Sulfide Dehydrogenase Research Articles

Hydrogen Sulfide Oxidizing Microbiome in Biogas-Stream Fed Biofilter in Palm Oil Factory

Hydrogen sulfide (H2S) is highly corrosive to electric generators, which is the main problem of biogas utilization. The industrial scale of the biofilter system relies on the performance of sulfide-oxidizing bacteria (SOB) via the activity of sulfur oxidation (soxABXYZ) and flavocytochrome sulfide dehydrogenase (fccAB) enzymes to reduce to a concentration below 100 ppm before using in industrial machinery. The main purpose of this research is to investigate the SOB community in full-scale H2S removal and their gene expression (fccAB and soxABXYZ) associated with H2S elimination efficiency. In this study, SOB communities were obtained from 2 sampling sites of the full-scale biofilter of palm oil factory (PPG), comprising starting sludge (PPG1) and recirculating sludge (PPG2). The abundance of SOB strains was examined by next-generation sequencing analysis (NGS) based on the 16S rRNA gene. The changes in the expression of genes involved in sulfur oxidation, namely soxABXYZ, and fccAB, between the 2 sampling sites were evaluated by using a comparative genomic hybridization (CGH) microarray. The results indicate that the high abundance of SOB genera that could play a vital role in biofilters belonged mainly to Sulfurovum, Paracoccus, Acidihalobacter, Acidithiobacillus, Thioalkalispira, Thiofaba, Caldisericum, Bacillus, were rapidly increased in the biofilter tank. Interestingly, expressions of soxAXYZ gene cluster at PPG2 were increased in Paracoccus pantotrophus J40 and Paracoccus alkenifer DSM 11593 for 1.1188 and 1.0518-fold, respectively, while in Acidihalobacter prosperus F5, the expression of fccAB genes was up to 1.3704 fold in comparison with PPG1. Increasing both relative abundance and gene expressions at PPG2 were correlated with 95% H2S removal efficiency. Hence, stabilization of the SOB microbiome is vital to H2S removal in industrial-scale biogas applications.

Diversity of Hydrogen Sulfide Oxidizers in Biogas-Stream Fed Bioscrubber in Seafood Factory

Corrosive and harmful hydrogen sulfide (H2S) are major obstacles to biogas utilization. Bioscrubber system is the one of the most popular tecnologies for removing H2S from biogas. Sulfur oxidizing bacteria (SOB) play an important role in controlling the efficiency and stability of bioscrubber systems through sulfur oxidation (soxABXYZ) and flavocytochrome sulfide dehydrogenase (fccAB) enzyme catalyses. The main purpose of this research is to analyse microbial diversity and gene expression pattern of SOBs related to H2S oxidation. In this study, SOB population were collected from the full-scale bioscrubber system of the seafood factory (TKF). The richness and diversity of SOB strains were analyzed using next-generation sequencing analysis (NGS) based on 16S rRNA gene. DNA microarray was used to examine the expression of soxABXYZ and fccAB genes involved in the H2S oxidation. A number of readable sequences indicate SOB diversity and richness that cover the entire population of bacteria in the TKF factory. The phylum proteobacteria was found covered up to 60% of total bacteria. Interestingly, the highest abundance genus of SOB coexisting in biocrubber to oxidize H2S in biogas was Pseudomonas, MWH-UniP1_aquatic, Smithella, Thioalkalimicrobium, Hydrogenophaga and Acinetobacter. Paracoccus sediminis, Pseudomonas stutzeri and Paracoccus denitrificans were found having the soxAXYZ and soxB gene expression level up to 0.2754-fold, 0.0808-fold and 0.3819-fold respectively. On the other hand, the expression of fccAB genes of P. denitrificans was increased up to 0.3819-fold. Altogether, the predominance of these species-strains was associated with the excellent H2S removal efficiency of 99.3% in the TKF factory.

