Microarray and functional gene analyses of sulfate-reducing prokaryotes in low-sulfate, acidic fens reveal cooccurrence of recognized genera and novel lineages

Low-sulfate, acidic (approximately pH 4) fens in the Lehstenbach catchment in the Fichtelgebirge mountains in Germany are unusual habitats for sulfate-reducing prokaryotes (SRPs) that have been postulated to facilitate the retention of sulfur and protons in these ecosystems. Despite the low in situ availability of sulfate (concentration in the soil solution, 20 to 200 μM) and the acidic conditions (soil and soil solution pHs, approximately 4 and 5, respectively), the upper peat layers of the soils from two fens (Schlöppnerbrunnen I and II) of this catchment displayed significant sulfate-reducing capacities. 16S rRNA gene-based oligonucleotide microarray analyses revealed stable diversity patterns for recognized SRPs in the upper 30 cm of both fens. Members of the family “Syntrophobacteraceae” were detected in both fens, while signals specific for the genus Desulfomonile were observed only in soils from Schlöppnerbrunnen I. These results were confirmed and extended by comparative analyses of environmentally retrieved 16S rRNA and dissimilatory (bi)sulfite reductase (dsrAB) gene sequences; dsrAB sequences from Desulfobacca-like SRPs, which were not identified by microarray analysis, were obtained from both fens. Hypotheses concerning the ecophysiological role of these three SRP groups in the fens were formulated based on the known physiological properties of their cultured relatives. In addition to these recognized SRP lineages, six novel dsrAB types that were phylogenetically unrelated to all known SRPs were detected in the fens. These dsrAB sequences had no features indicative of pseudogenes and likely represent novel, deeply branching, sulfate- or sulfite-reducing prokaryotes that are specialized colonists of low-sulfate habitats.The dissimilatory reduction of sulfate is carried out exclusively by prokaryotic organisms and is one of the most important mineralization processes in anoxic aquatic environments, especially marine sediments (29, 30). In contrast to well-studied sulfate-reducing communities in marine (18, 19, 38, 41, 53, 56, 57, 72) and freshwater habitats (39, 40, 59, 60), relatively little is known about the distribution, diversity, and in situ activities of sulfate-reducing prokaryotes (SRPs) in terrestrial ecosystems. The contribution of terrestrial SRPs to the overall turnover of organic matter is likely of minor importance on a global scale. However, SRPs contribute to the biodegradation of pollutants in soils and subsurface environments (1, 15, 49, 71) and are important to the geomicrobiology of specialized terrestrial habitats that are subject to flooding, such as rice fields (68, 76, 77) and fens (3, 5).δ³⁴S values and ³⁵S-labeling patterns indicate that the dissimilatory reduction of sulfate is an ongoing process in the acidic fens of a forested catchment in northern Bavaria, Germany (Lehstenbach, Fichtelgebirge) (3, 5). The deposition of sulfur that originated from the combustion of soft coal in Eastern Europe (10) led to accumulation of sulfur in the soils of this catchment (4). Although pollution controls have lessened the deposition in recent years, desorption of sulfate in aerated upland soils causes sulfate to enter fens at lower elevations. It was hypothesized that the dissimilatory reduction of sulfate in these mainly anoxic, waterlogged acidic fen soils (the pH of the fen soils is approximately 4) contributes to the retention of sulfur in this ecosystem (3, 4, 50). The reduction of sulfate in these fens is also a sink for protons and thus decreases the acidity of the soil solution and groundwater of this habitat.The acidity and low sulfate content of some of the fens in the Lehstenbach catchment provide an unusual habitat for SRPs, and the occurrence and activity of these organisms in such habitats have received little attention. The main objectives of this study were (i) to assess the capacity of the fen soils to reduce sulfate along vertical soil profiles in the upper peat layers, (ii) to determine the vertical community profiles for all known SRP lineages that inhabit the fens by the use of a 16S rRNA-based oligonucleotide microarray (SRP-PhyloChip) (44), (iii) to resolve the possible existence of novel SRP lineages in the fens by retrieval of dsrAB, which are genes that encode the alpha and beta subunits of the siroheme dissimilatory (bi)sulfite reductase (EC 1.8.99.3) (34, 66, 74), and (iv) to deduce the possible in situ functional relationships that can be inferred from this collective information.     

