Photolyase confers resistance to UV light but does not contribute to the symbiotic benefit of bioluminescence in Vibrio fischeri ES114

Abstract

Recent reports suggest that the selective advantage of bioluminescence for bacteria is mediated by light-dependent stimulation of photolyase to repair DNA lesions. Despite evidence for this model, photolyase mutants have not been characterized in a naturally bioluminescent bacterium, nor has this hypothesis been tested in bioluminescent bacteria under natural conditions. We have now characterized the photolyase encoded by phr in the bioluminescent bacterium Vibrio fischeri ES114. Consistent with Phr possessing photolyase activity, phr conferred light-dependent resistance to UV light. However, upon comparing ES114 to a phr mutant and a dark Delta luxCDABEG mutant, we found that bioluminescence did not detectably affect photolyase-mediated resistance to UV light. Addition of the light-stimulating autoinducer N-3-oxo-hexanoyl homoserine lactone appeared to increase UV resistance, but this was independent of photolyase or bioluminescence. Moreover, although bioluminescence confers an advantage for V. fischeri during colonization of its natural host, Euprymna scolopes, the phr mutant colonized this host to the same level as the wild type. Taken together, our results indicate that at least in V. fischeri strain ES114, the benefits of bioluminescence during symbiotic colonization are not mediated by photolyase, and although some UV resistance mechanism may be coregulated with bioluminescence, we found no evidence that light production benefits cells by stimulating photolyase in this strain.

FIG. 1.

Schematic representation of the genetic organization near phr in V. fischeri ES114. ORF designations (e.g., VFA0753) are taken from the V. fischeri genome project (34). The arrows represent ORFs and indicate the direction of gene transcription, as well as the gene size, which is presented relative to the scale bar. “#aa” indicates the number of amino acids encoded by each ORF. Protein similarities were identified using BLAST (1) and the BLOSUM62 scoring matrix (19). The identity and similarity to E. coli K12 ORFs are presented, although closer matches with the same gene designation were found for less well-characterized ORFs (e.g., from vibrios). Several vibrios contain closer matches to ORF VFA0756, and most of these have been designated hypothetical.

FIG. 2.

Effects of UV dose and subsequent recovery in the light (open symbols) or dark (filled symbols) on survival of V. fischeri ES114 (diamonds), the ΔluxCDABEG mutant EVS102 (triangles), or the Δphr mutant EW1 (squares). Percent survival was calculated relative to non-UV-exposed cells. The data represent means with standard errors for three independent cultures of each strain.

FIG. 3.

Complementation of UV sensivity in Δphr mutant EW1. Wild-type or EW1 (Δphr) cells carrying either pVSV105 or pEW1 (phr cloned in pVSV105) were exposed to 12,000 μJ/cm² UV and allowed to recover under fluorescent white lights for 2 h prior to being plated. Percent survival was calculated relative to non-UV-exposed cells. The data represent means with standard errors for three independent cultures.

FIG. 4.

Effects of pregrowth in 200 ng ml⁻¹ 3-oxo-C6-HSL (indicated by “+AI”) and outgrowth in the light (light bars) or dark (dark bars) on survival of V. fischeri ES114, the ΔluxCDABEG mutant EVS102, or the Δphr mutant EW1 following exposure to 12,000 μJ/cm² UV. Percent survival was calculated relative to non-UV-exposed cells. The data represent means with standard errors for three independent cultures of each strain.

FIG. 5.

Symbiotic colonization of E. scolopes hatchlings by the wild type, ΔluxCDABEG mutant EVS102, or Δphr mutant EW1. Average colonization levels 48 h after inoculation with the indicated strain are shown, with standard errors (n = 13 or 14).

FIG. 6.

Competition during mixed infection between V. fischeri EW1 (Δphr) and JRM100. JRM100 is a derivative of ES114 marked with an intergenic mini-Tn7-ermR insertion that does not affect competitiveness (26). Juvenile squid were exposed to an ∼1:1 mixture of the strains at a total concentration of 2,500 CFU ml⁻¹ for 14 h, and the RCI of EW1, defined as the ratio of EW1 to JRM100 in the squid divided by the ratio of these strains in the inoculum, was determined for each coinfected animal 48 h after inoculation. The RCI is plotted as a circle. A dotted line marks the RCI of 1, where the strain ratio in the squid matches that in the inoculum and there is no difference in strain competitiveness.