Phylogenetic analysis reveals multiple lateral transfers of adenosine-5'-phosphosulfate reductase genes among sulfate-reducing microorganisms.
ABSTRACT: Lateral gene transfer affects the evolutionary path of key genes involved in ancient metabolic traits, such as sulfate respiration, even more than previously expected. In this study, the phylogeny of the adenosine-5'-phosphosulfate (APS) reductase was analyzed. APS reductase is a key enzyme in sulfate respiration present in all sulfate-respiring prokaryotes. A newly developed PCR assay was used to amplify and sequence a fragment ( approximately 900 bp) of the APS reductase gene, apsA, from a taxonomically wide range of sulfate-reducing prokaryotes (n = 60). Comparative phylogenetic analysis of all obtained and available ApsA sequences indicated a high degree of sequence conservation in the region analyzed. However, a comparison of ApsA- and 16S rRNA-based phylogenetic trees revealed topological incongruences affecting seven members of the Syntrophobacteraceae and three members of the Nitrospinaceae, which were clearly monophyletic with gram-positive sulfate-reducing bacteria (SRB). In addition, Thermodesulfovibrio islandicus and Thermodesulfobacterium thermophilum, Thermodesulfobacterium commune, and Thermodesulfobacterium hveragerdense clearly branched off between the radiation of the delta-proteobacterial gram-negative SRB and the gram-positive SRB and not close to the root of the tree as expected from 16S rRNA phylogeny. The most parsimonious explanation for these discrepancies in tree topologies is lateral transfer of apsA genes across bacterial divisions. Similar patterns of insertions and deletions in ApsA sequences of donor and recipient lineages provide additional evidence for lateral gene transfer. From a subset of reference strains (n = 25), a fragment of the dissimilatory sulfite reductase genes (dsrAB), which have recently been proposed to have undergone multiple lateral gene transfers (M. Klein et al., J. Bacteriol. 183:6028-6035, 2001), was also amplified and sequenced. Phylogenetic comparison of DsrAB- and ApsA-based trees suggests a frequent involvement of gram-positive and thermophilic SRB in lateral gene transfer events among SRB.
Project description:A large fragment of the dissimilatory sulfite reductase genes (dsrAB) was PCR amplified and fully sequenced from 30 reference strains representing all recognized lineages of sulfate-reducing bacteria. In addition, the sequence of the dsrAB gene homologs of the sulfite reducer Desulfitobacterium dehalogenans was determined. In contrast to previous reports, comparative analysis of all available DsrAB sequences produced a tree topology partially inconsistent with the corresponding 16S rRNA phylogeny. For example, the DsrAB sequences of several Desulfotomaculum species (low G+C gram-positive division) and two members of the genus Thermodesulfobacterium (a separate bacterial division) were monophyletic with delta-proteobacterial DsrAB sequences. The most parsimonious interpretation of these data is that dsrAB genes from ancestors of as-yet-unrecognized sulfate reducers within the delta-Proteobacteria were laterally transferred across divisions. A number of insertions and deletions in the DsrAB alignment independently support these inferred lateral acquisitions of dsrAB genes. Evidence for a dsrAB lateral gene transfer event also was found within the delta-Proteobacteria, affecting Desulfobacula toluolica. The root of the dsr tree was inferred to be within the Thermodesulfovibrio lineage by paralogous rooting of the alpha and beta subunits. This rooting suggests that the dsrAB genes in Archaeoglobus species also are the result of an ancient lateral transfer from a bacterial donor. Although these findings complicate the use of dsrAB genes to infer phylogenetic relationships among sulfate reducers in molecular diversity studies, they establish a framework to resolve the origins and diversification of this ancient respiratory lifestyle among organisms mediating a key step in the biogeochemical cycling of sulfur.
