a fungal
metabolite, fusaric acid, are all mediated by PhlF, (10), which is directly activated by
2,4-DAPG and repressed by salicylic acid (1). Notz et al. (6) provided evidence for
the inhibition of 2,4-DAPG gene expression in the presence of fusaric-acid producing
strains of Fusarium oxysporum in soil. Although indirect mechanisms are possible,
one explanation for this effect is direct competition between 2,4-DAPG and fusaric
acid for a PhlF binding site.
5.3. Secondary metabolite production by Pf-5 is affected by the profile of
metabolites already present
In addition to PLT autoinduction, I demonstrated several other effects of Pf-5
metabolites. These are briefly listed below.
1.
2,4-DAPU positively regulates its own production (Chapter 3).
2.
Exogenous PLT suppresses 2,4-DAPG production (Chapter 2).
3.
Exogenous 24,-DAPG inhibits PLT production (Chapter 2).
4.
Exogenous PLT suppresses expression of a heterologous pyoverdine
gene in nutrient broth (Chapter 3).
159
5.
Exogenous PRN exerts a slight inhibitory effect on PLT gene
transcription and production (Chapter 3).
I failed to see a significant effect of exogenous PLT upon PRN, despite its
structural similarity (Chapter 3). Thus, PLT affects the production of at least three
(PLT itself, 2,4-DAPG, and pyoverdine, see above), but not all, secondary metabolites
in Pf-5 under the conditions I tested. The mutually repressive impacts of PLT and 2,4DAPG on one another's production occurs, at least in part, at the level of transcription
(Chapter 2; Press and Loper, unpublished data). The slight inhibitory effect of PRN
on PLT gene transcription and production (Chapter 3) cannot be explained by
precursor competition, as it is unlikely that these compounds share precursors (5, 7).
5.4. Site of perception of the PLT signal may be cytoplasmic or extracytoplasmic.
The data presented herein imply that PLT has, in addition to its role as an
extracellular toxin, a role with intracellular signaling repercussions. PLT could be
synthesized and perceived (during autoinduction) in either the cytoplasm, or the
periplasmic space. Some researchers argue that the membrane-bound periplasmic
space of Gram-negative bacteria is functionally akin to membrane-bound eukaryotic
organelles, and that the periplasmic "organelle" functions principally to isolate the
cytoplasm from molecular traffic entering from, or leaving for, the extracellular milieu
(Mario Pantoja, personal communication). Although this idea is appealing, there is
scant evidence to support such a broad proposal. However, it is noteworthy that all of
the genes in the PLT biosynthetic gene cluster are predicted by DAS analysis to have
transmembrane domains (data not shown), suggesting that PLT biosynthesis is a
membrane-associated process. Which side of the membrane is responsible for the
biosynthesis of PLT is not known. There is evidence of at least one instance (albeit in
a Gram-positive bacterium) in which an enzymatic reaction resulting in polyketide
synthesis occurs outside of the cytoplasmic membrane (2).
160
5.5 PLT accumulation requires a set of flanking transporter genes, which are in
turn regulated by PLT.
Circumstantial evidence supports a cytoplasmic location for PLT production.
A set of transport functions encoded by genes flanking the PLT biosynthetic gene
appears to be tightly associated with PLT production. Such a transport system would
seem unnecessary if PLT were produced in the periplasm. The transport system may
facilitate the passage of PLT across the membrane; alternatively, its role may involve
another substrate entirely. I identified two genes, pill and pltJ, that appear to be part
of a larger cluster (pitH, pill, pitf, orJK, and orfP) with potential for either i) PLTregulated PLT transport or ii) PLT-regulated transport of a different substrate.
Mutations inpitlorpklinhibited PLT production by Pf-5 approximately five-fold.
Moreover, apit.J mutation abolished PLT autoinduction. Optimal transcription of piti
and pltJ, in turn, requires the production of PLT by Pf-5 or its addition to the medium.
The upregulation of pill and pit.J expression by exogenous PLT was nearly immediate
(2 hours). Thus, it is evident that, besides affecting its own production and that of
other secondary metabolites, PLT also exerts tight control over the machinery for
transport of a substrate, possibly itself, across the cytoplasmic membrane. The
integrity of that substrate transport machinery, in return, is required for optimal PLT
production and for the PLT autoinduction phenomenon. A hypothesis awaiting testing
is that PLT autoinduction requires transport of PLT from the external medium across
the outer and inner membrane, into the cytoplasm, and that pitI and pilf are part of an
ABC transport apparatus that is involved in PLT import. In such a scenario, pitI and
pil.J mutations would be expected to lower, but not abolish, PLT production by
interrupting PLT autoinduction. This hypothesis is consistent with the data collected
thus far.
5.6. Significance
The data from the studies described herein demonstrate that the production of
PLT by Pf-5 can affect the production of PLT by neighboring cells. PLT and other
161
exogenous secondary metabolites were shown to have both autoregulatory and cross-
regulatory effects in vitro. Because Pf-5 derivatives engaged in PLT cross-feeding in
the rhizosphere, it is likely that cross-feeding occurs for other secondary metabolites
as well. If cross-feeding occurs, then production of PLT as well as other secondary
metabolites by one cell will profoundly affect the production of secondary metabolites
in neighboring cells. New evidence hints that PLT influences the iron status of cells
that perceive it. Thus, a new variable in the efficacy of biological control is now
apparent: inhibition of the production of certain antibiotics by others produced by the
biocontrol agent. For instance, when environmental conditions and the physio1ogica
status of Pf-5 favor the production of 2,4-DAPG, the production of PLT is inhibited,
and vice versa. Because the different antibiotics produced by Pf-5 do not have the
same spectrum of activity against target pathogens, (e.g. reference 3), knowledge of
environmental conditions that affect antibiotic production during application of a
biological control agent is essential. Strategies should be employed to maintain
expression of traits that are most important in the suppression of the target pathogen.
162
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APPENDICES
188
Appendix 1. Overexpression of PItR in Escherichia
coli
Al.!. Introduction.
In Pf-5 and related Pseudomonas species, complex regulatory cascades govern
the production of secondary metabolites. Participants in such regulatory cascades that
affect the production of pyoluteorin (PLT) were identified by transposon mutagenesis
(6) and have been found to encode proteins with global roles in regulation (5, 14, 18,
19). Biosynthetic genes have also been identified by transposon mutagenesis (6).
Sequencing of the region revealed the PLT biosynthetic genes to be clustered (11, 12).
Sequencing of the region also revealed a pathway-linked transcriptional regulator.
The gene, designated pltR, encodes a LysR transcriptional regulator, and is necessary
for the transcription of PLT biosynthetic genes from all known promoters (11). LysR
homologues are typically associated with, and activate transcription from, regulons
responsible for secondary metabolite biosynthesis. Many LysR proteins enhance
transcription from their own promoters. Many LysR proteins also are activated by
cofactor binding, and the cofactor is often a compound whose synthesis is regulated by
the LysR protein. Thus, the binding of cofactor can indirectly and simultaneously
induce transcription of genes for cofactor biosynthesis and the cofactor's receptor.
This scenario implies amplification of the cofactor and the LysR transcriptional
activator via autoregulation (15). I found that a Pf-5 derivative bearing a mutation in
pltR was
unable to produce PLT, and, therefore, also unable to undergo PLT
autoinduction (data not shown). Thus, 1 hypothesized that P1tR could act as the
receptor for PLT during autoinduction (Fig. A1.1).
transcription
PLT
. .
PItR
pltR
I
-+$_
pltR pitA
pltL
pltR
pltL
PItM
orfN
4_9
pltJ
rn
pltB
pitC
pltD
pit?
pita
Fig. A1.1. Proposal for P1tRIPLT interaction at PLT promoters. In this
scenario, P1tR does not activate transcription when not bound to cofactor (PLT).
However, cofactor (PLT) binding is thought to induce P1tR activation, perhaps by a
conformational change in the protein.
In P. fluorescens strain CHAO, 2,4-diacetylphloroglucinol (2,4-DAPG)
autoinduction and repression by PLT, salicylate and a fungal metabolite, fusaric acid,
are mediated by PhIF, a TetR type transcriptional regulator encoded by a gene linked
to the 2,4-DAPG biosynthetic gene cluster (16). Binding by Ph1F to the ph1ACBD
promoter inhibits transcription; PhlF binding is directly stabilized by salicylate but
alleviated by 2,4-DAPG (1). Other examples of dual regulation of a single
transcriptional regulatory protein by two metabolites also exist. For example, BenM, a
LysR-type transcriptional regulator from Acinetobacter sp. ADP1, regulates genes for
aromatic compound degradation. BenM is activated by synergistic binding of
benzoate, an aromatic compound, together with a breakdown product, cis,cis-
muconate (4). Based on such reports, I further hypothesized that PItR could act as a
receptor for both PLT (as a co-activator) and for 2,4-DAPG (as a co-repressor). If this
were the case, PltR would mediate the positive effects of exogenous PLT and the
negative effects of exogenous 2,4-DAPG at PLT promoters (Fig. Al .2).
190
O PItR°
pitH
j-.
ro° #}
..
piti
pltL
.-f-4-
orfN
.
llvu.I1lIuI
pitR pitA
p1W
pltC
P1W
or
K
pltF
pito
Fig. A1.2. Proposal for dual binding by PItR of PLT (co-activator) and 2,4DAPG (co-repressor). In this scenario, PItR does not activate transcription when
bound to corepressor (2,4-DAPG). However, PLT binding may activate PItR, perhaps
by a conformational change in the protein.
