Contribution of pilA to Competitive Colonization of the Squid Euprymna scolopes by Vibrio fischeri

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 squid are not colonized by V. fischeri but they rapidly acquire these symbionts from the surrounding seawater (28). The nascent light organ possesses elaborate ciliated appendages that entrain and concentrate planktonic V. fischeri in a mucus matrix, facilitating initial colonization (31). During this process, motile V. fischeri cells move from pores on the surface of the light organ, through ducts, into epithelium-lined crypt spaces (12, 31). Within hours after infection by V. fischeri, the squid also display a diurnal behavior in which they expel approximately 90% of their light organ symbionts each morning and supply the remaining bacteria with sufficient nutrients to repopulate the light organ by nightfall (6, 13). Therefore, V. fischeri strains may compete during initial entry into the light organ, as well as during subsequent daily expulsion and regrowth. Increased populations of V. fischeri in squid habitats appear to be a direct result of occupation of this symbiotic niche (24), illustrating an ecological advantage for strains that compete effectively for light organ colonization. Although most, if not all, wild-type V. fischeri isolates can colonize E. scolopes, isolates native to this host outcompete isolates from other sources for light organ colonization in mixed inoculations. For example, when presented to E. scolopes juveniles in equal ratios, strain ES114, which was isolated from E. scolopes, was more than 10-fold more competitive than strains EM17 and ET101, which were isolated from light organs of Euprymna morsei and Euprymna tasmanica, respectively (30). V. fischeri isolates not associated with squid may be even less competitive for colonization of E. scolopes (22). It is likely that several factors contribute to colonization competitiveness, and mutant analyses provide a means by which to reveal them. For example, Visick and Ruby demonstrated that a katA mutant, deficient in the production of a periplasmic catalase, was outcompeted by the parent strain (44). McFall-Ngai and colleagues found that addition of a mannose analog to seawater, or treatment of V. fischeri with extracellular protease, could partially block infection of E. scolopes and suggested that a V. fischeri mannose-binding cell surface protein contributes to the initiation of the V. fischeri-E. scolopes light organ symbiosis (16, 27). We therefore became interested in testing the role of cell surface molecules in colonization and colonization competitiveness. In this paper, we report the cloning and characterization of V. fischeri pilA, a gene that encodes a protein with similarity to type IV-A pilins and that contributes to the competitiveness of V. fischeri during colonization of the E. scolopes light organ.