Sulfate-reducing Bacterium Desulfovibrio Vulgaris Research Articles

The role of acetogens in microbially influenced corrosion of steel.

Microbially influenced corrosion (MIC) of iron (Fe0) by sulfate-reducing bacteria (SRB) has been studied extensively. Through a mechanism, that is still poorly understood, electrons or hydrogen (H2) molecules are removed from the metal surface and used as electron donor for sulfate reduction. The resulting ferrous ions precipitate in part with the sulfide produced, forming characteristic black iron sulfide. Hydrogenotrophic methanogens can also contribute to MIC. Incubation of pipeline water samples, containing bicarbonate and some sulfate, in serum bottles with steel coupons and a headspace of 10% (vol/vol) CO2 and 90% N2, indicated formation of acetate and methane. Incubation of these samples in serum bottles, containing medium with coupons and bicarbonate but no sulfate, also indicated that formation of acetate preceded the formation of methane. Microbial community analyses of these enrichments indicated the presence of Acetobacterium, as well as of hydrogenotrophic and acetotrophic methanogens. The formation of acetate by homoacetogens, such as Acetobacterium woodii from H2 (or Fe0) and CO2, is potentially important, because acetate is a required carbon source for many SRB growing with H2 and sulfate. A consortium of the SRB Desulfovibrio vulgaris Hildenborough and A. woodii was able to grow in defined medium with H2, CO2, and sulfate, because A. woodii provides the acetate, needed by D. vulgaris under these conditions. Likewise, general corrosion rates of metal coupons incubated with D. vulgaris in the presence of acetate or in the presence of A. woodii were higher than in the absence of acetate or A. woodii, respectively. An extended MIC model capturing these results is presented.

Membrane-bound oxygen reductases of the anaerobic sulfate-reducing Desulfovibrio vulgaris Hildenborough: roles in oxygen defence and electron link with periplasmic hydrogen oxidation

Cytoplasmic membranes of the strictly anaerobic sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough contain two terminal oxygen reductases, a bd quinol oxidase and a cc(b/o)o3 cytochrome oxidase (Cox). Viability assays pointed out that single Δbd, Δcox and double ΔbdΔcox deletion mutant strains were more sensitive to oxygen exposure than the WT strain, showing the involvement of these oxygen reductases in the detoxification of oxygen. The Δcox strain was slightly more sensitive than the Δbd strain, pointing to the importance of the cc(b/o)o3 cytochrome oxidase in oxygen protection. Decreased O2 reduction rates were measured in mutant cells and membranes using lactate, NADH, ubiquinol and menadiol as substrates. The affinity for oxygen measured with the bd quinol oxidase (Km, 300 nM) was higher than that of the cc(b/o)o3 cytochrome oxidase (Km, 620 nM). The total membrane activity of the bd quinol oxidase was higher than that of the cytochrome oxidase activity in line with the higher expression of the bd oxidase genes. In addition, analysis of the ΔbdΔcox mutant strain indicated the presence of at least one O2-scavenging membrane-bound system able to reduce O2 with menaquinol as electron donor with an O2 affinity that was two orders of magnitude lower than that of the bd quinol oxidase. The lower O2 reductase activity in mutant cells with hydrogen as electron donor and the use of specific inhibitors indicated an electron transfer link between periplasmic H2 oxidation and membrane-bound oxygen reduction via the menaquinol pool. This linkage is crucial in defence of the strictly anaerobic bacterium Desulfovibrio against oxygen stress.

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.

High-throughput Isolation and Characterization of Untagged Membrane Protein Complexes: Outer Membrane Complexes of Desulfovibrio vulgaris

Cell membranes represent the “front line” of cellular defense and the interface between a cell and its environment. To determine the range of proteins and protein complexes that are present in the cell membranes of a target organism, we have utilized a “tagless” process for the system-wide isolation and identification of native membrane protein complexes. As an initial subject for study, we have chosen the Gram-negative sulfate-reducing bacterium Desulfovibrio vulgaris. With this tagless methodology, we have identified about two-thirds of the outer membrane- associated proteins anticipated. Approximately three-fourths of these appear to form homomeric complexes. Statistical and machine-learning methods used to analyze data compiled over multiple experiments revealed networks of additional protein–protein interactions providing insight into heteromeric contacts made between proteins across this region of the cell. Taken together, these results establish a D. vulgaris outer membrane protein data set that will be essential for the detection and characterization of environment-driven changes in the outer membrane proteome and in the modeling of stress response pathways. The workflow utilized here should be effective for the global characterization of membrane protein complexes in a wide range of organisms.

