Contribution of pilA to competitive colonization of the squid Euprymna scolopes by Vibrio fischeri

Abstract

Vibrio fischeri colonizes the squid Euprymna scolopes in a mutualistic symbiosis. Hatchling squid lack these bacterial symbionts, and V. fischeri strains must compete to occupy this privileged niche. We cloned a V. fischeri gene, designated pilA, that contributes to colonization competitiveness and encodes a protein similar to type IV-A pilins. Unlike its closest known relatives, Vibrio cholerae mshA and vcfA, pilA is monocistronic and not clustered with genes associated with pilin export or assembly. Using wild-type strain ES114 as the parent, we generated an in-frame pilA deletion mutant, as well as pilA mutants marked with a kanamycin resistance gene. In mixed inocula, marked mutants were repeatedly outcompeted by ES114 (P < 0.05) but not by an unmarked pilA mutant, for squid colonization. In contrast, the ratio of mutant to ES114 CFUs did not change during 70 generations of coculturing. The competitive defect of pilA mutants ranged from 1.7- to 10-fold and was more pronounced when inocula were within the range estimated for V. fischeri populations in Hawaiian seawater (200 to 2,000 cells/ml) than when higher densities were used. ES114 also outcompeted a pilA mutant by an average of twofold at lower inoculum densities, when only a fraction of the squid became infected, most by only one strain. V. fischeri strain ET101, which was isolated from Euprymna tasmanica and is outcompeted by ES114, lacks pilA; however, 11 other diverse V. fischeri isolates apparently possess pilA. The competitive defect of pilA mutants suggests that cell surface molecules may play important roles in the initiation of beneficial symbioses in which animals must acquire symbionts from a mixed community of environmental bacteria.

FIG. 1.

Sequence analysis of the pilA region. The solid line and single-headed arrows represent V. fischeri genomic DNA and ORFs, respectively. The double-headed arrows flanking the accession numbers mark the sequences submitted to the GenBank database.

FIG. 2.

N-terminal comparison of V. fischeri PilA and type IV-A pilins. (A) N-terminal sequence of V. fischeri (V.f.) PilA. (B) Alignment of type-IV-A pilins relative to V. fischeri PilA. Bold white letters (on a black background) and bold black letters indicate residues that are identical or similar to those of V.f. PilA, respectively. Similarity was assigned by using the BLOSUM62 scoring matrix. The asterisk marks the N-terminal phenylalanine (F) of mature pilins. Abbreviations (GenBank database accession numbers): V.c., Vibrio cholerae (CAA54430); V.v., Vibrio vulnificus (AAL32049); P.a., Pseudomonas aeruginosa (AAK68038); N.g., Neisseria gonorrhoeae (CAA73472); A.sp., Azoarcus species (AAC27898); X.c., Xanthomonas citri (AAB97527); M.x., Myxococcus xanthus (S70530).

FIG. 3.

Colonization of squid by a V. fischeri pilA mutant. Twenty juvenile squid were inoculated with approximately 1,500 CFU of wild-type strain ES114 or strain EVS400 (ΔpilA) per ml. Twenty control animals received no inoculation. Luminescence was measured with an automated luminometer at hourly intervals, and the average values for animals inoculated with ES114 (circles), EVS400 (diamonds), or no V. fischeri (squares) are plotted. Error bars indicate standard errors.

FIG. 4.

Relative competitiveness of pilA mutants. Juvenile squid were exposed to a mixed inoculum of marked (kanR) and unmarked strains at a total concentration of approximately 3,000 CFU/ml, and the RCIs of the marked strains were determined for each animal (see Materials and Methods) and plotted as either circles (EVS401) or squares (EVS402). Marked pilA::kanR mutant strains EVS401 and EVS402 competed against wild-type ES114 (A) or unmarked ΔpilA mutant strain EVS400 (B). Symbols to the left or right of the frames indicate datum points where the RCI was less than 0.1 or greater than 10, respectively.

FIG. 5.

Relative competitiveness of pilA mutant EVS401 in culture. Marked pilA::kanR mutant EVS401 was cocultured with wild-type ES114 during growth in SWT, followed by growth in LBS. Error bars (some too small to visualize) indicate standard errors (n = 3).

FIG. 6.

Dose dependence and variability of the EVS401 competition defect. Squid were exposed to serially diluted mixtures of EVS401 and ES114, and the RCI of EVS401 was determined for each animal. For two experiments (filled circles and squares), animals were inoculated with cultured V. fischeri, and for two other experiments (open triangles and diamonds), V. fischeri cells vented from E. scolopes were used to inoculate fresh animals. Each datum point represents the mean value obtained by analysis of 15 to 20 animals.