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

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.     

Hybrid Cluster Proteins and Flavodiiron Proteins Afford Protection to Desulfovibrio vulgaris upon Macrophage Infection

Desulfovibrio species are Gram-negative anaerobic sulfate-reducing bacteria that colonize the human gut. Recently, Desulfovibrio spp. have been implicated in gastrointestinal diseases and shown to stimulate the epithelial immune response, leading to increased production of inflammatory cytokines by macrophages. Activated macrophages are key cells of the immune system that impose nitrosative stress during phagocytosis. Hence, we have analyzed the in vitro and in vivo responses of Desulfovibrio vulgaris Hildenborough to nitric oxide (NO) and the role of the hybrid cluster proteins (HCP1 and HCP2) and rubredoxin oxygen oxidoreductases (ROO1 and ROO2) in NO protection. Among the four genes, hcp2 was the gene most highly induced by NO, and the hcp2 transposon mutant exhibited the lowest viability under conditions of NO stress. Studies in murine macrophages revealed that D. vulgaris survives incubation with these phagocytes and triggers NO production at levels similar to those stimulated by the cytokine gamma interferon (IFN-γ). Furthermore, D. vulgaris hcp and roo mutants exhibited reduced viability when incubated with macrophages, revealing that these gene products contribute to the survival of D. vulgaris during macrophage infection.     

Mutualistic growth of the sulfate-reducer Desulfovibrio vulgaris Hildenborough with different carbohydrates

Desulfovibrio vulgaris Hildenborough genome presents a phosphotransferase system putatively involved in the transport of carbohydrates. However, utilization of sugars by this sulfate-reducing bacterium has never been reported. Herein, we have observed proliferation of D. vulgaris Hildenborough with some carbohydrates, in mutualism with Stenotrophomonas maltophilia, a non-fermentative, gram-negative gammaproteobacterium, or Microbacterium, a gram-positive actinobacterium. These results suggest the importance of feedback interactions between different heterotrophic bacterial species including the alternative for D. vulgaris of exploiting additional organic resources and novel habitats. Thus, D. vulgaris strongly participates in the mineralization of carbohydrates both in complex natural and artificial systems.     

Overexpression, purification and immunodetection of DsrD from Desulfovibrio vulgaris Hildenborough

Dissimilatory sulfite reductase (DsrAB) of the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough is an ₂₂ tetramer of 180 kDa, encoded by the dsr operon. In addition to the dsrA and dsrB genes, this operon contains a gene (dsrD) encoding a protein of only 78 amino acids. Although, the function of DsrD is currently unknown, the presence of a dsrD gene has been demonstrated in a variety of sulfate-reducing bacteria and archaea. DsrD was expressed in Escherichia coli at a very high level and purified to homogeneity. Protein blotting experiments, using antisera raised against purified DsrD, demonstrated that it is expressed constitutively in D. vulgaris and does not copurify with DsrAB. Spectroscopic analysis of DsrD indicated that it does not bind either sulfite or sulfide, the substrate and product, respectively of the reaction catalyzed by DsrAB. Thus, although the conservation of this protein and its demonstrated presence in D. vulgaris, suggest an essential function in dissimilatory sulfite reduction, this function remains to be elucidated.     

Amino acid sequence and function of rebredoxin from Desulfovibrio vulgaris Miyazaki

Rubredoxin was purified from Desulfovibrio vulgaris Miyazaki. It was sequenced and some of its properties determined. Rubredoxin is composed of 52 amino acids. It is highly homologous to that from D. vulgaris Hildenborough. Its N-methionyl residue is partially formalated. The millimolar absorption coefficients of the rubredoxin at 489 nm and 280 are 8.1 and 18.5, respectively, and the standard redox potential is +5 mB, which is slightly higher than those of other rubredoxins. Rubredoxin, as well as cytochrome c-553, was reduced with lactate by the action of lactate dehydrogenase of this organism, and the rection was stimulated with 2-methyl-1, 4-naphthoquinone. It is suggested that rubredoxin, in collaboration with membraous quinone, functions as natural electron carrier for cytoplasmic lactate dehydrogenase of this organism, whereas cytochrome c-553 plays the same role for periplasmic lactate dehydrogenase.     