P1tR is an attractive focus of study because, in contrast to several global
regulatory proteins studied in our laboratory that affect PLT production but have
undesirable pleiotropic effects (5, 14, 18, 19), P1tR bears potential to act as the final
member of regulatory cascades affecting PLT production. Therefore, PltR was
envisioned as a target protein that might be manipulated to overproduce PLT without
affecting other aspects of the physiology of Pf-5. Because PLT at high concentrations
is phytotoxic (9), I also reasoned that P1tR might eventually be engineered to respond
to a specific environmental signal such as Pythium exudates, so that PLT could be
selectively produced in the rhizosphere as appropriate for biocontrol.
A1.2. Characterization of P1tR
PltR had been characterized previously by mutating the pltR gene and
evaluating the phenotype of the mutant strain (11). The obvious phenotype was a
complete lack of transcription of PLT biosynthetic genes and resultant PLT
production. Overexpression of PltR in Pf-5 was also attempted. A 2.2 kb KpnIHin dill fragment containing the entire pltR gene was cloned into pME6000 (Brian
Nowak-Thompson, personal communication), an Escherichia coli -Pseudomonas
191
shuttle vector of the IncP incompatibility group, stable in P. fluorescens at a copy
number of 16-20 (Stephan Heeb, persona! communication). Mating this plasmid into
Pf-5 or Pf-5 pltR strains was unsuccessful, as colonies displayed unstable phenotypes
and became sectored on plates (Brian Nowak-Thompson, personal communication, M.
Brodhagen, unpublished data). When cultured in nutrient broth + 1% glycerol, a
medium conducive to PLT production, the complemented strains produced no PLT or
produced only trace amounts (data not shown), although they presumably carried
multiple copies of a transcriptional activator for PLT biosynthetic genes. Moreover,
nearly half of the strains I tested appeared to have acquired mutations in the
gacS/gacA two component regulatory genes during storage. Thus, I reasoned that
overproduction of P1tR in Pf-5 was not a favorable route to overproduce PLT.
However, I continued to characterize PItR as a potential model of pathway-specific
regulation and possible eventual target for protein engineering.
Two separate strategies were attempted in order to further characterize PltR.
These involved 1) attempts to generate a transcriptional fusion to pltR in order to test
various factors which might upregulate or repress its expression, and ii) attempts to
obtain pure P1tR protein. The goal of the latter work was to use PItR in protein-DNA
studies to identify P!tR binding sites. A second goal was to add PLT and 2,4-DAPG
to pure P1tR in DNA-binding assays, to test their potential as effector molecules by
assessing their ability to alter binding of PItR to its target promoter(s).
Al.2.1. A transcriptional reporter for pltR
The pltR gene was amplified using PCR, with primers designed to place EcoRI
and XbaI restriction sites at the 5' and 3' ends, respectively, of the open reading frame.
The primers pltREcoR-F (gtctagaatcctcaataagaatgcact) and pltRXba-R
(ggtctagatcattgacaacgcctag) were used to amplify a 475 bp fragment containing the
pltR promoter region. The fragment was then cloned into pMP22O, a broad host-range
plasmid of the IncP incompatibility group (17). The pMP22O plasmid contains a
192
promoterless lacZ gene downstream of the multiple cloning site, and thepltR promoter
was expected to drive its expression. The promoter probe vector containing the pltR
promoter mated into Pf-5 and derivatives, and tested for lacZ expression.
Disappointingly, Pf-5 containing the pltR promoter probe exhibited no difference in 13galactosidase activity, as determined by standard assays spectrophotometrically
measuring the conversion of o-nitrophenyl--D-galactopyranoside into the yellow onitrophenol (10), when grown on PLT-conducive medium (nutrient broth + 1%
glycerol) as opposed to PLT-repressive medium (nutrient broth + 1% glucose).
However, this was not entirely unexpected. The expression of PLT biosynthetic genes
had previously been measured using transposon insertion mutants bearing the
promoterless ice nucleation reporter gene (inaZ). The inaZ reporter system is sensitive
enough to detect expression of several transcripts per cell. In contrast, the lacZ system
is 100,000 times less sensitive. Thus, genes that have low expression levels may be
detected using the inaZ system while escaping detection by the lacZ system (8). PLT
biosynthetic genes were within the detection range of the inaZ reporter gene, and
recent DNA hybridization analysis has confirmed that the pltR transcript and the PLT
biosynthetic gene transcripts are present in low abundance when compared to those of
other genes in Pf-5 (Caroline Press, personal communication). Interestingly, the genes
for 2,4-DAPG biosynthesis presented the opposite problem. Whereas the highly
expressed phiA gene from P. fluorescens CHAO was detectable using the lacZ reporter
gene, aphlA::inaZ fusion was expressed so highly that detection was never below the
saturation limit (Regina Notz, personal communication). Future experiments to assess
transcriptional activity of pltR might take advantage of real time RT-PCR technology,
which can measure transcript abundance directly without the technical difficulties
associated with reporter genes.
193
A1.22. Overexpressjon of PItR in E. coli
A1.2.2.1. Design of a PltR peptide antibody
The protein expression systems (pTYB 1, pET1 5b) described below allow the
generation of fusion proteins, so that the fused affinity tag can be used in protein
purification. Both expression systems also allow the subsequent cleavage of the
affinity tag, leaving the native or near-native protein. Native protein was desirable in
assays to examine the DNA-binding and cofactor-binding activities of PItR, because a
native protein was more likely to retain such activities than a fusion protein.
However, subsequent detection of the PItR protein in Western analysis, or
"supershifts" in electromobility shift assays, required an antibody. Unfortunately,
cleavage of the affinity tag simultaneously cleaves a useful antigen for readily
available commercial antibodies. Therefore, an antibody to P1tR was needed.
A synthetic peptide comprising the final 17 amino acids
(CSALSRATTLQDKSQTS) of the predicted PItR protein was synthesized, and used
to raise polyclonal antibodies in rabbits (Alpha Diagnostic International, San Antonio,
TX). The C-terminal region of P1tR was chosen because of its hydrophilicity
(implying surface-exposed residues), antigenicity, and accessibility (as assessed by
Alpha Diagnostics International, San Antonio, TX). As is often the case, the extreme
C-terminus represented the most attractive choice for the generation of a peptide
antibody, as it is highly antigenic [e.g. Liang et al. (7)]. Antibodies were raised in two
separate rabbits (7074 and 7075) to the PItR peptide conjugated to the highly
immunogenic keyhole limpet hemocyanin carrier protein, and were affinity purified by
passing the rabbit serum over sepharose beads to which the PltR peptide had been
coupled. Purified antisera were tested for activity by standard ELISA procedures;
antibody titer for antisera from both rabbits was 1:100,000.
194
A1.2.2.2. Expression ofP1tR inpTYB1
The pltR gene was PCR-amplified with NruI and NdeI restriction sites
engineered at the 5' and 3' ends, respectively, of the gene. The following primers were
used: pltR_Nde (ggatttacatatgaaggcgctaggcg) and pltR_Nru (tactgattcgcgatgtctggctc).
The PCR-amplifiedpltR gene was cloned in-frame into the pTYB1 vector digested
with NdeI and Sapi. The resultant construct contained the pItR open reading frame,
flanked by a C-terminal fusion to an intein, followed in turn by a chitin-binding
domain (CBD) that served as an affinity tag for purification. The integrity of two
constructs was verified by sequence analysis (Central Services Laboratory, Oregon
State University). The final construct was moved into the expression strain, E. coli
ER2566. Protein expression in the pTYB1/ER2566 system (IMPACTTh1-CN, New
England Biolabs, Beverly, MA) is driven by a promoter containing the T7 polymerase
recognition sequence. A chromosomal copy of bacteriophage 17 RNA polymerase is
carried by expression hosts. The 17 RNA polymerase gene is under the control of the
lacUV5 promoter. Thus, expression of the T7 RNA polymerase is repressed by Lad
until IPTG is added. However, a low level of basal expression remains, which can be
eliminated in expression hosts carrying the pLysS plasmid, as discussed below. The
T7 polymerase is highly processive and highly specific. It recognizes a small (20 bp)
promoter sequence that E. coli RNA polymerase does not recognize. Thus, a target
gene behind the T7 promoter is not transcribed until the 17 polymerase gene
expression is induced by IPTG addition. Premature expression of the target gene
itself, like that of T7 polymerase, is controlled by a lac operator sequence within the
promoter; the Lad repressor protein is encoded elsewhere in the pTYB 1 vector.
After expression, the fusion protein was affinity-purified from lysed cells by
passing over chitin-sepharose beads, which bound the chitin-binding domain tag.
Addition of thiols [50 mM dithiothreitol (DTT)} to the resin-bound fusion protein
activated the intein, causing on-colunm cleavage of the fusion protein between the last
amino acid residue of P1tR and the first amino acid residue of the intein. Thus, native
P1tR was released from the column.
