An Expanded Transposon Mutant Library Reveals That Vibrio Fischeri δ-Aminolevulinate Auxotrophs Can Colonize Euprymna Scolopes

Libraries of defined mutants are valuable research tools but necessarily lack gene knockouts that are lethal under the conditions used in library construction. In this study, we augmented a mutant library generated on a rich medium (LBS, which contains [per liter] 10 g of tryptone, 5 g of yeast extract, 20 g of NaCl, and 50 mM Tris [pH 7.5]) by selecting transposon insertion mutants on supplemented LBS and screening for those unable to grow on LBS. We isolated strains with insertions in , (), , several heme biosynthesis genes, and , as well as a mutant disrupted 14 bp upstream of Mutants with insertions in or upstream of were recovered by addition of Mg to LBS, but their cell morphology and motility were affected. The mutant was more strongly affected and formed cells or chains of cells that appeared to wind back on themselves helically. Growth of mutants with insertions in , , or was recovered with -acetylglucosamine (NAG), d-alanine, or d-glutamate, respectively. We hypothesized that NAG, d-alanine, or d-glutamate might be available to in the light organ; however, none of these mutants colonized the host effectively. In contrast, and mutants, which are auxotrophic for δ-aminolevulinate (ALA), colonized at wild-type levels, although mutants later in the heme biosynthetic pathway were severely impaired or unable to colonize. Our findings parallel observations that legume hosts provide symbionts with ALA, but they contrast with virulence phenotypes of mutants in some pathogens. The results further inform our understanding of the symbiotic light organ environment. By supplementing a rich yeast-based medium, we were able to recover mutants with insertions in conditionally essential genes, and further characterization of these mutants provided new insights into this bacterium's symbiotic environment. Most notably, we show evidence that the squid host can provide with enough ALA to support its growth in the light organ, paralleling the finding that legumes provide ALA in symbiotic nodules. Taken together, our results show how a simple method of augmenting already rich media can expand the reach and utility of defined mutant libraries.