Sulfite Dehydrogenase Research Articles

Genome-based taxonomic classification of the genus Sulfitobacter along with the proposal of a new genus Parasulfitobacter gen. nov. and exploring the gene clusters associated with sulfur oxidation

BackgroundThe genus Sulfitobacter, a member of the family Roseobacteraceae, is widely distributed in the ocean and is believed to play crucial roles in the global sulfur cycle. However, gene clusters associated with sulfur oxidation in genomes of the type strains of this genus have been poorly studied. Furthermore, taxonomic errors have been identified in this genus, potentially leading to significant confusion in ecological and evolutionary interpretations in subsequent studies of the genus Sulfitobacter. This study aims to investigate the taxonomic status of this genus and explore the metabolism associated with sulfur oxidation.ResultsThis study suggests that Sulfitobacter algicola does not belong to Sulfitobacter and should be reclassified into a novel genus, for which we propose the name Parasulfitobacter gen. nov., with Parasulfitobacter algicola comb. nov. as the type species. Additionally, enzymes involved in the sulfur oxidation process, such as the sulfur oxidization (Sox) system, the disulfide reductase protein family, and the sulfite dehydrogenase (SoeABC), were identified in almost all Sulfitobacter species. This finding implies that the majority of Sulfitobacter species can oxidize reduced sulfur compounds. Differences in the modular organization of sox gene clusters among Sulfitobacter species were identified, along with the presence of five genes with unknown function located in some of the sox gene clusters. Lastly, this study revealed the presence of the demethylation pathway and the cleavage pathway used by many Sulfitobacter species to degrade dimethylsulfoniopropionate (DMSP). These pathways enable these bacteria to utilize DMSP as important source of sulfur and carbon or as a defence strategy.ConclusionsOur findings contribute to interpreting the mechanism by which Sulfitobacter species participate in the global sulfur cycle. The taxonomic rearrangement of S. algicola into the novel genus Parasulfitobacter will prevent confusion in ecological and evolutionary interpretations in future studies of the genus Sulfitobacter.

Evaluation of the effect of increasing the organic load in the thermophilic co-fermentation of sugarcane industry by-products on hydrogen, ethanol and lactic acid generation

The effects of increased organic loading rate (OLR) on H2 production and soluble metabolites in a thermophilic anaerobic fluidized bed reactor (AFBR-AT) at 55 °C were evaluated by the fermentation of vinasse and molasses from sugarcane (at a ratio of 1:1). The OLR varied between 30 and 135 kg COD.m−3.d−1, while the hydraulic retention time (HRT) was fixed at 4 h, and the initial co-substrate concentration varied between 5 and 22.5 g COD.L−1. The thermophilic acidogenic reactor showed a maximum hydrogen yield (HY) (2.49 mmol H2.g−1CODapp) at an OLR of 30 kg COD.m−3.d−1 and a higher hydrogen production rate (HPR) (2.8 L H2.L−1.d−1) at an OLR of 45 kg COD.m−3.d−1. Increasing the OLR negatively influenced H2 production. The results showed an increase in ethanol (EtOH) concentrations of up to 47.3 times and lactic acid (HLa) concentrations of up to 15.2 times when comparing OLRs of 30 and 135 kg COD.m−3.d−1, indicating changes in metabolic pathways for acid formation at organic loads ≥90 kg COD.m−3.d−1. Thermoanaerobacterium was the bacteria with higher relative abundance and was directly involved in H2 production, while Lactobacillus was mainly involved in HLa production in higher OLR, decreasing H2 production instead. Besides that, higher OLRs were favorable for sulfidogenesis and the development of sulfate-reducing bacteria, such as Desulfobacterota, due to the higher COD/sulfate ratio in OLR 135 kg COD.m−3.d−1. According to the metabolic inference in this condition, the enzyme activity of sulfite reductase and sulfite dehydrogenase may have occurred, consuming H+, thereby reducing H2 production.