195
A1.2.2.3. Expression of FuR
in pETl5b
The pitR open reading frame was also cloned into pETl5b, a vector that, like
pTYB1, drives expression of the target gene from the T7 promoter and controls leaky
expression with the Lad repressor encoded on the same plasmid. A fragment
containing the p/zR open reading frame was amplified using the primers pltR_Nde
(ggatttacatatgaaggcgctaggcg) and pitR_BamHI_1 416 (caggatcccgaaccgacgtgctg),
which created NdeI and BamHI sites at the 5' and 3' ends, respectively, of the gene.
The pltR gene was then cloned in-frame into pET1 5b digested with NdeI and BamHI,
creating a translational fusion to an N-terminal 6X-His tag separated from PItR by a
ten amino acid thrombin cleavage site. Although lack of success with this system
prevented the purification of large amounts of His-tagged PItR, the goal of this cloning
strategy had been to obtain a 6X-His affinity tagged PItR fusion protein, which could
be purified from crude cell lysate by passing over a nickel resin, which binds to the
His residues. Near-native PItR, containing one extra His residue at the N-terminus,
could then be obtained by cleavage from the affinity tag using thrombin.
A1.2.2.4. Host strain E.
coli BL21(DE3)pLysS
E. coil "B" strains are more robust than K-12 derivatives generally used in
molecular biology. Strain BL21 is deficient in proteases Lon and OmpT. The pLysS
plasmid carried by the expression hosts E. coil BL21(DE3)pLysS and the Rosetta
strains (see below) encodes T7 lysozyme, which degrades the T7 polymerase at a low
rate and thus prevents basal transcription of the target gene from the T7 promoter until
intentional expression is induced by IPTG addition. After induction, expression rates
are high and easily overpower the effect of the T7 lysozyme.
196
A1.2.2.5. Host strain E. coil B ER2566
E. coli B ER2566 (New England Biolabs, Beverly, MA) is a derivative of E.
co/i BL2 1. Like BL2 1, it carries a chromosomal copy of the T7 polymerase preceded
by the lacUV5 promoter, and lacks proteases Lon and OmpT. However, this strain
does not carry the pLysS plasmid; therefore, control over leaky expression of the
target gene is less stringent.
A1.2.2.6.
Host strain Rosetta
The Rosetta strain is also a BL21 derivative. It carries a pLysS plasmid for
stringent control of target gene expression. In addition, it carries plasmid-bome genes
for rare tRNAs. The predicted PItR protein contains codons for which the following
complementary tRNAs are not frequently found in E. co/i: cua (Leu), cga, aga, and
agg (Arg). TheE. coli codon frequencies for these were < 5%; a total of 3.2% of the
amino acids in PItR are encoded by rare E. coli codons. Thus, codon usage was
predicted to be a factor in the low rates of P1tR expression (described below) in E.
coli
B ER2566 and E. co/i BL21(DE3)pLysS.
Expression
different conditions
A1.2.2. 7.
ofP1tR
in djfferent vector - host combinations and under
The expression of P1tR was attempted first in the IMPACT-CN system
described above. Overnight 5 ml cultures grown at 37°C, with shaking at 200 rpm, in
Luria broth (LB) (13) containing 100 pg/mi ampicillin (Ap'°°) were used to inoculate
100 mis LB Ap10° in 500 ml Erlenmeyer flasks. Experimental cultures were grown
with shaking at 200 rpm to an OD
of 0.6, and then induced with 0.3 mM IPTG.
Following induction, cultures were grown under several different combinations of
induction times and temperatures. These conditions and the results are summarized in
Table Al. 1. PltR was harvested from cells using the protocol recommended in the
manufacturer's instruction manual (IMPACTTM-CN, New England Biolabs, Beverly,
197
MA). Because the predicted p1 of PItR lies between 8.0 and 9.0, buffers were made up
at pH 6.0, to keep P1tR in solution. After the indicated times, cells were harvested by
centrifugation, resuspended in lysing buffer, and sonicated to break open cells. Cell
lysates were clarified by centrifugation, and the clarified lysate was loaded onto preequilibrated columns containing a sepharose-chitin affinity resin. Once loaded, the
bound proteins were cleaved by activation of the intein in 50 mM DTT. The liberated,
native P1tR was eluted. Fractions collected at each step of the protein purification
process were separated using SDS-polyacrylamide gel electrophoresis (SDS-PAGE)
and visualized by Coomassie staining. Further examination of the fractions was done
via Western analysis, using an antibody to the chitin binding domain of the fusion
protein (New England Biolabs, Beverly, MA), and the PltR peptide antibody. The
expected sizes of the various protein products were as follows:
P1tR'-'intein'-'CBD
92 kDa
intein'-'CBD
55 kDa
PItR
38 kDa.
None of these protein products was seen in fractions from uninduced cultures.
The 92 kDa fusion protein was not seen on Coomassie-stained gels due to overloading
(data not shown), but a band corresponding to the 92 kDa fusion protein was detected
with the PltR peptide antibody in fractions of crude cell lysate and clarified lysate. In
fractions eluted from the sepharose-chitin bead resin, a 38 kDa band corresponding to
the expected size of native P1tR was detected with the PItR peptide antibody, and a 55
kDa band corresponding to the expected size of the residual intein'-'CBD was detected
with the CBD antibody in fractions eluted from the sepharose-chitin bead resin. Yield
of PltR was low in all treatments, but was optimal at 15°C and 20°C.
Table Al.!. Expression of PItR° from pTYB1 in E. coli B ER2566 cells, as
visualized by immunoblotting.
Temperature
Harvest time
15°C
15°C 20°C
20°C 27°C
(hours post-induction)
6 hours
l6hours
+1-
+
+1-
+
'Phis signs designate expression of PltR, as visualized by Western analysis. Minus signs denote the
absence of protein. +1- indicates that PItR was observed via Western analysis but in nearly
undetectable quantities.
Expression of P1tR in pTYB1/ER2566 was repeated, using the same culture
and extraction conditions as described above except that 2xYT (13) was used as the
culture medium in place of LB. Because yields were low in the first experiment,
protein degradation was suspected and cultures were harvested sooner after induction
(3.5 and 6 hours). Purification of P1tR from cells as described above yielded
approximately 0.4 j.tg of protein per ml of culture. Fractions eluted from the chitin
resin were corrected (based on OD6 of cells prior to lysis) by dilution to obtain an
approximately equivalent amount of total protein in each sample. Protein samples
were separated by SDS-PAGE and visualized by Coomassie staining. Although a
band corresponding to the predicted 38 kDa PltR protein was seen, another band of 55
kDa was also seen, indicating that fractions were contaminated with the intein!CBD
tag. Because protein yield was poor, the vectors containing pltR were expressed in
two other hosts, under a variety of conditions. The results are summarized in Table
Al .2, below.
199
Table A1.2. Expression° of PItR in various host/vector combinations's.
Temperature
Expression host.
E. coli
BL21 (DE3)pLysS
Expression
vector
pTYB1
Harvest
timec
Rosetta
pTYB1
pETl5b
-
6 hours
19 hours
pETl5b
15°C 20°C 27°C 30°C 37°C
3 hours
6 hours
19 hours
1 hour
2hours
3hours
4hours
Shours
6hours
+
-
+
+
+
*
*
+
*
+
*
+
+:
+
*
+
*
+
*
+
*
+
:
+
*
+
*
+
*
+
*
+
+:
+
*
+
*
+
*
+
*
+
+:
+
*
+
*
+
*
+
*
+
apis signs designate expression of P1tR, as visualized byCoomassie staining. Minus signs denote the
absence of protein.
bThe results indicated with asterisks were verified by immunoblotting.
dHOS after inducing with 1mM IPTG.
Western analysis of proteins expressed from pETl5b was done using an anti-
His antibody. No His-tagged protein was visible in E. coli BL21(DE3)pLysS cells.
Cell lysates from Rosetta cells carrying pltR in the pET 1 5b vector were separated on
SD S-PAGE and visualized with immtrnoblotting. A 38 kDa band that reacted to the
anti-His antibody was visible. However, a smear of smaller degradation products was
also apparent, increasing in intensity with increasing temperature. The only sample
lacking these degradation products were those from cells grown at 15°C for 1 or 2
hours. Protein yield in none of the samples described in this chapter exceeded 0.4 .ig
protein per ml of culture. Thus, I did not obtain sufficient quantities of pure PItR to
perform DNA-binding and cofactor-binding studies with pure protein. Due to time
constraints, the experiments were abandoned for the time being.
200
A1.3. Future recommendations.
From the experiments described above, it appears that PItR is not stable when
overexpressed in either Pf-5 or E. coli. It is logical from previous studies (19) to
hypothesize that PItR may be a target of Lon protease. Because the expression hosts
used in this study were all deficient in Lon, it appears that PltR suffers from
proteolysis by other routes. Expression in a host with fewer proteases might be useful,
although such hosts are not robust and are difficult to culture. In some instances,
expression of a target protein with a protein affinity tag is more stable than one that is
only tagged with 6xHis, because the larger protein can stabilize the target protein and
make it less susceptible to proteolysis. However, the PltR'-'intein'-'CBD fusion
protein, although stable in E. co/i B ER2566, was not expressed in E. co/i
BL21(DE3)pLysS or the Rosetta strain, so it is unlikely that expression could be
improved with a larger affinity tag.