Effects of biocides on gene expression in the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough

Although sulfate-reducing bacteria (SRB), such as Desulfovibrio vulgaris Hildenborough (DvH) are often eradicated in oil and gas operations with biocides, such as glutaraldehyde (Glut), tetrakis (hydroxymethyl) phosphonium sulfate (THPS), and benzalkonium chloride (BAC), their response to these agents is not well known. Whole genome microarrays of D. vulgaris treated with biocides well below the minimum inhibitory concentration showed that 256, 96, and 198 genes were responsive to Glut, THPS, and BAC, respectively, and that these three commonly used biocides affect the physiology of the cell quite differently. Glut induces expression of genes required to degrade or refold proteins inactivated by either chemical modification or heat shock, whereas BAC appears to target ribosomal structure. THPS appears to primarily affect energy metabolism of SRB. Mutants constructed for genes strongly up-regulated by Glut, were killed by Glut to a similar degree as the wild type. Hence, it is difficult to achieve increased sensitivity to this biocide by single gene mutations, because Glut affects so many targets. Our results increase understanding of the biocide's mode of action, allowing a more intelligent combination of mechanistically different agents. This can reduce stress on budgets for chemicals and on the environment.     

Ferric iron reduction by Desulfovibrio vulgaris Hildenborough wild type and energy metabolism mutants

Desulfovibrio vulgaris Hildenborough wild type and its hyn1, hyd and hmc mutants, lacking genes for periplasmic [NiFe] hydrogenase-1, periplasmic [FeFe] hydrogenase or the transmembrane high molecular weight cytochrome (Hmc) complex, respectively, were able to reduce Fe(III) chelated with nitrilotriacetic acid (NTA), but not insoluble ferric oxide, with lactate as the electron donor. The rate and extent of Fe(III)-NTA reduction followed the order hyn = WT > hmc >> hyd, suggesting that reduction of soluble Fe(III) is a periplasmic process that requires the presence of periplasmic [FeFe] hydrogenase. Reduction of Fe(III)-NTA was not coupled to cell growth. In fact cell concentrations declined when D. vulgaris was incubated with Fe(III)-NTA as the only electron acceptor. Wild type and mutant cells reducing a limiting concentration of sulfate (2 mM), reduced Fe(III)-NTA with similar rates. However, these were similarly incapable of catalyzing subsequent lactate-dependent reduction of Fe(III)-NTA to completion. Periplasmic reduction of Fe(III)-NTA appeared to inhibit the productive, sulfate-reducing metabolism of D. vulgaris, possibly because it prevents the cycling of reducing equivalents needed to achieve a net bioenergetic benefit.     

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

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.     

A direct demonstration of hydrogen cycling by Desulfovibrio vulgaris employing membrane-inlet mass spectrometry

Employing membrane-inlet mass spectrometry, direct evidence is presented that substrate amounts of hydrogen are simultaneously produced and consumed during the metabolism of pyruvate plus sulfate by washed intact cells of Desulfovibrio vulgaris (Hildenborough). The results clearly demonstrate that hydrogen cycling is an important bioenergetic mechanism for this sulfate-reducing bacterium when growing on pyruvate plus sulfate.     

Five-gene cluster in Clostridium thermoaceticum consisting of two divergent operons encoding rubredoxin oxidoreductase- rubredoxin and rubrerythrin-type A flavoprotein- high-molecular-weight rubredoxin

A five-gene cluster encoding four nonheme iron proteins and a flavoprotein from the thermophilic anaerobic bacterium Clostridium thermoaceticum (Moorella thermoacetica) was cloned and sequenced. Based on analysis of deduced amino acid sequences, the genes were identified as rub (rubredoxin), rbo (rubredoxin oxidoreductase), rbr (rubrerythrin), fprA (type A flavoprotein), and a gene referred to as hrb (high-molecular-weight rubredoxin). Northern blot analysis demonstrated that the five-gene cluster is organized as two subclusters, consisting of two divergently transcribed operons, rbr-fprA-hrb and rbo-rub. The rbr, fprA, and rub genes were expressed in Escherichia coli, and their encoded recombinant proteins were purified. The molecular masses, UV-visible absorption spectra, and cofactor contents of the recombinant rubrerythrin, rubredoxin, and type A flavoprotein were similar to those of respective homologs from other microorganisms. Antibodies raised against Desulfovibrio vulgaris Rbr reacted with both native and recombinant Rbr from C. thermoaceticum, indicating that this protein was expressed in the native organism. Since Rbr and Rbo have been recently implicated in oxidative stress protection in several anaerobic bacteria and archaea, we suggest a similar function of these proteins in oxygen tolerance of C. thermoaceticum.     

