PROJECT SUMMARY/ABSTRACT Cholera is a diarrheal disease characterized by life-threatening dehydration. It is caused by serotypes of the Gram-negative bacterium Vibrio cholerae, which produce cholera toxin. One of the strains linked to endemic cholera outbreaks is the O1 serotype El Tor. V. cholerae El Tor forms biofilms, which are protective communities in which the bacteria are shielded from environmental stress. The second messenger bis-(3'-5')- cyclic-diguanylate monophosphate (c-di-GMP) is key to the transition between motile and sessile (biofilm) lifestyles, with high levels of c-di-GMP associated with biofilm formation. Synthesis of c-di-GMP requires diguanylate cyclase enzymes. One such diguanylate cyclase, which is encoded by VCA0956 and named CdgF, has been linked to biofilm formation and to formation of the hyper-infective bacterial aggregates that form during late infection and are released from the intestinal tract in preparation for bacterial re-entry into the aqueous environment. Mechanisms by which cdgF expression is regulated are unknown. In this project, we will investigate the hypotheses that cdgF expression is controlled by the divergently encoded MarR family transcription factor that we named DgcR, and that DgcR responds to the ligand c-di-GMP, thus creating a positive feed-back loop that sustains c-di-GMP synthesis. We also propose that initial expression of cdgF occurs when DgcR is oxidized, an event that attenuates its ability to bind the cdgF promoter and repress transcription. DNA and ligand binding by DgcR will be determined in vitro by DNase I footprinting and by biophysical analyses of DgcR in absence and presence of ligand. Control of cdgF promoter activity in vivo will be addressed using cdgF promoter-reporter constructs in E. coli also expressing inducible dgcR, and the ability of c-di-GMP or oxidant to de-repress gene expression will be determined. In vitro transcription assays will be implemented to complement the in vivo assays. Completion of the proposed experiments is expected to delineate a novel feed-back system in which c-di-GMP sustains its own synthesis, and it is expected to define a direct link between oxidative stress and c-di-GMP accumulation. Knowing the mechanisms by which the clinically relevant CdgF enzyme is produced is important for a better understanding of V. cholerae El Tor pathogenicity, and it will open prospects for interfering with this regulation and hence prevent formation of the biofilm communities against which conventional antimicrobial agents are ineffective.

PROJECT NARRATIVE Cholera is a leading cause of mortality worldwide. Cholera epidemics are caused by specific strains of Vibrio cholerae, which persist in the environment and in the human host by forming biofilm. In these surface- associated communities, the bacteria are protected from host defenses and antibiotics. Biofilm formation depends on a key intracellular messenger, and in this program, we will determine mechanisms by which it is produced. Identification of this process is required for a comprehensive picture of V. cholerae pathogenesis and for the associated potential for disruption of antibiotic-resistant biofilm communities.