A novel bacterial sulfite dehydrogenase that requires three c-type cytochromes for electron transfer.

c-type Cytochromes (c-Cyts), primarily as electron carriers and oxidoreductases, play a key role in energy transduction processes in virtually all living organisms. Many bacteria, such as Shewanella oneidensis, are particularly rich in c-Cyts, supporting respiratory versatility not seen in eukaryotes. Unfortunately, a large number of c-Cyts are underexplored, and their biological functions remain unknown. In this study, we identify SorCABD of S. oneidensis as a novel sulfite dehydrogenase (SDH), which catalyzes the oxidation of sulfite to sulfate. In addition to catalytic subunit SorA, this enzymatic complex includes three c-Cyt subunits, which all together carry out electron transfer. The electrons extracted from sulfite oxidation are ultimately delivered to oxygen, leading to oxygen reduction, a process relying on terminal oxidase cyt cbb3. Genomic analysis suggests that the homologs of this SDH are present in a small number of bacterial genera, Shewanella and Vibrio in particular. Because these bacteria are generally capable of reducing sulfite under anaerobic conditions, the co-existence of a sulfite oxidation system implies that they may play especially important roles in the transformation of sulfur species in natural environments.Importancec-type Cytochromes (c-Cyts) endow bacteria with high flexibility in their oxidative/respiratory systems, allowing them to extracellularly transform diverse inorganic and organic compounds for survival and growth. However, a large portion of the bacterial c-Cyts remain functionally unknown. Here, we identify three c-Cyts that work together as essential electron transfer partners for the catalytic subunit of a novel SDH in sulfite oxidation in Shewanella oneidensis. This characteristic makes S. oneidensis the first organism known to be capable of oxidizing and reducing sulfite. The findings suggest that Shewanella, along with a small number of other aquatic bacteria, would serve as a particular driving force in the biogeochemical sulfur cycle in nature.

The Ion-Translocating NrfD-Like Subunit of Energy-Transducing Membrane Complexes.

Several energy-transducing microbial enzymes have their peripheral subunits connected to the membrane through an integral membrane protein, that interacts with quinones but does not have redox cofactors, the so-called NrfD-like subunit. The periplasmic nitrite reductase (NrfABCD) was the first complex recognized to have a membrane subunit with these characteristics and consequently provided the family's name: NrfD. Sequence analyses indicate that NrfD homologs are present in many diverse enzymes, such as polysulfide reductase (PsrABC), respiratory alternative complex III (ACIII), dimethyl sulfoxide (DMSO) reductase (DmsABC), tetrathionate reductase (TtrABC), sulfur reductase complex (SreABC), sulfite dehydrogenase (SoeABC), quinone reductase complex (QrcABCD), nine-heme cytochrome complex (NhcABCD), group-2 [NiFe] hydrogenase (Hyd-2), dissimilatory sulfite-reductase complex (DsrMKJOP), arsenate reductase (ArrC) and multiheme cytochrome c sulfite reductase (MccACD). The molecular structure of ACIII subunit C (ActC) and Psr subunit C (PsrC), NrfD-like subunits, revealed the existence of ion-conducting pathways. We performed thorough primary structural analyses and built structural models of the NrfD-like subunits. We observed that all these subunits are constituted by two structural repeats composed of four-helix bundles, possibly harboring ion-conducting pathways and containing a quinone/quinol binding site. NrfD-like subunits may be the ion-pumping module of several enzymes. Our data impact on the discussion of functional implications of the NrfD-like subunit-containing complexes, namely in their ability to transduce energy.

