Bacteria sense and respond to environmental cues to control important developmental processes. Decoding the language of chemical signaling in bacteria and the mechanisms by which these signals control coordinated behavior impacts our understanding of the role of bacteria in human health, the environment, and industrial processes. Bacteria exist in communities and often perform coordinated activities such as production and secretion of extracellular enzymes, luminescence, biofilm formation, and virulence. Vibrio cholerae, the causative agent of the diarrheal disease Cholera provides an excellent model system to study the effect of environmental signals on bacterial phenotypes. In V. cholerae, c-di-GMP affects transcriptome changes regulating many important phenotypes, such as biofilms and motility. I have identified two c-di-GMP binding transcription factors, VpsR and FlrA, involved in biofilm and motility, respectively. Currently, ten c-di-GMP binding transcription factors are known in bacteria, three of which are from V. cholerae. Three c-di-GMP binding transcription factors; FleQ, FlrA and VpsR belong to the NtrC-like enhancer binding protein family (EBPs). EBPs consist of an N-terminal receiver domain, central AAA+ domain (ATPase associated with diverse activities), and a C-terminal helix-turn-helix DNA binding domain. AAA+ domains are involved in ATP hydrolysis which drives open complex formation initiating transcription. The AAA+ domains are widespread in bacteria and are found in both transcription factors and other cellular machinery. The transcription factor VpsR binds c-di-GMP to induce biofilm gene expression. Alternatively, binding of c-di-GMP to FlrA, the master regulator of flagellar biosynthesis in V. cholerae, abrogates its ability to initiate downstream flagellar gene expression leading to a repression in motility. VpsR and FlrA exhibit the most homology in the AAA+ domain. I have discovered that the AAA+ domain of FlrA is important for interacting with c-di-GMP and demonstrated that two arginine residues are important for this binding. I have also isolated constitutively active mutants of VpsR which can be utilized to study the mechanism how c-di-GMP controls VpsR activity. Lastly, I have shown that other c-di-GMP regulated genes in V. cholerae are not regulated by known c-di-GMP binding transcription factors, suggesting that other unidentified machinery are involved in c-di-GMP signaling.
Read
