Effect of transposon-induced motility mutations on colonization of the host light organ by Vibrio fischeri

Abstract

Vibriofischeri is found both as a free-living bacterium in seawater and as the specific, mutualistic light organ symbiont of several fish and squid species. To identify those characteristics of symbiosis-competent strains that are required for successful colonization of the nascent light organ of juvenile Euprymna scolopes squids, we generated a mutant pool by using the transposon Mu dl 1681 and screened this pool for strains that were no longer motile. Eighteen independently isolated nonmotile mutants that were either flagellated or nonflagellated were obtained. In contrast to the parent strain, none of these nonmotile mutants was able to colonize the juvenile squid light organ. The flagellated nonmotile mutant strain NM200 possessed a bundle of sheathed polar flagella indistinguishable from that of the wild-type strain, indicating that the presence of flagella alone is not sufficient for colonization and that it is motility itself that is required for successful light organ colonization. This study identifies motility as the first required symbiotic phenotype of V. fischeri.A number of species in the genus Vibrio are found living in intimate association with specific animal hosts. The associations are often pathogenic, for example, those between Vibrio cholerae, V. vulnificus, or V anguillarum and various vertebrate and invertebrate species (13); however, mutualistic symbioses also exist, such as that between V. fischeri and the luminous squid Euprymna scolopes (6, 34). By examining genetic determinants that have evolved to play a role in mutualistic as well as pathogenic associations, it may be possible to uncover unifying principles that govern the establishment and development of bacterial colonizations of animal host tissues. In this study, we have used transposon mutagenesis to identify for the first time a required symbiotic determinant of a nonpathogenic, animal-associated bacterium.Upon hatching, the juvenile E. scolopes squid is aposymbiotic (i.e., its nascent light organ is devoid of bacteria); thus, the symbiosis needs to be reestablished with each successive generation (24,41). To initiate this benign infection, symbiosiscompetent V fischeri from the ambient seawater must enter the juvenile light organ through superficial pores and travel down narrow, ciliated ducts that lead into several epitheliumlined crypts (26). An inoculum of fewer than 10 bacterial cells enters the light organ and proliferates so rapidly that within 10 to 12 h the crypts are filled with an extracellular, monospecific culture of about 105 V fischeri cells whose luminescence can be easily detected (24,33).The natural occurrence of aposymbiotic juveniles, combined with the rapid and readily initiated colonization process, provides the opportunity to test mutant strains of V fischeri for the loss of symbiotic infectivity, thereby identifying genes required for the colonization of the juvenile E. scolopes light organ. Motility-and flagellum-associated structures have been shown to be important colonization or virulence determinan...

A genomic comparison of 13 symbiotic Vibrio fischeri isolates from the perspective of their host source and colonization behavior

“…All V. fischeri strains were grown at 28°C in either (i) seawater tryptone medium containing (per liter) 700 ml of a marine salts solution Instant Ocean (Spectrum Brands, Madison, WI, USA), 300 ml deionized water, 5 g Bacto-Tryptone, 3 g yeast extract and 3 ml of glycerol; or (ii) Luria-Bertani (LB) Salt media (Graf et al, 1994), containing (per liter) 20 g NaCl, 50 ml of 1 M Tris-HCl (pH 7.5), 10 g BactoTryptone and 5 g yeast extract, with the addition of erythromycin (5 μg ml − 1 ) for strains carrying a gene encoding a green or red fluorescent protein (RFP) in the Tn7 site (McCann et al, 2003). Derivatives of Escherichia coli DH5α were grown at 37°C in LB medium (10 g Bacto-Tryptone, 5 g yeast extract and 10 g NaCl per liter).…”

Role for Phosphoglucomutase in Vibrio fischeri-Euprymna scolopes Symbiosis

“…When a sufficient cell density is reached, the bacteria luminesce, providing the counterillumination ability believed to be an antipredation mechanism for the squid host (see reference 49 for a review). Various studies characterizing the Vibriosquid symbiosis have identified several genetic determinants involved in establishing this association (2,14,48,50,52); however, much remains to be learned about how these symbiotic partners interact.…”

“…The bacterial cells embed and aggregate in a mucuslike material sloughed off the appendages and secreted from pores located at the base of the light organ (36). As they enter the pores, they encounter mucus-filled ducts (27) and an outwardly directed current created by dense cilia lining the duct walls (see reference 49 for a review); not surprisingly, nonmotile V. fischeri are symbiosis defective (14,36). The ducts ultimately lead to the site of colonization, the internal crypts, where the microbes likely encounter a relatively hostile environment due to the presence of macrophage-like cells (35) and halide peroxidase, an enzyme implicated in oxidative stress (53).…”

Selective recruitment of bacteria during embryogenesis of an earthworm

“…After a waiting period (2-3 h), V. fischeri migrate into the nascent light organ through ciliated ducts that possess an oxidatively stressful environment to exclude other bacterial types (Small and McFall-Ngai, 1999;Davidson et al, 2004). Motility and resistance to oxidative stress are required for this migration (Graf et al, 1994;Visick and Ruby, 1998). Once the correct tissue is colonized, the ducts change to prevent further entrance, and the ciliated field that creates the current undergoes apoptosis and regresses (Foster et al, 2000).…”