Improving confirmed nanometric sulfur bioproduction using engineered Thioalkalivibrio versutus

Complicated production procedures and superior characteristics of nano-sized sulfur elevate its price to 25–40 fold higher than micrograde kind. Also, natural gas hydrogen sulfide levels are restricted because of its toxic environmental consequences. Thioalkalivibrio versutus is a polyextremophilic industrial autotroph with high natural gas desulfurization capability. Here, nanometric (>50 nm) sulfur bioproduction using T. versutus while desulfurizing natural gas was validated. Also, this production was enhanced by 166.7% via lowering sulfate production by 55.1%. A specially-developed CRISPR system, with 42% editing efficiency, simplified the genome editing workflow scheme for this challenging bacterium. In parallel, sulfur metabolism was uncovered using proteins mining and transcriptome studies for defining sulfate-producing key genes (heterodisulfide reductase-like complex, sulfur dioxygenase, sulfite dehydrogenase and sulfite oxidase). This study provided cost-effective nanometric sulfur production and improved this production using a novel CRISPR strategy, which could be suitable for industrial polyextremophiles, after uncovering sulfur pathways in T. versutus.

Sulfite oxidation by the quinone-reducing molybdenum sulfite dehydrogenase SoeABC from the bacterium Aquifex aeolicus

The microaerophilic bacterium Aquifex aeolicus is a chemolitoautotroph that uses sulfur compounds as electron sources. The model of oxidation of the energetic sulfur compounds in this bacterium predicts that sulfite would probably be a metabolic intermediate released in the cytoplasm. In this work, we purified and characterized a membrane-bound sulfite dehydrogenase, identified as an SoeABC enzyme, that was previously described as a sulfur reductase. It is a member of the DMSO-reductase family of molybdenum enzymes. This type of enzyme was identified a few years ago but never purified, and biochemical data and kinetic properties were completely lacking. An enzyme catalyzing sulfite oxidation using Nitro-blue tetrazolium as artificial electron acceptor was extracted from the membrane fraction of Aquifex aeolicus. The purified enzyme is a dimer of trimer (αβγ)2 of about 390 kDa. The KM for sulfite and kcat values were 34 μM and 567 s−1 respectively, at pH 8.3 and 55 °C. We furthermore showed that SoeABC reduces a UQ10 analogue, the decyl-ubiquinone, as well, with a KM of 2.6 μM and a kcat of 52.9 s−1. It seems to specifically oxidize sulfite but can work in the reverse direction, reduction of sulfur or tetrathionate, using reduced methyl viologen as electron donor. The close phylogenetic relationship of Soe with sulfur and tetrathionate reductases that we established, perfectly explains this enzymatic ability, although its bidirectionality in vivo still needs to be clarified. Oxygen-consumption measurements confirmed that electrons generated by sulfite oxidation in the cytoplasm enter the respiratory chain at the level of quinones.

Lack of Periplasmic Non-heme Protein SorA Increases Shewanella decolorationis Current Generation.

Bacterial extracellular electron transport (EET) plays an important role in many natural and engineering processes. Some periplasmic non-heme redox proteins usually coexist with c-type cytochromes (CTCs) during the EET process. However, in contrast to CTCs, little is known about the roles of these non-heme redox proteins in EET. In this study, the transcriptome of Shewanella decolorationis S12 showed that the gene encoding a periplasmic sulfite dehydrogenase molybdenum-binding subunit SorA was significantly up-regulated during electrode respiration in microbial fuel cells (MFCs) compared with that during azo-dye reduction. The maximum current density of MFCs catalyzed by a mutant strain lacking SorA (ΔsorA) was 25% higher than that of wild strain S12 (20 vs. 16 μA/cm2). Both biofilm formation and the current generation of the anodic biofilms were increased by the disruption of sorA, which suggests that the existence of SorA in S. decolorationis S12 inhibits electrode respiration. In contrast, disruption of sorA had no effect on respiration by S. decolorationis S12 with oxygen, fumarate, azo dye, or ferric citrate as electron acceptors. This is the first report of the specific effect of a periplasmic non-heme redox protein on EET to electrode and provides novel information for enhancing bacterial current generation.

Structural evidence for a reaction intermediate mimic in the active site of a sulfite dehydrogenase.