Role of Plant Residues in Determining Temporal Patterns of the Activity,Size, and Structure of Nitrate Reducer Communities in Soil

The incorporation of plant residues into soil not only represents an opportunity to limit soil organic matter depletion resulting from cultivation but also provides a valuable source of nutrients such as nitrogen. However, the consequences of plant residue addition on soil microbial communities involved in biochemical cycles other than the carbon cycle are poorly understood. In this study, we investigated the responses of one N-cycling microbial community, the nitrate reducers, to wheat, rape, and alfalfa residues for 11 months after incorporation into soil in a field experiment. A 20- to 27-fold increase in potential nitrate reduction activity was observed for residue-amended plots compared to the nonamended plots during the first week. This stimulating effect of residues on the activity of the nitrate-reducing community rapidly decreased but remained significant over 11 months. During this period, our results suggest that the potential nitrate reduction activity was regulated by both carbon availability and temperature. The presence of residues also had a significant effect on the abundance of nitrate reducers estimated by quantitative PCR of the narG and napA genes, encoding the membrane-bound and periplasmic nitrate reductases, respectively. In contrast, the incorporation of the plant residues into soil had little impact on the structure of the narG and napA nitrate-reducing community determined by PCR-restriction fragment length polymorphism (RFLP) fingerprinting. Overall, our results revealed that the addition of plant residues can lead to important long-term changes in the activity and size of a microbial community involved in N cycling but with limited effects of the type of plant residue itself.Modern agricultural practices include a return of plant residues to soil, as this is considered sustainable to the environment. It is now recognized that the conversion of native land into cultivated systems leads to carbon losses, which can be up to 20 to 40% (17). Postharvest plant residues therefore represent an important source of carbon, helping to replenish soil organic matter that decomposes as a result of cultivation. Decomposing plant residues are also a source of nutrients, such as nitrogen, with reduced nitrate leaching compared to mineral fertilizers, which is beneficial for water quality (3). In addition, leaving the plant residue on the soil surface limits water losses by evaporation and prevents soil erosion by wind or water (15).The biochemical composition of plant residues is one of the most important factors influencing their decomposition in soil (14, 28, 29, 51). Indeed, Manzoni et al. (28), using a data set of 2,800 observations, showed previously that the patterns of decomposition were regulated by the initial residue stoichiometry. Several other factors such as climatic conditions, soil type, or localization of the residue in the soil (incorporated or on the soil surface) were also reported previously to influence decomposition (2, 24, 29, 44). Microorganisms are the major decomposers of organic matter in soil, and therefore, the diversity and activity of the microbial community during plant residue decomposition has received much attention (6, 23, 26, 27, 35). It was shown previously that the biochemical composition of plant residues influences microbial respiration (8) and microbial community structure (7, 37). The recent development of carbon-labeling approaches has furthered our knowledge of the microorganisms that actively assimilate the carbon derived from various plant residues (10, 31). However, most of those studies focused on microorganisms involved in C mineralization, and in contrast, very little is known about the effect of plant residue decomposition on the microbial communities involved in biochemical cycles other than the carbon cycle. Thus, despite the influence of plant residues on nitrogen cycling (1, 4, 5, 16, 20), studies assessing the effect of the presence and composition of plant residues on the ecology of microbial communities involved in nitrogen cycling are rare (21, 32, 36).The dissimilatory reduction of nitrate into nitrite is the first step in the processes of denitrification and the dissimilatory reduction of nitrate to ammonium (33, 41). The reduction of nitrate by denitrification leads to losses of nitrogen, which is often a limiting nutrient for plant growth in agriculture. Two types of dissimilatory nitrate reductases, differing in location, have been characterized: a membrane-bound nitrate reductase (Nar) and a periplasmic nitrate reductase (Nap) (9, 53). Nitrate reducers can harbor either Nar, Nap, or both (40, 47). Nitrate reducers are probably the most taxonomically diverse functional community within the nitrogen cycle, with members in most bacterial phyla and also archaea (42). Because of this high level of diversity of heterotrophs sharing the ability to produce energy from nitrate reduction, nitrate reducers are an excellent model system to investigate the response of the N-cycling community to plant residue addition.The aim of this work was to determine how the incorporation of plant residues with contrasting biochemical compositions into soil affects the nitrate-reducing community. For this purpose, we monitored the dynamics of the potential activity, size, and structure of the nitrate-reducing community after the addition of wheat, rape, or alfalfa residues to soil in a field experiment. As the nature and availability of the substrate change during residue decomposition (38, 39, 48), the influence of the incorporation of different plant residues on the nitrate-reducing community was investigated at several sampling times for 11 months.     

Functional diversity and electron donor dependence of microbial populations capable of U(VI) reduction in radionuclide-contaminated subsurface sediments