Uranium Reduction by Desulfovibrio desulfuricans Strain G20 and a Cytochrome c3 Mutant

Previous in vitro experiments with Desulfovibrio vulgaris strain Hildenborough demonstrated that extracts containing hydrogenase and cytochrome c₃ could reduce uranium(VI) to uranium(IV) with hydrogen as the electron donor. To test the involvement of these proteins in vivo, a cytochrome c₃ mutant of D. desulfuricans strain G20 was assayed and found to be able to reduce U(VI) with lactate or pyruvate as the electron donor at rates about one-half of those of the wild type. With electrons from hydrogen, the rate was more severely impaired. Cytochrome c₃ appears to be a part of the in vivo electron pathway to U(VI), but additional pathways from organic donors can apparently bypass this protein.     

Deletion of two downstream genes alters expression of the hmc operon of Desulfovibrio vulgaris subsp. vulgaris Hildenborough

The hmc operon of Desulfovibrio vulgaris subsp. vulgaris Hildenborough consists of six genes (hmcA to hmcF) that encode structural components of the high-molecular-mass cytochrome redox protein complex (the Hmc complex). Two genes (rrf1 and rrf2) encoding regulatory proteins are present downstream of hmcF. Expression of the hmc operon, monitored by incubating protein blots with HmcA-specific or HmcF-specific antibodies, was found to be highest when hydrogen was the sole electron donor for sulfate reduction. Use of lactate or pyruvate as electron donor reduced expression of the hmc operon. A mutant with a deletion of the rrf1 and rrf2 genes was generated with the sacB mutagenesis method. This mutant overexpressed the hmc operon approximately threefold. It grew more rapidly than the wild type when hydrogen was used as the electron donor for sulfate reduction, but more slowly than the wild type when lactate was used. The results indicate that a physiological function of the Hmc complex is in electron flow from hydrogen to sulfate. At least one redox carrier is shared competitively by the hydrogen and lactate oxidation pathways in D. vulgaris. Received: 9 October 1996 / Accepted: 18 February 1997     

Growth of an anaerobic sulfate-reducing bacterium sustained by oxygen respiratory energy conservation after O2-driven experimental evolution

Desulfovibrio species are representatives of microorganisms at the boundary between anaerobic and aerobic lifestyles, since they contain the enzymatic systems required for both sulfate and oxygen reduction. However, the latter has been shown to be solely a protective mechanism. By implementing the oxygen-driven experimental evolution of Desulfovibrio vulgaris Hildenborough, we have obtained strains that have evolved to grow with energy derived from oxidative phosphorylation linked to oxygen reduction. We show that a few mutations are sufficient for the emergence of this phenotype and reveal two routes of evolution primarily involving either inactivation or overexpression of the gene encoding heterodisulfide reductase. We propose that the oxygen respiration for energy conservation that sustains the growth of the O₂-evolved strains is associated with a rearrangement of metabolite fluxes, especially NAD⁺/NADH, leading to an optimized O₂ reduction. These evolved strains are the first sulfate-reducing bacteria that exhibit a demonstrated oxygen respiratory process that enables growth.     

ISD1, an Insertion Element from the Sulfate-Reducing Bacterium Desulfovibrio vulgaris Hildenborough: Structure, Transposition, and Distribution

Insertion element ISD1, discovered when its transposition caused the insertional inactivation of an introduced sacB gene, is present in two copies in the genome of Desulfovibrio vulgaris Hildenborough. Southern blot analysis indicated at least two insertion sites in the sacB gene. Cloning and sequencing of a transposed copy of ISD1 indicated a length of 1,200 bp with a pair of 44-bp imperfect inverted repeats at the ends, flanked by a direct repeat of the 4-bp target sequence. AAGG and AATT were found to function as target sequences. ISD1 encodes a transposase from two overlapping open reading frames by programmed translational frameshifting at an A₆G shifty codon motif. Sequence comparison showed that ISD1 belongs to the IS3 family. Isolation and analysis of the chromosomal copies, ISD1-A and ISD1-B, by PCR and sequencing indicated that these are not flanked by direct repeats. ISD1-A is inserted in a region of the chromosome containing the gapdh-pgk genes (encoding glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase). Active transposition to other loci in the genome was demonstrated, offering the potential of a new tool for gene cloning and mutagenesis. ISD1 is the first transposable element described for the sulfate reducers, a large and environmentally important group of bacteria. The distribution of ISD1 in genomes of sulfate-reducing bacteria is limited. A single copy is present in the genome of D. desulfuricans Norway.