By combining X-ray crystallography, electron paramagnetic resonance techniques and density functional theory-based modelling, we provide evidence for a direct coordination of the product analogue, phosphate, to the molybdenum active site of a sulfite dehydrogenase. This interaction is mimicking the still experimentally uncharacterized reaction intermediate proposed to arise during the catalytic cycle of this class of enzymes. This work opens new perspectives for further deciphering the reaction mechanism of this nearly ubiquitous class of oxidoreductases.

The central active site arginine in sulfite oxidizing enzymes alters kinetic properties by controlling electron transfer and redox interactions

A central conserved arginine, first identified as a clinical mutation leading to sulfite oxidase deficiency, is essential for catalytic competency of sulfite oxidizing molybdoenzymes, but the molecular basis for its effects on turnover and substrate affinity have not been fully elucidated.We have used a bacterial sulfite dehydrogenase, SorT, which lacks an internal heme group, but transfers electrons to an external, electron accepting cytochrome, SorU, to investigate the molecular functions of this arginine residue (Arg78). Assay of the SorT Mo centre catalytic competency in the absence of SorU showed that substitutions in the central arginine (R78Q, R78K and R78M mutations) only moderately altered SorT catalytic properties, except for R78M which caused significant reduction in SorT activity. The substitutions also altered the Mo-centre redox potentials (MoVI/V potential lowered by ca. 60–80mV). However, all Arg78 mutations significantly impaired the ability of SorT to transfer electrons to SorU, where activities were reduced 17 to 46-fold compared to SorTWT, precluding determination of kinetic parameters. This was accompanied by the observation of conformational changes in both the introduced Gln and Lys residues in the crystal structure of the enzymes. Taking into account data collected by others on related SOE mutations we propose that the formation and maintenance of an electron transfer complex between the Mo centre and electron accepting heme groups is the main function of the central arginine, and that the reduced turnover and increases in KMsulfite are caused by the inefficient operation of the oxidative half reaction of the catalytic cycle in enzymes carrying these mutations.

Bioelectrocatalysis of Sulfite Dehydrogenase from Sinorhizobium meliloti with Its Physiological Cytochrome Electron Partner

AbstractWe demonstrate electrochemically driven catalytic voltammetry of the Mo‐dependent sulfite dehydrogenase (SorT) from the α‐Proteobacterium Sinorhizobium meliloti with its physiological electron acceptor, the c‐type cytochrome (SorU), with both proteins co‐adsorbed on a chemically modified Au working electrode. Both SorT and SorU were constrained under a perm‐selective dialysis membrane with the biopolymer chitosan as a co‐adsorbate, while the electrode was modified with a 3‐mercaptopropionate self‐assembled monolayer cast on the Au electrode. Cyclic voltammetry of the SorU protein reveals a well‐defined quasireversible FeIII/II redox couple at +130 mV versus NHE in 100 mM phosphate buffer solution (pH 7.0). Introduction of wild‐type sulfite dehydrogenase (SorTWT) and sulfite transforms this transient SorU voltammetric response into a sigmoidal catalytic wave, which increases with sulfite concentration before eventually saturating. In addition to the wild‐type enzyme, the variants SorTR78K, SorTR78M, and SorTR78Q were also examined electrochemically in an effort to better understand the role of amino acid residue Arg78, which is in the vicinity of the Mo active site of SorT.

Comparative Genome Analysis of Three Thiocyanate Oxidizing Thioalkalivibrio Species Isolated from Soda Lakes.