Project description:A comparative study of the kinetic characteristics (specific activity, initial and maximum rate, and affinity for substrates) of key enzymes of assimilatory sulfate reduction (APS reductase and dissimilatory sulfite reductase) in cell-free extracts of sulphate-reducing bacteria (SRB) from various biotopes was performed. The material for the study represented different strains of SRB from various ecotopes. Microbiological (isolation and cultivation), biochemical (free cell extract preparation) and chemical (enzyme activity determination) methods served in defining kinetic characteristics of SRB enzymes. The determined affinity data for substrates (i.e., sulfite) were 10 times higher for SRB strains isolated from environmental (soil) ecotopes than for strains from the human intestine. The maximum rate of APS reductase reached 0.282-0.862 µmol/min×mg-1 of protein that is only 10 to 28% higher than similar initial values. The maximum rate of sulfite reductase for corrosive relevant collection strains and SRB strains isolated from heating systems were increased by 3 to 10 times. A completely different picture was found for the intestinal SRB Vmax in the strains Desulfovibrio piger Vib-7 (0.67 µmol/min × mg-1 protein) and Desulfomicrobium orale Rod-9 (0.45 µmol/min × mg-1 protein). The determinant in the cluster distribution of SRB strains is the activity of the terminal enzyme of dissimilatory sulfate reduction-sulfite reductase, but not APS reductase. The data obtained from the activity of sulfate reduction enzymes indicated the adaptive plasticity of SRB strains that is manifested in the change in enzymatic activity.
Project description:Intestinal sulfate-reducing bacteria (SRB) growth and resultant hydrogen sulfide production may damage the gastrointestinal epithelium and thereby contribute to chronic intestinal disorders. However, the ecology and phylogenetic diversity of intestinal dissimilatory SRB populations are poorly understood, and endogenous or exogenous sources of available sulfate are not well defined. The succession of intestinal SRB was therefore compared in inbred C57BL/6J mice using a PCR-based metabolic molecular ecology (MME) approach that targets a conserved region of subunit A of the adenosine-5'-phosphosulfate (APS) reductase gene. The APS reductase-based MME strategy revealed intestinal SRB in the stomach and small intestine of 1-, 4-, and 7-day-old mice and throughout the gastrointestinal tract of 14-, 21-, 30-, 60-, and 90-day-old mice. Phylogenetic analysis of APS reductase amplicons obtained from the stomach, middle small intestine, and cecum of neonatal mice revealed that Desulfotomaculum spp. may be a predominant SRB group in the neonatal mouse intestine. Dot blot hybridizations with SRB-specific 16S ribosomal DNA (rDNA) probes demonstrated SRB colonization of the cecum and colon pre- and postweaning and colonization of the stomach and small intestine of mature mice only. The 16S rDNA hybridization data further demonstrated that SRB populations were most numerous in intestinal regions harboring sulfomucin-containing goblet cells, regardless of age. Reverse transcriptase PCR analysis demonstrated APS reductase mRNA expression in all intestinal segments of 30-day-old mice, including the stomach. These results demonstrate for the first time widespread colonization of the mouse intestine by dissimilatory SRB and evidence of spatial-specific SRB populations and sulfomucin patterns along the gastrointestinal tract.
Project description:Spore-forming, Gram-positive sulfate-reducing bacteria (SRB) represent a group of SRB that dominates the deep subsurface as well as niches in which resistance to oxygen and dessication is an advantage. Desulfotomaculum reducens strain MI-1 is one of the few cultured representatives of that group with a complete genome sequence available. The metabolic versatility of this organism is reflected in the presence of genes encoding for the oxidation of various electron donors, including three- and four-carbon fatty acids and alcohols. Synteny in genes involved in sulfate reduction across all four sequenced Gram-positive SRB suggests a distinct sulfate-reduction mechanism for this group of bacteria. Based on the genomic information obtained for sulfate reduction in D. reducens, the transfer of electrons to the sulfite and APS reductases is proposed to take place via the quinone pool and heterodisulfide reductases respectively. In addition, both H(2) -evolving and H(2) -consuming cytoplasmic hydrogenases were identified in the genome, pointing to potential cytoplasmic H(2) cycling in the bacterium. The mechanism of metal reduction remains unknown.