Contribution of rubredoxin:oxygen oxidoreductases and hybrid cluster proteins of Desulfovibrio vulgarisHildenborough to survival under oxygen and nitrite stress

A genomic island (GEI) of the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough, found to be able to migrate between two tRNA-Met loci of the genome, contains genes for rubredoxin:oxygen oxidoreductase-1 (roo1) and hybrid cluster protein-1 (hcp1) with additional copies for these genes (roo2 and hcp2) being found elsewhere on the chromosome. A suite of mutants was created in which roo2 and/or hcp2 and/or the GEI were either present or missing. The GEI and roo2 increased survival under microaerobic conditions and allowed growth in closer proximity to the air-water interface of soft agar tubes, two properties which appeared to be closely linked. When Hcp2(+) GEI(+) or Hcp2(-) GEI(+) cells, harbouring cytochrome c nitrite reductase (NrfHA) and growing on lactate and sulfate, were amended with 10 mM nitrite at mid-log phase (8-10 mM sulfide), all nitrite was reduced within 30 h with a rate of 3.0 mmol (g biomass)(-1) h(-1) after which sulfate reduction resumed. However, Hcp2(+) GEI(-) or Hcp2(-) GEI(-) cells were unable to use lactate, causing sulfide to be used as electron donor for nitrite reduction at a sixfold lower rate. Complementation studies indicated that hcp1, not roo1, enhanced the rate of nitrite reduction under these conditions. Hcp2 enhanced the rate of nitrite reduction when, in addition to lactate, hydrogen was also present as an electron donor. These results indicate a critical role of Hcps in alleviating nitrite stress in D. vulgaris Hildenborough by maintaining the integrity of electron transport chains from lactate or H(2) to NrfHA through removal of reactive nitrogen species. It thus appears that the GEI contributes considerably to the fitness of the organism, allowing improved growth in microaerobic environments found in sulfide-oxygen gradients and in environments, containing both sulfide and nitrite, through the action of Roo1 and Hcp1 respectively.

Deletion of the Desulfovibrio vulgaris Carbon Monoxide Sensor Invokes Global Changes in Transcription

The carbon monoxide-sensing transcriptional factor CooA has been studied only in hydrogenogenic organisms that can grow using CO as the sole source of energy. Homologs for the canonical CO oxidation system, including CooA, CO dehydrogenase (CODH), and a CO-dependent Coo hydrogenase, are present in the sulfate-reducing bacterium Desulfovibrio vulgaris, although it grows only poorly on CO. We show that D. vulgaris Hildenborough has an active CO dehydrogenase capable of consuming exogenous CO and that the expression of the CO dehydrogenase, but not that of a gene annotated as encoding a Coo hydrogenase, is dependent on both CO and CooA. Carbon monoxide did not act as a general metabolic inhibitor, since growth of a strain deleted for cooA was inhibited by CO on lactate-sulfate but not pyruvate-sulfate. While the deletion strain did not accumulate CO in excess, as would have been expected if CooA were important in the cycling of CO as a metabolic intermediate, global transcriptional analyses suggested that CooA and CODH are used during normal metabolism.

Bacterioferritin protects the anaerobe Desulfovibrio vulgaris Hildenborough against oxygen

Intracellular free iron, is under aerobic conditions and via the Fenton reaction a catalyst for the formation of harmful reactive oxygen species. In this article, we analyzed the relation between intracellular iron storage and oxidative stress response in the sulfate reducing bacterium Desulfovibrio vulgaris Hildenborough, an anaerobe that is often found in oxygenated niches. To this end, we investigated the role of the iron storage protein bacterioferritin using transcriptomic and physiological approaches. We observed that transcription of bacterioferritin is strongly induced upon exposure of cells to an oxygenated atmosphere. When grown in the presence of high concentrations of oxygen the D. vulgaris bacterioferritin mutant exhibited, in comparison with the wild type strain, lower viability and a higher content of intracellular reactive oxygen species. Furthermore, the bacterioferritin gene is under the control of the oxidative stress response regulator D. vulgaris PerR. Altogether the data revealed a previously unrecognized ability for the iron storage bacterioferritin to contribute to the oxygen tolerance exhibited by D. vulgaris.