Thiocyanate is a C1 compound containing carbon, nitrogen, and sulfur. It is a (by)product in a number of natural and industrial processes. Because thiocyanate is toxic to many organisms, including humans, its removal from industrial waste streams is an important problem. Although a number of bacteria can use thiocyanate as a nitrogen source, only a few can use it as an electron donor. There are two distinct pathways to use thiocyanate: (i) the “carbonyl sulfide pathway,” which has been extensively studied, and (ii) the “cyanate pathway,” whose key enzyme, thiocyanate dehydrogenase, was recently purified and studied. Three species of Thioalkalivibrio, a group of haloalkaliphilic sulfur-oxidizing bacteria isolated from soda lakes, have been described as thiocyanate oxidizers: (i) Thioalkalivibrio paradoxus (“cyanate pathway”), (ii) Thioalkalivibrio thiocyanoxidans (“cyanate pathway”) and (iii) Thioalkalivibrio thiocyanodenitrificans (“carbonyl sulfide pathway”). In this study we provide a comparative genome analysis of these described thiocyanate oxidizers, with genomes ranging in size from 2.5 to 3.8 million base pairs. While focusing on thiocyanate degradation, we also analyzed the differences in sulfur, carbon, and nitrogen metabolism. We found that the thiocyanate dehydrogenase gene is present in 10 different Thioalkalivibrio strains, in two distinct genomic contexts/genotypes. The first genotype is defined by having genes for flavocytochrome c sulfide dehydrogenase upstream from the thiocyanate dehydrogenase operon (present in two strains including the type strain of Tv. paradoxus), whereas in the second genotype these genes are located downstream, together with two additional genes of unknown function (present in eight strains, including the type strains of Tv. thiocyanoxidans). Additionally, we found differences in the presence/absence of genes for various sulfur oxidation pathways, such as sulfide:quinone oxidoreductase, dissimilatory sulfite reductase, and sulfite dehydrogenase. One strain (Tv. thiocyanodenitrificans) lacks genes encoding a carbon concentrating mechanism and none of the investigated genomes were shown to contain known bicarbonate transporters. This study gives insight into the genomic variation of thiocyanate oxidizing bacteria and may lead to improvements in the application of these organisms in the bioremediation of industrial waste streams.

Mechanism of electron transport during thiosulfate oxidation in an obligately mixotrophic bacterium Thiomonas bhubaneswarensis strain S10 (DSM 18181T).

This study describes the thiosulfate-supported respiratory electron transport activity of Thiomonas bhubaneswarensis strain S10 (DSM 18181T). Whole-genome sequence analysis revealed the presence of complete sox (sulfur oxidation) gene cluster (soxCDYZAXB) including the sulfur oxygenase reductase (SOR), sulfide quinone reductase (SQR), sulfide dehydrogenase (flavocytochrome c (fcc)), thiosulfate dehydrogenase (Tsd), sulfite dehydrogenase (SorAB), and intracellular sulfur oxidation protein (DsrE/DsrF). In addition, genes encoding respiratory electron transport chain components viz. complex I (NADH dehydrogenase), complex II (succinate dehydrogenase), complex III (ubiquinone-cytochrome c reductase), and various types of terminal oxidases (cytochrome c and quinol oxidase) were identified in the genome. Using site-specific electron donors and inhibitors and by analyzing the cytochrome spectra, we identified the shortest thiosulfate-dependent electron transport chain in T. bhubaneswarensis DSM 18181T. Our results showed that thiosulfate supports the electron transport activity in a bifurcated manner, donating electrons to quinol (bd) and cytochrome c (Caa 3 ) oxidase; these two sites (quinol oxidase and cytochrome c oxidase) also showed differences in their phosphate esterification potential (oxidative phosphorylation efficiency (P/O)). Further, it was evidenced that the substrate-level phosphorylation is the major contributor to the total energy budget in this bacterium.

Catalytic voltammetry of the molybdoenzyme sulfite dehydrogenase from Sinorhizobium meliloti.