Heterotrophic bacteria dominate the sulfide oxidation process in coastal sediments

Sulfate reduction and sulfur oxidation are very active in coastal sediments. Together they shape the biogeochemistry and microbial ecology at hot places of organic matter metabolism. Compared to the well-studied sulfate reduction process, sulfur oxidation process is relatively lack of investigation. For a long time, autotrophic/mixotrophic bacteria were thought to be the main sulfur oxidation strains in sediments. Herein, we investigated the distribution of reduced sulfur compounds in differently layers of coastal sediments at the Yellow sea. We found that sulfide (H2S), sulfane sulfur (S0), and thiosulfate mainly accumulated in deep anaerobic sediments and were mostly oxidized in surface sediments (∼3 cm in depth). Bacterial community analysis indicated that heterotrophic bacteria were dominating species in surface sediments. Metatranscriptome analysis showed that transcripts of flavocytochrome c sulfide dehydrogenase (FCC), persufide dioxygenase (PDO), and sulfite-oxidizing enzyme (SOE) were abundant in surface sediments. In addition, using a Roseobacterium (Ruegeria pomeroyi DSS-3) as the model strain, we investigated how FCC, PDO, and SOE work together to oxidize H2S to sulfate. This study clarified that heterotrophic bacteria are the main sulfur oxidation strains in coastal sediments.

Enhancement of bio-S0 recovery and revealing the inhibitory effect on microorganisms under high sulfide loading

Biodesulfurization is a mature technology, but obtaining biosulfur (S0) that can be easily settled naturally is still a challenge. Increasing the sulfide load is one of the known methods to obtain better settling of S0. However, the inhibitory effect of high levels of sulfide on microbes has also not been well studied. We constructed a high loading sulfide (1.55–10.86 kg S/m3/d) biological removal system. 100% sulfide removal and 0.56–2.53 kg S/m3/d S0 (7.0 ± 0.09–16.4 ± 0.25 μm) recovery were achieved at loads of 1.55–7.75 kg S/m3/d. Under the same load, S0 in the reflux sedimentation tank, which produced larger S0 particles (24.2 ± 0.73–53.8 ± 0.70 μm), increased the natural settling capacity and 45% recovery. For high level sulfide inhibitory effect, we used metagenomics and metatranscriptomics analyses. The increased sulfide load significantly inhibited the expression of flavin cytochrome c sulfide dehydrogenase subunit B (fccB) (Decreased from 615 ± 75 to 30 ± 5 TPM). At this time sulfide quinone reductase (SQR) (324 ± 185–1197 ± 51 TPM) was mainly responsible for sulfide oxidation and S0 production. When the sulfide load reached 2800 mg S/L, the SQR (730 ± 100 TPM) was also suppressed. This resulted in the accumulation of sulfide, causing suppression of carbon sequestration genes (Decreased from 3437 ± 842 to 665 ± 175 TPM). Other inhibitory effects included inhibition of microbial respiration, production of reactive oxygen species, and DNA damage. More sulfide-oxidizing bacteria (SOB) and newly identified potential SOB (99.1%) showed some activity (77.6%) upon sulfide accumulation. The main microorganisms in the sulfide accumulation environment were Thiomicrospiracea and Burkholderiaceae, whose sulfide oxidation capacity and respiration were not significantly inhibited. This study provides a new approach to enhance the natural sedimentation of S0 and describes new microbial mechanisms for the inhibitory effects of sulfide.

Reconstructing hepatic metabolic profile and glutathione-mediated metabolic fate of acrylamide

The toxicity of acrylamide (AA) has continuously attracted wide concerns as its extensive presence from both environmental and dietary sources. However, its hepatic metabolic transformation and metabolic fate still remain unclear. This study aims to unravel the metabolic profile and glutathione (GSH) mediated metabolic fate of AA in liver of rats under the dose-dependent exposure. We found that exposure to AA dose-dependently alters the binding of AA and GSH and the generation of mercapturic acid adducts, while liver as a target tissue bears the metabolic transformation of AA via regulating GSH synthesis and consumption pathways, in which glutamine synthase (GSS), cytochrome P450 2E1 (CYP2E1), and glutathione S-transferase P1 (GSTP1) play a key role. In response to high- and low-dose exposures to AA, there were significant differences in liver of rats, including the changes in GSH and cysteine (CYS) activities and the conversion ratio of AA to glycidamide (GA), and liver can affect the transformation of AA by regulating the GSH-mediated metabolic pathway. Low-dose exposure to AA activates GSH synthesis pathway in liver and upregulates GSS activity and CYS content with no change in γ-glutamyl transpeptidase 1 (GGT1) activity. High-dose exposure to AA activates the detoxification pathway of GSH and increases GSH consumption by upregulating GSTP1 activity. In addition, molecular docking results showed that most of the metabolic molecules transformed by AA and GA other than themselves can closely bind to GSTP1, GSS, GGT1, N-acetyltransferase 8, and dimethyl sulfide dehydrogenase 1. The binding of AA-GSH and GA-GSH to GSTP1 and CYP2E1 enzymes determine the tendentiousness between toxicity and detoxification of AA, which exerts a prospective avenue for targeting protective role of hepatic enzymes against in vivo toxicity of AA.