Microbial sulfidogenesis in ferrihydrite-rich environments: Effects on iron mineralogy and arsenic mobility

Microbial sulfidogenesis plays a potentially important role in Fe and As biogeochemistry within wetland soils, sediments and aquifers. This study investigates the specific effects of microbial sulfidogenesis on Fe mineralogy and associated As mobility in mildly acidic (pH 6) and mildly basic (pH 8) advective-flow environments. A series of experiments were conducted using advective-flow columns, with an initial solid-phase comprising As(III)-bearing ferrihydrite-coated quartz sand. Columns for each pH treatment were inoculated with the sulfate-reducing bacteria Desulfovibrio vulgaris, and were compared to additional abiotic control columns. Over a period of 28 days, microbial sulfidogenesis (as coupled to the incomplete oxidation of lactate) caused major changes in Fe mineralogy, including replacement of ferrihydrite by mackinawite and magnetite at the in-flow end of the inoculated columns. At pH 8, the Fe 2+ produced by electron transfer between sulfide and ferrihydrite was mainly retained near its zone of formation. In contrast, at pH 6, much of the produced Fe 2+ was transported with advecting groundwater, facilitating the downstream Fe 2+-catalyzed transformation of ferrihydrite to goethite. At both pH 6 and pH 8, the sulfide-driven reductive dissolution of ferrihydrite and its replacement by mackinawite at the in-flow end of the inoculated columns resulted in substantial mobilization of As into the pore-water. At pH 8, this caused the downstream As concentrations within the inoculated columns to be greater than the corresponding abiotic column. However, the opposite occurred under pH 6 conditions, with the Fe 2+-catalyzed transformation of ferrihydrite to goethite in the inoculated columns causing a decrease in downstream As concentrations compared to the abiotic column. Although thermodynamically favorable at intermediate times and depth intervals within the inoculated columns, solid As sulfide phases were undetectable by As XANES spectroscopy. Our findings show that microbial sulfidogenesis can trigger significant As mobilization in subsurface environments with advective groundwater flow. The results also demonstrate that formation of mackinawite by sulfidization of ferric (hydr)oxides is not effective for the immobilization of As, whereas the Fe 2+-catalyzed transformation of ferrihydrite to goethite under mildly acidic conditions may mitigate As mobility.

Response of Desulfovibrio vulgaris Hildenborough to hydrogen peroxide: enzymatic and transcriptional analyses

We studied the effect of hydrogen peroxide (H(2)O(2)) stress on the anaerobic sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough. In a lactate/sulfate medium, growth was affected from 0.1 mM H(2)O(2) and totally inhibited at 0.7 mM. Surprisingly, transcript analyses revealed that the PerR regulon exhibited opposite regulation in the presence of 0.1 and 0.3 mM H(2)O(2). The variations in peroxidase- and superoxide dismutase-specific activities in the cell-free extracts of H(2)O(2)-stressed cultures were related to changes in the corresponding transcript abundance. Our data suggest that sod, sor, ngr and tpx genes, in addition to the PerR regulon, belong to the H(2)O(2) stimulon.

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.

Arsenic repartitioning during biogenic sulfidization and transformation of ferrihydrite

