Specificity of localization and phosphotransfer in the CheA proteins of Rhodobacter sphaeroides

Specificity of protein–protein interactions plays a vital role in signal transduction. The chemosensory pathway of Rhodobacter sphaeroides comprises multiple homologues of chemotaxis proteins characterized in organisms such as Escherichia coli. Three CheA homologues are essential for chemotaxis in R. sphaeroides under laboratory conditions. These CheAs are differentially localized to two chemosensory clusters, one at the cell pole and one in the cytoplasm. The polar CheA, CheA2, has the same domain structure as E. coli CheA and can phosphorylate all R. sphaeroides chemotaxis response regulators. CheA3 and CheA4 independently localize to the cytoplasmic cluster; each protein has a subset of the CheA domains, with CheA3 phosphorylating CheA4 together making a functional CheA protein. Interestingly, CheA3‐P can only phosphorylate two response regulators, CheY6 and CheB2. R. sphaeroides CheAs exhibit two interesting differences in specificity: (i) the response regulators that they phosphorylate and (ii) the chemosensory cluster to which they localize. Using a domain‐swapping approach we investigated the role of the P1 and P5 CheA domains in determining these specificities. We show that the P1 domain is sufficient to determine which response regulators will be phosphorylated in vitro while the P5 domain is sufficient to localize the CheAs to a specific chemosensory cluster.

[1]  Judith P Armitage,et al.  A bifunctional kinase-phosphatase in bacterial chemotaxis , 2008, Proceedings of the National Academy of Sciences.

[2]  Rodrigo Gouveia-Oliveira,et al.  MaxAlign: maximizing usable data in an alignment , 2007, BMC Bioinformatics.

[3]  J M Pemberton,et al.  An improved suicide vector for construction of chromosomal insertion mutations in bacteria. , 1992, Gene.

[4]  Judith P Armitage,et al.  Requirements for chemotaxis protein localization in Rhodobacter sphaeroides , 2005, Molecular microbiology.

[5]  J. S. Parkinson,et al.  Liberation of an interaction domain from the phosphotransfer region of CheA, a signaling kinase of Escherichia coli. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[6]  M. Simon,et al.  The carboxy-terminal portion of the CheA kinase mediates regulation of autophosphorylation by transducer and CheW , 1993, Journal of bacteriology.

[7]  J P Armitage,et al.  TlpC, a novel chemotaxis protein in Rhodobacter sphaeroides, localizes to a discrete region in the cytoplasm , 2002, Molecular microbiology.

[8]  M. Homma,et al.  Attractant binding alters arrangement of chemoreceptor dimers within its cluster at a cell pole. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[9]  J. Kalinowski,et al.  Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutamicum. , 1994, Gene.

[10]  R C Stewart,et al.  Rapid phosphotransfer to CheY from a CheA protein lacking the CheY-binding domain. , 2000, Biochemistry.

[11]  Robert C. Edgar,et al.  MUSCLE: multiple sequence alignment with high accuracy and high throughput. , 2004, Nucleic acids research.

[12]  J. Falke,et al.  CheA Kinase of bacterial chemotaxis: chemical mapping of four essential docking sites. , 2006, Biochemistry.

[13]  Judith P Armitage,et al.  Chemotaxis in Rhodobacter sphaeroides Requires an Atypical Histidine Protein Kinase* , 2004, Journal of Biological Chemistry.

[14]  J. Maddock,et al.  Polar Clustering of the Chemoreceptor Complex inEscherichia coli Occurs in the Absence of Complete CheA Function , 2000, Journal of bacteriology.

[15]  M. Simon,et al.  Structure of CheA, a Signal-Transducing Histidine Kinase , 1999, Cell.

[16]  J P Armitage,et al.  Identification of a chemotaxis operon with two cheY genes in Rhodobacter sphaeroides , 1995, Molecular microbiology.

[17]  Gabriela Gonzalez-Bonet,et al.  Reconstruction of the chemotaxis receptor–kinase assembly , 2006, Nature Structural &Molecular Biology.

[18]  G. Wadhams,et al.  Identification and localization of a methyl‐accepting chemotaxis protein in Rhodobacter sphaeroides , 2000, Molecular microbiology.

[19]  Judith P. Armitage,et al.  Molecular Biology of the Rhodobacter Sphaeroides Flagellum , 1990 .

[20]  J. S. Parkinson,et al.  Chemotactic signaling by the P1 phosphorylation domain liberated from the CheA histidine kinase of Escherichia coli , 1996, Journal of bacteriology.

[21]  Judith P. Armitage,et al.  Using Structural Information to Change the Phosphotransfer Specificity of a Two-Component Chemotaxis Signalling Complex , 2010, PLoS biology.

[22]  Davi R. Ortega,et al.  Universal architecture of bacterial chemoreceptor arrays , 2009, Proceedings of the National Academy of Sciences.

