Vibrio fischeri flavohaemoglobin protects against nitric oxide during initiation of the squid-Vibrio symbiosis

Abstract

Nitric oxide (NO) is implicated in a wide range of biological processes, including innate immunity against pathogens, signal transduction and protection against oxidative stress. However, its possible roles in beneficial host-microbe associations are less well recognized. During the early stages of the squid-vibrio symbiosis, the bacterial symbiont Vibrio fischeri encounters host-derived NO, which has been hypothesized to serve as a specificity determinant. We demonstrate here that the flavohaemoglobin, Hmp, of V. fischeri protects against NO, both in culture and during colonization of the squid host. Transcriptional analyses indicate that hmp expression is highly responsive to NO, principally through the repressor, NsrR. Hmp protects V. fischeri from NO inhibition of aerobic respiration, and removes NO under both oxic and anoxic conditions. A Δhmp mutant of V. fischeri initiates squid colonization less effectively than wild type, but is rescued by the presence of an NO synthase inhibitor. The hmp promoter is activated during the initial stage of colonization, during which the Δhmp strain fails to form normal-sized aggregates of colonizing cells. Taken together, these results suggest that the sensing of host-derived NO by NsrR, and the subsequent removal of NO by Hmp, influence aggregate size and, thereby, V. fischeri colonization efficiency.

Fig. 1

Expression of V. fischeri hmp responds to NO under both oxic and anoxic conditions, while norV responds only when oxygen is absent. Wild-type cells were grown in minimal-salts medium plus GlcNAc, either in the presence or absence of O₂. The relative expression levels (NO-treated/untreated) of either hmp (A) or norV (B) transcripts were determined by qRT-PCR. Data points are the means (±1 SEM) calculated from three biological replicate experiments.

Fig. 2

Hmp and NorV confer protection from NO inhibition on growth, depending on the oxygen availability. Growth in minimal-salts medium plus GlcNAc, was monitored at OD₆₀₀. Aerobic cultures (A and B) were challenged with 100 μM of the NO-donor DEA-NONOate (thick arrow), when growth reached early log phase. To pre-adapt the cells (B), a pretreatment of 40 μM DEA-NONOate was added (thin arrow) 45 min before the challenge. Similarly, anaerobic cultures (C and D) were challenged with 40 μM DEA-NONOate (thick arrow). To pre-adapt the cells (D), 5 μM DEA-NONOate was added (thin arrow) 45 min before the challenge. Growth, either without (filled symbols) or with (open symbols) pretreatment, was monitored for the wild-type (circle), Δhmp (triangle), ΔnorV (dash line), Δhmp-norV (cross) and ΔnsrR (square) strains. Experiments were repeated three times with similar results. One representative experiment is shown here. Data points are the means calculated from the three technical replicates of that experiment.

Fig. 3

V. fischeri Hmp protects aerobic respiration from NO inhibition. Measurements of oxygen consumption by wild type (trace 1), ΔnsrR (trace 2) and Δhmp (trace 3) were made before and after addition (arrow) of the NO-generator Proli-NONOate (arrow). The measurements were performed on cells either without (A) or with (B) pretreatment with 80 μM DEA-NONOate. The dashed lines in trace 3 of panel A define how the period of inhibition was derived. The experiment was repeated three times with similar results. One representative experiment is shown here.

Fig. 4

The hmp promoter responds to host-derived NO during the aggregation stage of the symbiosis. Cells of wild type (black bars) or ΔnsrR (white bars), carrying the hmp promoter-reporter plasmid pYLW45, were added to seawater, or used to inoculate newly hatched squids. To induce the hmp promoter in the seawater experiment, 80μM DEA-NONOate was added to seawater. To lower the intrinsic level of NO production in the mucus, some squids were pretreated with 100 μM NOS inhibitor SMTC for 2 to 3 hrs before inoculation with V. fischeri. Between 3 to 4 hrs after inoculation, the levels of induced GFP fluorescence and constitutively expressed RFP fluorescence in both strains were monitored using confocal microscopy. The ratio of GFP/RFP fluorescence generated under different conditions was calculated. Significant differences between the ratio found in seawater and the other treatments are indicated: *, p<0.05; **, p<0.01. The experiments were repeated three times with similar results. Data points are means (±1 SEM) calculated from three biological replicates.

Fig. 5

The size of aggregates formed by V. fischeri during squid colonization correlates with the potential of the bacteria to remove NO. (A) Orientation figure indicating the position of the juvenile’s nascent light organ. A confocal image of the light organ is placed on the cartoon in its approximate position within the mantle cavity (scale bar = 300 μm). (B) Enlarged confocal micrograph of the region of the light organ located within the dotted square of A. An aggregate of V. fischeri cells is indicated by the arrow (scale bar = 10 μm). (C) The bacterial aggregates formed in host-derived mucus by wild-type, ΔnsrR or Δhmp strains carrying GFP-encoded pVSV102, and observed using confocal microscopy (at 400X) between 2.5 and 4 hrs after inoculation (scale bar = 20 μm). To lower the intrinsic level of NO production in the mucus, some squids were pretreated with the nitric-oxide synthesis inhibitor SMTC for 2 to 3 hrs before inoculation with V. fischeri. The experiments were repeated three times with similar results.