Sulfite dehydrogenase from the soil bacterium Sinorhizobium meliloti (SorT) is a periplasmic, homodimeric molybdoenzyme with a molecular mass of 78 kDa. It differs from most other well studied sulfite oxidizing enzymes, as it bears no heme cofactor. SorT does not readily reduce ferrous horse heart cytochrome c which is the preferred electron acceptor for vertebrate sulfite oxidases. In the present study, ferrocene methanol (FM) (in its oxidized ferrocenium form) was utilized as an artificial electron acceptor for the catalytic SorT sulfite oxidation reaction. Cyclic voltammetry of FM was used to generate the active form of the mediator at the electrode surface. The FM-mediated catalytic sulfite oxidation by SorT was investigated by two different voltammetric methods, namely, (i) SorT freely diffusing in solution and (ii) SorT confined to a thin layer at the electrode surface by a semipermeable dialysis membrane. A single set of rate and equilibrium constants was used to simulate the catalytic voltammograms performed under different sweep rates and with various concentrations of sulfite and FM which provides new insights into the kinetics of the SorT catalytic mechanism. Further, we were able to model the role of the dialysis membrane in the kinetics of the overall catalytic system.

Sulfite oxidation in the purple sulfur bacterium Allochromatium vinosum: identification of SoeABC as a major player and relevance of SoxYZ in the process

In phototrophic sulfur bacteria, sulfite is a well-established intermediate during reduced sulfur compound oxidation. Sulfite is generated in the cytoplasm by the reverse-acting dissimilatory sulfite reductase DsrAB. Many purple sulfur bacteria can even use externally available sulfite as a photosynthetic electron donor. Nevertheless, the exact mode of sulfite oxidation in these organisms is a long-standing enigma. Indirect oxidation in the cytoplasm via adenosine-5'-phosphosulfate (APS) catalysed by APS reductase and ATP sulfurylase is neither generally present nor essential. The inhibition of sulfite oxidation by tungstate in the model organism Allochromatium vinosum indicated the involvement of a molybdoenzyme, but homologues of the periplasmic molybdopterin-containing SorAB or SorT sulfite dehydrogenases are not encoded in genome-sequenced purple or green sulfur bacteria. However, genes for a membrane-bound polysulfide reductase-like iron-sulfur molybdoprotein (SoeABC) are universally present. The catalytic subunit of the protein is predicted to be oriented towards the cytoplasm. We compared the sulfide- and sulfite-oxidizing capabilities of A. vinosum WT with single mutants deficient in SoeABC or APS reductase and the respective double mutant, and were thus able to prove that SoeABC is the major sulfite-oxidizing enzyme in A. vinosum and probably also in other phototrophic sulfur bacteria. The genes also occur in a large number of chemotrophs, indicating a general importance of SoeABC for sulfite oxidation in the cytoplasm. Furthermore, we showed that the periplasmic sulfur substrate-binding protein SoxYZ is needed in parallel to the cytoplasmic enzymes for effective sulfite oxidation in A. vinosum and provided a model for the interplay between these systems despite their localization in different cellular compartments.

Sinorhizobium meliloti Sigma Factors RpoE1 and RpoE4 Are Activated in Stationary Phase in Response to Sulfite

Rhizobia are soil bacteria able to establish a nitrogen-fixing symbiosis with legume plants. Both in soil and in planta, rhizobia spend non-growing periods resembling the stationary phase of in vitro-cultured bacteria. The primary objective of this work was to better characterize gene regulation in this biologically relevant growth stage in Sinorhizobium meliloti. By a tap-tag/mass spectrometry approach, we identified five sigma factors co-purifying with the RNA polymerase in stationary phase: the general stress response regulator RpoE2, the heat shock sigma factor RpoH2, and three extra-cytoplasmic function sigma factors (RpoE1, RpoE3 and RpoE4) belonging to the poorly characterized ECF26 subgroup. We then showed that RpoE1 and RpoE4 i) are activated upon metabolism of sulfite-generating compounds (thiosulfate and taurine), ii) display overlapping regulatory activities, iii) govern a dedicated sulfite response by controlling expression of the sulfite dehydrogenase SorT, iv) are activated in stationary phase, likely as a result of endogenous sulfite generation during bacterial growth. We showed that SorT is required for optimal growth of S. meliloti in the presence of sulfite, suggesting that the response governed by RpoE1 and RpoE4 may be advantageous for bacteria in stationary phase either by providing a sulfite detoxification function or by contributing to energy production through sulfite respiration. This paper therefore reports the first characterization of ECF26 sigma factors, the first description of sigma factors involved in control of sulphur metabolism, and the first indication that endogenous sulfite may act as a signal for regulation of gene expression upon entry of bacteria in stationary phase.

