Roles of the Regulatory Proteins FlhF and FlhG in the Vibrio cholerae Flagellar Transcription Hierarchy

Vibrio cholerae , the causative agent of the human diarrheal disease cholera, is a motile bacterium with a single polar flagellum, and motility has been inferred to be an important aspect of virulence. TheV. cholerae flagellar hierarchy is organized into four classes of genes. The expression of each class of genes within a flagellar hierarchy is generally tightly regulated in other bacteria by both positive and negative regulatory elements. To further elucidate flagellar biogenesis inV. cholerae , we characterized the roles of the three putative regulatory genes,flhF ,flhG , and VC2061.V. cholerae flhF andflhG mutants appeared nonmotile in a soft agar assay. Electron microscopy revealed that theflhF mutant lacked a polar flagellum, while interestingly, theflhG mutant possessed multiple (8 to 10) polar flagella per cell. The transcriptional activity of class III and class IV gene promoters in theflhF mutant was decreased, suggesting that FlhF acts as a positive regulator of class III gene transcription. The transcription of all four classes of flagellar promoters was increased in theflhG mutant, suggesting that FlhG acts as a negative regulator of class I gene transcription. Additionally, the ability to colonize the infant mouse intestine was reduced for theflhG mutant (∼10-fold), indicating that the negative regulation of class I flagellar genes enhances virulence. TheV. cholerae VC2061 mutant was motile and produced a polar flagellum indistinguishable from that of the wild type, and the transcriptional activities of the four classes of flagellar promoters were similar to that of the wild type. Our results indicate that FlhG and FlhF regulate class I and class III flagellar transcription, respectively, while VC2061 plays no detectable role inV. cholerae flagellar biogenesis.