SummaryThe soxB gene encodes the SoxB component of the periplasmic thiosulfate-oxidizing Sox enzyme complex, which has been proposed to be widespread among the various phylogenetic groups of sulfur-oxidizing bacteria (SOB) that convert thiosulfate to sulfate with and without the formation of sulfur globules as intermediate. Indeed, the comprehensive genetic and genomic analyses presented in the present study identified the soxB gene in 121 phylogenetically and physiologically divergent SOB, including several species for which thiosulfate utilization has not been reported yet. In first support of the previously postulated general involvement of components of the Sox enzyme complex in the thiosulfate oxidation process of sulfur-storing SOB, the soxB gene was detected in all investigated photoand chemotrophic species that form sulfur globules during thiosulfate oxidation (Chromatiaceae, Chlorobiaceae, Ectothiorhodospiraceae, Thiothrix, Beggiatoa, Thiobacillus, invertebrate symbionts and free-living relatives). The SoxB phylogeny reflected the major 16S rRNA gene-based phylogenetic lineages of the investigated SOB, although topological discrepancies indicated several events of lateral soxB gene transfer among the SOB, e.g. its independent acquisition by the anaerobic anoxygenic phototrophic lineages from different chemotrophic donor lineages. A putative scenario for the proteobacterial origin and evolution of the Sox enzyme system in SOB is presented considering the phylogenetic, genomic (sox gene cluster composition) and geochemical data.

Phylogeny of the alpha and beta subunits of the dissimilatory adenosine-59-phosphosulfate (APS) reductase from sulfate-reducing prokaryotesorigin and evolution of the dissimilatory sulfate-reduction pathway Newly developed PCR assays were used to PCR-amplify and sequence fragments of the dissimilatory adenosine-59-phosphosulfate (APS) reductase genes (aprBA) comprising nearly the entire gene locus (2?2-2?4 kb, equal to 92-94 % of the protein coding sequence) from 75 sulfate-reducing prokaryotes (SRP) of a taxonomically wide range. Comparative phylogenetic analysis included all determined and publicly available AprBA sequences from SRP and selected homologous sequences of sulfur-oxidizing bacteria (SOB). The almost identical AprB and AprA tree topologies indicated a shared evolutionary path for the aprBA among the investigated SRP by vertical inheritance and concomitant lateral gene transfer (LGT). The topological comparison of AprB/A-and 16S rRNA gene-based phylogenetic trees revealed novel LGT events across the SRP divisions. Compositional gene analysis confirmed Thermacetogenium phaeum to be the first validated strain affected by a recent lateral transfer of aprBA as a putative effect of long-term co-cultivation with a Thermodesulfovibrio species. Interestingly, the Apr proteins of SRP and SOB diverged into two phylogenetic lineages, with the SRP affiliated with the green sulfur bacteria, e.g. Chlorobaculum tepidum, while the Allochromatium vinosum-related sequences formed a distinct group. Analysis of genome data indicated that this phylogenetic separation is also reflected in the differing presence of the putative proteins functionally associated with Apr, QmoABC complex (quinone-interacting membrane-bound oxidoreductase) and AprM (transmembrane protein). Scenarios for the origin and evolution of the dissimilatory APS reductase are discussed within the context of the dissimilatory sulfite reductase (DsrAB) phylogeny, the appearance of QmoABC and AprM in the SRP and SOB genomes, and the geochemical setting of Archean Earth.

The dissimilatory adenosine-5-phosposulfate reductase is a key enzyme of the microbial sulfate reduction and sulfur oxidation processes. Because the alpha-and beta-subunit-encoding genes, aprBA, are highly conserved among sulfate-reducing and sulfur-oxidizing prokaryotes, they are most suitable for molecular profiling of the microbial community structure of the sulfur cycle in environment. In this study, a new aprA gene-targeting assay using a combination of PCR and denaturing gradient gel electrophoresis is presented. The screening of sulfate-reducing and sulfur-oxidizing reference strains as well as the analyses of environmental DNA from diverse habitats (e.g., microbial mats, invertebrate tissue, marine and estuarine sediments, and The sulfur cycle is predominated by reductive and oxidative processes of microorganisms: the dissimilatory sulfate reduction is considered as the main anaerobic process in the biomineralization of organic matter in the environment, accounting for up to 50% of its degradation in marine sediments (29), while dissimilatory sulfur oxidation processes occur wherever reduced inorganic sulfur compounds are available from the activity of the sulfate-reducing prokaryotes or from geological sources (5, 16). The sulfate-reducing and sulfur-oxidizing prokaryotes (SRP and SOP, respectively) are phylogenetically and physiologically diverse groups (5,16,25,40,47). Their polyphyletic nature restricts the concomitant detection of all recognized members by the use of a single 16S rRNA genetargeting probe or primer pair in environmental analyses and limits the identification of novel lineages. In addition, the 16S rRNA gene-based analysis cannot provide an unambiguous link between the genetic identity of an uncultured microorganism and its physiological or metabolic capacity. Alternative molecular approaches circumvent these limitations by using functional genes that encode key enzymes of the dissimilatory sulfate reduction and sulfur oxidation pathways (ATP sulfurylase, Sat; adenosine-5Ј-phosphosulfate [APS] reductase, AprBA; sulfite reductase, DsrAB) and thus are much more suited to analyze and determine the phylogenetic complexity of the microbial sulfur cycle in nature.Indeed, PCR assays have been developed for the amplification of dsrAB genes, but up to now these approaches are restricted to diversity analyses of the SRP community (2,3,15,42,43,57). Although multiple events of lateral gene transfer (LGT) have affected the Dsr phylogeny of certain SRP lineages, the usefulness of dsrAB as functional gene markers for environmental analysis was confirmed (32,62,67). Recently, new PCR assays have been developed for the amplification of the aprBA gene locus from SRP and sulfur-oxidizing bacteria (SOB). The ubiquitous presence of aprBA genes in SRP was confirmed (38); however, the PCR-based screening among SOB reference strains revealed its restricted distribution to photo-and chemotrophs with strict anaerobic or at least facultative anaerobic lifestyles, e.g., several Chlorobiaceae and most Chromatiac...

