Martinez-Wilson Hector F, Tamayo Rita, Tischler Anna D, Lazinski David W, Camilli Andrew
Howard Hughes Medical Institute and the Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts 02111, USA.
J Bacteriol. 2008 Oct;190(19):6439-47. doi: 10.1128/JB.00541-08. Epub 2008 Aug 1.
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.
磷酸化信号转导系统是细菌适应新环境过程中信号转导的重要介质。此前我们描述了vieSAB操纵子,它编码一种假定的三蛋白组分磷酸化信号转导系统,参与调控霍乱弧菌毒力基因的表达。VieSAB的调控活性至少部分是通过假定的应答调节因子VieA的环二鸟苷酸(c-di-GMP)降解活性来发挥的。到目前为止,尚无直接证据表明VieSAB编码一个磷酸化信号转导系统。此外,VieS在调节VieA活性中所起的作用仍不清楚。为了解决这些问题,我们表达并纯化了VieA和VieS的可溶性胞质部分,并将它们用于自磷酸化和磷酸转移测定。这些测定表明,VieS在体外具有激酶活性,并且能够选择性地磷酸化VieA。表型比较显示,缺失vieS会导致生物膜产量增加,与缺失vieA时相当,而与在DeltavieA突变体中观察到的严重缺陷相比,DeltavieS突变体中的运动性仅略有下降。我们还发现,DeltavieS菌株的vieA转录水平较低,并且与DeltavieA突变体类似,细胞内c-di-GMP水平升高。使用定点vieA突变体的进一步分析表明,观察到的一些表型是由于VieA的磷酸化状态所致。本报告中提供的证据首次在生化和遗传方面将VieS和VieA联系起来,支持了这些蛋白质在信号系统中共同发挥作用的假说。