Metagenomic and Biochemical Characterizations of Sulfur Oxidation Metabolism in Uncultured Large Sausage-Shaped Bacterium in Hot Spring Microbial Mats

So-called “sulfur-turf” microbial mats in sulfide containing hot springs (55–70°C, pH 7.3–8.3) in Japan were dominated by a large sausage-shaped bacterium (LSSB) that is closely related to the genus Sulfurihydrogenibium. Several previous reports proposed that the LSSB would be involved in sulfide oxidation in hot spring. However, the LSSB has not been isolated yet, thus there has been no clear evidence showing whether it possesses any genes and enzymes responsible for sulfide oxidation. To verify this, we investigated sulfide oxidation potential in the LSSB using a metagenomic approach and subsequent biochemical analysis. Genome fragments of the LSSB (a total of 3.7 Mb sequence including overlapping fragments) were obtained from the metagenomic fosmid library constructed from genomic DNA of the sulfur-turf mats. The sequence annotation clearly revealed that the LSSB possesses sulfur oxidation-related genes coding sulfide dehydrogenase (SD), sulfide-quinone reductase and sulfite dehydrogenase. The gene encoding SD, the key enzyme for sulfide oxidation, was successfully cloned and heterologously expressed in Escherichia coli. The purified recombinant enzyme clearly showed SD activity with optimum temperature and pH of 60°C and 8.0, respectively, which were consistent with the environmental conditions in the hot spring where the sulfur-turf thrives. Furthermore, the affinity of SD to sulfide was relatively high, which also reflected the environment where the sulfide could be continuously supplied. This is the first report showing that the LSSB harbors sulfide oxidizing metabolism adapted to the hot spring environment and can be involved in sulfide oxidation in the sulfur-turf microbial mats.

Roseobacter clade bacteria are abundant in coastal sediments and encode a novel combination of sulfur oxidation genes

Roseobacter clade bacteria (RCB) are abundant in marine bacterioplankton worldwide and central to pelagic sulfur cycling. Very little is known about their abundance and function in marine sediments. We investigated the abundance, diversity and sulfur oxidation potential of RCB in surface sediments of two tidal flats. Here, RCB accounted for up to 9.6% of all cells and exceeded abundances commonly known for pelagic RCB by 1000-fold as revealed by fluorescence in situ hybridization (FISH). Phylogenetic analysis of 16S rRNA and sulfate thiohydrolase (SoxB) genes indicated diverse, possibly sulfur-oxidizing RCB related to sequences known from bacterioplankton and marine biofilms. To investigate the sulfur oxidation potential of RCB in sediments in more detail, we analyzed a metagenomic fragment from a RCB. This fragment encoded the reverse dissimilatory sulfite reductase (rDSR) pathway, which was not yet found in RCB, a novel type of sulfite dehydrogenase (SoeABC) and the Sox multi-enzyme complex including the SoxCD subunits. This was unexpected as soxCD and dsr genes were presumed to be mutually exclusive in sulfur-oxidizing prokaryotes. This unique gene arrangement would allow a metabolic flexibility beyond known sulfur-oxidizing pathways. We confirmed the presence of dsrA by geneFISH in closely related RCB from an enrichment culture. Our results show that RCB are an integral part of the microbial community in marine sediments, where they possibly oxidize inorganic and organic sulfur compounds in oxic and suboxic sediment layers.