Project description:Sediment samples were collected worldwide from 16 locations on four continents (in New York, California, New Jersey, Virginia, Puerto Rico, Venezuela, Italy, Latvia, and South Korea) to assess the extent of the diversity and the distribution patterns of sulfate-reducing bacteria (SRB) in contaminated sediments. The SRB communities were examined by terminal restriction fragment (TRF) length polymorphism (TRFLP) analysis of the dissimilatory sulfite reductase genes (dsrAB) with NdeII digests. The fingerprints of dsrAB genes contained a total of 369 fluorescent TRFs, of which <20% were present in the GenBank database. The global sulfidogenic communities appeared to be significantly different among the anthropogenically impacted (petroleum-contaminated) sites, but nearly all were less diverse than pristine habitats, such as mangroves. A global SRB indicator species of petroleum pollution was not identified. However, several dsrAB gene sequences corresponding to hydrocarbon-degrading isolates or consortium members were detected in geographically widely separated polluted sites. Finally, a cluster analysis of the TRFLP fingerprints indicated that many SRB microbial communities were most similar on the basis of close geographic proximity (tens of kilometers). Yet, on larger scales (hundreds to thousands of kilometers) SRB communities could cluster with geographically widely separated sites and not necessarily with the site with the closest proximity. These data demonstrate that SRB populations do not adhere to a biogeographic distribution pattern similar to that of larger eukaryotic organisms, with the greatest species diversity radiating from the Indo-Pacific region. Rather, a patchy SRB distribution is encountered, implying an initially uniform SRB community that has differentiated over time.
Project description:Here we describe the diversity and activity of sulfate-reducing bacteria (SRB) in sulfidogenic bioreactors by using the simultaneous analysis of PCR products obtained from DNA and RNA of the 16S rRNA and dissimilatory sulfite reductase (dsrAB) genes. We subsequently analyzed the amplified gene fragments by using denaturing gradient gel electrophoresis (DGGE). We observed fewer bands in the RNA-based DGGE profiles than in the DNA-based profiles, indicating marked differences in the populations present and in those that were metabolically active at the time of sampling. Comparative sequence analyses of the bands obtained from rRNA and dsrB DGGE profiles were congruent, revealing the same SRB populations. Bioreactors that received either ethanol or isopropanol as an energy source showed the presence of SRB affiliated with Desulfobulbus rhabdoformis and/or Desulfovibrio sulfodismutans, as well as SRB related to the acetate-oxidizing Desulfobacca acetoxidans. The reactor that received wastewater containing a diverse mixture of organic compounds showed the presence of nutritionally versatile SRB affiliated with Desulfosarcina variabilis and another acetate-oxidizing SRB, affiliated with Desulfoarculus baarsii. In addition to DGGE analysis, we performed whole-cell hybridization with fluorescently labeled oligonucleotide probes to estimate the relative abundances of the dominant sulfate-reducing bacterial populations. Desulfobacca acetoxidans-like populations were most dominant (50 to 60%) relative to the total SRB communities, followed by Desulfovibrio-like populations (30 to 40%), and Desulfobulbus-like populations (15 to 20%). This study is the first to identify metabolically active SRB in sulfidogenic bioreactors by using the functional gene dsrAB as a molecular marker. The same approach can also be used to infer the ecological role of coexisting SRB in other habitats.
