The vibrio cholerae hybrid sensor kinase VieS contributes to motility and biofilm regulation by altering the cyclic diguanylate level.
Phosphorelay systems are important mediators of signal transduction during bacterial adaptation to new environments. Previously we described the vieSAB operon, encoding a putative three-protein component phosphorelay involved in regulating Vibrio cholerae virulence gene expression. At least part of the regulatory activity of VieSAB is exerted through the cyclic diguanylate (c-di-GMP)-degrading activity of the putative response regulator VieA. So far no direct evidence that VieSAB encodes a phosphorelay system exists. In addition, the role VieS plays in modulating VieA activity remains unclear. To address these questions, we expressed and purified VieA and a soluble cytoplasmic portion of VieS and used them in autophosphorylation and phosphotransfer assays. These assays showed that VieS has kinase activity in vitro and is able to selectively phosphorylate VieA. A phenotypic comparison revealed that deletion of vieS results in increased biofilm production comparable to that seen for deletion of vieA, whereas motility was decreased only slightly in the DeltavieS mutant compared to the profound defect observed in a DeltavieA mutant. We also found that the DeltavieS strain has a lower level of vieA transcript and, similar to a DeltavieA mutant, an increased intracellular level of c-di-GMP. Further analysis using site-directed vieA mutants showed that some of the phenotypes observed were due to the phosphorylation status of VieA. The evidence presented in this report is the first to link VieS and VieA biochemically and genetically, lending support to the hypothesis that these proteins function together in a signaling system.
Martinez-Wilson
Hector F
HF
Howard Hughes Medical Institute and the Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts 02111, USA.
Tamayo
Rita
R
Tischler
Anna D
AD
Lazinski
David W
DW
Camilli
Andrew
A
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D018441:Biofilms / Q000254:growth & development*; D002118:Calcium / Q000494:pharmacology; D006152:Cyclic GMP / Q000031:analogs & derivatives* / Q000235:genetics / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D000071677:Histidine Kinase; D007700:Kinetics; D008345:Manganese / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014734:Vibrio cholerae / Q000235:genetics / Q000378:metabolism / Q000502:physiology*
D001426:Bacterial Proteins; C112165:VieA protein, Vibrio cholerae; C112167:VieS protein, Vibrio cholerae; D008345:Manganese; C062025:bis(3',5')-cyclic diguanylic acid; D011494:Protein Kinases; D000071677:Histidine Kinase; D006152:Cyclic GMP; D002118:Calcium