An expression vector resulting in a 6xHis tagged target protein, and suitable
for use in Pseudomonas, exists (3). The expression of PItR could be attempted using
this vector expressed in Pf-5. In that case, proper coclon usage, correct folding, and
the availability of necessary prosthetic groups/cofactors would be guaranteed from the
parent strain. Because the vectors are inducible through the lac promoter, and the
expression vector carries the lad repressor gene, overexpression is controlled, and the
unsatisfactory results previously obtained from overexpressing P1tR from pME6000
(described above) might be circumvented using these vectors (pLAH3O, pLAH31, and
pLAH32).
It is also possible to perform DNA-binding studies using bacterial cell lysates
rather than purified proteins. This strategy has been successfully used in both
electromobility shift assays (EMSAs) and in DNAse I footprinting studies (2).
Cofactor binding during DNAse I footprinting analysis has also been done using crude
201
bacterial cell lysates, resulting in the successful identification of both benzoate and
muconate as cofactors for the LysR type transcriptional regulator BenM (Becky
Bundy, personal communication). Future attempts to determine the site of PltR
binding, and to determine whether PLT and 2,4-DAPG might act as co-effectors, are
expected to provide insights about mechanisms by which Pseudomonas antibiotics
might exert their intracellular regulatory influences.
Al .4. Acknowledgements
I am grateful to Dr. Linda Thomashow for inviting me to perform the PItR
expression studies in her laboratory, and to members of Dr. Thomashows lab for
invaluable assistance and advice. I am especially grateful to Dr. Dmitri Mavrodi and
Greg Phillips, who were my mentors in protein expression.
202
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Delany, and F. O'Gara. 2002. Characterization of interactions between
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Ausubel., F., R. Brent, R. E. Kingston, D. E. Moore, J. G. Seidman, J.
A. Smith, and K. Struhi (eds.). 1998. Current Protocols in Molecular
Biology. Wiley and Sons, Inc. New York, NY.
3.
Bertani, 1., G. Devescovi, and V. Venturi. 1999. Controlled specific
expression and purification of 6xllis-tagged proteins for Pseudomonas.
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4.
Bundy, B. M., L. S. Collier, T. R. Hoover, and E. L. Neidle. 2002.
Synergistic transcriptional activation by one regulatory protein in response
to two metabolites. Proc. Nati. Acad. Sci. USA 99:7693-7698.
5.
Corbell, N., and J. E. Loper. 1995. A global regulator of secondary
metabolite production in Pseudomonasfiuorescens Pf-5. J. Bacteriol.
177:6230-6236.
6.
Kraus, J., and J.E. Loper. 1992. Lack of evidence for a role of antifungal
metabolite production by Pseudomonasfluorescens Pf-5 in the biological
control of Pythium damping-off of cucumber. Phytopathology 82:264-271.
7.
Liang, T. C., W. Luo, J.-T. Hsieh, and S.-H. Lin. 1996. Antibody
binding to a peptide but not the whole protein by recognition of the Cterminal carboxy group. Arch. Biochem. Biophys. 329:208-2 14.
8.
Lindgren, P. B., R. Frederick, A. G. Govindarajan, N. J. Panopoulos, B.
J. Staskawicz, and S. E. Lindow. 1989. An ice nucleation reporter gene
system: identification of inducible pathogenicity genes in Pseudomonas
syringae pv. phaseolicola. EMBO J. 8:1291-1301.
9.
Maurhofer, M., C. Keel., U. Schnider, C. Voisard, D. Haas, and G.
Défago. 1992. Influence of enhanced antibiotic production in
Pseudomonasfluorescens strain CHAO on its disease suppressive capacity.
Phytopathology 82:190-195.
10.
Miller, J. H. 1992. A short course in bacterial genetics: A laboratory
manual and handbook for Escherichia coli and related bacteria. Cold
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11.
Nowak-Thompson, B., N. Chaney, S.J. Gould, and J.E. Loper. 1999.
Characterization of the pyoluteorin biosynthetic gene cluster of
Pseudomonasfluorescens Pf-5. Journal of Bacteriology 181 (7):2 166-1274.
12.
Nowak-Thompson, B., S. J. Gould, and J. E. Loper. 1997. Identification
and sequence analysis of the genes encoding a polyketide synthase required
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204:17-24.
13.
Sambrook, J. and D. W. Russell. 2001. Molecular Cloning: A
Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, NY.
14.
Sarniguet, A., J. Kraus, M. D. Ilenkels, A. M. Muehichen, and J. E.
Loper. 1995. The sigma factor o affects antibiotic production and
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Acad. Sci. USA 92:1225-12259.
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Schell, M.A. 1993. Molecular biology of the LysR family of
transcriptional regulators. Annual Review of Microbiology 47:597-626.
16.
Schnider-Keel, U., A. Seematter, M. Maurhofer, C. Blumer, B. Duffy,
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Spaink, H. P., R. J. H. Okker, C. A. Wijffelman, E. Pees, and B. J. J.
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Whistler, C. A., N. A. Corbell, A. Sarniguet, W. Ream, and J. E. Loper.
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Whistler, C. A., V. 0. Stockwell, and J. E. Loper. 2000. Lon protease
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204
Appendix 2. Nucleotide and Amino Acid Sequence for pitH, pill, pit.!, or/K, or/F,
orfQ, or/U, orJX, and orfY.
A2.1. Predicted amino acid sequences and positions of open reading frames in
nucleotide sequence
The deduced amino acid sequences for pitH, pit!, pit.J, orfK, orJP, orfQ,
orfU, orJX, and orfY are listed below. All genes are transcribed in the same direction
as p1tLABCDEFG unless noted as "reverse complement". The numbering scheme for
nucleotide sequences is continued from that of published pyoluteorin biosynthetic
gene region (accession number AFO8 1920) for convenience. Asterisks designate stop
codons. Where sequence information was taken from the incomplete Pf-5 genome
sequenced by The Institute for Genomic Research, the open reading frame designation
is preceded by "TIGR".
pitH, reverse complement
Nucleotide 23,340-22,705.
An alternative start codon allows an open reading frame from nucleotide 23,376 to
nucleotide 22,705; the resultant additional amino acids are underlined.
MKQPPAQTTPNIM!JTRRRTSRSDGEHTKIRILEVAARLFAQHGYANTASK
LICEEAGADLAAINYHFGSREALYKAVLIEGHKQLVSFEALSQLAQSEEP
ALIKLDSFIDAIVTRVLDEQSWQSKVCAREILAPTVHFTSLVQEEVMPKF
RLLEALISEITGFPIGDPALARCTISIIAPCLMLA\TI]DRQQPSPLQAVLQ
HDZNALIKAHFKLFARSGLAAIAQ *
205
piti
Nucleotide 23,452 to 24,465
MKKQLIVGVAVLLVATAGVSWFLLRPEKQNDHLKLHGNVDIRQVSLAFDG
SERIAALYAEEGDLVQPGQVLAEIJDTRTLRLEINRSKARIGAQEQALLRL
KNGTRPQEVEQSKARFDAAQAQMQLAQLHMQRLRRIADDTQGKGVSQQRL
DQAAARLQVAKAQSQEQRESWTLAKIGPRNEDIAQATADLQASRDLDLL
EHYLABAQLKAPTQARIRTRLLEPGDMS PQRPVFALALT]DPKWVRAYVN
ERQLGHIRADQMARVYTDSFPDQAIDGKVGYI SSVAEFTPKSVETEDLRT
SLIVYEIRVLVKDPDDRLRLGMPATVYL]DQAPVAGATP *
pltJ
Nucleotide 24,462 to 26,231
