H-NOX-mediated nitric oxide sensing modulates symbiotic colonization by Vibrio fischeri

Abstract

The bioluminescent bacterium Vibrio fischeri initiates a specific, persistent symbiosis in the light organ of the squid Euprymna scolopes. During the early stages of colonization, V. fischeri is exposed to host-derived nitric oxide (NO). Although NO can be both an antimicrobial component of innate immunity and a key signaling molecule in eukaryotes, potential roles in beneficial host-microbe associations have not been described. V. fischeri hnoX encodes a heme NO/oxygen-binding (H-NOX) protein, a member of a family of bacterial NO- and/or O(2)-binding proteins of unknown function. We hypothesized that H-NOX acts as a NO sensor that is involved in regulating symbiosis-related genes early in colonization. Whole-genome expression studies identified 20 genes that were repressed in an NO- and H-NOX-dependent fashion. Ten of these, including hemin-utilization genes, have a promoter with a putative ferric-uptake regulator (Fur) binding site. As predicted, in the presence of NO, wild-type V. fischeri grew more slowly on hemin than a hnoX deletion mutant. Host-colonization studies showed that the hnoX mutant was also 10-fold more efficient in initially colonizing the squid host than the wild type; similarly, in mixed inoculations, it outcompeted the wild-type strain by an average of 16-fold after 24 h. However, the presence of excess hemin or iron reversed this dominance. The advantage of the mutant in colonizing the iron-limited light-organ tissues is caused, at least in part, by its greater ability to acquire host-derived hemin. Our data suggest that V. fischeri normally senses a host-generated NO signal through H-NOX(Vf) and modulates the expression of its iron uptake capacity during the early stages of the light-organ symbiosis.

Fig. 1.

Electronic absorption spectra of protein complexes of the H-NOX protein from V. fischeri. Purified H-NOX_Vf-Fe(II) (solid), -Fe(II)-CO (dotted), and -Fe(II)-NO (dashed) at concentrations of ∼1 μM are shown.

Fig. 2.

Heat map summarizing the expression profile of all genes of V. fischeri ES114 whose promoter regions are predicted to contain a Fur-binding site. Genes are identified by their locus tags (rows), and experimental treatment triplicates are grouped in vertical columns. WT, wild-type cells; hnoX, hnoX-insertion mutant cells; NO, nitric-oxide treatment. The expression-level values were normalized to their Z scores for each row (red, low expression level; white, high expression level). The hemin-related genes are indicated by gray boxes.

Fig. 3.

Responses of wild-type V. fischeri and the ΔhnoX mutant to the addition of NO during the growth on hemin. (A) An iron-depleted, minimal-salts medium containing hemin (10 μM) as the sole iron source was inoculated with either the wild-type strain ES114 (circles) or the ΔhnoX-insertion mutant (squares), and growth was monitored by optical density (OD₆₀₀) at 28 °C with shaking. One-half of each culture (open symbols) was treated two times with the NO generator DEA-NONOate (arrows) as growth was monitored. Error bars indicate the SEM of triplicate cultures in a single experiment. Similar results were obtained in two other experiments. (B) The growth rates of the cultures in panel A were determined for the period between 2 and 5 h after inoculation in either the absence (solid bars) or presence (open bars) of DEA-NONOate. Growth rates were also determined for the wild type and ΔhnoX mutant, carrying either the plasmid vector (pVSV105) or the vector with an intact copy of the hnoX gene (pComhnoX; hnoX+). Plasmid carriage itself had no effect on the growth rates of either of the strains (Fig. S6). Error bars indicate the SEM of the mean growth rates calculated from three separate experiments; t test analysis indicated a significantly faster growth rate in the presence of NO for the ΔhnoX and ΔhnoX + vector.

Fig. 4.

Colonization by the V. fischeri ΔhnoX mutant. (A) Newly hatched juvenile squid (n = 22) were inoculated with either wild-type (circles) or mutant (squares) bacteria, and the appearance of luminescence was monitored periodically. Uninoculated squid (X) did not produce light above background (dashed line). One luminescence unit is equivalent to ∼4 × 10⁵ quanta/sec. (B) Juvenile squid were exposed to different dosages (Inset) of either the wild-type (circles) or mutant (squares) bacteria. Colonization efficiency was determined by mathematically estimating the inoculation dosage at which 50% of the host animals became colonized (arrows) as indicated by the appearance of luminescence by 48 h (Materials and Methods). The r² values of both regressions (solid lines) were >0.8. (C) Juvenile squid (n = 30) were exposed to a mixed inoculum (∼1:1) of the wild type and mutant at a total concentration of 3,000 cfu/mL. The ratio of the two strains in the light-organ population (relative competitive index) (39) at 24 and 48 h postinoculation was measured with no additions (closed circles), 0.2 μM hemin (closed square), 10 μM FeCl₃ (open circles), or 100 μM S-methyl-L-thiocitrulline (SMTC), an inhibitor of host NO synthase (2) (open squares) added at the time of inoculation. Open or closed triangles indicate carriage of either a complementing hnoX-encoding plasmid or the vector control, respectively.