Two-component sensor required for normal symbiotic colonization of euprymna scolopes by Vibrio fischeri

Abstract

The light organ of the squid Euprymna scolopes is specifically colonized to a high density by the marine bacterium Vibrio fischeri. To date, only a few factors contributing to the specificity of this symbiosis have been identified. Using a genetic screen for random transposon mutants defective in initiating the symbiotic association or in colonizing the light organ to high density, we identified a mutant of V. fischeri that exhibited an apparent defect in symbiosis initiation. This mutant was not defective in motility, luminescence, or growth in minimal medium, suggesting that it lacks an essential, previously unidentified symbiotic function. By sequence analysis, we showed that the locus inactivated in this mutant encodes a predicted 927-amino-acid protein with a high degree of similarity to the sensor component of hybrid two-component regulatory systems. We have therefore designated this locus rscS, for regulator of symbiotic colonization-sensor. Sequence analysis revealed two hydrophobic regions which may result in the formation of a periplasmic loop involved in signal recognition; PhoA fusion data supported this proposed membrane topology. We have investigated the start site of rscS transcription by primer extension and identified a putative promoter region. We hypothesize that RscS recognizes a signal associated with the light organ environment and responds by stimulating a putative response regulator that controls protein function or gene expression to coordinate early colonization events. Further studies on RscS, its cognate response regulator, and the signaling conditions will provide important insight into the interaction between V. fischeri and E. scolopes.

FIG. 1

Luminescence levels of colonization mutant KV712 and its parent. (A) Newly hatched juvenile E. scolopes squid were incubated in artificial seawater containing no V. fischeri (solid triangles, top panel) or either the parent strain ESR1 (solid circles, top panel) or the colonization mutant KV712 (solid squares, bottom panel). Bioluminescence was measured over time. The light levels of 10 animals inoculated with ESR1 (indicated by n = 10) and of 7 animals inoculated with KV712 (indicated by n = 7), were averaged, while three squid colonized by KV712 are represented singly (indicated by n = 1). Error bars are shown, representing the standard deviation of luminescence readings for ESR1-colonized animals; the level of light observed for seven of the KV712-inoculated squid was not above the limit of detection for the machine, and thus error bars are not displayed. (B) ESR1 (circles) and KV712 (squares) were grown in CM in the absence (open symbols) or presence (solid symbols) of an autoinducer of bioluminescence (3-oxo-hexanoyl-l-homoserine lactone). The level of luminescence was measured using a Turner 20/20 luminometer. The two strains grew at essentially the same rate.

FIG. 2

Symbiotic colonization by V. fischeri strain KV712 and its parent, ESR1. Newly hatched juvenile E. scolopes were exposed for 15 h to either KV712 or ESR1. The level of colonization achieved by these strains was determined by homogenization and plating 19 h after the organisms were placed together. Each circle represents the number of V. fischeri cells in an individual squid, and the bar indicates the average colonization level of the 10 squid in each inoculation condition (2,300 cells per ml). The level of colonization of four squid inoculated with KV712 was below the limit of detection, as indicated by the dashed line.

FIG. 3

Schematic diagram of the rscS locus and flanking genes. (A) The region of the chromosome flanking the transposon insertion in KV712 is indicated. The transposon (solid triangle) inserted in a large ORF, rscS, which is flanked by genes with high sequence similiarity to the E. coli glpR and glpK genes. The direction of transcription is indicated. (B) Noteworthy regions of the 927-amino-acid RscS protein are diagrammed. RscS exhibits sequence similarity to the V. harveyi (Vh) LuxQ, Bordetella pertussis (Bp) BvgS, and E. coli (Ec) ArcB proteins, particularly, as indicated below the black bars, in the regions flanking the histidine and aspartic acid residues (H1, D1, and H2) thought to be involved in the phosphorelay cascade. RscS also exhibits sequence similarity to the nucleotide-binding sites depicted by the gray boxes and labeled N, G1, F, and G2. Two putative transmembrane (TM) regions are indicated by striped bars and are designated TM1 and TM2. The locations of active phoA transposon insertions are indicated by open triangles.

FIG. 4

Complementation of the colonization defect in KV712. Newly hatched juvenile E. scolopes were inoculated with one of four strains: KV712 or its parent ESR1, each carrying either vector pKV69 or rscS⁺ plasmid pLMS26. The squid were exposed to approximately 1,000 cells of V. fischeri per ml of seawater for 3 h. The level of colonization achieved by these strains 17 h after the organisms were placed together was determined by homogenization and plating as described in Materials and Methods. Each circle represents the number of V. fischeri cells in an individual squid, and the bar indicates the average colonization level of the six squid in each inoculation condition.

FIG. 5

Promoter mapping by primer extension. (A) The primer extension products obtained using a primer complementary to the rscS coding sequence (see Materials and Methods) appear in lane 1 and are indicated by arrows. Contained within the adjacent lanes, labeled A, C, G, and T, are the bands obtained by DNA sequencing using the same primer. (B) The DNA sequence at the beginning of the rscS ORF and upstream is shown. The putative ATG translational start is indicated by the bold ATG. Centered 11 bp upstream of the possible translational start is a potential Shine-Dalgarno site, boldface and underlined. The possible transcriptional starts identified by primer extension are Mboldface; the more significant product is also indicated by an asterisk. A possible promoter region is indicated by a dashed underline and labeled above with −10 and −35.