Vibrio fischeri flagellin A is essential for normal motility and for symbiotic competence during initial squid light organ colonization

Abstract

The motile bacterium Vibrio fischeri is the specific bacterial symbiont of the Hawaiian squid Euprymna scolopes. Because motility is essential for initiating colonization, we have begun to identify stage-specific motility requirements by creating flagellar mutants that have symbiotic defects. V. fischeri has six flagellin genes that are uniquely arranged in two chromosomal loci, flaABCDE and flaF. With the exception of the flaA product, the predicted gene products are more similar to each other than to flagellins of other Vibrio species. Immunoblot analysis indicated that only five of the six predicted proteins were present in purified flagella, suggesting that one protein, FlaF, is unique with respect to either its regulation or its function. We created mutations in two genes, flaA and flaC. Compared to a flaC mutant, which has wild-type flagellation, a strain having a mutation in the flaA gene has fewer flagella per cell and exhibits a 60% decrease in its rate of migration in soft agar. During induction of light organ symbiosis, colonization by the flaA mutant is impaired, and this mutant is severely outcompeted when it is presented to the animal as a mixed inoculum with the wild-type strain. Furthermore, flaA mutant cells are preferentially expelled from the animal, suggesting either that FlaA plays a role in adhesion or that normal motility is an advantage for retention within the host. Taken together, these results show that the flagellum of V. fischeri is a complex structure consisting of multiple flagellin subunits, including FlaA, which is essential both for normal flagellation and for motility, as well as for effective symbiotic colonization.

FIG. 1.

Chromosomal arrangement of flagellin genes of V. fischeri (Vf) (A) and other Vibrio species, including V. anguillarum (Va), V. cholerae (Vc), V. parahaemolyticus (Vp), and V. vulnificus (Vv) (B) (21, 23, 28). The arrows indicate the direction of transcription, and, when known, the presence of promoter consensus sequences is indicated by arrowheads. Empirical evidence that transcription requires the alternative sigma factors σ⁵⁴ and σ²⁸ is indicated by solid arrowheads, while the presence of putative promoter consensus sequences upstream of the corresponding genes is indicated by open arrowheads.

FIG. 2.

Phylogenetic analysis of flagellin proteins in the genus Vibrio. Predicted amino acid sequences were obtained either from sequencing (V. fischeri [Vf]) (this study) or from the GenBank database (V. anguillarum [Va], V. cholerae [Vc], V. parahaemolyticus [Vp], and V. vulnificus [Vv]). Both maximum-parsimony (data shown) and distance-based (data not shown) analyses were performed by using PAUP (45), and similar results were obtained. Bootstrap values greater than 70 are indicated at nodes. The Pseudomonas aeruginosa (Pa) flagellin sequence of fliC served as an outgroup.

FIG. 3.

Electrophoretic separation of V. fischeri flagellar filament proteins. (A) Flagellar proteins from cells of strain ES114 (lane 1), strain DM143 (lane 2), and nonmotile strain DM127 (lane 3) immobilized on nitrocellulose and immunoblotted with a polyclonal flagellin antibody (see Materials and Methods). (B and C) Flagellar proteins were prepared from either strain ES114 (B) or strain DM143 (C), separated by 2D gel electrophoresis, and detected by silver staining. Immunoblotting with the polyclonal flagellin antibody detected the five protein species indicated; a to e indicate the assignments shown in Table 2.

FIG. 4.

Motility agar plates showing the decreased rate of migration of flaA mutant strain DM143 (B) compared to the rate of migration of the wild type (A). Mid-exponential cells were inoculated into motility agar plates containing 0.25% agar and incubated for 8 h, at which time the plates were photographed. Although not clearly visible, the flaA mutant (B) exhibited rings of chemotaxis similar to those of the parent (A). (C) Rates of migration of strains DM143 (gray bars), DM143 carrying flaA in trans (solid bars), and DM143 carrying a control plasmid (open bars) relative to the rates of migration of the wild type through media containing different concentrations of agar. Measurements were made in triplicate, and pairs of strains (strains ES114 and DM143, strains ES114/pDM104 and DM143/pDM104, and strains ES114/pVO8 and DM143/pVO8) were tested on the same plates. The error bars indicate standard errors; not all error bars are visible. The experiment was repeated three times, and similar results were obtained in all experiments.

FIG. 5.

Transmission electron micrographs of V. fischeri cells in the mid-exponential growth phase. (A and B) Cells of wild-type strain ES114 (A) have more sheathed flagella than cells of the FlaA mutant DM143 (B). Bars = 500 nm. (C and D) At a higher magnification, the presence of a sheath, the diameter of the filament (arrows), and the presence of unknown structures at the distal end of the filament were similar for wild-type flagella (C) and FlaA mutant flagella (D). Bars = 50 nm.

FIG. 6.

Colonization of squid by different V. fischeri strains, as indicated by the development of symbiotic luminescence. Newly hatched E. scolopes juveniles were exposed to seawater containing either no V. fischeri (♦), strain ES114 (▪), flaC strain DM138 (▴), or flaA strain DM143 (•). The bioluminescence emission values are averages for 10 animals for each treatment. The error bars indicate the standard errors of the means. Similar results were obtained in two separate experiments.

FIG. 7.

Relative levels of colonization by wild-type strain ES114 (solid bars) and FlaA mutant strain DM143 (open bars) 24 h after juvenile squid were exposed to an inoculum of each strain for either 3 or 14 h. The error bars indicate the standard errors of the means. Similar results were obtained in two additional experiments.

FIG. 8.

Confocal microscopy of GFP-labeled V. fischeri cells during the early stages of light organ colonization. (A) Schematic drawing of one half of a juvenile light organ, showing the pore and duct through which V. fischeri must enter to colonize the crypts. Animals colonized with GFP-labeled wild-type strain ES114 for 16 h contained fluorescent cells in the deep regions of crypts 1 and 2 (data not shown), as well as of crypt 3 (B). In contrast, colonization of the same deep crypt regions by the FlaA mutant DM143 was delayed for an additional 8 h, and low numbers of the mutant were present in crypt 3 24 h after inoculation (C). b, GFP-labeled bacteria; h, host cells stained with Cell-Tracker Red.