In order to elucidate the potential mechanisms of U(VI) reduction for the optimization of bioremediation strategies, the structure-function relationships of microbial communities were investigated in microcosms of subsurface materials cocontaminated with radionuclides and nitrate. A polyphasic approach was used to assess the functional diversity of microbial populations likely to catalyze electron flow under conditions proposed for in situ uranium bioremediation. The addition of ethanol and glucose as supplemental electron donors stimulated microbial nitrate and Fe(III) reduction as the predominant terminal electron-accepting processes (TEAPs). U(VI), Fe(III), and sulfate reduction overlapped in the glucose treatment, whereas U(VI) reduction was concurrent with sulfate reduction but preceded Fe(III) reduction in the ethanol treatments. Phyllosilicate clays were shown to be the major source of Fe(III) for microbial respiration by using variable-temperature Mössbauer spectroscopy. Nitrate- and Fe(III)-reducing bacteria (FeRB) were abundant throughout the shifts in TEAPs observed in biostimulated microcosms and were affiliated with the genera Geobacter, Tolumonas, Clostridium, Arthrobacter, Dechloromonas, and Pseudomonas. Up to two orders of magnitude higher counts of FeRB and enhanced U(VI) removal were observed in ethanol-amended treatments compared to the results in glucose-amended treatments. Quantification of citrate synthase (gltA) levels demonstrated a stimulation of Geobacteraceae activity during metal reduction in carbon-amended microcosms, with the highest expression observed in the glucose treatment. Phylogenetic analysis indicated that the active FeRB share high sequence identity with Geobacteraceae members cultivated from contaminated subsurface environments. Our results show that the functional diversity of populations capable of U(VI) reduction is dependent upon the choice of electron donor.Uranium contamination in subsurface environments is a widespread problem at mining and milling sites across North America, South America, and Eastern Europe (1). Uranium in the oxidized state, U(VI), is highly soluble and toxic and thus is a potential contaminant to local drinking-water supplies (46). Nitrate is often a cocontaminant with U(VI) as a result of the use of nitric acid in the processing of uranium and uranium-bearing waste (6, 45). Oxidized uranium can be immobilized in contaminated groundwater through the reduction of U(VI) to insoluble U(IV) by indirect (abiotic) and direct (enzymatic) processes catalyzed by microorganisms. Current remediation practices favor the stimulation of reductive uranium immobilization catalyzed by indigenous microbial communities along with natural attenuation and monitoring (5, 24, 40, 44, 65, 68, 69). Microbial uranium reduction activity in contaminated subsurface environments is often limited by carbon or electron donor availability (13, 24, 44, 69). Previous studies have indicated that U(VI) reduction does not proceed until nitrate is depleted (13, 16, 24, 44, 68, 69), as high nitrate concentrations inhibit the reduction of U(VI) by serving as a competing and more energetically favorable terminal electron acceptor for microorganisms (11, 16). The fate and transport of uranium in groundwater are also strongly linked through sorption and precipitation processes to the bioreduction of Fe minerals, including oxides, layer-silicate clay minerals, and sulfides (7, 23, 53).In order to appropriately design U(VI) bioremediation strategies, the potential function and phylogenetic structure of indigenous subsurface microbial communities must be further understood (24, 34, 46). Conflicting evidence has been presented on which microbial groups, Fe(III)- or sulfate-reducing bacteria (FeRB or SRB), effectively catalyze the reductive immobilization of U(VI) in the presence of amended electron donors (5, 44, 69). The addition of acetate to the subsurface at a uranium-contaminated site in Rifle, Colorado, initially stimulated FeRB within the family Geobacteraceae to reduce U(VI) (5, 65). However, with long-term acetate addition, SRB within the family Desulfobacteraceae, which are not capable of U(VI) reduction, increased in abundance and a concomitant reoxidation of U(IV) was observed (5, 65). At a uranium-contaminated site in Oak Ridge, Tennessee, in situ and laboratory-based experiments successfully employed ethanol amendments to stimulate denitrification followed by the reduction of U(VI) by indigenous microbial communities (13, 24, 44, 48, 50, 57, 68). In these studies, ethanol amendments stimulated both SRB and FeRB, with SRB likely catalyzing the reduction of U(VI). This suggests that the potential for bioremediation will be affected by the choice of electron donor amendment through effects on the functional diversity of U(VI)-reducing microbial populations. As uranium reduction is dependent on the depletion of nitrate, the microbial populations mediating nitrate reduction are also critical to the design of bioremediation strategies. Although nitrate-reducing bacteria (NRB) have been studied extensively in subsurface environments (2, 15, 19, 24, 56, 58, 70), the mechanisms controlling the in situ metabolism of NRB remain poorly understood.The dynamics of microbial populations capable of U(VI) reduction in subsurface sediments are poorly understood, and the differences in the microbial community dynamics during bioremediation have not been explored. Based on the results of previous studies (13, 44, 49, 57, 68, 69), we hypothesized that the activity of nitrate- and Fe(III)-reducing microbial populations, catalyzing the reductive immobilization of U(VI) in subsurface radionuclide-contaminated sediments, would be dependent on the choice of electron donor. The objectives of the present study were (i) to characterize structure-function relationships for microbial groups likely to catalyze or limit U(VI) reduction in radionuclide-contaminated sediments and (ii) to further develop a proxy for the metabolic activity of FeRB. Microbial activity was assessed by monitoring terminal electron-accepting processes (TEAPs), electron donor utilization, and Fe(III) mineral transformations in microcosms conducted with subsurface materials cocontaminated with high levels of U(VI) and nitrate. In parallel, microbial functional groups (i.e., NRB and FeRB) were enumerated and characterized using a combination of cultivation-dependent and -independent methods.     

