Transcript E.coli
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