Rubrerythrin and rubredoxin oxidoreductase in Desulfovibrio vulgaris: a novel oxidative stress protection system

Abstract

Evidence is presented for an alternative to the superoxide dismutase (SOD)-catalase oxidative stress defense system in Desulfovibrio vulgaris (strain Hildenborough). This alternative system consists of the nonheme iron proteins, rubrerythrin (Rbr) and rubredoxin oxidoreductase (Rbo), the product of the rbo gene (also called desulfoferrodoxin). A Deltarbo strain of D. vulgaris was found to be more sensitive to internal superoxide exposure than was the wild type. Unlike Rbo, expression of plasmid-borne Rbr failed to restore the aerobic growth of a SOD-deficient strain of Escherichia coli. Conversely, plasmid-borne expression of two different Rbrs from D. vulgaris increased the viability of a catalase-deficient strain of E. coli that had been exposed to hydrogen peroxide whereas Rbo actually decreased the viability. A previously undescribed D. vulgaris gene was found to encode a protein having 50% sequence identity to that of E. coli Fe-SOD. This gene also encoded an extended N-terminal sequence with high homologies to export signal peptides of periplasmic redox proteins. The SOD activity of D. vulgaris is not affected by the absence of Rbo and is concentrated in the periplasmic fraction of cell extracts. These results are consistent with a superoxide reductase rather than SOD activity of Rbo and with a peroxidase activity of Rbr. A joint role for Rbo and Rbr as a novel cytoplasmic oxidative stress protection system in D. vulgaris and other anaerobic microorganisms is proposed.

FIG. 1

Viabilities of D. vulgaris wild type and L2 strains following exposure to either air or air plus 10 μM PQ. (A) Surviving CFU versus the time of the [air+PQ]-exposed cells normalized to the surviving CFU of the air-only exposed cells. (B) The actual CFU/ml versus the time for both air only- and [air + PQ]-exposed cells.

FIG. 2

Amino acid sequence alignment of the putative sod gene product from D. vulgaris with the Fe-containing SOD from E. coli (GenBank accession no. J03511.1). The putative leader sequence cleavage site in the D. vulgaris SOD sequence is shown in italics between residues A34 and A35. Asterisks (∗) indicate iron ligand residues in E. coli SOD (22). +, amino acids are similar to each other.

FIG. 3

Sequence alignment of export signal peptides from selected bacterial and archaeal redox proteins with the putative signal peptide of the D. vulgaris (Hildenborough) SOD (DvH_SOD). Alignment of the sequences follows that in reference . Sequence sources (GenBank accession numbers) are as follows: Db_HysB, Desulfomicrobium baculatum NiFeSe hydrogenase (P13063); Ws_HydA, Wolinella succinogenes NiFe hydrogenase (S33852); Sa_SoxF, Sulfolobus acidocaldarius Rieske iron-sulfur protein (S56156); Ec_HybA, E. coli hydrogenase (P37179); Ec_NrfC, E. coli putative iron-sulfur protein (P32708); Hi_NrfC, Haemophilus influenzae putative iron-sulfur protein (P45015); Sm_NosZ Sinorhizobium meliloti nitrous oxide reductase (Q59746); and Ps_NosZ, Pseudomonas stutzeri nitrous oxide reductase (P19573).

FIG. 4

Aerobic growth versus the time at 37°C in M63 medium of E. coli QC774 (sodA sodB) containing pCYB1-based plasmids expressing D. vulgaris Rbo and Rbr and D. gigas Nlr.

FIG. 5

Viability of aerobically grown E. coli NC202 (katG katE) expressing plasmid-borne D. vulgaris Rbr, Ngr, or Rbo genes following a 30-min exposure to 2.5 mM H₂O₂. IPTG (0.4 mM) was added to induce overexpression of the genes prior to H₂O₂ exposure.

FIG. 6

Proposed model for an Rbo/Rbr oxidative stress protection system in D. vulgaris.

FIG. 7

Diagrams of open reading frames encoding adjacent rbo, rbr, and rub homologs from anaerobic bacteria and archaea. rub, rubredoxin gene; rbo, Rbo homolog gene; nlr, neelaredoxin homolog gene; rdl, rubredoxin-like protein gene; fur, Fur homolog gene. Horizontal distances between boxes are proportional to the relative spacings of the genes. Arrow-shaped orientations of boxes show putative transcription directions. Open reading frames were derived from sequences deposited in GenBank with the following accession numbers: X99543 (D. baarsii), AE001047 (Archaeoglobus fulgidus), AF156097 (P. furiosus), AJ248285 (Pyrococcus abysii), AE001739 (Thermotoga maritima), AF202316 (Moorella thermoacetica), U67520 (Methanococcus jannaschii), AE000854 (Methanobacterium thermoautotrophicum), M28848 [Desulfovibrio vulgaris (Hildenborough) rbo-rub] and U82323 [D. vulgaris (Hildenborough) fur-rbr-rdl].