Diversity of Dissimilatory Sulfite Reductase Genes (dsrAB) in a Salt Marsh Impacted by Long-Term Acid Mine Drainage

Sulfate-reducing bacteria (SRB) play a major role in the coupled biogeochemical cycling of sulfur and chalcophilic metal(loid)s. By implication, they can exert a strong influence on the speciation and mobility of multiple metal(loid) contaminants. In this study, we combined DsrAB gene sequencing and sulfur isotopic profiling to identify the phylogeny and distribution of SRB and to assess their metabolic activity in salt marsh sediments exposed to acid mine drainage (AMD) for over 100 years. Recovered dsrAB sequences from three sites sampled along an AMD flow path indicated the dominance of a single Desulfovibrio species. Other major sequence clades were related most closely to Desulfosarcina, Desulfococcus, Desulfobulbus, and Desulfosporosinus species. The presence of metal sulfides with low δ³⁴S values relative to δ³⁴S values of pore water sulfate showed that sediment SRB populations were actively reducing sulfate under ambient conditions (pH of ∼2), although possibly within less acidic microenvironments. Interestingly, δ³⁴S values for pore water sulfate were lower than those for sulfate delivered during tidal inundation of marsh sediments. 16S rRNA gene sequence data from sediments and sulfur isotope data confirmed that sulfur-oxidizing bacteria drove the reoxidation of biogenic sulfide coupled to oxygen or nitrate reduction over a timescale of hours. Collectively, these findings imply a highly dynamic microbially mediated cycling of sulfate and sulfide, and thus the speciation and mobility of chalcophilic contaminant metal(loid)s, in AMD-impacted marsh sediments.Salt marshes exhibit high primary production rates (1, 101) and form biogeochemical “transition zones” for nutrient production, transport, and cycling between terrestrial and coastal marine environments (41, 66, 100). These zones also serve to reduce the flux of potentially toxic metals in contaminated groundwater to estuaries (12, 99, 106). Both functions depend strongly on microbial activity, especially that of sulfate-reducing bacteria (SRB) (42, 62, 67). SRB recycle much of the sedimentary organic carbon pool in marsh sediments (42-44) and indirectly inhibit production of the greenhouse gas methane (37, 71). They can restrict the mobility of dissolved contaminant metals by inducing precipitation of poorly soluble metal sulfides, and studies have examined their use in constructed wetlands to bioremediate acid mine drainage (AMD) and other metalliferous waste streams (11, 35, 40, 46, 50, 76, 90, 94, 104). However, the high acidity and metal concentrations inherent to AMD can inhibit SRB growth (15, 88, 98), and preferential growth of iron- and sulfur-oxidizing bacteria over SRB has been observed in some treatment wetlands (39).For natural salt marshes, 16S ribosomal nucleic acid- and phospholipid fatty acid (PLFA)-based analyses have shown that SRB commonly comprise a significant fraction of the microbial community (13, 24, 31, 34, 51, 58). Studies of salt marsh dissimilatory sulfite reductase genes (dsrAB), a highly conserved functional phylogenetic marker of prokaryotic sulfate reducers (49, 57, 102, 103, 107), have revealed both novel and deeply branching clades (3). Studies of mining-impacted sites at pH 2.0 to 7.8 (5, 7, 39, 70, 72, 77, 84), of soils and geothermal settings at a pH of ∼4 (55, 68), of metal-contaminated estuaries at pH 6.8 to 7.2 (65), and of hypersaline lakes at pH 7.5 (56) further outline the distribution and tolerance of specific groups and species of SRB under geochemically stringent conditions. Other findings point toward the existence of deltaproteobacteria in environments at a pH of ∼1 (10), although it is unknown if these include SRB. SRB diversity in salt marshes under long-term contamination by AMD has not been well investigated. Such studies may provide useful information for bioremediation projects in estuarine environments, as well as general insights into relationships between SRB physiology and the geochemistry of AMD.We studied the diversity of SRB, based on phylogenetic analysis of recovered DsrAB gene sequences (∼1.9 kb), in natural salt marsh sediments of the San Francisco Bay impacted by AMD for over 100 years. Sulfur isotope ratio and concentration measurements of pore water sulfate and metal sulfide minerals provided information about the spatial and temporal extent of active bacterial sulfate reduction (BSR) in sediment cores taken from specific sites along an AMD flow path. Collectively, the results revealed a tidal marsh system characterized by rapidly cycling bacterial sulfate reduction and sulfide reoxidation associated with oscillating tidal inundation and groundwater infiltration.     