Comparative genomics reveals extensive intra-species genetic divergence of the prevalent gut commensal Ruminococcus gnavus.

Ruminococcus gnavus is prevalent in the intestines of humans and animals, and ambiguities have been reported regarding its relations with the development of diseases and host well-being. We postulate the ambiguities of its function in different cases may be attributed to strain-level variability of genomic features of R. gnavus. We performed comparative genomic and pathogenicity prediction analysis on 152 filtered high-quality genomes, including 4 genomes of strains isolated from healthy adults in this study. The mean G+C content of genomes of R. gnavus was 42.73±0.33 mol%, and the mean genome size was 3.46±0.34 Mbp. Genome-wide evolutionary analysis revealed R. gnavus genomes were divided into three major phylogenetic clusters. Pan-core genome analysis revealed that there was a total of 28 072 predicted genes, and the core genes, soft-core genes, shell genes and cloud genes accounted for 3.74 % (1051/28 072), 1.75 % (491/28 072), 9.88 % (2774/28 072) and 84.63 % (23 756/28 072) of the total genes, respectively. The small proportion of core genes reflected the wide divergence among R. gnavus strains. We found certain coding sequences with determined health benefits (such as vitamin production and arsenic detoxification), whilst some had an implication of health adversity (such as sulfide dehydrogenase subunits). The functions of the majority of core genes were unknown. The most widespread genes functioning in antibiotic resistance and virulence are tetO (tetracycline-resistance gene, present in 75 strains) and cps4J (capsular polysaccharide biosynthesis protein Cps4J encoding gene, detected in 3 genomes), respectively. Our results revealed genomic divergence and the existence of certain safety-relevant factors of R. gnavus. This study provides new insights for understanding the genomic features and health relevance of R. gnavus, and raises concerns regarding predicted prevalent pathogenicity and antibiotic resistance among most of the strains.

Metagenomics and metatranscriptomics uncover the microbial community associated with high S0 production in a denitrifying desulfurization granular sludge reactor

The denitrification desulfurization process is a promising technology for elemental sulfur (S0) production from sulfide containing wastewater. However, the microbial community associated with high S0 production still is not well studied. This study describes an efficient denitrification S0 production bioreactor based on inoculation with anaerobic granular sludge. At an optimal S/N molar ratio of 7:2, 80 % of the influent sulfide was transformed to high quality elemental sulfur with a purity of 92.5% while the total inorganic nitrogen removal efficiency was stable at ∼80%. Metatranscriptomic analysis found that community expression of the gene encoding the sulfide-quinone reductase (SQR) was 10-fold greater than that of the flavocytochrome-c sulfide dehydrogenase subunit B (fccB). Moreover, the expression level of SQR was also significantly higher than the Dsr gene encoding for dissimilatory sulfate reductase, which encodes a critical S0 oxidation enzyme. Metagenomic binning analysis confirmed that sulfide-oxidizing bacteria (SOB) utilizing SQR were common in the community and most likely accounted for high S0 production. An unexpected enrichment in methanogens and high expression activity of bacteria carrying out Stickland fermentation as well as in other bacteria with reduced genomes indicated a complex community supporting stable sulfide oxidation to S0, likely aiding in performance stability. This study establishes this treatment approach as an alternative biotechnology for sulfide containing wastewater treatment and sheds light on the microbial interactions associated with high S0 production.

Catalytic Properties of Flavocytochrome c Sulfide Dehydrogenase from Haloalkaliphilic Bacterium Thioalkalivibrio paradoxus.

