Characterizing Mutant Forms of Flagellum Regulatory Protein FlrC in Vibrio cholerae and Identification of Motility Phenotypes
Vibrio cholerae is a highly motile gram-negative, comma-shaped bacterium with a single polar flagellum. V. cholerae causes the fatal human gastrointestinal disease cholera, an acute diarrheal illness caused by infection of the small intestine with the toxigenic bacterium. Flagellar synthesis, motility, and chemotaxis are virulence determinants in V. cholerae. The Two-component Regulators FlrB and FlrC are necessary for the expression of Class III flagellar genes. The histidine kinase FlrB phosphorylates FlrC at a conserved aspartic acid residue (D54), and FlrC-P is the transcriptional activator of the sigma-54-dependent Class III promoters. Spontaneous motile mutants were isolated from a non-motile ΔflrB strain, and mutations within flrC were identified that were responsible for this phenotype. Four of these "constitutive" mutations (A107T, M114I, G208D, and C213Y) were introduced in various combinations into FlrC, and the resultant proteins analyzed for transcriptional activation. In general the greatest transcriptional activity resulted from a combination of all four mutations, and inactivation of the site of phosphorylation (D54A) within this quad mutant did not reduce transcriptional activity, suggesting that this represents FlrC in the "on" state. This constitutive form of flrC is being introduced into the V. cholerae genome, in order to determine the effect of unregulated FlrC activity on the ability of V. cholerae to swim and to colonize the intestinal tract.