MSAPAVTVQAVELVKRFRSGTREQLALEQVSLEVPSGKLSALVGPDGAGK
TTLLRMIAGLLKADEGLLKVLGLD\TAADPQQVQDL ISYMPQKFGLYEDLT
I QENLELYADLHGVRAQQREERFSRLIJQMMDLTRFRDRIJAGQLSGGMKQK
LGLACTLVRS PQLLLLDEPTVGVDPLSRRELWSIIEQLIEQENLTVLI ST
AYMDEAQRCAQVFVLYQGRLIAQGPPAQLCIjLAQGLCYGVGPEAGTPARL
LQARLIJDDNEHI I DAVPEGGMVRFIRSAQVPI1QAI PSLAGQAS PQALEAR
LEDGFMVLLRQQVGSVASLDEPAQAPASYGQG]DAASGPAVVI EVRDLVRK
FGDFTAVASTSFEVLKGEIFGLLGPNGAGKTTTFRMLCGLLPAS SGYLEV
AGVNLRTARAAARRKIGYVSQKFALYANLSALENLRFFGGAYGLHGAKLK
ARVALMLEQFDLDEHKHLRSGELPGGYKQRLAMAVALLHE PQILFLDEPT
SGIDPLARRAFWRRITALAQSGTTIVITTHFMEEAEYCDRIVI QDAGRLL
ALGTPEQVRHQGSESLADMNSAFIAVVERARAQASAPVQ *
TIGR orJK
Nucleotide 26,235-27,384
MTMTDSNRSASGFRRRLIALTRKELRQLMRDKSNLAIGIVLPIVLILIFG
YGLSLDIRNTPLAVVLEDSS PSARSVVAKLASSDYFSVVQVTSMAEARAIi
MDKRRVDGIVRVPADFSRRLEQQDARLQLLLHGADANSAATLGRYVRGAL
NVWQQQQFDRSRQPQSAGRVLVVERTyrWFNAANS STWYLVPGLIALIMTLV
GAFLTSLVLAREWERGTLESLFVTPVRSIEILLAKII PYFLVGLILGLVNC
LVSARLLFEVPIQGSLVLLLLSSMLYLLVTLGIGLLI SAKTRNQFLLASQI
All FSFLPALMLSGFLFDLR11VPTFIRVVGSI LPATYFMELVKTLIFLAGN
NWPLIAKNLAILAAYA\TFLLNAARLCTRKKLD *
TIGR orfp
Nucleotide 27,389-28,507
MDKLJMJFLRELLFLFQKEFLAIVKDPANRVILIAPAIVQSLLFGYGATYD
LNYVPYAVLDQSQGRASTELL,ARFDASGVFARVSTLTSVDEIAPVIDRGS
ALJLVLHIAADFDERLSRHENAGLQLILDGRNSTTAGMAAGHIANLVADFN
QQFLAVDNAPVRLETPAWFNPNLQTRWNIVPGLIAALSMIQTLMLAALSV
AREREQGTF]DQLLVTPLTPFVILLGKALPSVLIGLLQSSLILLIGLFWFK
I PMIGSVLDLYLGLFVFTSACVGIGLSI SALSANMQQANVYTFVLI4NPLI
LLSGLITPVHSMPEALQLLTYLDPLRFAIDLVRRIYLEGATLADVSWNLV
PMIJAVALVTLPLAAWLFRNRLV*
TIGR orfQ
Nucleotide 28,519-30,016
MKTTHSGYLRNGLTLANLLAITACTVGPDFKAPAVTS PERWTDWHSGPPA
S SVPGKAPATTSQTADGNWWQVFGDPVLDQLQAQVREGSPDLHTALLRFA
QVRLQRQIVASLETPEVSFSAP.ASRNRQSRYAPNNRMLEALGSDSDALEK
VLTDPYSLYQAGFDVSWELDLWGRVSRLGEAAQAEVDGAAATLDDVLLSV
SSELARNYFEVRTAQRQTRLLEQEAQI LAkHLQWAVQAREGEEDGFAVE
RQEARLASLHAQI PGWKALHTQAGNRIALLLGEHPGALETLLAERPDNAL
SRPLPNLQLGLPGDLARERPDIRAAQARLHRATAGIGVAEAELYPSVNLG
ADFGFESYKSGQFADWSSRTWSVGpRLDLPLFDRGRRSKTVVLRTLEQQE
AAVAFHRTVLAAWQEVDDAMSRYHNEYQRAHLKDSYDSKARTYEWTRVR
YAAGEASYLEELEAQRTVLEAQPDIJVNSDSQLRTHLI SIYKSMGGHST *
TIGR o,-JU
Nucleotide 30,622-31,227
MQMDDLYKIALFS SLILVVPGPSNTLL.LASGFKFGVLRSLPLVLI EMLGY
TVS ICTWGWGLVLLSHDYPWLIHLIKLLCALYIAALALKTWKTSTTGAQH
TERFE SWPRHLLIATLLNPKGLIFASVIFPAS SFASLGDFLP SLSAFFLL
LAPIALLWVSLGAGIRAQYLGRVSGPL,FSRGTAVVLALFSATLTYTVVRA
v*
207
TIGR orJX
reverse complement
Nucleotide 32,065-33,210
MRTPGTAAFS STTRTSWPRSGRSLTTEPHDSNPTQAPPTPI PTDKSRTTM
KAFLIDRYGKNSGRMGEAPTPTLGAHDVLIEVHAS SVNVLDSKIBNGEFK
LILPYALPLVLGNDLAGVVVEVGPQVQRFKPGPEVYARPPETRIGTFAQW
IAVNENAIALKPANTTMTEAAS I PLVALTAWQVLVETAQLKKGQKVLIHZ
GAGGVGTL,AI QLAKHLGAFVATTTSThNVEWVKALGADQVIDYKQQNFES
L1LHGYDLVLNSLGSDVLEKSLKVLKPGGQLI SI SGPPTVQFAQEQGLAWP
LQQVMRLLSFGIRRKARQRDVRYAFVFMIADGAQLQQITALI EAGI IKPV
VDRSF SFESTAEALQYVEQGRTKGKVVVRIK *
TIGR orfY
reverse complement
Nucleotide 33,122-34,000
MIWTQTTFPPALQTSFINAANQSIMVRGI PFAYRDTGPAGGVPLVLFNHW
GAVLDNFDPAI IDGLAQTRRVITTDYRGIGGSGGTAPLJTVGDMAQDAIEL
IQALGFKSVDILGFSLGGFVAQDIALQAPQLVHRLILTGTGPAGGIGIDI(
VGSVTWPLMLIKGLLTLRDPKYYLFFTSTPHGRRAASEYLQRLKARSKHRD
KGPTPGAFLRQLQAITAWGKQAPQDLGRLIRAPTLIVNGDN]DIMVPSANSH
ALAKRVPNAQLVMYEDAGHGGI FQYHADFVAKVRAFLDDRAP *
A2.2. Relevant nucleotide sequence
The nucleotide sequence for the region including pitH, pitl, pit.1, orJK, orJP,
orfQ, orftf, orJX, and orfY is displayed below. For convenient reference, the
numbering sequence is continued from that of published pyoluteorin biosynthetic gene
region (accession number AFO8 1920). Sequence information derived from this thesis
is highlighted in bolded type. Sequence information generously provided by Ian
Paulsen (The Institute for Genomic Research, Rockville, MD) comprises the
remaining sequence for the section, in normal typeface.
22701
22751
22801
22 851
22901
22951
23001
23051
23101
23151
23201
23251
23301
23351
23401
23451
23501
23551
23601
23651
23701
23751
23801
23851
23901
CCGACTATTG GGCATGGCC GCGAGGCCGG ACCGGCGAA CAGCTTGAAG
TGGGCTTTCA GGGCGTTGGC GTCGTGTGC AGG&CGGCT GCkGCGGGCT
GGGCTGC!GG CGACTATCA CCGCCAGCAT CAGGCAGGGC GCGATGATGC
TGATGGTGCA ACGCGCCAGC GCCGGGTCAC CGATCGGAAA GCCGGTGTC
TCGCTGAGA GCGCTCCAG CAGACGGAAT TTGGGATCA CTTCTTCTTG
CACAAGGCTG GTGAAkTGGA CAGTGGGCGC CAGTATTTCC CGTGCACAGA
CTTTGCTTG CCAGCTCTGC TCATCGGCA CCCGGGTGAC GATGGCATCG
ATGAAGCTAT CGAGCTTGAT CAGCGCCGGC TCTTCGCTT GCGCCAGCTG
CGACAGGGCT TCGAAGCCA CAGCTGCTT GTGTCCCTCG ATCAGCACCG
CCTTGTACAG GGCTTCCCGG CTACCGAGT GGTAGTTGAT GGCGGCGAGG
TCGGCGCCGG CTTCTTCACA GATGGCTTG CTCGCGGTGT TGGCATAGCC
ATGCTGGGCA AACAGGCGGG CAGCCACTTC GAGAATGCGG ATTTTAGTGT
GTTCGCCGTC GCTTCTGCTG GTTCTGCGTC GCGTGTTCAT GATGTTCcTC
GTGGTCTGAG CGGGGGGTTG CTTCATGGCC AGTATCTGTT ATTTCTAT
TTAA.ATTCAA ATTGAATTTT AkTTAGGCCT TGGCAAACA AGGACTCATG
GATGAAGAAG CAGTGTTG TAGGGGTTGC CGTACTGCTG GTGGCCACAG
CGGGTGTTTC CTGGTTTCTC CTTCGCCCCG AGAAGCAGA CGAecACCTC
AAGCTCCATG GCAACGTCGA CATTCGCCAG GTGTCACTGG CGTTCGACGG
CAGTGAGCGA ATCGCCGCGC TGTATGCCGA AGAGGCGAC CTGGGCAGC
CAGGCCAGGT CCTGGCGGAA CTCGACkCAC GCACACTGCG CCTGGA.AATA
AATCGCTCCA AGGCCAGGAT CGGCGCCCAG GAACAGGCGC TGCTGCGCTT
GAAAAATGGC ACACGGCCTC AGAGGTCGA GCAGTCCAAG GCACGTTTG
ATGCCGCCCA GGCGCAAATG CAGC'GGCTC AACTGCACAT GCAGCGCCTG
CGCAGGATCG CCGACGACAC CCAGGGCAAG GGCGTCAGCC AGCACGCCT
GGACCAGGCA GCCGCCCGCT TGCAGTGGC CAAGGCCCAG TCGCAGGAGC
23951 AGCGCGATC CTGGACCCTG GCCAAGATCG GCCCACGCAA TGAGCATC
24001
24051
24 101
24151
24201
24251
24301
24351
24401
24451
24501
24 551
24601
24651
24701
24751
24 801
24851
GCCCAGGCCA CGGCCGACCT GCAAGCCTCC AGGGCCGACC TGGACCTGCT
GGIACATTAC CTGGCGCGCG CTCAGCTCAA GGCGCCGACC CAGGCCCGGA
CCGCACGCG CCTGCTGGAG CCGGGGGATA TGGCCTCGCC CCAGCGCCCG
GTGTTTGCCC TGGCCCTGAC CGACCCCAAG TGGGTACGGG CCTATGTCAA
CGAACGCCAG TTGGGCCACA TACGTGCGGA CCAGATGGCG CGGGTCTACA
CCGACAGCTT TCCTGACCAG GCCATCGACG GCAAGTCGG CTACATCTCC
PCGGTTGCCG AATTCACCCC CAAGTCGGTG GAACGGAAG ACCTGCGCAC
CAGCCTGGTC TACGAGATCC GGGTGCTGGT CAAGGACCC GACGATCGCC
TGCGGCTCGG TATGCCGGCG ACCGTCTACC TTGACCAGGC ACCGGTGGCC
GGGGCCACGC CATGAGTGCG CCAGCGGTAA CGGTTCAGGC TGTTGAACTG
GTCAAGCGCT TTCGCAGTGG AACGCGAGAA CAGPTGGCTC TGGAGCAGG
TTCTCTGGAG GTGCCCAGTG GCAATTGAG GCCCTGGTG GGGCCGGACG
GGGCCGGAA AACCACCCTG TTGCGGATGA TTGCCGGCTT GCTCAAGGCC
GACGAAGGGC TGTTGAAGGT GCTGGGGCTG GACGTCGCCG CAGATCCGCA
GCAGGTGCAG GACCTCATCA GCTACATGCC CAGAAGTTC GGCCTGTACG
AAGACCTGAC GATCCAGGA.A AAccTcGAAc TCTACGCCGP CCTGCATGGC
GTGCGCGCCC AGCAGCGCGA GGAGCGCTTT TCCCGGTTGC TGCAGTGAT
GGACCTGACG CGTTTCGCG ACCGGCTGGC CGGACAGCTT CCGGCGGCA
24901 TGWICAGAA ACTGGGGTG GCCTGTACCC TGGTCCGTTC
24951 TTGCTGCTGG ACGAGCCCAC CGTGGGGGTG GATCCGCGT
25001 ACTGTGGAGC ATCATCGAAC AACTGATCGA GCAGGAAAAC
25051 TGATCAGCAC TGCCTACATG GACGAGGCCC AGCGTTGCGC
25101 GTGCTATACC AGGGGCGGCT GATCGCCCAG GGGCCACCGG
25151 CCATCTGGCG CAGGGCCTGT GCTATGGCGT CGGCCCCGAG
25201 CGGCCAGGC GTTGCAGGCG CGCTTGCTTG ACGCATGA
25251 GATGCCGTGC CCGAAGGCGG CATGGTTCGC TTTATCCGTA
25301 GCCTCTGCAG GCCAT!CCAT CCCTGGC!GG CCAGGCCTCT
25351 TGGAGGCCCG CCTCGAAGAT GGCTTCATGG TGTTGCTCCG
25401 GGCAGCGTTG CAAGCCTGGA CGAGCCGGCG CAGGCGCCCG
ACCGCAACTG
CGCGGCGGGA
CTCACGGTGC
CCAGGTCTT?
