Characterization of constitutively active flagellar regulatory protein FlrC of Vibrio cholerae

Date

2010

Authors

Villarreal, Steven M.

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Abstract

The human pathogen Vibrio cholerae is a gram-negative bacterium that is highly motile because of its polar flagellum. V. cholerae thrives in an aquatic environment and once it has established a residence in the human host it produces a diarrheal disease known as cholera. V.cholerae is motile due to the presence of a single polar flagellum. It has been established that motility, flagellar synthesis, and chemotaxis are virulence determinants in V.cholerae. Genes that encode the flagellum are transcribed in the form of a hierarchy of classes of genes. Expression of one class of genes leads to the expression of subsequent classes of genes. Class II gene products FlrB and FlrC are needed for expression of Class III genes and Class IV genes. Phosphorylation of FlrC by FlrB activates FlrC to a transcriptionally active form: FlrC-P then activates sigma 54-dependent transcription of Class III genes. FlrC is phosphorylated at an aspartic acid residue (D54) in the N-terminal-response regulator domain. Mutation of the aspartic acid residue to an alanine prevents phosphorylation and V.cholerae strains with D54A mutant FlrC are non-flagellated and non-motile. We performed a genetic selection to identify mutant forms of FlrC able to activate transcription in the absence of FlrB. We identified two mutant FlrC alleles, A107T + M114V, and C215Y, able to provide motility to a DeltaflrB strain. The individual mutations were introduced into plasmid-borne FlrC, and assessed for transcriptional activation of flaA. Each mutation caused elevated FlrC-dependent transcription compared to the native protein, in both V. cholerae and S. typhimurium reporter strains, demonstrating that the mutations enhance FlrC activity; notably the A107T and M114V mutations, although isolated together, individually lead to enhanced FlrC activity. The nonmotile Delta flrB V. cholerae strain is defective for intestinal colonization in the infant mouse, and although the introduction of the C215Y mutation into flrC restores motility, this strain remains defective for intestinal colonization. These results indicate that regulation of flagellar synthesis by FlrC is critical for the virulence of V.cholera.

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Department

Integrative Biology