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

MCP CheA CheY/BHistidine protein kinase signalling

Rhodobacter sphaeroides•Member of -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?

cheA2 tlpCcheW2cheY3 cheW3 cheR2 cheB1

cheY2cheW1cheY1 cheR1mcpAmcpB cheA1

mcpGcheY4

tlpScheY5

cheA4 tlpTcheW4 cheY6cheR3 cheB2 cheA3

cheBRA

slp

cheD

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

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 CheA2 CheA A3 4

CheY1 CheY2 CheY4CheY3 CheY6CheY5 CheB2CheB1

Horribly complex!

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

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

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

Localisation requires two CheOp3 proteins

cheA4 tlpTcheW4 cheY6cheR3 cheB2 cheA3ppfA

PpfA regulates the number and position of cytoplasmic clusters

Cephalexin treated WS8N ppfA

PpfA: a protein partitioning factor

PpfA (Protein)• signal for new cluster

formation, and anchoring midcell, ¼ and ¾ positioning.

• ATP dependent (Walker box mutants=null)

• Partner/interactions?

ParA (DNA)• characteristic midcell, ¼ and

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

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 stateKinase vs phosphatase

CheY6-P

A3A4External worldA2

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

CheB2-P

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

CheA4

CheA3

CheA1P1 P2 P3 P4 P5

P1 P2 P3 P4 P5

CheA2P1 P2 P3 P4 P5

P1 P2 P3 P4 P5

P3 P4 P5

P3 P4 P5

P1 P5

Swapped P1 domains and looked at phosphotransferSwapped P5 domains and looked at localisationCreated 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 ScottNicolas Delalez

Mostyn Brown

David Wilkinson

Christian BellYo-Cheng Chang

Murray Tipping

Gareth Davies

Elaine Byles

COLLABORATORS

Dave Stuart

Philip Maini

Marcus Tindall

Charlotte Deane

Rebecca Hamer