Differential Responses of Nitrate Reducer Community Size,Structure, and Activity to Tillage Systems

The main objective of this study was to determine how the size, structure, and activity of the nitrate reducer community were affected by adoption of a conservative tillage system as an alternative to conventional tillage. The experimental field, established in Madagascar in 1991, consists of plots subjected to conventional tillage or direct-seeding mulch-based cropping systems (DM), both amended with three different fertilization regimes. Comparisons of size, structure, and activity of the nitrate reducer community in samples collected from the top layer in 2005 and 2006 revealed that all characteristics of this functional community were affected by the tillage system, with increased nitrate reduction activity and numbers of nitrate reducers under DM. Nitrate reduction activity was also stimulated by combined organic and mineral fertilization but not by organic fertilization alone. In contrast, both negative and positive effects of combined organic and mineral fertilization on the size of the nitrate reducer community were observed. The size of the nitrate reducer community was a significant predictor of the nitrate reduction rates except in one treatment, which highlighted the inherent complexities in understanding the relationships the between size, diversity, and structure of functional microbial communities along environmental gradients.The transition from intensive tillage to various forms of conservation tillage began more than 50 years ago with the development of herbicides which have replaced mechanical cultivation. Since then, the principles of no-till cropping have been extensively adopted by farmers worldwide. This cropping system, also known as direct seeding, mimics natural systems by leaving the soil mostly undisturbed and permanently covered with crop residues or living plants. The benefits of reducing tillage in sustainable agriculture are now well recognized for various environmental and economic reasons (14). Leaving all residues of the previous crop on the soil surface protects against evaporative water loss, wind erosion, and surface water runoff. Concomitant with reduced erosion, no-till cropping can also result in enhanced soil carbon storage in the topsoil layer, with estimated carbon sequestration rates of 30 to 60 g C m² year⁻¹ (27, 50). In turn, these changes in soil organic matter and soil structure under a no-till cropping system can affect microbial communities (20). Thus, the microbial biomass is most often higher in no-till systems than in conventional tillage systems (11, 26). Analysis of the structure or activity of soil microbial communities has also revealed significant differences between conventional tillage and minimal tillage or no-tillage systems (25, 29). However, although the effect of tillage practices on the total soil microbial community in relation to soil organic matter management has frequently been investigated, knowledge of the changes in N-cycling microbial communities induced by no-till management is limited and is mainly focused on N process rates (3, 11, 32).The aim of this work was to determine how conversion from conventional tillage to no-till affects microorganisms involved in the N cycle. For this purpose, we used the nitrate reducing community as a model functional guild (40). Prokaryote nitrate reducers constitute a wide taxonomic group with a shared ability to produce energy from the dissimilatory reduction of nitrate to nitrite, the first step of denitrification and of the dissimilatory processes of reduction of nitrate to ammonium (39). Nitrate reduction by denitrification is of great importance, since the resulting nitrite is then reduced to N₂O or N₂ gases, which can lead to considerable nitrogen losses in agriculture and emissions of the N₂O greenhouse gas (4, 13). We hypothesized that higher C and N contents in the no-till system will result in increased nitrate reduction rates and nitrate reducer abundance combined with shifts in the community composition. Relationships between the size, activity, and structure of the nitrate reducer community in the studied cropping systems were also investigated. The structure and size of the nitrate reducer community were assessed by fingerprinting and real-time PCR using the narG and napA genes, encoding the membrane-bound and periplasmic nitrate reductases, respectively, as molecular markers (40, 41). The potential activity of the nitrate reducing community was determined by colorimetric measurement of the nitrite produced during nitrate reduction.     

Thioredoxin Is Involved in U(VI) and Cr(VI) Reduction in Desulfovibrio desulfuricans G20