Dissimilatory adenosine-59-phosphosulfate (APS) reductase (AprBA) is a key enzyme of the dissimilatory sulfate-reduction pathway. Homologues have been found in photo-and chemotrophic sulfur-oxidizing prokaryotes (SOP), in which they are postulated to operate in the reverse direction, oxidizing sulfite to APS. Newly developed PCR assays allowed the amplification of 92-93 % (2.1-2.3 kb) of the APS reductase locus aprBA. PCR-based screening of 116 taxonomically divergent SOP reference strains revealed a distribution of aprBA restricted to photo-and chemotrophs with strict anaerobic or at least facultative anaerobic lifestyles, including Chlorobiaceae, Chromatiaceae, Thiobacillus, Thiothrix and invertebrate symbionts. In the AprBAbased tree, the SOP diverge into two distantly related phylogenetic lineages, Apr lineages I and II, with the proteins of lineage II (Chlorobiaceae and others) in closer affiliation to the enzymes of the sulfate-reducing prokaryotes (SRP). This clustering is discordant with the dissimilatory sulfite reductase (DsrAB) phylogeny and indicates putative lateral aprBA gene transfer from SRP to the respective SOB lineages. In support of lateral gene transfer (LGT), several beta-and gammaproteobacterial species harbour both aprBA homologues, the DsrAB-congruent 'authentic' and the SRP-related, LGT-derived gene loci, while some relatives possess exclusively the SRP-related apr genes as a possible result of resident gene displacement by the xenologue. The two-gene state might be an intermediate in the replacement of the resident essential gene. Collected genome data demonstrate the correlation between the AprBA tree topology and the composition/arrangement of the apr gene loci (occurrence of qmoABC or aprM genes) from SRP and SOP of lineages I and II. The putative functional role of the SRP-related APS reductases in photo-and chemotrophic SOP is discussed.

bMineralization of organic matter in anoxic environments relies on the cooperative activities of hydrogen producers and consumers linked by interspecies electron transfer in syntrophic consortia that may include sulfate-reducing species (e.g., Desulfovibrio). Physiological differences and various gene repertoires implicated in syntrophic metabolism among Desulfovibrio species suggest considerable variation in the biochemical basis of syntrophy. In this study, comparative transcriptional and mutant analyses of Desulfovibrio alaskensis strain G20 and Desulfovibrio vulgaris strain Hildenborough growing syntrophically with Methanococcus maripaludis on lactate were used to develop new and revised models for their alternative electron transfer and energy conservation systems. Lactate oxidation by strain G20 generates a reduced thiol-disulfide redox pair(s) and ferredoxin that are energetically coupled to H ؉ /CO 2 reduction by periplasmic formate dehydrogenase and hydrogenase via a flavin-based reverse electron bifurcation process (electron confurcation) and a menaquinone (MQ) redox loop-mediated reverse electron flow involving the membrane-bound Qmo and Qrc complexes. In contrast, strain Hildenborough uses a larger number of cytoplasmic and periplasmic proteins linked in three intertwining pathways to couple H ؉ reduction to lactate oxidation. The faster growth of strain G20 in coculture is associated with a kinetic advantage conferred by the Qmo-MQ-Qrc loop as an electron transfer system that permits higher lactate oxidation rates under elevated hydrogen levels (thereby enhancing methanogenic growth) and use of formate as the main electron-exchange mediator (>70% electron flux), as opposed to the primarily hydrogen-based exchange by strain Hildenborough. This study further demonstrates the absence of a conserved gene core in Desulfovibrio that would determine the ability for a syntrophic lifestyle. In anoxic environments depleted in inorganic electron acceptors (e.g., freshwater and marine sediments, flooded soils, landfills, and sewage digesters), the complete mineralization of complex organic matter to CO 2 and methane relies on the cooperative activities of phylogenetically and metabolically distinct microbial groups assembled in syntrophic consortia. In these assemblages, sulfate-reducing bacteria (SRB) function as secondary fermenters obligately linked via interspecies electron transfer to the metabolic activity of methanogenic archaea since the oxidation of common substrates (organic acids and alcohols) yields sufficient energy only for cell maintenance and growth when the methanogens maintain low concentrations of the products of SRB metabolism (acetate, hydrogen, and formate) (1-3). As a result, the metabolism of one community member is directly influenced by and dependent upon the activity of the other. Hydrogen and formate are considered the primary shuttle compounds for interspecies electron transfer (1-3). Additionally, single reports suggest the involvement of cysteine or exogenous carriers such as humi...