Tuning Bacterial Behaviour

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Tuning Bacterial Behaviour
Judy Armitage University of Oxford Department of
Biochemistry and Oxford Centre for Integrative
Systems Biology StoMP 2009
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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

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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
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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

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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?

• 4 CheAs
• 8 membrane spanning MCPs
• 4 cytoplasmic Tlps
• 6 CheYs
• 2 CheBs
• NO CheZ

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R.sphaeroides uses a brake to stop
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Activity of the chemotaxis proteins in vitroIs
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
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Pattern of in vitro phosphotransfer
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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?

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Chemotaxis in vitro phosphotransfer
Horribly complex!
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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

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Pathways targeted to different part of cell
Cytoplasmic general CheB1, CheB2, CheY3, CheY4,
CheY6
Red CheOp2 Blue CheOp3
.
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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

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Localisation requires two CheOp3 proteins

• PpfA (Slp)
• Homology to ParA family type 1 DNA partitioning
  proteins, contains Walker type ATPase domain
• Deletion results in reduced taxis to a range of
  organic acids, but normal growth

• TlpT
• Putative cytoplasmic chemoreceptor
• Essential to chemotaxis to a range of organic
  acids
• Co-localises in the cytoplasm with CheA3, A4 and
  CheW4, TlpC, TlpS

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PpfA regulates the number and position of
cytoplasmic clusters
Cephalexin treated WS8N DppfA
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PpfA a protein partitioning factor

• PpfA (Protein)
• signal for new cluster formation, and anchoring
  midcell, ¼ and ¾ positioning.
• ATP dependent (Walker box mutantsnull)
• Partner/interactions?

• ParA (DNA)
• characteristic midcell, ¼ and ¾ positioning of
  plasmids
• Polymerisation? Oscillation?
• ATP/ADP ParA switch
• ParB and parC(S) partners

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Cytoplasmic chemoreceptor TlpT
TlpT nucleating protein for cytoplasmic cluster?
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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

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R.sphaeroides chemosensory pathway the happiness
centre?
Metabolic state Kinase vs phosphatase
CheB2-P
External world
A3A4
A2
CheY6-P
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
  kinasephosphatase activity

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How do these pathways control the single motor?
How is discrimination achieved?
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What determines localisation

• Is it operon position on chromosome?
• Are there specific interaction domains?

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Rhodobacter sphaeroides CheA Proteins
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
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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)

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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