Project description:Sulfate is the predominant electron acceptor for anaerobic oxidation of methane (AOM) in marine sediments. This process is carried out by a syntrophic consortium of anaerobic methanotrophic archaea (ANME) and sulfate reducing bacteria (SRB) through an energy conservation mechanism that is still poorly understood. It was previously hypothesized that ANME alone could couple methane oxidation to dissimilatory sulfate reduction, but a genetic and biochemical basis for this proposal has not been identified. Using comparative genomic and phylogenetic analyses, we found the genetic capacity in ANME and related methanogenic archaea for sulfate reduction, including sulfate adenylyltransferase, APS kinase, APS/PAPS reductase and two different sulfite reductases. Based on characterized homologs and the lack of associated energy conserving complexes, the sulfate reduction pathways in ANME are likely used for assimilation but not dissimilation of sulfate. Environmental metaproteomic analysis confirmed the expression of 6 proteins in the sulfate assimilation pathway of ANME. The highest expressed proteins related to sulfate assimilation were two sulfite reductases, namely assimilatory-type low-molecular-weight sulfite reductase (alSir) and a divergent group of coenzyme F420-dependent sulfite reductase (Group II Fsr). In methane seep sediment microcosm experiments, however, sulfite and zero-valent sulfur amendments were inhibitory to ANME-2a/2c while growth in their syntrophic SRB partner was not observed. Combined with our genomic and metaproteomic results, the passage of sulfur species by ANME as metabolic intermediates for their SRB partners is unlikely. Instead, our findings point to a possible niche for ANME to assimilate inorganic sulfur compounds more oxidized than sulfide in anoxic marine environments.
Project description:In this study, the sulfate-reducing bacteria, (SRB) were identified and reported for the first time through analysis of functional gene dsrAB, from the DNA of sediment samples collected from 10 sites of the Chilika lake. The finding illustrates Forty six Operational Taxonomic Units (OTUs), identified from the DGGE which were obtained from the 10 sediment samples. Of these, 34 OTUs exhibited around 78-96% sequence similarity and 12 OTUs showed 97 to 100% sequence similarity to the dsrAB gene of reported type strains of SRB. The sequence information obtained revealed the presence and distribution of diverse types of SRB which include phylotypes related to Desulfovibrio, Desulfonatronovibrio, Desulfomicrobium, Desulfobotulous and Desulfobacca. Upon comparison of dsrAB gene sequences of SRB obtained through this study with those collected from the GenBank, and through the dendrogram constructed, it was observed that except 13 OTUs that clustered closely with the reported type strains, all other 36 OTUs clustered distantly and had no representative member of SRB. This indicated the presence of phylogenetically diverse groups of SRB inhabiting the lake Chilika.
Project description:Patterns of microbial biogeography result from a combination of dispersal, speciation and extinction, yet individual contributions exerted by each of these mechanisms are difficult to isolate and distinguish. The influx of endospores of thermophilic microorganisms to cold marine sediments offers a natural model for investigating passive dispersal in the ocean. We investigated the activity, diversity and abundance of thermophilic endospore-forming sulfate-reducing bacteria (SRB) in Aarhus Bay by incubating pasteurized sediment between 28 and 85?°C, and by subsequent molecular diversity analyses of 16S rRNA and of the dissimilatory (bi)sulfite reductase (dsrAB) genes within the endospore-forming SRB genus Desulfotomaculum. The thermophilic Desulfotomaculum community in Aarhus Bay sediments consisted of at least 23 species-level 16S rRNA sequence phylotypes. In two cases, pairs of identical 16S rRNA and dsrAB sequences in Arctic surface sediment 3000?km away showed that the same phylotypes are present in both locations. Radiotracer-enhanced most probable number analysis revealed that the abundance of endospores of thermophilic SRB in Aarhus Bay sediment was ca. 10(4) per cm(3) at the surface and decreased exponentially to 10(0) per cm(3) at 6.5?m depth, corresponding to 4500 years of sediment age. Thus, a half-life of ca. 300 years was estimated for the thermophilic SRB endospores deposited in Aarhus Bay sediments. These endospores were similarly detected in the overlying water column, indicative of passive dispersal in water masses preceding sedimentation. The sources of these thermophiles remain enigmatic, but at least one source may be common to both Aarhus Bay and Arctic sediments.
Project description: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 delta(34)S values relative to delta(34)S values of pore water sulfate showed that sediment SRB populations were actively reducing sulfate under ambient conditions (pH of approximately 2), although possibly within less acidic microenvironments. Interestingly, delta(34)S 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.