CGCAGTTGTG
GCCGGCACCC
ACACATCATC
GCGCCCAGGT
CCCCAGGCGC
GCAACAGGTT
CCAGCTACGG
25451
25501
25551
25601
25651
25701
25751
25801
25851
25901
25951
26001
26051
26101
CCAGGGCGAC GCTGCCTCTG GGCGTGCGGT GGTCATCGA GTCCGCGPCC
TGGTGCGCAA GTTCGGGGAC TTCACCGCGG TTGCCAGCAC CTCGTTCGAG
GTGTTGAAGG GCGAGATATT CGGCTTGCTG GGCCCCAACG GAGCCGGCAA
GACCACCACG TTTCGCATGC TCTGCGGTTT GCTCCCGCC TCCAGCGGGT
ACCTTGAGGT GGCTGGCGTC AACCTGCGCA CCGCACGGGC GGCGGCGCGG
CGCAAGACG GCTACGTCTC GCAGAAGTTC GCCCTGThCG CGAACCTGTC
GGCCCTGGAG AACCTGCGCT TCTTCGGCGG CGCCTATGGC CTGCACGGCG
CGAAGCTGAA AGCGCGGGTG GCGTTGATGC TCGAACAGTT CGACCCGAC
GAACACAAGC ACCTGCGCAG CGGCGAGCTG CCCGGTGGC ACAAGCAGCG
CCTGGCCATG GCGGTCGCGC TGCTGCACGA GCCACAGATC CTGTTTCTCG
ACGAACCCAC CAGCGGTATC G&CCCCTTGG CCCGCCGGGC GTTCTGGCGC
CGTATCACCG CCCTGGCGCA AAGCGGCACG ACGATCGTGA TCACC?CCCA
CTTCATGGAA GAGGCCGAGT ACTGCGACCG TATCG!CPLTC CA.GGATGCCG
GACGGCTGCT GGCCCTGGGC ACTCCGGA.AC AGGTACGCCA CCAGGGCAGC
26 151
GAGTCGCTG CCGACATGAA CAGCGCCTTC ATTGCCGGG TCGAGCGTGC
2620 1
TCGGGCCCkG GCCAGCGCCC CGGTGCAGTG AGGAATGACC ATGACTGACT
26251 CCATCGATC CGCCTCAGGG TTCAGGCGCA GGCTCATTGC GCTCACCCGA
26301 AAGGAGTTGC GCCAATTGAT GCGTGACAAG AGCAACCTGG CGATAGGCAT
26351 TGTCCTGCCC ACGTACTG TCCTGATAT TGGCTACGGC CTGTCCCTGG
26401 ACATTCGCAA CACGCCGCTG GCCGTGGTCC TGGAAGACAG CTCCCCCAGC
264 51
GCCCGCAGCG TGGGGCGAA GCTTGCGAGT TCGGACTACT TCTCGGTGGT
26501 CCAGGTGACC AGCATGGCCG AGGCCCGAGC GCTGATGGAC AAGCGTCGGG
26551 TCGACGGCAT CGTGCGCGTG CCGGCGGATT TTTCCAGGCG CCTCGAACAG
26601 CAGGATGCGC GCTTGCAATT GCTCCTGCAT GGCGCCGATG CCAACAGCGC
26651 GGCGACCCTT GGACGCTATG TCAGGGGCGC GCTGAJTGTC TGGCAACAGC
26701 AGCAGTTCGA CCGCTCCCGG CAGCCCCAGT CGGCCGGTCG GGTGCTGGTC
26751 GTCGAACGCA TGTGGTTCAA CGCCGCCAAC AGCAGTACCT GGTACCTGGT
26801 GCCCGGGCTC ATCGCCCTGA TCATGACCCT GGTGGGTGCC TTCCTGACCT
26851 CTCTGGTACT GGCCCGGGAG TGGGAGCGGG GCACGCTCGA ATCACTGTTC
26901 GTCACCCCGG TCAGGTCGAT CGAAATACTC CTGGCCAAGA TCATTCCCTA
26951 TTTCCTGGTG GGCCTGCTGG GCCTGGTGAT GTGCCTGGTT TCGGCGCGGT
27001 TGCTGTTCGA GGTACCGATC CAGGGTTCGC TGGTGCTCCT GCTGTTGAGC
27051 TCCATGCTCT ACCTGCTGGP CACCCTGGGC ATTGGCCTGC TGATTTCGGC
27101 GAAGACACGC AACCAGTTCC TGGCCAGCCA GATCGCGATC ATCTTCAGCT
27 151
TCCTGCCGGC GCTGATGCTC TCGGGGTTCC TCTTCGATCT GCGCAATGTG
27201 CCGACGTTCA TCCGCGTGGT GGGCTCGATC CTGCCGGCGA CTTACTTCAT
272 51
GGAACTGGTC AAGACCCTGT TCCTGGCTGG CAACAACTGG CCATTGATCG
27301 CCAAGAACCT CGCAATCCTC GCGGCCTACG CCGTGTTCCT GTTGAACGCT
27351 GCACGCCTGT GCACCCGCAA GAAGCTGGAT TGAAGCCATG GACAAGCTGA
27401 TGAATTTCCT ACGCGAGTTG CTGTTCCTGT TTCAGAAGGA GTTCCTGGCC
27451 ATCGTCAAGG ACCCGGCCAA CCGGGTGATC CTGATTGCCC CGGCGATCGT
27501 CCAGTCATTG CTGTTCGGCT ACGGCGCCAC CTACGACCTG AACTACGTGC
27551 CCTATGCCGT GCTGGATCAG AGCCAGGGGC GCGCTTCGAC AGAGCTTTTG
27601 GCACGCTTCG ATGCCTCCGG GGTATTTGCC CGAGTGTCGA CCCTGACCAG
27651 CGTCGATGAG ATCGCCCCGG TGATCGACCG GGGCAGTGCC TTGCTGGTGC
27701 TGCATATCGC CGCAGACTTC GACGAGCGCC TGAGCCGGCA TGAAAACGCC
27751 GGCTTGCAGT TGATCCTGGA TGGGCGTAAC TCGACGACCG CGGGAATGGC
27801 GGCCGGGCAT ATCGCCAACC TGGTGGCTGA CTTCAACCAG CAGTTCCTGG
27851 CGGTGGACAA CGCGCCGGTA CGCCTGGAGA CCCGTGCCTG GTTCAACCCC
27901 AACCTGCAAA CCCGCTGG? CATAGTGCCG GGCCTGATCG CCGCGCTGAG
27951 CATGATCCAG ACCCTGATGC TGGCGGCGTT GTCGGTAGCC AGGGAGCGCG
28001 AGCAGGGCAC CTTCGACCAA CTGCTGGTGA CTCCGCTGAC GCCTTTCGTG
28051 ATCCTGCTGG GCAAGGCCCT GCCGTCGGTA CTGATCGGTC TCTTGCAGTC
28101 CAGCCTGATC CTCCTCATCG GCCTGTTCTG GTTCAAGATC CCCATGATCG
28151 GCTcCGTGCT GGACCTGTAC CTGGGGCTGT TCGTCTTCAC CAGCGCCTGC
210
28201
28251
28301
28351
28401
28451
28501
28551
28601
28651
28701
28751
28801
28851
28901
28951
29001
29051
29101
29151
29201
29251
29301
29351
29401
29451
29501
29551
29601
29651
29701
29751
29801
29851
2 9901
29951
30001
30051
30101
30151
30201
30251
30301
30351
30401
30451
30501
30551
30601
30651
30701
30751
30801
30851
30901
GTGGGCATCG GGCTGTCGAT CTCGGCGCTG TCGGCCAACA TGCAGCAGGC
CATGGTGTAC ACCTTCGTGC TGATGATGCC GCTGATCCTG CTCTCCGGCC
TGATCACCCC GGTGCACAGC ATGCCCGAGG CCCTGCAACT GCTGACCTAT
CTCGATCCGC TGCGCTTTGC CATCGACCTG GTCAGGAGGA TCTACCTGGA
GGGGGCAACG CTGGCGGATG TGTCCTGGAA CCTGGTGCCC ATGCTTGCCG
TTGCGCTGGT GACCCTGCCG CTGGCTGCGT GGCTGTTTCG CAATCGATTG
