Johanna K. Voordouw

Physiological and Gene Expression Analysis of Inhibition of Desulfovibrio vulgaris Hildenborough by Nitrite

A Desulfovibrio vulgaris Hildenborough mutant lacking the nrfA gene for the catalytic subunit of periplasmic cytochrome c nitrite reductase (NrfHA) was constructed. In mid-log phase, growth of the wild type in medium containing lactate and sulfate was inhibited by 10 mM nitrite, whereas 0.6 mM nitrite inhibited the nrfA mutant. Lower concentrations (0.04 mM) inhibited the growth of both mutant and wild-type cells on plates. Macroarray hybridization indicated that nitrite upregulates the nrfHA genes and downregulates genes for sulfate reduction enzymes catalyzing steps preceding the reduction of sulfite to sulfide by dissimilatory sulfite reductase (DsrAB), for two membrane-bound electron transport complexes (qmoABC and dsrMKJOP) and for ATP synthase (atp). DsrAB is known to bind and slowly reduce nitrite. The data support a model in which nitrite inhibits DsrAB (apparent dissociation constant K(m) for nitrite = 0.03 mM), and in which NrfHA (K(m) for nitrite = 1.4 mM) limits nitrite entry by reducing it to ammonia when nitrite concentrations are at millimolar levels. The gene expression data and consideration of relative gene locations suggest that QmoABC and DsrMKJOP donate electrons to adenosine phosphosulfate reductase and DsrAB, respectively. Downregulation of atp genes, as well as the recorded cell death following addition of inhibitory nitrite concentrations, suggests that the proton gradient collapses when electrons are diverted from cytoplasmic sulfate to periplasmic nitrite reduction.

Function of oxygen resistance proteins in the anaerobic, sulfate-reducing bacterium Desulfovibrio vulgaris hildenborough.

Two mutant strains of Desulfovibrio vulgaris Hildenborough lacking either the sod gene for periplasmic superoxide dismutase or the rbr gene for rubrerythrin, a cytoplasmic hydrogen peroxide (H(2)O(2)) reductase, were constructed. Their resistance to oxidative stress was compared to that of the wild-type and of a sor mutant lacking the gene for the cytoplasmic superoxide reductase. The sor mutant was more sensitive to exposure to air or to internally or externally generated superoxide than was the sod mutant, which was in turn more sensitive than the wild-type strain. No obvious oxidative stress phenotype was found for the rbr mutant, indicating that H(2)O(2) resistance may also be conferred by two other rbr genes in the D. vulgaris genome. Inhibition of Sod activity by azide and H(2)O(2), but not by cyanide, indicated it to be an iron-containing Sod. The positions of Fe-Sod and Sor were mapped by two-dimensional gel electrophoresis (2DE). A strong decrease of Sor in continuously aerated cells, indicated by 2DE, may be a critical factor in causing cell death of D. vulgaris. Thus, Sor plays a key role in oxygen defense of D. vulgaris under fully aerobic conditions, when superoxide is generated mostly in the cytoplasm. Fe-Sod may be more important under microaerophilic conditions, when the periplasm contains oxygen-sensitive, superoxide-producing targets.

Massive dominance of Epsilonproteobacteria in formation waters from a Canadian oil sands reservoir containing severely biodegraded oil

Summary The subsurface microbiology of an Athabasca oil sands reservoir in western Canada containing severely biodegraded oil was investigated by combining 16S rRNA gene- and polar lipid-based analyses of reservoir formation water with geochemical analyses of the crude oil and formation water. Biomass was filtered from formation water, DNA was extracted using two different methods, and 16S rRNA gene fragments were amplified with several different primer pairs prior to cloning and sequencing or community fingerprinting by denaturing gradient gel electrophoresis (DGGE). Similar results were obtained irrespective of the DNA extraction method or primers used. Archaeal libraries were dominated by Methanomicrobiales (410 of 414 total sequences formed a dominant phylotype affiliated with a Methanoregula sp.), consistent with the proposed dominant role of CO2-reducing methanogens in crude oil biodegradation. In two bacterial 16S rRNA clone libraries generated with different primer pairs, > 99% and 100% of the sequences were affiliated with Epsilonproteobacteria (n = 382 and 72 total clones respectively). This massive dominance of Epsilonproteobacteria sequences was again obtained in a third library (99% of sequences; n = 96 clones) using a third universal bacterial primer pair (inosine-341f and 1492r). Sequencing of bands from DGGE profiles and intact polar lipid analyses were in accordance with the bacterial clone library results. Epsilonproteobacterial OTUs were affiliated with Sulfuricurvum, Arcobacter and Sulfurospirillum spp. detected in other oil field habitats. The dominant organism revealed by the bacterial libraries (87% of all sequences) is a close relative of Sulfuricurvum kujiense – an organism capable of oxidizing reduced sulfur compounds in crude oil. Geochemical analysis of organic extracts from bitumen at different reservoir depths down to the oil water transition zone of these oil sands indicated active biodegradation of dibenzothiophenes, and stable sulfur isotope ratios for elemental sulfur and sulfate in formation waters were indicative of anaerobic oxidation of sulfur compounds. Microbial desulfurization of crude oil may be an important metabolism for Epsilonproteobacteria indigenous to oil reservoirs with elevated sulfur content and may explain their prevalence in formation waters from highly biodegraded petroleum systems.

