Roles of the regulatory proteins FlhF and FlhG in the Vibrio cholerae flagellar transcription hierarchy

Abstract

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. The V. 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 in V. cholerae, we characterized the roles of the three putative regulatory genes, flhF, flhG, and VC2061. V. cholerae flhF and flhG mutants appeared nonmotile in a soft agar assay. Electron microscopy revealed that the flhF mutant lacked a polar flagellum, while interestingly, the flhG mutant possessed multiple (8 to 10) polar flagella per cell. The transcriptional activity of class III and class IV gene promoters in the flhF 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 the flhG 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 the flhG mutant (approximately 10-fold), indicating that the negative regulation of class I flagellar genes enhances virulence. The V. 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 in V. cholerae flagellar biogenesis.

FIG. 1.

Motility phenotypes and electron micrographs of V. cholerae wild-type and mutant strains. (A) V. cholerae strains KKV598 (wild type, WT), KKV1560 (flhF), KKV1701 (flhG), KKV1721 (VC2061), KKV1694 (flhF/pflhF1, complementing plasmid containing a truncated flhF gene lacking the last 42 bp), and KKV1696 (flhF/pflhF2, complementing plasmid containing the entire flhF gene) were inoculated into motility agar plus 0.05% arabinose and incubated at 30°C for 15 h. (B) KKV598 (wild type, WT); (C) KKV1560 (flhF); (D and E) KKV1701 (flhG), first day; (F) KKV1701 (flhG), second day. Bars, 500 nm.

FIG. 2.

Expression of representative class I, class II, class III, and class IV flagellar promoters in flhF and VC2061 mutant V. cholerae strains. V. cholerae strains KKV598 (wild type), KKV1560 (flhF), and KKV1721 (VC2061) carrying plasmids pKEK73 (flrAp-lacZ), pKEK72 (flrBp-lacZ), pKEK327 (fliEp-lacZ), pKEK80 (flaAp-lacZ), pKEK331 (flgKp-lacZ), pKEK79 (flaBp-lacZ), pKEK76 (flaCp-lacZ), pKEK77 (flaDp-lacZ), and pKEK81 (flaEp-lacZ) were assayed for β-galactosidase activity during logarithmic growth in LB. Assays were performed in triplicate, and standard deviations are shown.

FIG. 3.

Expression of representative class I, class II, class III, and class IV flagellar promoters in the flhG mutant V. cholerae strain. V. cholerae strains KKV598 (wild type) and KKV1701 (flhG) were transformed with the plasmids pKEK73 (flrAp-lacZ), pKEK72 (flrBp-lacZ), pKEK80 (flaAp-lacZ), pKEK331 (flgKp-lacZ), pKEK79 (flaBp-lacZ), pKEK76 (flaCp-lacZ), pKEK77 (flaDp-lacZ) and pKEK81 (flaEp-lacZ). β-Galactosidase activity was assayed during logarithmic growth in LB on the first and second days after transformation of the flhG mutant. Assays were performed in triplicate, and standard deviations are shown.

FIG. 4.

Intestinal colonization of flhF, flhG, and VC2061 mutant V. cholerae strains. Mutant strains KKV1560 (flhF), KKV1701 (flhG), and KKV1721 (VC2061) were coinoculated with O395 (wild type) perorally into infant mice at a ratio of ∼1:1. The competitive index is given as the output mutant/wild-type ratio divided by the input mutant/wild-type ratio. Each data point represents an individual mouse.

FIG. 5.

Proposed model of regulation of the V. cholerae flagellar transcription hierarchy. The results presented here indicate that FlhG acts as a negative regulator of class I (flrA) transcription, while FlhF acts as positive regulator of class III flagellar promoters, perhaps exerting its function through the modulation of FlrC activity. We have previously shown that FlrB is a positive regulator of class III gene transcription by facilitating the phosphorylation of FlrC (5) and that FlgM is a negative regulator of class IV gene transcription through its function as an anti-σ²⁸ factor (4). flgM is transcribed from a class IV promoter (as shown here) and also from a flagellum-independent promoter (37).