Rubredoxin:oxygen oxidoreductase enhances survival of Desulfovibrio vulgaris hildenborough under microaerophilic conditions

Abstract

Genes for superoxide reductase (Sor), rubredoxin (Rub), and rubredoxin:oxygen oxidoreductase (Roo) are located in close proximity in the chromosome of Desulfovibrio vulgaris Hildenborough. Protein blots confirmed the absence of Roo from roo mutant and sor-rub-roo (srr) mutant cells and its presence in sor mutant and wild-type cells grown under anaerobic conditions. Oxygen reduction rates of the roo and srr mutants were 20 to 40% lower than those of the wild type and the sor mutant, indicating that Roo functions as an O2 reductase in vivo. Survival of single cells incubated for 5 days on agar plates under microaerophilic conditions (1% air) was 85% for the sor, 4% for the roo, and 0.7% for the srr mutant relative to that of the wild type (100%). The similar survival rates of sor mutant and wild-type cells suggest that O2 reduction by Roo prevents the formation of reactive oxygen species (ROS) under these conditions; i.e., the ROS-reducing enzyme Sor is only needed for survival when Roo is missing. In contrast, the sor mutant was inactivated much more rapidly than the roo mutant when liquid cultures were incubated in 100% air, indicating that O2 reduction by Roo and other terminal oxidases did not prevent ROS formation under these conditions. Competition of Sor and Roo for limited reduced Rub was suggested by the observation that the roo mutant survived better than the wild type under fully aerobic conditions. The roo mutant was more strongly inhibited than the wild type by the nitric oxide (NO)-generating compound S-nitrosoglutathione, indicating that Roo may also serve as an NO reductase in vivo.

FIG. 1.

Maps of the sor-rub-roo region of the wild type and the ROO100 (roo) and SRR100 (srr) mutants. The hybridization positions of primers p212-f, p213-r, p214-r, p215-f, p120-r, p122-f, p123-r, and p242-f and the locations of the sor, rub, roo, and cat genes are indicated. The scale of 300 bp applies to all of the maps. A map of the sor mutant has been shown elsewhere (31). Relative to the start of the sor coding region (+1), SalI sites yielding a DNA fragment hybridizing with the p212f and p214r or p122f and p123r labeled probes are present at positions −546 and +547 (wild type), −546 and +4500 (roo mutant), and −546 and +3957 (srr mutant).

FIG. 2.

Protein blot of purified Roo (lane 1) and total cell extracts of the D. vulgaris wild-type (lane 2), roo mutant (lane 3), srr mutant (lane 4), and sor mutant (lane 5) strains probed with anti-Roo polyclonal antibodies. The positions of three molecular mass markers are shown. Native Roo is a dimer of 43-kDa subunits.

FIG. 3.

Change in the specific ORRs (micromoles of O₂ per minute per milligram of dry biomass) of the wild-type and roo, sor, and srr mutant strains with time. These data were used to obtain the specific ORRs at 2 h.

FIG. 4.

Specific ORRs of mutants relative to the wild type (100%) in either medium C (hatched bars) or WP medium (solid bars). Data for the wild type in medium C or in WP with lactate or acetate are shown.

FIG. 5.

Survival of D. vulgaris wild-type (WT) and mutant strains under microaerophilic conditions. (A) Survival in liquid cultures bubbled with 150 ml/min of 1% (vol/vol) air. The fraction of surviving cells (logN_t/N₀) is plotted against time t (h) for the wild-type (closed diamonds) and roo mutant (closed squares), sor mutant (crosses), and srr mutant (closed triangles) strains. The data shown are averages of three or four independently conducted experiments; standard errors from the mean are shown. (B) Survival of single cells on plates following incubation in 1% (vol/vol) air for 5 days. Survival was calculated as the fraction (F) of surviving cells relative to the wild type (100%). The actual survival of the wild type in 1% (vol/vol) air relative to anaerobic conditions was 30.6% ± 3.4%. The standard errors of the means are for three independently conducted experiments.

FIG. 6.

Loss of cell viability (logN_t/N₀) with time t (hours) upon exposure of liquid cultures to 100% (vol/vol) air. Data are presented for the wild type (wt), as well as for the roo, sor, and srr mutants, as indicated. Data are averages for three or four independently conducted experiments; standard errors of the means are also shown.

FIG. 7.

Disk diffusion assay of inhibition of D. vulgaris wild-type and roo mutant strains by various concentrations of the NO-generating agent S-nitrosoglutathione. S-Nitrosoglutathione (30 μl of the indicated concentration) was added to a paper disk placed on a plate spread with 100 μl of exponentially growing wild-type or roo mutant cells. Following incubation for 3 days at 32°C, the inhibition zones surrounding the disks were quantitated. Duplicates were done for each concentration and strain.

FIG. 8.

Proposed pathway for cytoplasmic oxygen, NO, and ROS reduction in D. vulgaris. NADH resulting from glycolytic breakdown of polyglucose is used by NADH:rubredoxin oxidoreductase (Nro) to reduce the oxidized rubredoxin pool (Rub_ox). The reduced rubredoxin pool (Rub_red) serves as the electron donor for oxygen and NO reduction by Roo for superoxide reduction by Sor and for H₂O₂ reduction by the rubrerythrins (Rbrs).