[23]  Judith P Armitage,et al.  Phosphotransfer in Rhodobacter sphaeroides chemotaxis. , 2002, Journal of molecular biology.

[24]  J P Armitage,et al.  Evidence for two chemosensory pathways in Rhodobacter sphaeroides , 1997, Molecular microbiology.

[25]  G. Wadhams,et al.  Making sense of it all: bacterial chemotaxis , 2004, Nature Reviews Molecular Cell Biology.

[26]  J. S. Parkinson,et al.  Mutational Analysis of the Chemoreceptor-Coupling Domain of the Escherichia coli Chemotaxis Signaling Kinase CheA , 2006, Journal of bacteriology.

[27]  Laura Camarena,et al.  Chemotactic Control of the Two Flagellar Systems of Rhodobacter sphaeroides Is Mediated by Different Sets of CheY and FliM Proteins , 2007, Journal of bacteriology.

[28]  Judith P Armitage,et al.  In vivo and in vitro analysis of the Rhodobacter sphaeroides chemotaxis signaling complexes. , 2007, Methods in enzymology.

[29]  W R SISTROM,et al.  A requirement for sodium in the growth of Rhodopseudomonas spheroides. , 1960, Journal of general microbiology.

[30]  J. S. Parkinson,et al.  Cysteine-Scanning Analysis of the Chemoreceptor-Coupling Domain of the Escherichia coli Chemotaxis Signaling Kinase CheA , 2006, Journal of bacteriology.

[31]  Judith P Armitage,et al.  The CheYs of Rhodobacter sphaeroides* , 2006, Journal of Biological Chemistry.

[32]  Victor Sourjik,et al.  Receptor clustering and signal processing in E. coli chemotaxis. , 2004, Trends in microbiology.

[33]  M. Simon,et al.  Expression of CheA fragments which define domains encoding kinase, phosphotransfer, and CheY binding activities. , 1993, Biochemistry.

[34]  M. Homma.  Chemoreceptor signaling involves dimer-to-dimer interaction with its cluster at a cell pole , 2004 .

[35]  J. Stock,et al.  Organization of the Receptor-Kinase Signaling Array That Regulates Escherichia coli Chemotaxis* , 2002, The Journal of Biological Chemistry.

[36]  Pablo Vinuesa,et al.  A Complete Set of Flagellar Genes Acquired by Horizontal Transfer Coexists with the Endogenous Flagellar System in Rhodobacter sphaeroides , 2007, Journal of bacteriology.

[37]  Michael T. Laub,et al.  Rewiring the Specificity of Two-Component Signal Transduction Systems , 2008, Cell.

[38]  Judith P Armitage,et al.  The third chemotaxis locus of Rhodobacter sphaeroides is essential for chemotaxis , 2002, Molecular microbiology.

[39]  Geoffrey J. Barton,et al.  Jalview Version 2—a multiple sequence alignment editor and analysis workbench , 2009, Bioinform..

[40]  Sung-Hou Kim,et al.  Four-helical-bundle structure of the cytoplasmic domain of a serine chemotaxis receptor , 1999, Nature.

[41]  D. C. Harris,et al.  Identification of a fourth cheY gene in Rhodobacter sphaeroides and interspecies interaction within the bacterial chemotaxis signal transduction pathway , 2000, Molecular microbiology.

[42]  J. S. Parkinson,et al.  Collaborative signaling by bacterial chemoreceptors. , 2005, Current opinion in microbiology.

[43]  S. L. Porter,et al.  Rhodobacter sphaeroides: complexity in chemotactic signalling. , 2008, Trends in microbiology.

[44]  S. L. Porter,et al.  CheR- and CheB-Dependent Chemosensory Adaptation System of Rhodobacter sphaeroides , 2001, Journal of bacteriology.

[45]  G. Wadhams,et al.  Targeting of two signal transduction pathways to different regions of the bacterial cell , 2003, Molecular microbiology.

[46]  Judith P. Armitage,et al.  Inducible-Expression Plasmid for Rhodobacter sphaeroides and Paracoccus denitrificans , 2009, Applied and Environmental Microbiology.

[47]  J P Armitage,et al.  Fine tuning bacterial chemotaxis: analysis of Rhodobacter sphaeroides behaviour under aerobic and anaerobic conditions by mutation of the major chemotaxis operons and cheY genes , 2000, The EMBO journal.

[48]  L. Shapiro,et al.  Polar location of the chemoreceptor complex in the Escherichia coli cell. , 1993, Science.

[49]  J. S. Parkinson,et al.  Chemotactic Signaling by an Escherichia coli CheA Mutant That Lacks the Binding Domain for Phosphoacceptor Partners , 2004, Journal of bacteriology.

[50]  Dennis Bray,et al.  Molecular model of a lattice of signalling proteins involved in bacterial chemotaxis , 2000, Nature Cell Biology.