A transposon insertion mutant has been identified in a Desulfovibrio desulfuricans G20 mutant library that does not grow in the presence of 2 mM U(VI) in lactate-sulfate medium. This mutant has also been shown to be deficient in the ability to grow with 100 μM Cr(VI) and 20 mM As(V). Experiments with washed cells showed that this mutant had lost the ability to reduce U(VI) or Cr(VI), providing an explanation for the lower tolerance. A gene encoding a cyclic AMP (cAMP) receptor protein (CRP) was identified as the site of the transposon insertion. The remainder of the mre operon (metal reduction) contains genes encoding a thioredoxin, thioredoxin reductase, and an additional oxidoreductase whose substrate has not been predicted. Expression studies showed that in the mutant, the entire operon is downregulated, suggesting that the CRP may be involved in regulating expression of the whole operon. Exposure of the cells to U(VI) resulted in upregulation of the entire operon. CdCl₂, a specific inhibitor of thioredoxin activity, inhibits U(VI) reduction by washed cells and inhibits growth of cells in culture when U(VI) is present, confirming a role for thioredoxin in U(VI) reduction. The entire mre operon was cloned into Escherichia coli JM109 and the transformant developed increased U(VI) resistance and the ability to reduce U(VI) to U(IV). The oxidoreductase protein (MreG) from this operon was expressed and purified from E. coli. In the presence of thioredoxin, thioredoxin reductase, and NADPH, this protein was shown to reduce both U(VI) and Cr(VI), providing a mechanism for the cytoplasmic reduction of these metals.Previous studies have shown that soluble U(VI) can be reduced to the less-soluble U(IV) by pure cultures of bacteria (19, 20, 25). This process can be useful for in situ reduction, which results in uranium precipitation and therefore decreased mobility in groundwater (8, 33). Desulfovibrio desulfuricans G20 and Desfulovibrio vulgaris, neither of which can use U(VI) as a respiratory electron acceptor, have been shown to directly reduce U(VI) (19, 24), and the mechanism for U(VI) reduction has been addressed. A purified hydrogenase and periplasmic cytochrome c₃ from cell extracts of D. vulgaris will reduce U(VI) to U(IV) with hydrogen as the electron donor (19), suggesting that cytochrome c₃ of D. vulgaris may be directly involved in U(VI) reduction. When a cytochrome c₃ mutant of D. desulfuricans G20 was generated, it would not reduce U(VI) with H₂ as the electron donor (25); however, growth and U(VI) reduction occurred with lactate as the electron donor, although at lower rates than the wild type. Cytochrome c₃ was also found to be bound to insoluble U(IV), providing further evidence that this protein may be involved in U(VI) reduction (24). Electron microscopic images showed that reduced U(IV) was not only present in the periplasm but also in the cytoplasm (28), indicating that the periplasmic cytochrome c₃ may be only partially responsible for the in vivo U(VI) reduction process, with an additional pathway in the cytoplasm.In order to identify this additional mechanism, transposon insertion mutants were generated. This mutant library has also been used to identify genes involved in sediment fitness (10, 21) and syntrophic growth (16). In this study, the mutants were screened for loss of U(VI) resistance. A mutant was identified that was sensitive to U(VI) and would not grow with 2 mM U(VI) or reduce it in suspensions of washed cells. This was the only mutant identified that would not reduce U(VI) in both tests. The disrupted operon (named mre, for metal reduction) was characterized, and it is shown here that the mechanism for the U(VI) reduction process involves at least three genes, including thioredoxin, thioredoxin reductase, and an additional metal oxidoreductase. Some or all of these components are likely also responsible for Cr(VI) and As(V) reduction by this organism.     

Microorganisms with novel dissimilatory (bi)sulfite reductase genes are widespread and part of the core microbiota in low-sulfate peatlands