Iron (hydr)oxides are strong sorbents of arsenic (As) that undergo reductive dissolution and transformation upon reaction with dissolved sulfide. Here we examine the transformation and dissolution of As-bearing ferrihydrite and subsequent As repartitioning amongst secondary phases during biotic sulfate reduction. Columns initially containing As(V)–ferrihydrite coated sand, inoculated with the sulfate reducing bacteria Desulfovibrio vulgaris (Hildenborough), were eluted with artificial groundwater containing sulfate and lactate. Rapid and consistent sulfate reduction coupled with lactate oxidation is observed at low As(V) loading (10% of the adsorption maximum). The dominant Fe solid phase transformation products at low As loading include amorphous FeS within the zone of sulfate reduction (near the inlet of the column) and magnetite downstream where Fe(II) (aq) concentrations increase; As is displaced from the zone of sulfidogenesis and Fe(III) (s) depletion. At high As(V) loading (50% of the adsorption maximum), sulfate reduction and lactate oxidation are initially slow but gradually increase over time, and all As(V) is reduced to As(III) by the end of experimentation. With the higher As loading, green rust(s), as opposed to magnetite, is a dominant Fe solid phase product. Independent of loading, As is strongly associated with magnetite and residual ferrihydrite, while being excluded from green rust and iron sulfide. Our observations illustrate that sulfidogenesis occurring in proximity with Fe (hydr)oxides induce Fe solid phase transformation and changes in As partitioning; formation of As sulfide minerals, in particular, is inhibited by reactive Fe(III) or Fe(II) either through sulfide oxidation or complexation.

On hydrolysis of cellulose and nitrocellulose under sulfate-reducing conditions

Hydrolysis of cellulose and nitrocellulose in the presence of sulfate-reducing bacterium Desulfovibrio vulgaris 1388 was studied. The cellulolytic activity was found in culture medium after D. vulgaris growth (1.45 ± 0.04 nmol/mg protein/min). In the presence of cellulose or nitrocellulose the activity accounted for 4.82 ± 0.23 and 2.35 ± 0.11 nmol/mg protein/min, respectively. The initial rates of cellulose decomposition were measured using toluene to inhibit the microbial uptake of hydrolysis product—glucose. It was established that 7.6% of initially added cellulose was hydrolyzed in 3 weeks. The highest rate of glucose accumulation was observed on day 10 (2.13 μmol glucose/g dry-wt cellulose/h). At the same time only 3.3% of nitrocellulose was hydrolyzed, since nitro-groups of polymer exerted negative influence on the hydrolysis process. It is supposed that nonspecific extracellular hydrolases participate in the polymers hydrolysis.

Functional characterization of the early steps of tetrapyrrole biosynthesis and modification in Desulfovibrio vulgaris Hildenborough

The biosynthesis of the tetrapyrrole framework has been investigated in the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough by characterization of the enzymes required for the transformation of aminolaevulinic acid into sirohydrochlorin. PBG (porphobilinogen) synthase (HemB) was found to be a zinc-dependent enzyme that exists in its native state as a homohexamer. PBG deaminase (HemC) was shown to contain the dipyrromethane cofactor. Uroporphyrinogen III synthase is found fused with a uroporphyrinogen III methyltransferase (HemD-CobA). Both activities could be demonstrated in this amalgamated protein and the individual enzyme activities were separated by dissecting the relevant gene to allow the production of two distinct proteins. A gene annotated in the genome as a bifunctional precorrin-2 dehydrogenase/sirohydrochlorin ferrochelatase was in fact shown to act only as a dehydrogenase and is simply capable of synthesizing sirohydrochlorin rather than sirohaem. Genome analysis also reveals a lack of any uroporphyrinogen III decarboxylase, an enzyme necessary for the classical route to haem synthesis. However, the genome does encode some predicted haem d1 biosynthetic enzymes even though the bacterium does not contain the cd1 nitrite reductase. We suggest that sirohydrochlorin acts as a substrate for haem synthesis using a novel pathway that involves homologues of the d1 biogenesis system. This explains why the uroporphyrinogen III synthase is found fused with the methyltransferase, bypassing the need for uroporphyrinogen III decarboxylase activity.

Expression profiling of hypothetical genes in Desulfovibrio vulgaris leads to improved functional annotation

Hypothetical (HyP) and conserved HyP genes account for >30% of sequenced bacterial genomes. For the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough, 347 of the 3634 genes were annotated as conserved HyP (9.5%) along with 887 HyP genes (24.4%). Given the large fraction of the genome, it is plausible that some of these genes serve critical cellular roles. The study goals were to determine which genes were expressed and provide a more functionally based annotation. To accomplish this, expression profiles of 1234 HyP and conserved genes were used from transcriptomic datasets of 11 environmental stresses, complemented with shotgun LC–MS/MS and AMT tag proteomic data. Genes were divided into putatively polycistronic operons and those predicted to be monocistronic, then classified by basal expression levels and grouped according to changes in expression for one or multiple stresses. One thousand two hundred and twelve of these genes were transcribed with 786 producing detectable proteins. There was no evidence for expression of 17 predicted genes. Except for the latter, monocistronic gene annotation was expanded using the above criteria along with matching Clusters of Orthologous Groups. Polycistronic genes were annotated in the same manner with inferences from their proximity to more confidently annotated genes. Two targeted deletion mutants were used as test cases to determine the relevance of the inferred functional annotations.

