title: 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 aVibrio fischerimutant 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 inalr,glr(murI),glmS, several heme biosynthesis genes, andftsA, as well as a mutant disrupted 14 bp upstream offtsQMutants with insertions inftsAor upstream offtsQwere recovered by addition of Mg2+to LBS, but their cell morphology and motility were affected. TheftsAmutant was more strongly affected and formed cells or chains of cells that appeared to wind back on themselves helically. Growth of mutants with insertions inglmS,alr, orglrwas recovered withN-acetylglucosamine (NAG), d-alanine, or d-glutamate, respectively. We hypothesized that NAG, d-alanine, or d-glutamate might be available toV. fischeriin theEuprymna scolopeslight organ; however, none of these mutants colonized the host effectively. In contrast,hemAandhemLmutants, 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 provideBradyrhizobiumsymbionts with ALA, but they contrast with virulence phenotypes ofhemAmutants in some pathogens. The results further inform our understanding of the symbiotic light organ environment.IMPORTANCEBy supplementing a rich yeast-based medium, we were able to recoverV. fischerimutants 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 provideV. fischeriwith enough ALA to support its growth in the light organ, paralleling the finding that legumes provideBradyrhizobiumALA 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.
authors.affiliation: Department of Microbiology, University of Georgia, Athens, Georgia, USA.
authors.forename: Noreen L
authors.initials: NL
authors.lastname: Lyell
authors.affiliation: Department of Microbiology, University of Georgia, Athens, Georgia, USA.
authors.forename: Alecia N
authors.initials: AN
authors.lastname: Septer
authors.affiliation: Department of Microbiology, University of Georgia, Athens, Georgia, USA.|Department of Microbiology and Plant Biology, University of Oklahoma, Norman, Oklahoma, USA.
authors.forename: Anne K
authors.initials: AK
authors.lastname: Dunn
authors.affiliation: Department of Microbiology, University of Georgia, Athens, Georgia, USA.
authors.forename: Drew
authors.initials: D
authors.lastname: Duckett
authors.affiliation: Department of Microbiology, University of Georgia, Athens, Georgia, USA.
authors.forename: Julie L
authors.initials: JL
authors.lastname: Stoudenmire
authors.affiliation: Department of Microbiology, University of Georgia, Athens, Georgia, USA estabb@uga.edu.
authors.forename: Eric V
authors.initials: EV
authors.lastname: Stabb
mesh_terms: D000409:Alanine / Q000378:metabolism; D059706:Aliivibrio / Q000235:genetics / Q000254:growth & development; D048908:Aliivibrio fischeri / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism* / Q000502:physiology; D000622:Aminolevulinic Acid / Q000378:metabolism; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics; D004251:DNA Transposable Elements / Q000235:genetics*; D049832:Decapodiformes / Q000382:microbiology* / Q000502:physiology; D015723:Gene Library; D005798:Genes, Bacterial / Q000235:genetics; D018698:Glutamic Acid / Q000378:metabolism; D006427:Hemin / Q000378:metabolism; D054884:Host-Pathogen Interactions / Q000502:physiology; D008027:Light; D008565:Membrane Proteins / Q000235:genetics; D009154:Mutation; D010457:Peptidoglycan / Q000378:metabolism; D010641:Phenotype; D010776:Photobacterium / Q000235:genetics / Q000378:metabolism; D013559:Symbiosis / Q000235:genetics* / Q000502:physiology*; D014774:Virulence
chemical_list: D001426:Bacterial Proteins; D004251:DNA Transposable Elements; C065103:FtsA protein, Bacteria; D008565:Membrane Proteins; D010457:Peptidoglycan; D018698:Glutamic Acid; D006427:Hemin; D000622:Aminolevulinic Acid; D000409:Alanine
keywords: Aliivibrio; Photobacterium; aminolevulinic acid; hemin; photobacteria; symbiosis