Sulfate-reducing bacteria (SRB) are anaerobic prokaryotes found ubiquitously in nature. SRB were the first nonphotosynthetic, anaerobic bacteria shown to generate energy (ATP) through electron transfer-coupled phosphorylation. For this process, the SRB typically use sulfate as the terminal electron acceptor for respiration of hydrogen or various organic acids, which results in the production of sulfide, a highly reactive and toxic end-product. Beyond their obvious function in the sulfur cycle, SRB play an important role in global cycling of numerous other elements 1 . For example, in the carbon cycle, the SRB form part of microbial consortia that completely mineralize organic carbon in anaerobic environments; polymeric materials (e.g., cellulose) are first depolymerized and metabolized by fermentative microorganisms, and the resulting organic acid and reduced gas (that is, CO and H 2 ) end-products are further fermented or oxidized by other microbes, including SRB. The latter are particularly active in sulfate-rich (e.g., marine) environments, where they effectively link the global sulfur and carbon cycles 1,2 .Beyond these ecological roles, SRB also have a major economic impact because of their involvement in biocorrosion of ferrous metals in anaerobic environments 3 , described as "industrial venereal disease-it's expensive, everybody has it, and nobody wants to talk about it" 4 . For example, because SRB are abundant in oil fields, their metabolism has many negative consequences for the petroleum industry (e.g., corrosion of drilling and pumping machinery and storage tanks, souring of oil by sulfide production, plugging of machinery and rock pores with biomass and sulfide precipitates). The SRB also contribute to bioremediation of toxic metal ions 5,6 . Their metabolism increases the pH, causing toxic metal ions like copper (II), nickel (II) and cadmium (II) to precipitate as metal sulfides in acidic aquatic environments (e.g., mine effluents). Additionally, SRB can deliver electrons directly to oxidized toxic metal ions, including uranium (VI), technetium (VII), and chromium (VI), converting these into less soluble, reduced forms. Hence, SRB-mediated reduction represents a potentially useful mechanism for the bioremediation of metal ion contaminants from anaerobic sediments 6 .Most research on the metabolism and biochemistry of SRB has been done on the genus Desulfovibrio, a member of the δ-proteobacteria The genome sequence of the anaerobic, sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough Desulfovibrio vulgaris Hildenborough is a model organism for studying the energy metabolism of sulfate-reducing bacteria (SRB) and for understanding the economic impacts of SRB, including biocorrosion of metal infrastructure and bioremediation of toxic metal ions. The 3,570,858 base pair (bp) genome sequence reveals a network of novel c-type cytochromes, connecting multiple periplasmic hydrogenases and formate dehydrogenases, as a key feature of its energy metabolism. The relative arrangement of genes encod...

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.Sulfate-reducing bacteria are frequently exposed to nitrite by interaction with nitrate-reducing, sulfide-oxidizing bacteria in anoxic environments. Because sulfate is often the predominant electron acceptor in anoxic environments (e.g., marine sediments), sulfate-reducing bacteria are primarily responsible for organic carbon oxidation. The sulfide produced is then targeted by nitrate-reducing, sulfide-oxidizing bacteria, which generally use CO 2 as their sole carbon source (4). Thus, sulfate-reducing bacteria and nitrate-reducing, sulfide-oxidizing bacteria symbiotically catalyze the oxidation of organic matter with nitrate through a sulfide intermediate. This symbiosis can potentially be stalled by production of nitrite by the nitratereducing, sulfide-oxidizing bacteria, which is a powerful inhibitor of sulfate-reducing bacteria. Some sulfate-reducing bacteria have a periplasmic nitrite reductase to prevent and/or overcome this inhibition. Cocultures of a Desulfovibrio sp. and the nitrate-reducing, sulfide-oxidizing bacterium Thiomicrospira sp. strain CVO were strongly or transiently inhibited, depending on the absence or presence of nitrite reductase activity in the Desulfovibrio sp. (6). Desulfovibrio vulgaris Hildenborough was very resistant to inhibition by either nitrite or strain CVO and nitrate and had high nitrite reductase activity through the presence of a periplasmic cytochrome c nitrite reductase (NrfHA) that reduces nitrite to ammonium. This reaction is purely detoxifying; no cell growth is associated with t...

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.The role of the cytoplasmic membrane in the conservation of chemiosmotic energy is fairly well understood for aerobic bacteria, because of similarities to the mitochondrial inner membrane paradigm. However, this role is much less clear for anaerobes. For instance, chemiosmotic energy conservation is crucial for sulfate-reducing bacteria (SRB), when growing with lactate as the electron donor, because the ATP yield of substrate-level phosphorylation exactly balances what is required for sulfate activation (19,21,33). Odom and Peck proposed that SRB of the genus Desulfovibrio conserve chemiosmotic energy by hydrogen cycling (17). In their model, hydrogen generated from cytoplasmic lactate oxidation was proposed to diffuse across the cytoplasmic membrane, where it is oxidized by periplasmic hydrogenase to form a proton gradient with the electrons being conducted back to the cytoplasmic sulfate reduction pathway. Analysis of the genome sequence for Desulfovibrio vulgaris Hildenborough (http://www.tigr.org) has indicated the presence of two cytoplasm-facing, membrane-bound hydrogenases, four periplasmic hydrogenases, and a variety of transmembrane electron-transporting complexes. Hence, all components required for a hydrogen cycling mechanism of energy conservation are ...

