Genetically altered or tagged Vibrio fischeri strains can be observed in association with their mutualistic host Euprymna scolopes, providing powerful experimental approaches for studying this symbiosis. Two limitations to such in situ analyses are the lack of suitably stable plasmids and the need for a fluorescent tag that can be used in tandem with green fluorescent protein (GFP). Vectors previously used in V. fischeri contain the p15A replication origin; however, we found that this replicon is not stable during growth in the host and is retained by fewer than 20% of symbionts within a day after infection. In contrast, derivatives of V. fischeri plasmid pES213 were retained by ϳ99% of symbionts even 3 days after infection. We therefore constructed pES213-derived shuttle vectors with a variety of selectable and visual markers. To include a visual tag that can be used in conjunction with GFP, we compared seven variants of the DsRed2 red fluorescent protein (RFP): mRFP1, tdimer2(12), DsRed.T3, DsRed.T4, DsRed.M1, DsRed.T3_S4T, and DsRed.T3(DNT). The last variant was brightest, displaying >20-fold more fluorescence than DsRed2 in V. fischeri. RFP expression did not detectably affect the fitness of V. fischeri, and cells were readily visualized in combination with GFP-expressing cells in mixed infections. Interestingly, even when inocula were dense enough that most E. scolopes hatchlings were infected by two strains, there was little mixing of the strains in the light organ crypts. We also used constitutive RFP in combination with the luxICDABEG promoter driving expression of GFP to visualize the spatial and temporal induction of this bioluminescence operon during symbiotic infection. Our results demonstrate the utility of pES213-based vectors and RFP for in situ experimental approaches in studies of the V. fischeri-E. scolopes symbiosis.Vibrio fischeri is a useful model for studies of bioluminescence, quorum sensing, and beneficial animal-bacterium interactions. Genetic tools have advanced each of these fields and have been especially important for studying the light organ symbiosis between V. fischeri and the Hawaiian bobtail squid, Euprymna scolopes. This squid acquires V. fischeri from its surroundings after hatching, allowing reconstitution of this association with wild-type or genetically altered V. fischeri. In such experiments, mutant analyses have revealed colonization mechanisms (1, 8, 11, 14-16, 38, 42, 44) and visualization of wild-type cells tagged with green fluorescent protein (GFP) has elucidated early infection events (28,30,31). GFP also holds great promise as an in situ reporter of gene activity, because cells expressing GFP can be visualized in the light organ. However, despite the utility of genetic tools in this model symbiosis, researchers still lack suitably stable plasmids and a useful fluorescent tag that is compatible with GFP.To date, plasmids used to maintain genes in V. fischeri have contained the Escherichia coli plasmid p15A origin of replication (17,39,43). Although p15A-derived pl...

Vibrio fischeri belongs to the Vibrionaceae, a large family of marine ␥-proteobacteria that includes several dozen species known to engage in a diversity of beneficial or pathogenic interactions with animal tissue. Among the small number of pathogenic Vibrio species that cause human diseases are Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus, the only members of the Vibrionaceae that have had their genome sequences reported. Nonpathogenic members of the genus Vibrio, including a number of beneficial symbionts, make up the majority of the Vibrionaceae, but none of these species has been similarly examined. Here we report the genome sequence of V. fischeri ES114, which enters into a mutualistic symbiosis in the light organ of the bobtail squid, Euprymna scolopes. Analysis of this sequence has revealed surprising parallels with V. cholerae and other pathogens.genomics ͉ pili ͉ symbiosis ͉ toxins ͉ toxin-coregulated pilus