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 Mg²⁺ 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 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 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: Aliivibrio; Photobacterium; aminolevulinic acid; hemin; photobacteria; symbiosis.

FIG 1

Cell morphology of fts mutants. Phase-contrast images show wild-type strain ES114 (A), Tn-ftsQ mutant SV11 (B and C), and ftsA::Tn mutant RP5 (D, E, and F). Bars indicate size standards of 10 μm (A to E) and 100 μm (F). White arrows in panels D and E indicate where mutant cells or strings of cells have looped back on themselves. Cells were grown to mid-log phase in SWTO at 28°C with shaking at 200 rpm.

FIG 2

Colonization of squid by representative mini-Tn5 insertion mutants. Squid were inoculated in parallel with either a representative mutant or ES114, and the CFU in each of the squid was determined 48 h later. Colonization by ES114 ranged from 0.7 × 10⁵ to 3.8 × 10⁵ CFU/squid across experiments, and mutant CFU/squid data were therefore normalized to those for the wild type and presented as the percentage of the wild-type CFU/squid in the same experiment. Each symbol indicates results (average CFU/squid for multiple squid) of an independent experiment. Each mutant was tested on at least 7 and no more than 19 squid in each experiment. Open boxes indicate the limit of detection in experiments where no mutant colonies were recovered from inoculated squid. Boxes indicate that mutant colonization and wild-type colonization were significantly different (P < 0.01) in a Mann-Whitney test, and circles indicate that the values for the mutant and wild type were not significantly different (P > 0.01).

FIG 3

Effects of a ΔhemA mutation on squid colonization. (A) CFU per squid 72 h after inoculation with V. fischeri ES114 (wt), AKD610 (ΔhemA), AKD910 (ΔhutD-hutZ), or AKD917 (ΔhemA ΔhutD-hutZ). Bars indicate standard errors of the means for colonization levels in 20 animals (n = 20). A one-way analysis of variance (ANOVA) indicated that there was no statistical difference in colonization levels for the tested strains (P > 0.05). (B) Relative symbiotic competitiveness of ΔhemA mutant AKD610 43 h after inoculation in a mix with ES114. Each circle indicates the AKD610:ES114 ratio in an individual animal divided by the ratio of these strains in the inoculum. The average log RCI of −0.6 (RCI = 0.25) indicated a statistically significant competitive disadvantage for the mutant (P < 0.0001).

FIG 4

Concentration-dependent recovery of ΔhemA mutant growth by ALA. Wild-type strain ES114 and the ΔhemA mutant AKD610 were grown in LBS supplemented with ALA as indicated. Bars indicate standard errors (n = 3). For clarity, cultures of ES114 with added ALA were not graphed, but addition of ALA to ES114 did not affect its growth significantly.