INTRODUCTIONThe Ocean Drilling Program (ODP) is committed to deep-biosphere research and has constructed a new microbiological laboratory on board the JOIDES Resolution. The use of the JOIDES Resolution as a platform for deep-biosphere research requires that the recovered cores are suitable for microbiological study. The major concern is whether microbes from the drilling fluid are introduced into the recovered core material during coring. Therefore, it is critical to verify whether recovered cores are contaminated. Here we present details of two tracer methods used to quantify the amount of contamination. These methods were modified from land-based drilling operations for use on the JOIDES Resolution (see review by Griffin et al., 1997). Tracer experiments were first conducted during ODP Leg 185 (Plank, Ludden, Escutia, et al., in press) and involve the delivery of both chemical and particulate tracers during drilling and their quantification in the ODP cores. These tracers were introduced while drilling unconsolidated sediments using the advanced hydraulic piston corer (APC), sedimentary rock using the extended core barrel and rotary core barrel (RCB), and igneous rock using the RCB and diamond core barrel. This technical note presents details on the characteristics, preparation, and delivery of the tracers and their quantification in cores. Suggestions are made regarding sample handling with the goal of minimizing sample contamination. It is strongly recommended that these contamination tests be routinely conducted when coring for microbiological studies. D.C. SMITH ET AL. TRACER TESTS FOR MICROBIOLOGICAL STUDIES 2 MATERIALS AND METHODS Chemical Tracer: Perfluorocarbon CharacteristicsPerfluorocarbon tracers (PFT) have been used extensively in landbased drilling applications (Senum and Dietz, 1991;Russell et al. 1992;McKinley and Colwell, 1996) because they are inert and can be detected with high sensitivity. Perfluoro(methylcyclohexane) is the tracer that has been tested on the JOIDES Resolution. This perfluorocarbon (Aldrich 30293-7) has a molecular weight of 350.05, a boiling point of 76°C, and a density of 1.76 g/mL. Its solubility is ~1 mg/L in water and is 10 g/L in methanol (Colwell et al., 1992). The low solubility in water facilitates gas phase partitioning and quantitative headspace analysis. Preparation and DeliveryThe stock PFT is shipped in sealed ampoules, and it is not necessary to dilute it prior to use. Because the PFT is volatile and can be detected at extremely low concentrations, it is necessary to open the ampoules and transfer the PFT to the carboy used for delivery in a ventilated area well away from the core handling and PFT detection areas. Gloves should be worn during this process and discarded afterward. It is recommended that this transfer be performed on the helicopter deck while the JOIDES Resolution is under way and that all materials that may have been in contact with the PFT be disposed of immediately. These precautions will minimize the probability of obtaining false positi...

Although Pelobacter species are closely related to Geobacter species, recent studies suggested that Pelobacter carbinolicus may reduce Fe(III) via a different mechanism because it lacks the outer-surface c-type cytochromes that are required for Fe(III) reduction by Geobacter sulfurreducens. Investigation into the mechanisms for Fe(III) reduction demonstrated that P. carbinolicus had growth yields on both soluble and insoluble Fe(III) consistent with those of other Fe(III)-reducing bacteria. Comparison of whole-genome transcript levels during growth on Fe(III) versus fermentative growth demonstrated that the greatest apparent change in gene expression was an increase in transcript levels for four contiguous genes. These genes encode two putative periplasmic thioredoxins; a putative outer-membrane transport protein; and a putative NAD(FAD)-dependent dehydrogenase with homology to disulfide oxidoreductases in the N terminus, rhodanese (sulfurtransferase) in the center, and uncharacterized conserved proteins in the C terminus. Unlike G. sulfurreducens, transcript levels for cytochrome genes did not increase in P. carbinolicus during growth on Fe(III). P. carbinolicus could use sulfate as the sole source of sulfur during fermentative growth, but required elemental sulfur or sulfide for growth on Fe(III). The increased expression of genes potentially involved in sulfur reduction, coupled with the requirement for sulfur or sulfide during growth on Fe(III), suggests that P. carbinolicus reduces Fe(III) via an indirect mechanism in which (i) elemental sulfur is reduced to sulfide and (ii) the sulfide reduces Fe(III) with the regeneration of elemental sulfur. This contrasts with the direct reduction of Fe(III) that has been proposed for Geobacter species.

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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 acetoinfermenting 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 cells 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.

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.