An Expanded Transposon Mutant Library Reveals that Vibrio fischeri δ-Aminolevulinate Auxotrophs Can Colonize Euprymna scolopes

Abstract

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 Vibrio fischeri 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 alr, glr (murI), glmS, several heme biosynthesis genes, and ftsA, as well as a mutant disrupted 14 bp upstream of ftsQ. Mutants with insertions in ftsA or upstream of ftsQ were recovered by addition of Mg 2ϩ to LBS, but their cell morphology and motility were affected. The ftsA 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 glmS, alr, or glr was recovered with N-acetylglucosamine (NAG), D-alanine, or D-glutamate, respectively. We hypothesized that NAG, D-alanine, or D-glutamate might be available to V. fischeri in the Euprymna scolopes light organ; however, none of these mutants colonized the host effectively. In contrast, hemA and hemL mutants, which are auxotrophic for ␦-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 Bradyrhizobium symbionts with ALA, but they contrast with virulence phenotypes of hemA mutants in some pathogens. The results further inform our understanding of the symbiotic light organ environment.IMPORTANCE By supplementing a rich yeast-based medium, we were able to recover V. fischeri 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 V. fischeri with enough ALA to support its growth in the light organ, paralleling the finding that legumes provide Bradyrhizobium 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.KEYWORDS Photobacterium, Aliivibrio, symbiosis, aminolevulinic acid, hemin, photobacteria D efined knockout mutant libraries are useful resources that have been generated to promote research in several bacterial experimental models and pathogens (1-10). In such studies, researchers have often listed essential genes, the knockouts of which were apparently lethal under the conditions of library construction and therefore not represented by mutants in the library. In order to recover insertion mutants collectively

Vibrio fischeri DarR Directs Responses to d -Aspartate and Represents a Group of Similar LysR-Type Transcriptional Regulators

“…A mutation in darR suppresses the D-Glu auxotrophy of a murI mutant. In a recent search for transposon insertions in conditionally essential genes, we recovered the murI::miniTn5-erm mutant AN3, which requires D-Glu supplementation for growth (36). The D-Glu auxotrophy of this murI mutant is consistent with the established function of MurI in other bacteria as a glutamate racemase required to provide the D-Glu moiety in PG.…”

Uneven distribution of cobamide biosynthesis and dependence in bacteria predicted by comparative genomics

“…Based on the ecosystem assignment information available for 48% of the genomes, 78% of tetrapyrrole precursor salvagers are categorized as host-associated bacteria compared to 41% in the complete filtered data set. One interpretation of this finding is that tetrapyrrole precursors are provided by the host, either from host cells that produce them as intermediates in heme biosynthesis [89, 90] or, for gut-associated microbes, as part of the host’s diet. Alternatively, these precursors may be provided by other microbes, as was observed in a coculture of Fibrobacter species [91].…”

Genetic Manipulation of Vibrio fischeri

“…Vibrio fischeri is a Gram‐negative, comma‐shaped, bioluminescent marine bacterium (Allen & Baumann, 1971; Lyell et al., 2017). It serves as a model system for studying a variety of traits, including light production and biofilm formation as well as the regulatory processes that control these energy‐intensive phenotypes (Christensen & Visick, 2020; Tischler, Hodge‐Hanson, & Visick, 2019).…”