Application of molecular techniques to evaluate the methanogenic archaea and anaerobic bacteria in the presence of oxygen with different COD:Sulfate ratios in a UASB reactor

In this paper, the microbial characteristics of the granular sludge in the presence of oxygen (3.0±0.7 mg O 2 l −1) were analyzed using molecular biology techniques. The granules were provided by an upflow anaerobic sludge blanket (UASB) operated over 469 days and fed with synthetic substrate. Ethanol and sulfate were added to obtain different COD/SO 4 2− ratios (3.0, 2.0, and 1.6). The results of fluorescent in situ hybridization (FISH) analyses showed that archaeal cells, detected by the ARC915 probe, accounted for 77%, 84%, and 75% in the COD/SO 4 2− ratios (3.0, 2.0, and 1.6, respectively). Methanosaeta sp. was the predominant acetoclastic archaea observed by optical microscopy and FISH analyses, and confirmed by sequencing of the excised bands of the DGGE gel with a similarity of 96%. The sulfate-reducing bacterium Desulfovibrio vulgaris subsp. vulgaris (similarity of 99%) was verified by sequencing of the DGGE band. Others identified microorganism were similar to Shewanella sp. and Desulfitobacterium hafniense, with similarities of 95% and 99%, respectively. These results confirmed that the presence of oxygen did not severely affect the metabolism of microorganisms that are commonly considered strictly anaerobic. We obtained mean efficiencies of organic matter conversion and sulfate reducing higher than 74%.

Crystallization and preliminary X-ray analysis of a class II release factor RF3 from a sulfate-reducing bacterium.

Class II release factor 3 (RF3) from the sulfate-reducing bacterium Desulfovibrio vulgaris Miyazaki F, which promotes rapid dissociation of a class I release factor, has been overexpressed, purified and crystallized in complex with GDP at 293 K using the sitting-drop vapour-diffusion method. A data set was collected to 1.8 A resolution from a single crystal at 100 K using synchrotron radiation. The crystal belongs to space group P1, with unit-cell parameters a = 47.39, b = 82.80, c = 148.29 A, alpha = 104.21, beta = 89.78, gamma = 89.63 degrees . The asymmetric unit contains four molecules of the RF3-GDP complex. The Matthews coefficient was calculated to be 2.3 A(3) Da(-1) and the solvent content was estimated to be 46.6%.

Structural and functional relationships in the hybrid cluster protein family: structure of the anaerobically purified hybrid cluster protein fromDesulfovibrio vulgarisat 1.35 Å resolution

The hybrid cluster protein (HCP) from the sulfate-reducing bacterium Desulfovibrio vulgaris strain Hildenborough has been isolated and crystallized anaerobically. The phase problem was solved for a P2(1)2(1)2(1) crystal form using multiple-wavelength anomalous diffraction data collected in the vicinity of the Fe K absorption edge. Although the overall protein structure is essentially the same as that previously obtained, it shows that the nature of the hybrid cluster has particular differences when isolated and crystallized in the absence of oxygen and this provides insight into the structural features associated with changes in the oxidation state. A comparison between HCPs and carbon monoxide dehydrogenases (CoDs) shows that they possess a similar fold and that the dehydrogenases have a related cluster at the equivalent HCP hybrid cluster position. This helps to understand the nature of the hybrid cluster and to predict a dimeric structure for class 3 HCPs, which lack the N-terminal region.