The X-ray crystal structure of a pseudoazurin from Sinorhizobium meliloti

The X-ray crystal structure of oxidised pseudoazurin from the denitrifying plant symbiotic bacterium Sinorhizobium meliloti (SmPAz2) has been solved to a resolution of 2.0Å. The pseudoazurin from Sinorhizobium sp. is unusual as it forms an operon with a sulfite dehydrogenase enzyme, rather than a Cu nitrite reductase. Examination of the structure reveals that the geometric parameters of the Type I Cu site in SmPAz2 correlate with observed features in the electronic spectrum of the protein. Comparison of the structure of SmPAz2 with those of pseudoazurins from five other bacterial species shows that the surface of SmPAz2 bears a conserved hydrophobic patch encircled by positively-charged residues, which may serve as a recognition site for its redox partners.

Molecular genetics and biochemistry of N-acetyltaurine degradation by Cupriavidus necator H16

Cupriavidus necator H16 (DSM 428), whose genome has been sequenced, was found to degrade N-acetyltaurine as a sole source of carbon and energy for growth. Utilization of the compound was quantitative. The degradative pathway involved an inducible N-acetyltaurine amidohydrolase (NaaS), which catalysed the cleavage of N-acetyltaurine to acetate and taurine. The degradation of the latter compound is via an inducible, degradative pathway that involves taurine dehydrogenase [EC 1.4.2.-], sulfoacetaldehyde acetyltransferase [EC 2.3.3.15], phosphotransacetylase [EC 2.4.1.8], a sulfite exporter [TC 9.A.29.2.1] and sulfite dehydrogenase [EC 1.8.2.1]. Induction of the expression of representative gene products, encoded by at least four gene clusters, was confirmed biochemically. The acetate released by NaaS was activated to acetyl-CoA by an inducible acetate-CoA ligase [EC 6.2.1.1]. NaaS was purified to homogeneity; it had a K(m) value of 9.4 mM for N-acetyltaurine, and it contained tightly bound Zn and Fe atoms. The denatured enzyme has a molecular mass of about 61 kDa (determined by SDS-PAGE) and the native enzyme was apparently monomeric. Peptide-mass fingerprinting identified the locus tag as H16_B0868 in a five-gene cluster, naaROPST (H16_B0865-H16_B0869). The cluster presumably encodes a LysR-type transcriptional regulator (NaaR), a membrane protein (NaaO), a solute : sodium symporter-family permease [TC 2.A.21] (NaaP), the metal-dependent amidohydrolase (NaaS) and a putative metallochaperone (COG0523) (NaaT). Reverse-transcription PCR indicated that naaOPST were inducibly transcribed.

Five novel acid-tolerant oligotrophic thiosulfate-metabolizing chemolithotrophic acid mine drainage strains affiliated with the genus Burkholderia of Betaproteobacteria and identification of two novel soxB gene homologues

Five acid-tolerant thiosulfate-metabolizing bacteria were isolated from acid mine drainage samples from Garubathan, India. 16S rRNA gene analysis revealed that the strains were affiliated with the genus Burkholderia of the class of Betaproteobacteria. Comparative 16S rRNA gene sequence analyses indicated that the strains designated as GAH1 and GAH2 produced a separate phylogenetic branch having Burkholderia pyrrocinia ATCC 51958 T (96–98%) as the closest relative. Strains GAH4 and Burkholderia tropica Ppe8 T (93%) branched out separately in the phylogenetic tree. Strain GMX2 was most closely related to Burkholderia cepacia ATCC 25417 T (99.6%) and Burkholderia vietnamiensis LMG 10929 T (99%). Strain GAH5 was most closely related to B. pyrrocinia ATCC 51958 T (98%). Oligotrophy has been demonstrated in all AMD strains of Burkholderia spp. All strains showed chemolithoautotrophic and mixotrophic growth in thiosulfate. Furthermore, cell-free extracts of all test strains possessed thiosulfate and sulfite dehydrogenase activities. Phylogenetic analysis of the soxB gene revealed that GAH4 and GAH2 strains formed a novel cluster, Betaproteobacteria II, having highest similarity with Allochromatium vinosum, a member of Gammaproteobacteria II.