GTGTGATAAG GAACGTCATG AAAACTACTC ACTCTGGATA CCTGCGTAAT
GGGCTGACGC TGGCCATGCT GCTGGCCATC ACCGCCTGCA CCGTCGGGCC
CGATTTCAAG GCGCCGGCAG TTACCAGCCC GGAACGCTGG ACGGATTGGC
ACAGTGGCCC GCCAGCGTCT TCGGTGCCGG GCAAAGCCCC GGCGACCACC
TCGCAPCCG CAGACGGCAA CTGGTGGCAG GTGTTTGGCG ACCCGGTGCT
GGACCAGCTG CAGGCCCAGG TGCGCGAAGG CAGCCCGGAC CTGCACACGG
CCTTGCTGCG GTTTGCCCAG GTGCGGTTGC AACGCCAGAT AGTCGCTTCC
CTGGAAACGC CCGAAGTGTC CTTTTCCGCG GCGGCTTCGC GAAACCGGCA
AAGCCGGTAT GCGCCGAACA ATCGAATGCT TGAAGCCTTG GGCAGCGACA
GTGATGCCCT GGAAAAGGTG CTTACCGACC CTTATAGCCT GTACCAGGCC
GGGTTCGACG TTTCATGGGA GCTGGATCTC TGGGGGCGGG TGTCACGCCT
GGGTGAAGCC GCCCAGGCCG AGGTCGATGG TGCGGCCGCG ACCCTGGACG
ACGTGTTGCT CAGTGTCTCC AGCGAGCTGG CGCGAAACTA CTTCGAAGTG
CGTACCGCAC AGCGCCAGAC CCGTCTTCTG GAGCAGGAGG CACAGATCCT
GGCCGCGCAC CTGCAAGTGG TTGCCGTCCA GGCCCGGGAG GGCGAGGAAG
ACGGGTTCGC GGTGGAACGC CAAGAGGCAC GGCTGGCCTC GCTGCATGCA
CAGATACCGG GTTGGAAGGC GCTGCACACC CAGGCGGGCA ACCGTATTGC
GCTGTTGCTG GGGGAGCATC CCGGAGCCCT GGAAACCTTG CTCGCGGAGC
GGCCTGATAA TGCCTTGAGC CGCCCGCTGC CCAACCTGCA GTTGGGCCTG
CCTGGCGACC TGGCCCGGGA GCGTCCGGAC ATCCGCGCGG CCCAGGCCCG
CTTGCATCGG GCGACGGCGG GCATCGGAGT GGCCGAGGCC GAACTGTACC
CGAGCGTGAT GCTGGGGGCC GACTTCGGCT TCGAATCCTA CAAGAGCGGC
CAGTTTGCCG ACTGGAGCAG CCGCACCTGG TCGGTAGGGC CGCGCCTGGA
CCTGCCGCTG TTCGACCGTG GGCGGCGCAG CAAGACCGTG GTCCTGCGCA
CACTCGAACA GCAAGAGGCG GCCGTGGCAT TTCACCGGAC CGTACTGGCA
GCCTGGCAGG AAGTGGACGA TGCGATGAGC CGCTACCACA ACGAATACCA
GCGTGCCGCG CACCTCAAGG ACAGCTATGA CAGCAAGGCC CGCACCTATG
AGTGGACCCG CGTCCGGTAC GCGGCCGGGG AGGCCAGCTA CCTGGAGGAA
CTGGAAGCGC AGCGCACCGT GCTGGAGGCG CAGCGCGACC TGGTCAATAG
CGACAGTCAA CTGCGTACTC ACCTGATCAG CATTTACAAA TCCATGGGTG
GGCATTCCAC CTGAGCCCCT GAAAQGCTGC CTCTGGCAGC ACTCCCCCCA
GACCCACTTT CCTAACGGAA TAGGAAGCCA TGCAAATGGA TGACCTCTAC
AAGATCGCGC TTTTTTCGTC CCTGATACTG GTAGTGCCGG GGCCCTCAAA
CACCTTGCTG CTGGCCTCGG GCTTCAAGTT CGGCGTGTTG CGCTCGCTGC
CACTGGTGTT GATCGAAATG CTCGGCTACA CCGTTTCGAT CTGCACCTGG
GGCTGGGGGC TTGTGCTGCT GTCCCACGAC TACCCGTGGC TGATCCACCT
GATCAAGCTG CTGTGCGCCT TGTACATCGC GGCGCTGGCC TTGAAGACCT
GGAAGACCTC CACCACCGGA GCCCAGCACA CCGAGCGCTT TGAGAGCTGG
CCACGGCACC TGTTGATTGC GACCTTGCTC AACCCCAAGG GGCTGATCTT
CGCCAGTGTG ATCTTCCCGG CCAGCTCATT CGCCAGCCTG GGGGATTTCC
TGCCGTCATT GAGCGCTTTC TTCCTGGCAC TGGCGCCAAT AGCCTTGCTC
TGGGTCTCGC TGGGGGCGGG CATTCGAGCC CAATACCTGG GGCGGGTTTC
TGGACCGCTG TTTTCCCGGG GCACCGCCGT CGTTCTGGCG CTGTTTTCCG
CCACCCTCAC TTACACCGTG GTTCGTGCGG TGTAGGTGTT TTTGCCGCGG
CTTTCAGCGC CCAAGTGAGG CTGGGGCAAT GGCGCTGCTG AGAGAGGCCC
TTGTACCCCC CGTGTTCCAG CACCACTCTG TTGTCGCCAA CACCCGGGGG
GGCTCATTCC ACGTAGCGCA ACCACAAATG GGTGCGACCG CGAACAGCCT
CGACAACCGT TGAAACCACG GCTTGCCGGT TTTCCTCGGA GGTGTTGTAG
AGCGCACAAA AAACGCTCGC CACCGCGCTG GCGACGCAAA GGTCGGCAGG
211
30951
31001
31051
31101
31151
31201
31251
31301
31351
31401
31451
31501
31551
3 1601
31651
31701
31751
31801
31851
31901
31951
32001
32051
32101
32151
32201
32251
32301
32351
32401
32451
32501
32551
32601
32651
32701
32751
32801
32851
32901
32951
33001
33051
33101
33151
33201
33251
33301
33351
33401
33451
33501
33551
33601
33651
CCCTGCACGC AGGAAACCGA GGTAAGACGC GCCAATCCCC ATCATCCGGG
ATTTTGCTCG CCAGTGGGGC GGTTTTTGGG CAGGCGCGTA CCCGCGGGCG
CTGAGCGGTG GCCTTGATCC GATGAACAGG GCGTTTGGCG GATGTTAATC
TGCGCCTCCC GGACAAACGG TGGGCCAGTG GATGCAGAGG ATGTTGAGTG
AAGCCGAGCT TCACAAGGTG CTGGAATCGA TCGAAAGCGA CTCCTGCCAT
GACCTGAATC TTCTGTATGA CGGGATGCAG ATAGACCGGT GTGCTCTTTT
CAATCAAGTC GCCATGGCGG TGGCCCGGCT CTTTATCGAG GGGCAGCGCG
ACTTCCATTA CGGCGATGCG GTCATGTGGG TCGCGGCCAG AACCGCCGGC
GAACTCACGC GGTTTCGCTA CGGCTCCAGC ATCAAGCGGC GCCAAAGAAT
CGGCGGCGCG ACAGCAGCGG CCGGCGCTTG CGGCTCCGGC TTCTCCGGCG
CCTGGGCTGA CGTTGCCTCG CGGGGCTCAG GCGCTCTTGT CGACTCGGTC
GGGTCATCGG TCGGCGATGG GGTCATTTGA TCCTGACCAC CACCTTGCCC
TTGGTTCGTC CCTGCTCGAC GTACTGCAAG GCCTCTGCCG TCGATTCGAA
ACTGAAGCTG CGGTCAACCA CAGGCTTGAT GATTCCGGCC TCGATCAGCG
CGGTGATCTG TTGCAGCTGG GCGCCATCGG CCCGCATGAA CACAAAGGCG
TAGCGGACGT CCCGCTGGCG CGCCTTGCGC CGGATACCGA AGCTCAAGAG
GCGCATGACC TGCTGCAACG GCCAGGCCAG CCCTTGCTCC TGGGCGAATT
GCACCGTGGG TGGCCCGGAG ATGGAAATCA GCTGTCCGCC GGGCTTGAGG