Two Distinct Roles for Two Functional Cobaltochelatases (CbiK) in Desulfovibrio vulgaris Hildenborough

The sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough possesses a large number of porphyrin-containing proteins whose biosynthesis is poorly characterized. In this work, we have studied two putative CbiK cobaltochelatases present in the genome of D. vulgaris. The assays revealed that both enzymes insert cobalt and iron into sirohydrochlorin, with specific activities with iron lower than that measured with cobalt. Nevertheless, the two D. vulgaris chelatases complement an E. coli cysG mutant strain showing that, in vivo, they are able to load iron into sirohydrochlorin. The results showed that the functional cobaltochelatases have distinct roles with one, CbiK(C), likely to be the enzyme associated with cytoplasmic cobalamin biosynthesis, while the other, CbiK(P), is periplasmic located and possibly associated with an iron transport system. Finally, the ability of D. vulgaris to produce vitamin B 12 was also demonstrated in this work.

Presence and expression of terminal oxygen reductases in strictly anaerobic sulfate-reducing bacteria isolated from salt-marsh sediments

In the anaerobic sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough genes were found encoding membrane terminal oxygen reductases of two types: a cytochrome c oxidase and a cytochrome bd oxidase, both enzymes are terminal oxidases typical of facultative or aerobic microorganisms (Heidelberg JF, et al., The genome sequence of the anaerobic, sulfate-reducing bacterium D. vulgaris Hildenborough. Nat Biotechnol 2004; 22: 554–9). To apprehend the presence of both oxidases in other sulfate-reducing bacteria (SRB), several assays were performed on isolates recovered from salt-marsh sediments in Portugal, representative of the different phylogenetic groups identified. Hybridization and PCR experiments for DNA sequencing were performed on the chosen isolates. Primers were selected to amplify conserved regions of cytochrome c oxidases and cytochrome bd oxidases taking into consideration alignment of corresponding subunit I sequences. The results showed that both oxidase genes are present on the chromosome of several isolates characterized as Desulfovibrio. These genes were shown to be transcribed, as demonstrated by Reverse Transcriptase-PCR experiments on total RNA. In order to assess the relative contribution of each oxidase to oxygen consumption, oxygen uptake was measured for each isolate and further characterized by the effect of cyanide on oxygen consumption. It was concluded that cytochrome bd oxidase was the terminal membrane oxygen reductase allowing oxygen consumption. In addition, it was observed that isolates containing cytochrome bd oxidase had higher resistance to air exposure, suggesting an important role of this enzyme in survival to air exposure. The pattern for the presence of oxygen reductase genes was compared to the physiological pattern of substrate use, which was determined for each isolate. Salinity tolerance, pH and temperature growth of each isolate were also analyzed.

Evolution of the syntrophic interaction between Desulfovibrio vulgaris and Methanosarcina barkeri: Involvement of an ancient horizontal gene transfer

The sulfate reducing bacteria Desulfovibrio vulgaris and the methanogenic archaea Methanosarcina barkeri can grow syntrophically on lactate. In this study, a set of three closely located genes, DVU2103, DVU2104, and DVU2108 of D. vulgaris, was found to be up-regulated 2- to 4-fold following the lifestyle shift from syntroph to sulfate reducer; moreover, none of the genes in this gene set were differentially regulated when comparing gene expression from various D. vulgaris pure culture experiments. Although exact function of this gene set is unknown, the results suggest that it may play roles related to the lifestyle change of D. vulgaris from syntroph to sulfate reducer. This hypothesis is further supported by phylogenomic analyses showing that homologies of this gene set were only narrowly present in several groups of bacteria, most of which are restricted to a syntrophic lifestyle, such as Pelobacter carbinolicus, Syntrophobacter fumaroxidans, Syntrophomonas wolfei, and Syntrophus aciditrophicus. Phylogenetic analysis showed that all three individual genes in the gene set tended to be clustered with their homologies from archaeal genera, and they were rooted on archaeal species in the phylogenetic trees, suggesting that they were horizontally transferred from archaeal methanogens. In addition, no significant bias in codon and amino acid usages was detected between these genes and the rest of the D. vulgaris genome, suggesting the gene transfer may have occurred early in the evolutionary history so that sufficient time has elapsed to allow an adaptation to the codon and amino acid usages of D. vulgaris. This report provides novel insights into the origin and evolution of bacterial genes linked to the lifestyle change of D. vulgaris from a syntrophic to a sulfate-reducing lifestyle.