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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.sub.m] for nitrite = 0.03 mM), and in which NrfHA ([K.sub.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.

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.sub.2]-heterodisulfide oxidoreductase complex, which is then oxidized by periplasmic Fe-only hydrogenase to generate a proton gradient.

Abstract Background The bacterium Pelobacter carbinolicus is able to grow by fermentation, syntrophic hydrogen/formate transfer, or electron transfer to sulfur from short-chain alcohols, hydrogen or formate; it does not oxidize acetate and is not known to ferment any sugars or grow autotrophically. The genome of P. carbinolicus was sequenced in order to understand its metabolic capabilities and physiological features in comparison with its relatives, acetate-oxidizing Geobacter species. Results Pathways were predicted for catabolism of known substrates: 2,3-butanediol, acetoin, glycerol, 1,2-ethanediol, ethanolamine, choline and ethanol. Multiple isozymes of 2,3-butanediol dehydrogenase, ATP synthase and [FeFe]-hydrogenase were differentiated and assigned roles according to their structural properties and genomic contexts. The absence of asparagine synthetase and the presence of a mutant tRNA for asparagine encoded among RNA-active enzymes suggest that P. carbinolicus may make asparaginyl-tRNA in a novel way. Catabolic glutamate dehydrogenases were discovered, implying that the tricarboxylic acid (TCA) cycle can function catabolically. A phosphotransferase system for uptake of sugars was discovered, along with enzymes that function in 2,3-butanediol production. Pyruvate:ferredoxin/flavodoxin oxidoreductase was identified as a potential bottleneck in both the supply of oxaloacetate for oxidation of acetate by the TCA cycle and the connection of glycolysis to production of ethanol. The P. carbinolicus genome was found to encode autotransporters and various appendages, including three proteins with similarity to the geopilin of electroconductive nanowires. Conclusions Several surprising metabolic capabilities and physiological features were predicted from the genome of P. carbinolicus, suggesting that it is more versatile than anticipated.

The levels of transcripts for dsrA, a gene encoding a subunit of the dissimilatory sulfite reductase, are examined with chemostat-grown Desulfovibrio vulgaris. The studies have shown a direct correspondence between dsrA transcript levels and the rates of sulfate reduction per cell when both electron donor- and electron acceptor-limiting conditions are considered.

A study was conducted to determine the mechanisms for Fe(III) reduction in Pelobacter carbinolicus which have growth yields on both soluble and insoluble Fe(III) consistent with those of other Fe(III)-reducing bacteria. The results showed that P. carbinolicus reduced Fe(III) indirectly via sulfur cycling rather than by the direct reduction of Fe(III) found in Geobacter species.

The addition of organic compounds to groundwater in order to promote bioremediation may represent a new selective pressure on subsurface microorganisms. The ability of Geobacter sulfurreducens, which serves as a model for the Geobacter species that are important in various types of anaerobic groundwater bioremediation, to adapt for rapid metabolism of lactate, a common bioremediation amendment, was evaluated. Serial transfer of five parallel cultures in a medium with lactate as the sole electron donor yielded five strains that could metabolize lactate faster than the wild-type strain. Genome sequencing revealed that all five strains had non-synonymous single-nucleotide polymorphisms in the same gene, GSU0514, a putative transcriptional regulator. Introducing the single-base-pair mutation from one of the five strains into the wild-type strain conferred rapid growth on lactate. This strain and the five adaptively evolved strains had four to eight-fold higher transcript abundance than wild-type cells for genes for the two subunits of succinyl-CoA synthase, an enzyme required for growth on lactate. DNA-binding assays demonstrated that the protein encoded by GSU0514 bound to the putative promoter of the succinyl-CoA synthase operon. The binding sequence was not apparent elsewhere in the genome. These results demonstrate that a single-base-pair mutation in a transcriptional regulator can have a significant impact on the capacity for substrate utilization and suggest that adaptive evolution should be considered as a potential response of microorganisms to environmental change(s) imposed during bioremediation. [ABSTRACT FROM AUTHOR]

