Tuning Bacterial Behaviour
Judy Armitage
University of Oxford
Department of
Biochemistry and Oxford
Centre for Integrative
Systems Biology
StoMP 2009
E.coli chemotaxis-the best
understood “system” in Biology
•E.coli has one constitutive chemosensory pathway.
•Biases swimming direction by regulating motor switching
•Not essential and phenotype obvious
•All components known, kinetics of all reactions, copy number of all proteins,
structures of most
•Cells respond to ~2 molecules over 6 orders of magnitude
•Paradigm for 2 component pathways
E.coli chemotaxis
• 4 dedicated constitutive membrane
spanning receptors (MCPs) plus Aer
• One sensory pathway via CheW
(linker), CheA (histidine protein
kinase), CheY (response regulator)
• Chemotaxis is via biasing a
normally random swimming pattern
• Adaptation of MCPs via single
CheB/R methylation system
• Mutations give either smooth
swimming or tumbling phenotypes
• Unusual HPK pathway
• Termination of CheY-P through
CheZ-not HPK phosphatase
Histidine protein kinase signalling
MCP 
CheA  CheY/B
Rhodobacter sphaeroides
•Member of a-subgroup proteobacteria
•Heterotrophic, photoheterotrophic, anaerobic
respiration, CO2- N2- fixation, hydrogenase, fermentation
•Quorum sensing, biofilm forming
•Membrane differentiation-aerobic vs photoheterotrophic
•Targeting-flagellum, cell division proteins,
chemotaxis proteins
Chemotaxis in R.sphaeroides
• Single unidirectional flagellum (under lab
conditions)
• Stopping involves a molecular
brake
• 3 chemosensory operons
• Need transport and possibly partial
metabolism for chemotactic response
• Why have 3 chemosensory pathways to
control on flagellar motor?
cheY5 mcpB tlpS mcpA cheD cheY1 cheA1
• 4 CheAs
•8 membrane spanning MCPs
•4 cytoplasmic Tlps
•6 CheYs
•2 CheBs
• NO CheZ
cheY3
cheA4
cheR3
cheA2
cheW1 cheR1 cheY2
cheW2 cheW3 cheR2
cheB2 cheW4 slp
cheBRA
cheY4 mcpG
cheB1
tlpT cheY6
tlpC
cheA3
R.sphaeroides uses a brake to stop
Activity of the chemotaxis
proteins in vitro
Is there “cross talk” between apparently
homologous proteins encoded by the
different operons?
In vitro phosphotransfer measured
between 4 CheA HPKs and the 6 CheY and
2 CheB RRs
CheA has H on Hpt domain
Pattern of in vitro phosphotransfer
Kinase and Response Regulators
• CheA2 will phosphotransfer to all Che
Response Regulators-wherever encoded
(CheOp1, CheOp2 or CheOp3)
• CheA1 will only phosphotransfer to proteins
encoded in own operon (CheOp1)
• CheA3/4 will only phosphotransfer to proteins
encoded in its operon (CheOp3)
• How is discrimination achieved?
Chemotaxis: in vitro
phosphotransfer
CheA1
CheY1
CheY2
Horribly complex!
CheY3
CheA2
CheY4
CheA3A4
CheY5
CheY6
CheB1
CheB2
Where are the gene
products?
• Do the genes encode proteins that make separate or
cross-talking pathways in vivo ?
• G(C,Y)FP –(N and C terminal) fusions to all che
genes; replaced in genome behind native promoters
and tested for normal behaviour
• Confirmed by immuno-elecronmicroscopy
Pathways targeted to different part
of cell
Red: CheOp2
Blue: CheOp3
Cytoplasmic
general:
CheB1, CheB2,
CheY3, CheY4,
CheY6
.
Localisation
• Chemosensory proteins are physically separate in the cell
• CheOp2 encoded proteins with MCPs at poles and CheOp3
with Tlps in cell centre
• CheAs physically separate and therefore do not cross
phosphotransfer in vivo ?
• What controls localisation?
• Why have 2 physically separate chemosensing pathways?
• Is this common? Does it only apply to taxis pathways?
Would not have been identified without in vivo investigations
Localisation requires two CheOp3
proteins
cheA4
cheR3
cheB2 cheW4 ppfA tlpT
cheY6
cheA3
TlpT
PpfA (Slp)
• Putative cytoplasmic
chemoreceptor
• Homology to ParA family type
1 DNA partitioning proteins,
contains “Walker” type ATPase
domain
• Essential to chemotaxis to a
range of organic acids
•Co-localises in the cytoplasm
with CheA3, A4 and CheW4,
TlpC, TlpS
•Deletion results in reduced
taxis to a range of organic acids,
but normal growth
PpfA regulates the number and
position of cytoplasmic clusters
Cephalexin treated WS8N
DppfA
PpfA: a protein partitioning factor
ParA (DNA)
• characteristic midcell, ¼ and
¾ positioning of plasmids
• Polymerisation? Oscillation?
• ATP/ADP ParA switch
• ParB and parC(S) partners
PpfA (Protein)
• signal for new cluster
formation, and anchoring
midcell, ¼ and ¾
positioning.
• ATP dependent (Walker box
mutants=null)
• Partner/interactions?
Cytoplasmic chemoreceptor TlpT
TlpT :nucleating protein for cytoplasmic cluster?
How common is this protein
segregating system?
•53% of complete genomes in databases have more than one putative chemotaxis pathway (max 8)
•60% of these have putative ppfA in one Che operon
•Of these 83% also have putative cytoplasmic chemoreceptor gene adjacent and all have disordered N-terminal
domain
R.sphaeroides chemosensory pathway: the happiness
centre?
Metabolic state
Kinase vs phosphatase
CheB2-P
A3 A 4
A2
CheY6-P
External
world
CheY3/4-P
•CheA3 is a kinase and specific phosphatase for CheY6
• Model prediction: phosphoryl groups originating from CheA3A4 can end up on CheY3 and
CheY4 using CheB2 and CheA2 as a phosphoconduit.
•His-asp-his-asp phosphorelay between clusters is route to integrating and balancing the
signals from metabolism and the external environment.
•Dominant CheY6-P level regulated by CheA3 kinase:phosphatase activity
How do these pathways control the single
motor?
How is discrimination achieved?
What determines
localisation
•Is it operon position on chromosome?
•Are there specific interaction domains?
Rhodobacter sphaeroides CheA
Proteins
CheA1
CheA2
CheA3
CheA4
P1
P2
P3
P4
P5
P1
P2
P3
P4
P5
P1
P2
P3
P4
P5
P1
P2
P3
P4
P5
P1
P5
P3
P4
P5
P3
P4
P5
Swapped P1 domains and looked at phosphotransfer
Swapped P5 domains and looked at localisation
Created chimeras with same P1 domains in CheAs at both cell locations
Conclusions
• There is internal organisation in bacteria with
apparent homologues targeted to specific sites in
the cell (high throughput in vitro analysis may give
misleading interaction patterns)
• Interaction between cognate HPK-RR depend on
very few amino acids (motifs may allow
engineering of novel interactions)
The people who did the work
George Wadhams
Steven Porter
Mark Roberts
Sonja Pawelczyk
Mila Kojadinovic
Kathryn Scott
Nicolas Delalez
Mostyn Brown
David Wilkinson
Christian Bell
Yo-Cheng Chang
Murray Tipping
Gareth Davies
Elaine Byles
COLLABORATORS
Dave Stuart
Philip Maini
Marcus Tindall
Charlotte Deane
Rebecca Hamer