Peatlands of the Lehstenbach catchment (Germany) house as-yet-unidentified microorganisms with phylogenetically novel variants of the dissimilatory (bi)sulfite reductase genes dsrAB. These genes are characteristic of microorganisms that reduce sulfate, sulfite, or some organosulfonates for energy conservation but can also be present in anaerobic syntrophs. However, nothing is currently known regarding the abundance, community dynamics, and biogeography of these dsrAB-carrying microorganisms in peatlands. To tackle these issues, soils from a Lehstenbach catchment site (Schlöppnerbrunnen II fen) from different depths were sampled at three time points over a 6-year period to analyze the diversity and distribution of dsrAB-containing microorganisms by a newly developed functional gene microarray and quantitative PCR assays. Members of novel, uncultivated dsrAB lineages (approximately representing species-level groups) (i) dominated a temporally stable but spatially structured dsrAB community and (ii) represented “core” members (up to 1% to 1.7% relative abundance) of the autochthonous microbial community in this fen. In addition, denaturing gradient gel electrophoresis (DGGE)- and clone library-based comparisons of the dsrAB diversity in soils from a wet meadow, three bogs, and five fens of various geographic locations (distance of ∼1 to 400 km) identified that one Syntrophobacter-related and nine novel dsrAB lineages are widespread in low-sulfate peatlands. Signatures of biogeography in dsrB-based DGGE data were not correlated with geographic distance but could be explained largely by soil pH and wetland type, implying that the distribution of dsrAB-carrying microorganisms in wetlands on the scale of a few hundred kilometers is not limited by dispersal but determined by local environmental conditions.Peatlands contain 15% to 30% of the global soil carbon (13, 79) and represent a net carbon sink that has contributed to global cooling in the past 8,000 to 11,000 years (21). While peatlands are generally resilient to external perturbation, it is predicted that long-term global changes such as warming, decreased precipitation, and increased atmospheric deposition of reactive nitrogen and sulfur compounds will transform peatlands into new ecosystem types, accompanied by unforeseeable changes in the carbon balance (17). The carbon loss from peatlands is mediated largely by the anaerobic microbial decomposition of organic matter to the greenhouse gases carbon dioxide and methane (36), and it is estimated that 10 to 20% of the globally emitted methane is derived from peatlands (30, 87). Primary and secondary fermentation and subsequent methanogenesis are considered to be the main carbon degradation processes because of the absence or limited availability of alternative electron acceptors. However, other microbial processes, such as denitrification and dissimilatory iron and sulfate reduction, can occur together with methanogenesis in the same peat soil fraction and contribute considerably to anaerobic carbon mineralization (4, 5, 43, 44). Fluctuations in environmental conditions on short- and long-term scales govern trophic interdependencies among microorganisms. Transitions between synergistic (e.g., the syntrophic interspecies transfer of hydrogen/formate) and antagonistic (e.g., competition for the same substrates) microbial interactions determine the extent of carbon flow diversion away from methanogenesis. A prime example is the suppression of microorganisms catalyzing methanogenic carbon degradation by sulfate-reducing microorganisms (SRM) that are energetically favored in the competition for substrates such as acetate, alcohols, and hydrogen (22, 81, 82). While sulfate concentrations are generally low in peatlands (10 to 300 μM), ongoing sulfate reduction proceeds at rates (2.5 to 340 nmol cm⁻³ day⁻¹) that are comparable to rates in sulfate-rich environments such as marine sediments (5, 40, 41). It was previously proposed that such high sulfate reduction rates are fueled by an anoxic recycling of reduced sulfur compounds via the so-called “thiosulfate shunt” (5). The alternative replenishment of the sulfate pool by the reoxidation of reduced sulfur species in the presence of oxygen is dependent on the vegetation type and alternating periods of precipitation and drought (14, 18, 64, 68, 86). In addition, increasing global atmospheric sulfur pollution and acid precipitation contribute to terrestrial sulfate pools and are predicted to repress methane emissions from peatlands by up to 15% within the first third of this century (22).Given the significance of dissimilatory sulfate reduction in peatlands, it is surprising that most information about the identity of microorganisms catalyzing this process in peatlands is derived from studies of a single model fen system (Schlöppnerbrunnen) located in the forested Lehstenbach catchment (Bavaria, Germany). Different redox processes such as fermentation (25), methanogenesis (29), denitrification (63), Fe(III) reduction (69), and sulfate reduction (2, 51) are present and have been studied at this site (4). The atmospheric deposition of sulfur originating from the combustion of soft coal in Eastern Europe until the 1990s led to the accumulation of sulfur species in the soils of this catchment. Although air pollution affecting this site has decreased in recent years (39), historically deposited sulfate stored in upland soils can desorb and is then transported via groundwater flow into the fen, where it drives dissimilatory sulfate reduction (1). DNA stable isotope probing using in situ concentrations of typical ¹³C-labeled degradation intermediates (mixture of lactate, acetate, formate, and propionate) has shown that a low-abundance Desulfosporosinus species, representing on average only 0.006% of the total bacterial and archaeal 16S rRNA genes, has the potential to be responsible for a substantial part of the sulfate reduction in the studied fen. However, a large fraction of the sulfate reduction observed in situ still remains unexplained (67). Other microorganisms that are potentially involved in sulfate reduction were previously detected in this fen by using 16S rRNA gene- and dsrAB-based diversity analyses. Few of these dsrAB sequences were affiliated with the previously described SRM genera Desulfomonile and Syntrophobacter, but most of the retrieved dsrAB sequences may derive from new taxa, as they represent novel lineages without cultivated representatives (51, 67, 73). Microorganisms that respire sulfite or sulfate anaerobically depend on the dsrAB-encoded key enzyme dissimilatory (bi)sulfite reductase for energy conservation, and thus, these genes have been widely used as markers for PCR-based molecular diversity studies of this guild (16, 38, 46, 84). However, some organisms that are phylogenetically related to SRM but that have seemingly lost the ability for sulfite/sulfate reduction can also harbor dsrAB. The dsrAB sequences of these organosulfonate reducers (45) or syntrophs (32) can be amplified by the commonly used DSR1F-DSR4R PCR primer mix (50). DNA stable isotope probing experiments targeting dsrAB in incubations with a mixture of ¹³C-labeled lactate, acetate, formate, and propionate could therefore not unambiguously link members of the novel dsrAB lineages to sulfate reduction in the Schlöppnerbrunnen peatland (67). Besides their unknown identity and ecophysiological function, additional important questions regarding the ecology of these enigmatic dsrAB-containing microorganisms remain unanswered: what is their actual abundance in peatlands, are they a stable part of the microbial peatland community or do they occur only sporadically, and are they endemic to the Schlöppnerbrunnen fen site or more widely distributed in different types of wetlands? Using a set of molecular ecology tools, we address these questions in this study and demonstrate that some dsrAB-containing microorganisms are widespread in peatlands and can thrive in these systems in considerable numbers.     