Flavocytochrome c sulfide dehydrogenase (FCC) is one of the central enzymes of the respiratory chain in sulfur-oxidizing bacteria. FCC catalyzes oxidation of sulfide and polysulfide ions to elemental sulfur accompanied by electron transfer to cytochrome c. The catalytically active form of the enzyme is a non-covalently linked heterodimer composed of flavin- and heme-binding subunits. The Thioalkalivibrio paradoxus ARh1 genome contains five copies of genes encoding homologous FCCs with an amino acid sequence identity from 36 to 54%. When growing on thiocyanate or thiosulfate as the main energy source, the bacterium synthesizes products of different copies of FCC genes. In this work, we isolated and characterized FCC synthesized during the growth of Tv. paradoxus on thiocyanate. FCC was shown to oxidize exclusively sulfide but not other reduced sulfur compounds, such as thiosulfate, sulfite, tetrathionate, and sulfur, and it also does not catalyze the reverse reaction of sulfur reduction to sulfide. Kinetic parameters of the sulfide oxidation reaction are characterized.

The Heterotrophic Bacterium Cupriavidus pinatubonensis JMP134 Oxidizes Sulfide to Sulfate with Thiosulfate as a Key Intermediate.

Heterotrophic bacteria actively participate in the biogeochemical cycle of sulfur on Earth. The heterotrophic bacterium Cupriavidus pinatubonensis JMP134 contains several enzymes involved in sulfur oxidation, but how these enzymes work together to oxidize sulfide in the bacterium has not been studied. Using gene-deletion and whole-cell assays, we determined that the bacterium uses sulfide:quinone oxidoreductase to oxidize sulfide to polysulfide, which is further oxidized to sulfite by persulfide dioxygenase. Sulfite spontaneously reacts with polysulfide to produce thiosulfate. The sulfur-oxidizing (Sox) system oxidizes thiosulfate to sulfate. Flavocytochrome c sulfide dehydrogenase enhances thiosulfate oxidation by the Sox system but couples with the Sox system for sulfide oxidation to sulfate in the absence of sulfide:quinone oxidoreductase. Thus, C. pinatubonensis JMP134 contains a main pathway and a contingent pathway for sulfide oxidation.IMPORTANCE We establish a new pathway of sulfide oxidation with thiosulfate as a key intermediate in Cupriavidus pinatubonensis JMP134. The bacterium mainly oxidizes sulfide by using sulfide:quinone oxidoreductase, persulfide dioxygenase, and the Sox system with thiosulfate as a key intermediate. Although the purified and reconstituted Sox system oxidizes sulfide, its rate of sulfide oxidation in C. pinatubonensis JMP134 is too low to be physiologically relevant. The findings reveal how these sulfur-oxidizing enzymes participate in sulfide oxidation in a single bacterium.

Genomic and Metabolic Insights into Denitrification, Sulfur Oxidation, and Multidrug Efflux Pump Mechanisms in the Bacterium Rhodoferax sediminis sp. nov.

This genus contains both phototrophs and nonphototrophic members. Here, we present a high-quality complete genome of the strain CHu59-6-5T, isolated from a freshwater sediment. The circular chromosome (4.39 Mbp) of the strain CHu59-6-5T has 64.4% G+C content and contains 4240 genes, of which a total of 3918 genes (92.4%) were functionally assigned to the COG (clusters of orthologous groups) database. Functional genes for denitrification (narGHJI, nirK and qnor) were identified on the genomes of the strain CHu59-6-5T, except for N2O reductase (nos) genes for the final step of denitrification. Genes (soxBXAZY) for encoding sulfur oxidation proteins were identified, and the FSD and soxF genes encoding the monomeric flavoproteins which have sulfide dehydrogenase activities were also detected. Lastly, genes for the assembly of two different RND (resistance-nodulation division) type efflux systems and one ABC (ATP-binding cassette) type efflux system were identified in the Rhodoferax sediminis CHu59-6-5T. Phylogenetic analysis based on 16S rRNA sequences and Average Nucleotide Identities (ANI) support the idea that the strain CHu59-6-5T has a close relationship to the genus Rhodoferax. A polyphasic study was done to establish the taxonomic status of the strain CHu59-6-5T. Based on these data, we proposed that the isolate be classified to the genus Rhodoferax as Rhodoferax sediminis sp. nov. with isolate CHu59-6-5T.

Metabolic Adaptation to Sulfur of Hyperthermophilic Palaeococcus pacificus DY20341T from Deep-Sea Hydrothermal Sediments.

