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.All animals and plants are hosts to a native microbiota. These microbial symbionts often contribute to the normal health and development of their respective hosts in exchange for a relatively privileged niche. Many plant and animal symbionts are transmitted horizontally, through the environment after embryogenesis, and are not directly inherited through germplasm from the previous generation. Therefore, with each host generation, environmental bacteria must compete to colonize the empty niches within new host individuals. In perhaps the best-studied example of such competition, mixed communities of rhizobia compete for access to the roots and nodules of leguminous plants. Many complex traits, including antibiotic production, motility, and specific cell surface attributes contribute to the nodulation competitiveness of rhizobial strains (3,9,41,46). Less is known about the competition between environmental bacteria for colonization of animal hosts, especially the natural modes of infection by the native, nonpathogenic microbiota.The light organ symbiosis between the luminescent bacterium Vibrio fischeri and the nocturnal Hawaiian squid Euprymna scolopes serves as a model chronic, mutualistic association between extracellular bacteria and animal epithelia (32,43). At the time of hatching, the light organs of juvenile...

Complete genome sequence of Vibrio fischeri : A symbiotic bacterium with pathogenic congeners

“…The first is the Flp1 paralog, which is required for achieving normal colonization levels in the light organ. ‡ ‡ The second is the PilA2 pilus, which facilitates efficient light organ colonization by bacteria (7). The presence of multiple pilus gene clusters in the V. fischeri genome suggests that different pili may be expressed to aid this bacterium either in the diverse environments it inhabits or during the multiple stages of its development as a symbiont (27).…”

“…Members of this evolutionarily diverse family are found in many bacteria (24), including V. parahaemolyticus and V. vulnificus, but not the sequenced V. cholerae strain. The last two type-IV pilus loci of V. fischeri have homologs only in V. cholerae: one encodes a highly diverged PilA-like gene (7), and the other is a toxincoregulated pilus (TCP)-encoding locus (25) (Fig. 4).…”

“…Unlike many symbiotic bacteria, V. fischeri has been studied at the physiological and genetic levels for decades, has a well described environmental biology, and is easily examined by using molecular genetics (7). Thus, this particular nonpathogenic member of the Vibrionaceae is an ideal candidate for comparative genome analyses with pathogenic vibrios.…”

Verminephrobacter eiseniae type IV pili and flagella are required to colonize earthworm nephridia

“…Similar to E. fetida , hatchling squid acquire Vibrio spp. symbionts from a mixed microbial community, and pilA mutation in Vibrio fisheri generated a competitive disadvantage when challenged with wild type for colonization of light organ (Stabb and Ruby, 2003). For endophytic N 2 -fixing Azoarcus sp.…”

“…Binding can be charge-dependent, or mediated by highly specific adhesins for binding to select receptors (Mattick, 2002). There are a few examples demonstrating TFP importance for symbiont colonization, but literature is sparse (Stabb and Ruby, 2003;Bohm et al, 2007).…”