Previous studies failed to detect c-type cytochromes in Pelobacter species despite the fact that other close relatives in the Geobacteraceae, such as Geobacter and Desulfuromonas species, have abundant c-type cytochromes. Analysis of the recently completed genome sequence of Pelobacter carbinolicus revealed 14 open reading frames that could encode c-type cytochromes. Transcripts for all but one of these open reading frames were detected in acetoin-fermenting and/or Fe(III)-reducing cells. Three putative c-type cytochrome genes were expressed specifically during Fe(III) reduction, suggesting that the encoded proteins may participate in electron transfer to Fe(III). One of these proteins was a periplasmic triheme cytochrome with a high level of similarity to PpcA, which has a role in Fe(III) reduction in Geobacter sulfurreducens. Genes for heme biosynthesis and system II cytochrome c biogenesis were identified in the genome and shown to be expressed. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels of protein extracted from acetoin-fermenting P. carbinolicus ceils contained three heme-staining bands which were confirmed by mass spectrometry to be among the 14 predicted c-type cytochromes. The number of cytochrome genes, the predicted amount of heme c per protein, and the ratio of heme-stained protein to total protein were much smaller in P. carbinolicus than in G. sulfurreducens. Furthermore, many of the c-type cytochromes that genetic studies have indicated are required for optimal Fe(III) reduction in G. sulfurreducens were not present in the P. carbinolicus genome. These results suggest that further evaluation of the functions of c-type cytochromes in the Geobacteraceae is warranted. [ABSTRACT FROM AUTHOR]

Progress in studying the active biosphere in the deep seafloor is currently limited by the unknown amount of drilling-induced microbial contamination. Contamination tests were conducted during Leg 185 of the Ocean Drilling Program on the drillship JOIDES Resolution to assess the suitability of this platform for deep biosphere research. Tests using both a chemical tracer [perfluoro(methylcyclohexane)] and a particulate tracer (0.5-μm-diameter fluorescent microspheres) were conducted during coring with the advanced hydraulic piston core in unconsolidated sediments and with the rotary core barrel and diamond core barrel in igneous rock. Detection of both types of tracers on the exterior of recovered cores confirmed successful delivery. The particulate tracer was never detected in the interior of unconsolidated sediment (n = 24). The average concentration of the chemical tracer (n = 12) was equivalent to 0.35μL of drilling fluid per gram of sediment. The particulate tracer was not found in the interiors of igneous rock samples that were crushed (n = 4) but was found in the interiors of 64% of the thin sections examined (n = 12), indicating that the samples were contaminated during sectioning rather than drilling. Perfluorocarbon data indicate that drilling fluid in the igneous rock samples averaged 0.01 μL g[sup -1] rock. Based on the abundance of bacteria in the surface seawater (4.2 10[sup 8]L[sup -1]), which was used as the drilling fluid, the potential contamination of both sample types is on the order of a few bacteria per gram of cored material. This estimate is conservative, given the high pervasiveness of the perfluorocarbon tracer relative to microbes. [ABSTRACT FROM AUTHOR]

Groundwater samples from 200- to 950-m depths in four igneous rock sites in Finland were investigated for different metabolic groups of microorganisms, and the data were compared with the available geochemical record. Samples were collected with a pressurized groundwater sampling system developed for gas and microbiological sampling. Two of the sites had groundwater that was fresh, with < 0.2 g/l dissolved solids, whereas that at the two other sites was much more saline, reaching a maximum of 24 g/l dissolved solids. The groundwater contained gases, 33 to 340 ml/l, with nitrogen or methane dominating. Total cell numbers were 10 5 to 10 6 cells/ml, which is typical for deep igneous rock aquifers. Growth media were designed to mimic the actual groundwater chemistry at each sampling point and used for most probable number enumeration of methanogens, acetogens, sulfate-reducing bacteria (SRB), and iron-reducing bacteria (IRB). SRB predominated in sites where iron sulfide fracture-filling minerals are common. IRB were the main population in one site where iron sulfide fracture minerals are not present, but iron hydroxide fracture minerals predominate. Fracture-filling minerals were a better indicator of microbial populations than was groundwater chemistry. Low numbers of autotrophic methanogens were cultured. One of several possible interpretations of stable isotope data suggested that most of the detected methane is thermogenic, which would correlate with few active methanogens. However, we concluded other interpretations were also possible. [ABSTRACT FROM AUTHOR]