ACCTTGAGGG ACTTTTCCAG TACGTCGCTG CCCAGGCTGT TCAACACCAG
GTCGTAGCCA TGCAAGAGGC TTTCGAAGTT CTGCTGCTTG TAGTCGATCA
CCTGATCGGC GCCCAGGGCT TTGACCCATT CGACATTCGC CGTGCTGGTG
GTGGTCGCGA CGAM.GCGCC GAGGTGTTTG GCCAGCTGGA TGGCAAGGGT
GCCGACACCG CCTGCGCCTG CGTGGATCAG CACTTTCTGG CCCTTTTTCA
GTTGGGCGGT TTCCACCAGC ACTTGCCAGG CGGTGAGTGC GACCAAGGGG
APCGACGCGG CCTCGGTCAT GGTGGTGTTG GCAGGTTTCA GGGCGATTGC
GTTTTCGTTC ACGGCGATCC ACTGGGCGAA CGTCCCGATC CGTGTTTCAG
GCGGGCGCGC GTAGACTTCG TCTCCCGGCT TGAAGCGTTG CACTTGGGGG
CCAACCTCAA CCACGACGCC CGCCAGGTCG TTGCCCAGTA CCAGCGGCAA
TGCATAGGGC AGGATCAGCT TGAATTCGCC GTTGCGGATC TTCGAGTCCA
GCACGTTGAC GCTGCTGGCG TGGACCTCGA TCAACACGTC GTGGGCACCC
AGCGTGGGCG TGGGCGCTTC GCCCATGCGC CCACTGTTCT TGCCATAGCG
ATCAATTAGA AAGGCTTTCA TCGTGGTGCG GCTCTTGTCG GTTGGAATAG
GTGTGGGCGG TGCTTGGGTG GGGTTGCTGT CATGGGGCTC TGTCGTCAAG
GAACGCCCGG ACCTTGGCCA CGAAGTCCGC GTGGTACTGG AAAATGCCGC
CGTGCCCGGC GTCCTCATAC ATCACTAGCT GTGCGTTGGG GACACGCTTG
GCCAGGGCAT GGGAGTTGGC GCTGGGCACC ATGATGTCGT TGTCGCCATT
GACGATCAGC GTCGGCGCCC GCAAGCGCCC GAGATCCTGC GGCGCCTGTT
TGCCCCACGC GGTGATGGCT TGCAGCTGGC GCAGGAAAGC GCCGGGCGTT
GGGCCCTTGT CGCGATGCTT GCTGCGGGCT TTCAGGCGTT GCAGATATTC
CGAGGCGGCT CGACGACCAT GGGGCGTCGA GGTAAAGAAC AGGTAGTACT
TGGGATCGCG CAAGGTCAGC AAGCCTTTGA GCATCAAGGG CCAGGTCACC
GAGCCGACCT TGTCGATACC GATGCCGCCG GCCGGTCCGG TGCCGGTGAG
AATCAAQCGG TGCACCAATT GGGGGGCTTG CAGGGCGATG TCCTGAGCGA
CGAAGCCGCC GAGTGAGAAG CCCAGGATAT CGACGCTCTT GAACCCCAGG
GCCTGTATCA GCTCAATGGC GTCTTGCGCC ATGTCGCCGA CCGTCAGGGG
GGCGGTTCCA CCCGAGCCGC CGATGCCGCG ATAGTCGGTG GTGATGACTC
GCCGGGTCTG TGCCAGCCCA TCGATGATTG CCGGGTCGAA GTTGTCCAGC
ACCGCGCCCC AGTGGTTAAA CAGCACCAGG GGCACGCCGC CCGCGGGACC
GGTATCGCGA TAGGCGAAGG GAATGCCCCT GACCATGATC GACTGGTTCG
CCGCGTTGAT GAIICGACGTC TGCAAAGCCG GGGGGAAGGT TGTCTGCGTT
GGATTCATTG CTACTCCGAA CAGCGGACCG GATTCGTCGC AGTGCCTGCC
GCGAATCCGC CGCGAATGCT CGCTACCTGA AGATCACTCT TGGGTCTGGT
AACGCTGGGC AACCTCGGAT TGACGGATGT GCCCCAGCAG GCCCTGACGT
JCGCTCTGCA GGTCATCCCA GGGTTCAGCC TGTTGCAGTG GCGGGATGGT
CACCGGTTCG CGCCGATCGA AACCGACCAG TGCGGCATCC ACCAGATCGC
212
33701
33751
33801
33851
33901
33951
34001
34051
34101
34151
34201
34251
34301
34351
34401
34451
34501
34551
34601
34651
34701
34751
34801
34851
34901
34951
35001
35051
35101
35151
35201
35251
35301
35351
CCACTTCCAT GATTTCGTCC AGGGTATTGA TGTCGACGCC GGAGCGGTCC
CAGATTTCCG TGCGGGTGGC GGCCGGCAGC ACGGCCTGCA CGTACACGCC
CCGGGGCGAC AGTTCCAGGC TCAGGCCCTG AGACAGGAAC AGCACGAAAG
CCTTGGTTGC ACCATAGACC GTCATGCCGA ACTCCGGCGC CAGGCCCACC
ACCGAACCGA TGTTGATGAT CGCGCCGTCA CCGGCTTTGG CCAGGCGCGG
GGCAATGGCA CTGGCCAGCC GCACCAGTGC CGTGGTGTTG AGGGCAACCA
GTTGCGCTAC GCTGTCGGTG CTTTGCTCGA TGAAGTTGCC GGACAGCGCG
GCGCCGGCGT TATTGATGAG GATGCCGATA CGGGCGTCGT CGCGCAGGCG
GCTTTCAACG GTTGTCAGAT CGCTGAGTTG GGTCAAATCC GCTTTCATCA
CCTCGACGGC GACCCCGTGT TCGCCGCGCA GTCGGGCTGC AAGTGCGTCC
AGGCGTGCCC GGTCGCGGGC AACCAGGACC AGATCATGGC CACGTTGCGC
GAAGCGCTCG GCGTAGATGG CTCCGATGCC AGTGGAGGCG CCAGTGATGA
GAACGGTAGG GCGAGTGTTC ATGGGGTCAT CTCTCTTTCA AAATGGGTGA
AAACGGACAG CTCGGTTTCT GCTTGCTCAG CTTGCGCTGG CGCTCGAATC
AACCAGCGTC GGGTAGTCGA TGTAGCCTGC CGCGCCGCCG CCGTAGAGGG
TGGCGGGATT GAATGCGGAC AAGGCGGCGC CGGTCTTGAG ACGCTCCACC
AGATCCGGGT TGCCGATGAA CGGGCGACCG AACGCGATCA GATCGGCCTG
GTCTTCGGCG AGCCGTGAGG TTGCCAGCTC CAGGTCATAG CCGTTGTTGG
CGATGTAGGT GTTTTTGAAA CGCTGGCGCA GGGCGGTGAA ATCCAATGGC
GCCACGTCAC GTGGGCCACC GGTCGCGCCT TCGACCATGT GCAGGTAAAC
GACGTCGAGG GCGTCGAGTT GATCGACGAC GTAATTGAAT TGCGCCTGCG
GATTGCTGCT GGAGACACCG TTGGCCGGCG ACACCGGCGA CAAGCGCACC
CCGGTGCGAT CCGCGCCGAT TTCATTGACC ACGGCAGCGG TGACTTCGAG
CAACAGACGC GCACGATTTT CAATCGAGCC GCCGTAAGCA TCGGTGCGTA
CGTTGGCGCT GTCCTTGAGG ACTGATCCA GCAPTAGCC GTTCGCGCCG
TGAATTTCCA CGCCATCGAA CCCGGCAGCG ATAGCGTTTG CCGCGGCCTG
GCGGAAATCG GCGACGATCC CCGGCAGCTC GCTGATGTCC AGTGCGCGGG
GCTCGCTGGC GTCTTCAAAG CGGTTGTTGA CGAACACCTT GGTGGCCGCA
CGCAGCGCGG AAGGGGCCAC GGGGGCGGCG CCGTTTTCTT GCAGATCAAC
GTGGGACACG CGGCCGACAT GCCACAGTTG CACAAAGATC TTTGCGCCCT
GGGCGTGGAC CGCGTCGGTC ACCGTGCGCC AGCCATCAAT CTGCGCCTGG
GTGTAGATCC CGGGGGTGTC CTGATAGCCC TGGCCCTGTT GGGAAATCTG
CGTGGCTTCG CTGATCAGCA AGCCTGCGCT AGCGCGTTGG CTGTAATAGG
TGGCGGCGAA TTCGCTGGGA ACAAAGCCTG CGC