Global discovery of colonization determinants in the squid symbiont Vibrio fischeri

Abstract

Animal epithelial tissue becomes reproducibly colonized by specific environmental bacteria. The bacteria (microbiota) perform critical functions for the host's tissue development, immune system development, and nutrition; yet the processes by which bacterial diversity in the environment is selected to assemble the correct communities in the host are unclear. To understand the molecular determinants of microbiota selection, we examined colonization of a simplified model in which the light organ of Euprymna scolopes squid is colonized exclusively by Vibrio fischeri bacteria. We applied high-throughput insertion sequencing to identify which bacterial genes are required during host colonization. A library of over 41,000 unique transposon insertions was analyzed before and after colonization of 1,500 squid hatchlings. Mutants that were reproducibly depleted following squid colonization represented 380 genes, including 37 that encode known colonization factors. Validation of select mutants in defined competitions against the wild-type strain identified nine mutants that exhibited a reproducible colonization defect. Some of the colonization factors identified included genes predicted to influence copper regulation and secretion. Other mutants exhibited defects in biofilm development, which is required for aggregation in host mucus and initiation of colonization. Biofilm formation in culture and in vivo was abolished in a strain lacking the cytoplasmic chaperone DnaJ, suggesting an important role for protein quality control during the elaboration of bacterial biofilm in the context of an intact host immune system. Overall these data suggest that cellular stress responses and biofilm regulation are critical processes underlying the reproducible colonization of animal hosts by specific microbial symbionts.

Keywords: bacterial colonization; biofilm; chaperone; functional genomics; symbiosis.

Fig. 1.

Use of insertion sequencing to identify putative essential genes in Vibrio fischeri. (A) Overall approach. Dense transposon mutagenesis was performed in V. fischeri. This “input library” was then passaged, either through colonization in the hatchling squid during a 3-h inoculation and 48-h total colonization time or for an equivalent number of generations (n = 15) in LBS medium in vitro. Identification of genes that were missing in the input library and comparison of the output libraries to the input library enabled categorization of V. fischeri genes as putative colonization factors, as putative growth factors (i.e., required for normal medium growth), or as essential genes. (B) The mariner transposon developed for this purpose inserted randomly across the V. fischeri chromosomes and plasmid. Normalized transposon counts per 10,000-bp bin are plotted from the input library. (C) Data points represent individual V. fischeri ES114 genes, with the normalized transposon counts per million (cpm) and transposon sites (TA dinucleotides) in the 5′-most 90% of each gene. Orange circles are predicted nonessential genes and genes for which there is not sufficient information. Blue circles are predicted essential genes that lack an E. coli ortholog (n = 244). Plus signs are genes for which the E. coli ortholog is essential and the V. fischeri ortholog is predicted to be essential (n = 211). Solid black circles represent genes for which the E. coli ortholog is essential and the V. fischeri ortholog is predicted to be nonessential (n = 19).

Fig. 2.

INSeq analysis identifies squid colonization factor candidates. To identify putative colonization factors, the dynamics of the 41,897 mariner transposon library was followed during squid colonization. (A) Flowchart of the manner by which V. fischeri genes were categorized during the analysis. The 155 rRNA and tRNA genes were removed before the analysis shown. Genes that were classified as essential or that grew poorly in LBS medium (depleted by >1 log over 15 generations) were excluded from analysis in the squid host. The remaining 3,258 genes included 2,291 genes with mutants that were sampled in all squid output libraries. This conservative filter ensured that the genes assayed were abundant enough to colonize squid in the experimental conditions provided. (B) These genes were plotted by their best colonizing replicate (i.e., highest count for the mutant), and 380 genes had transposon counts that were depleted in all squid output libraries by at least 1.8-fold. This set of 380 genes represents the colonization factor candidates presented in this report. (C) For each category of colonization mutant, the percentage of the genes that were identified in the screen is shown by the black bars. The gray bars represent the percentage of genes that did not display depletion, and the remaining percentage indicates genes that were not assayed fully in squid (e.g., in vitro growth defect). Additional detail is provide in Fig. S3. (D) COG categorization of depleted genes. COG categories that are present in V. fischeri are shown, and plotted for each, the number of essential genes (blue) and the number of significantly squid-depleted genes (red).

Fig. 3.

Validation of host colonization factors identified by INSeq analysis. (A) Competition (1:1) of the indicated mutant strain versus a LacZ-marked wild-type strain in squid. Hatchling squid were inoculated with 2 × 10³ cfu/mL bacteria, washed at 3 and 24 h, and assayed at 48 h. The competitive index (CI) is the Log₁₀(mutant/wild type), where the mutant/wild-type output ratio is normalized to the input ratio as detailed in SI Materials and Methods. Dots represent the CI for individual squid, and bars represent the median. The value below the strain represents the fold depletion of the strain (e.g., a median CI of −1 would equal a 10-fold depletion). (B) Competition (1:1) of the indicated mutant strain versus a LacZ-marked wild-type strain in culture. Log phase cells were mixed, diluted, and grown for 15 generations in LBS medium. Dots represent the CI for three independent replicates, and bars represent the median. The competitive fold depletion was normalized to the in vitro competition as described in the SI Materials and Methods, and the normalized in vivo depletion is shown in the gray shaded box. (C) Wrinkled colony biofilm analysis at 24 h and 48 h after plating the indicated strains containing the biofilm-inducing pKG11 plasmid on LBS–tetracycline. (D) Single-strain growth curves in LBS medium. Strains were grown in a microplate for 30 h. The gray filled curve is shown similarly in all plots and represents the wild-type growth. Mutant growth curves are shown with the black lines.

Fig. 4.

The DnaJ/DnaK chaperone system regulates Syp biofilm formation. (A) Biofilm formation, as assayed by wrinkled colony formation, is not observed in dnaJ or dnaK mutant strains but is complemented when the interrupted gene is provided in trans. (B) Single-strain colonization of E. scolopes squid by each of the indicated strains; Apo, aposymbiotic. Hatchling squid were inoculated with 2 × 10³ cfu/mL bacteria, washed at 3 h and 24 h, and assayed at 48 h. (C) Aggregate formation in the squid ciliated epithelial field by strains of the indicated genotype. Hatchling squid were inoculated with 2 × 10⁶ cfu/mL bacteria that constitutively express GFP from the pVSV102 plasmid and assayed at 3 h by epifluorescence microscopy. Large aggregates are readily apparent in the wild-type colonized animals (arrow) but only isolated cells are observed in the isogenic ΔdnaJ mutant (arrow). (Scale bars, 50 μm.) (D) Syp polysaccharide immunoblot with biofilm-specific antibodies. (E) QRT-PCR demonstrating the relative abundance for rscS and sypG transcripts in the indicated strains.