Effects of Deletion of Genes Encoding Fe-Only Hydrogenase of Desulfovibrio vulgaris Hildenborough on Hydrogen and Lactate Metabolism

The physiological properties of a hyd mutant of Desulfovibrio vulgaris Hildenborough, lacking periplasmic Fe-only hydrogenase, have been compared with those of the wild-type strain. Fe-only hydrogenase is the main hydrogenase of D. vulgaris Hildenborough, which also has periplasmic NiFe- and NiFeSe-hydrogenases. The hyd mutant grew less well than the wild-type strain in media with sulfate as the electron acceptor and H(2) as the sole electron donor, especially at a high sulfate concentration. Although the hyd mutation had little effect on growth with lactate as the electron donor for sulfate reduction when H(2) was also present, growth in lactate- and sulfate-containing media lacking H(2) was less efficient. The hyd mutant produced, transiently, significant amounts of H(2) under these conditions, which were eventually all used for sulfate reduction. The results do not confirm the essential role proposed elsewhere for Fe-only hydrogenase as a hydrogen-producing enzyme in lactate metabolism (W. A. M. van den Berg, W. M. A. M. van Dongen, and C. Veeger, J. Bacteriol. 173:3688-3694, 1991). This role is more likely played by a membrane-bound, cytoplasmic Ech-hydrogenase homolog, which is indicated by the D. vulgaris genome sequence. The physiological role of periplasmic Fe-only hydrogenase is hydrogen uptake, both when hydrogen is and when lactate is the electron donor for sulfate reduction.

Function of Periplasmic Hydrogenases in the Sulfate-Reducing Bacterium Desulfovibrio vulgaris Hildenborough

The sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough possesses four periplasmic hydrogenases to facilitate the oxidation of molecular hydrogen. These include an [Fe] hydrogenase, an [NiFeSe] hydrogenase, and two [NiFe] hydrogenases encoded by the hyd, hys, hyn1, and hyn2 genes, respectively. In order to understand their cellular functions, we have compared the growth rates of existing (hyd and hyn1) and newly constructed (hys and hyn-1 hyd) mutants to those of the wild type in defined media in which lactate or hydrogen at either 5 or 50% (vol/vol) was used as the sole electron donor for sulfate reduction. Only strains missing the [Fe] hydrogenase were significantly affected during growth with lactate or with 50% (vol/vol) hydrogen as the sole electron donor. When the cells were grown at low (5% [vol/vol]) hydrogen concentrations, those missing the [NiFeSe] hydrogenase suffered the greatest impairment. The growth rate data correlated strongly with gene expression results obtained from microarray hybridizations and real-time PCR using mRNA extracted from cells grown under the three conditions. Expression of the hys genes followed the order 5% hydrogen>50% hydrogen>lactate, whereas expression of the hyd genes followed the reverse order. These results suggest that growth with lactate and 50% hydrogen is associated with high intracellular hydrogen concentrations, which are best captured by the higher activity, lower affinity [Fe] hydrogenase. In contrast, growth with 5% hydrogen is associated with a low intracellular hydrogen concentration, requiring the lower activity, higher affinity [NiFeSe] hydrogenase.

