Dec 18, 2015
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