A single regulatory gene is sufficient to alter bacterial host range

Abstract

Microbial symbioses are essential for the normal development and growth of animals. Often, symbionts must be acquired from the environment during each generation, and identification of the relevant symbiotic partner against a myriad of unwanted relationships is a formidable task. Although examples of this specificity are well-documented, the genetic mechanisms governing it are poorly characterized. Here we show that the two-component sensor kinase RscS is necessary and sufficient for conferring efficient colonization of Euprymna scolopes squid by bioluminescent Vibrio fischeri from the North Pacific Ocean. In the squid symbiont V. fischeri ES114, RscS controls light-organ colonization by inducing the Syp exopolysaccharide, a mediator of biofilm formation during initial infection. A genome-level comparison revealed that rscS, although present in squid symbionts, is absent from the fish symbiont V. fischeri MJ11. We found that heterologous expression of RscS in strain MJ11 conferred the ability to colonize E. scolopes in a manner comparable to that of natural squid isolates. Furthermore, phylogenetic analyses support an important role for rscS in the evolution of the squid symbiosis. Our results demonstrate that a regulatory gene can alter the host range of animal-associated bacteria. We show that, by encoding a regulator and not an effector that interacts directly with the host, a single gene can contribute to the evolution of host specificity by switching 'on' pre-existing capabilities for interaction with animal tissue.

Figure 1. rscS is sufficient to confer efficient colonization of E. scolopes

(a) Mauve output shows each chromosome as one locally colinear block. (b) rscS is absent in MJ11 despite a high level of conservation in the surrounding locus. RscS-controlled biofilm phenotypes, including colony wrinkling (c) and pellicle formation (d) of strains harboring either the vector control (pKV69) or the vector carrying rscS1 (pKG11). (e) Squid-colonization assay of strains harboring either the vector control or the vector carrying rscS⁺ (pLMS33), following 3 h inoculation and assayed at 48 h post-inoculation. Data points are individual animals.

Figure 2. The presence of rscS is correlated to natural squid-association and to the ability to experimentally colonize E. scolopes

(a) PCR assay for the presence of rscS and syp genes with primer sets (top to bottom) rscS-flank, rscS-internal1, rscS-internal2, sypB-internal, sypC-internal, or sypR-internal. M = markers, N = negative control. (b) Colonization competence of strains in (a), colonized as described in Fig. 1, at a concentration of approximately 5 × 10³ CFU/ml. Entries in red indicate rscS⁻ derivatives; entry in blue indicates a strain carrying a plasmid (pLMS33) encoding rscS⁺ from ES114.

Figure 3. rscS entered the V. fischeri lineage prior to colonization of squids in the North Pacific Ocean

Bayesian phylogeny of 26 V. fischeri strains using the concatenation of three loci (recA, mdh, katA). The tree was rooted with Aeromonas salmonicida subsp. salmonicida A449. Support for branches is indicated in cases where support is >50% for all three: Bayesian posterior probabilities (top left); Maximum Likelihood bootstrap (top right); and Maximum Parsimony bootstrap (bottom). rscS DNA was detected in all strains in the grey box and in none of the strains outside the box. Bar: 0.01 expected changes per site.

Figure 4. Reconstruction of the evolution of V. fischeri symbioses in the North Pacific Ocean

All strains are genotypically syp⁺; strains which are phenotypically Syp⁺ in squid are illustrated with the grey wrinkled outline. The model reconstructs host range expansion into squid by (1) acquisition of rscS_A, and subsequent (2) developmentally-appropriate expression of the ancestral syp polysaccharide genes. Gene flow (3) into planktonic strains and (4) into fish symbionts accounts for the present of rscS_A in fish isolates, but (5) rscS⁻ strains maintain a colonization incompatibility with regard to squid. (6) We postulate that rscS_B evolved from rscS_A in fish.