Role of Geobacter sulfurreducens Outer Surface c-Type Cytochromes in Reduction of Soil Humic Acid and Anthraquinone-2,6-Disulfonate

Deleting individual genes for outer surface c-type cytochromes in Geobacter sulfurreducens partially inhibited the reduction of humic substances and anthraquinone-2,6,-disulfonate. Complete inhibition was obtained only when five of these genes were simultaneously deleted, suggesting that diverse outer surface cytochromes can contribute to the reduction of humic substances and other extracellular quinones.Humic substances can play an important role in the reduction of Fe(III), and possibly other metals, in sedimentary environments (6, 34). Diverse dissimilatory Fe(III)-reducing microorganisms (3, 5, 7, 9, 11, 19-22, 25) can transfer electrons onto the quinone moieties of humic substances (38) or the model compound anthraquinone-2,6-disulfonate (AQDS). Reduced humic substances or AQDS abiotically reduces Fe(III) to Fe(II), regenerating the quinone. Electron shuttling in this manner can greatly increase the rate of electron transfer to insoluble Fe(III) oxides, presumably because soluble quinone-containing molecules are more accessible for microbial reduction than insoluble Fe(III) oxides (19, 22). Thus, catalytic amounts of humic substances have the potential to dramatically influence rates of Fe(III) reduction in soils and sediments and can promote more rapid degradation of organic contaminants coupled to Fe(III) reduction (1, 2, 4, 10, 24).To our knowledge, the mechanisms by which Fe(III)-reducing microorganisms transfer electrons to humic substances have not been investigated previously for any microorganism. However, reduction of AQDS has been studied using Shewanella oneidensis (17, 40). Disruption of the gene for MtrB, an outer membrane protein required for proper localization of outer membrane cytochromes (31), inhibited reduction of AQDS, as did disruption of the gene for the outer membrane c-type cytochrome, MtrC (17). However, in each case inhibition was incomplete, and it was suggested that there was a possibility of some periplasmic reduction (17), which would be consistent with the ability of AQDS to enter the cell (40).The mechanisms for electron transfer to humic substances in Geobacter species are of interest because molecular studies have frequently demonstrated that Geobacter species are the predominant Fe(III)-reducing microorganisms in sedimentary environments in which Fe(III) reduction is an important process (references 20, 32, and 42 and references therein). Geobacter sulfurreducens has routinely been used for investigations of the physiology of Geobacter species because of the availability of its genome sequence (29), a genetic system (8), and a genome-scale metabolic model (26) has made it possible to take a systems biology approach to understanding the growth of this organism in sedimentary environments (23).     

Syntrophic Degradation of Cadaverine by a Defined Methanogenic Coculture

A novel, strictly anaerobic, cadaverine-oxidizing, defined coculture was isolated from an anoxic freshwater sediment sample. The coculture oxidized cadaverine (1,5-diaminopentane) with sulfate as the electron acceptor. The sulfate-reducing partner could be replaced by a hydrogenotrophic methanogenic partner. The defined coculture fermented cadaverine to acetate, butyrate, and glutarate plus sulfide or methane. The key enzymes involved in cadaverine degradation were identified in cell extracts. A pathway of cadaverine fermentation via 5-aminovaleraldehyde and crotonyl-coenzyme A with subsequent dismutation to acetate and butyrate is suggested. Comparative 16S rRNA gene analysis indicated that the fermenting part of the coculture belongs to the subphylum Firmicutes but that this part is distant from any described genus. The closest known relative was Clostridium aminobutyricum, with 95% similarity.Cadaverine is a biogenic primary aliphatic amine. Together with other biogenic amines, like putrescine or spermidine, it is formed during oxygen-limited decomposition of protein-rich organic matter by decarboxylation of amino acids or by amination of aldehydes and ketones (8, 27, 30, 42, 53, 54). These putrid-smelling and, at higher concentrations (100 to 400 mg per kg), often toxic compounds play a major role in food microbiology, e.g., as flavoring constituents in the ripening of cheese or as contaminants of fish and meat products, wine, and beer (24, 29, 49).Little is known about the degradation of primary amines. Mono- and diamine oxidases of higher organisms and bacteria (23, 41, 64) initiate aerobic degradation, leading to the respective formation of aldehyde, ammonia, and hydrogen peroxide as products (28). Alternatively, in a putrescine-degrading mutant of Escherichia coli, putrescine is degraded by a putrescine-2-oxoglutarate transaminase and a subsequent dehydrogenase to form 4-aminobutyrate, which is further metabolized via succinate (43).Anaerobic degradation of primary amines could follow basically similar pathways. The released reducing equivalents can be disposed of in a manner similar to that described for primary alcohols (9, 15, 16). In the absence of external electron acceptors, such as sulfate or nitrate, incomplete oxidation of cadaverine to fatty acids or dicarboxylic acids could be coupled to syntrophic methane production, homoacetogenesis, or reductive synthesis of long-chain fatty acids (1, 25, 31).In the present study, we describe a new isolate of strictly anaerobic bacteria which oxidizes cadaverine syntrophically with the methanogen Methanospirillum hungatei and forms acetate, butyrate, glutarate, and methane as products. The enzymes involved in the degradation of cadaverine were identified, and a catabolic pathway is proposed.