Gene Expression Analysis of Energy Metabolism Mutants of Desulfovibrio vulgaris Hildenborough Indicates an Important Role for Alcohol Dehydrogenase

Comparison of the proteomes of the wild-type and Fe-only hydrogenase mutant strains of Desulfovibrio vulgaris Hildenborough, grown in lactate-sulfate (LS) medium, indicated the near absence of open reading frame 2977 (ORF2977)-coded alcohol dehydrogenase in the hyd mutant. Hybridization of labeled cDNA to a macroarray of 145 PCR-amplified D. vulgaris genes encoding proteins active in energy metabolism indicated that the adh gene was among the most highly expressed in wild-type cells grown in LS medium. Relative to the wild type, expression of the adh gene was strongly downregulated in the hyd mutant, in agreement with the proteomic data. Expression was upregulated in ethanol-grown wild-type cells. An adh mutant was constructed and found to be incapable of growth in media in which ethanol was both the carbon source and electron donor for sulfate reduction or was only the carbon source, with hydrogen serving as electron donor. The hyd mutant also grew poorly on ethanol, in agreement with its low level of adh gene expression. The adh mutant grew to a lower final cell density on LS medium than the wild type. These results, as well as the high level of expression of adh in wild-type cells on media in which lactate, pyruvate, formate, or hydrogen served as the sole electron donor for sulfate reduction, indicate that ORF2977 Adh contributes to the energy metabolism of D. vulgaris under a wide variety of metabolic conditions. A hydrogen cycling mechanism is proposed in which protons and electrons originating from cytoplasmic ethanol oxidation by ORF2977 Adh are converted to hydrogen or hydrogen equivalents, possibly by a putative H(2)-heterodisulfide oxidoreductase complex, which is then oxidized by periplasmic Fe-only hydrogenase to generate a proton gradient.

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 genomic island of the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough promotes survival under stress conditions while decreasing the efficiency of anaerobic growth

A 47 kb genomic island (GEI) bracketed by 50 bp direct repeats, containing 52 annotated genes, was found to delete spontaneously from the genome of Desulfovibrio vulgaris Hildenborough. The island contains genes for site-specific recombinases and transposases, rubredoxin:oxygen oxidoreductase-1 (Roo1) and hybrid cluster protein-1 (Hcp1), which promote survival in air and nitrite stress. The numbering distinguishes these from the Roo2 and Hcp2 homologues for which the genes are located elsewhere in the genome. Cells with and without the island (GEI(+) and GEI(-) cells respectively) were obtained by colony purification. GEI(-) cells arise in anaerobic cultures of colony-purified GEI(+) cells, indicating that the site-specific recombinases encoded by the island actively delete this region. GEI(+) cells survive better in microaerophilic conditions due to the presence of Roo1, whereas the Hcps appear to prevent inhibition by sulfur and polysulfide, which are formed by chemical reaction of sulfide and nitrite. Hence, the island confers resistance to oxygen and nitrite stress. However, GEI(-) cells have a higher growth rate in anaerobic media. Microarrays and enzyme activity stains indicated that the GEI(-) cells have increased expression of genes, which promote anaerobic energy conservation, explaining the higher growth rate. Hence, while lowering the efficiency of anaerobic metabolism, the GEI increases the fitness of D. vulgaris under stress conditions, a feature reminiscent of pathogenicity islands which allow more effective colonization of environments provided by the targeted hosts.

Function of formate dehydrogenases in Desulfovibrio vulgaris Hildenborough energy metabolism

The genome of the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough encodes three formate dehydrogenases (FDHs), two of which are soluble periplasmic enzymes (FdhAB and FdhABC3) and one that is periplasmic but membrane-associated (FdhM). FdhAB and FdhABC3 were recently shown to be the main enzymes present during growth with lactate, formate or hydrogen. To address the role of these two enzymes, ΔfdhAB and ΔfdhABC3, mutants were generated and studied. Different phenotypes were observed in the presence of either molybdenum or tungsten, since both enzymes were important for growth on formate in the presence of Mo, whereas in the presence of W only FdhAB played a role. Both ΔfdhAB and ΔfdhABC3 mutants displayed defects in growth with lactate and sulfate providing the first direct evidence for the involvement of formate cycling under these conditions. In support of this mechanism, incubation of concentrated cell suspensions of the mutant strains with lactate and limiting sulfate also gave elevated formate concentrations, as compared to the wild-type strain. In contrast, both mutants grew similarly to the wild-type with H2 and sulfate. In the absence of sulfate, the wild-type D. vulgaris cells produced formate when supplied with H2 and CO2, which resulted from CO2 reduction by the periplasmic FDHs. The conversion of H2 and CO2 to formate allows the reversible storage of reducing power in a much more soluble molecule. Furthermore, we propose this may be an expression of the ability of some sulfate-reducing bacteria to grow by hydrogen oxidation, in syntrophy with organisms that consume formate, but are less efficient in H2 utilization.

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 biocides mode of action, allowing a more intelligent combination of mechanistically different agents. This can reduce stress on budgets for chemicals and on the environment.