The hyperthermo-piezophilic archaeon Palaeococcus pacificus DY20341T, isolated from East Pacific hydrothermal sediments, can utilize elemental sulfur as a terminal acceptor to simulate growth. To gain insight into sulfur metabolism, we performed a genomic and transcriptional analysis of Pa. pacificus DY20341T with/without elemental sulfur as an electron acceptor. In the 2001 protein-coding sequences of the genome, transcriptomic analysis showed that 108 genes increased (by up to 75.1 fold) and 336 genes decreased (by up to 13.9 fold) in the presence of elemental sulfur. Palaeococcus pacificus cultured with elemental sulfur promoted the following: the induction of membrane-bound hydrogenase (MBX), NADH:polysulfide oxidoreductase (NPSOR), NAD(P)H sulfur oxidoreductase (Nsr), sulfide dehydrogenase (SuDH), connected to the sulfur-reducing process, the upregulation of iron and nickel/cobalt transfer, iron–sulfur cluster-carrying proteins (NBP35), and some iron–sulfur cluster-containing proteins (SipA, SAM, CobQ, etc.). The accumulation of metal ions might further impact on regulators, e.g., SurR and TrmB. For growth in proteinous media without elemental sulfur, cells promoted flagelin, peptide/amino acids transporters, and maltose/sugar transporters to upregulate protein and starch/sugar utilization processes and riboflavin and thiamin biosynthesis. This indicates how strain DY20341T can adapt to different living conditions with/without elemental sulfur in the hydrothermal fields.

Identification of Proteins and Genes Expressed by Methylophaga thiooxydans During Growth on Dimethylsulfide and Their Presence in Other Members of the Genus.

Dimethylsulfide is a volatile organic sulfur compound that provides the largest input of biogenic sulfur from the oceans to the atmosphere, and thence back to land, constituting an important link in the global sulfur cycle. Microorganisms degrading DMS affect fluxes of DMS in the environment, but the underlying metabolic pathways are still poorly understood. Methylophaga thiooxydans is a marine methylotrophic bacterium capable of growth on DMS as sole source of carbon and energy. Using proteomics and transcriptomics we identified genes expressed during growth on dimethylsulfide and methanol to refine our knowledge of the metabolic pathways that are involved in DMS and methanol degradation in this strain. Amongst the most highly expressed genes on DMS were the two methanethiol oxidases driving the oxidation of this reactive and toxic intermediate of DMS metabolism. Growth on DMS also increased expression of the enzymes of the tetrahydrofolate linked pathway of formaldehyde oxidation, in addition to the tetrahydromethanopterin linked pathway. Key enzymes of the inorganic sulfur oxidation pathway included flavocytochrome c sulfide dehydrogenase, sulfide quinone oxidoreductase, and persulfide dioxygenases. A sulP permease was also expressed during growth on DMS. Proteomics and transcriptomics also identified a number of highly expressed proteins and gene products whose function is currently not understood. As the identity of some enzymes of organic and inorganic sulfur metabolism previously detected in Methylophaga has not been characterized at the genetic level yet, highly expressed uncharacterized genes provide new targets for further biochemical and genetic analysis. A pan-genome analysis of six available Methylophaga genomes showed that only two of the six investigated strains, M. thiooxydans and M. sulfidovorans have the gene encoding methanethiol oxidase, suggesting that growth on methylated sulfur compounds of M. aminisulfidivorans is likely to involve different enzymes and metabolic intermediates. Hence, the pathways of DMS-utilization and subsequent C1 and sulfur oxidation are not conserved across Methylophaga isolates that degrade methylated sulfur compounds.

Comparative Genomics of Thiohalobacter thiocyanaticus HRh1T and Guyparkeria sp. SCN-R1, Halophilic Chemolithoautotrophic Sulfur-Oxidizing Gammaproteobacteria Capable of Using Thiocyanate as Energy Source.

The genomes of Thiohalobacter thiocyanaticus and Guyparkeria (formerly known as Halothiobacillus) sp. SCN-R1, two gammaproteobacterial halophilic sulfur-oxidizing bacteria (SOB) capable of thiocyanate oxidation via the “cyanate pathway”, have been analyzed with a particular focus on their thiocyanate-oxidizing potential and sulfur oxidation pathways. Both genomes encode homologs of the enzyme thiocyanate dehydrogenase (TcDH) that oxidizes thiocyanate via the “cyanate pathway” in members of the haloalkaliphilic SOB of the genus Thioalkalivibrio. However, despite the presence of conservative motives indicative of TcDH, the putative TcDH of the halophilic SOB have a low overall amino acid similarity to the Thioalkalivibrio enzyme, and also the surrounding genes in the TcDH locus were different. In particular, an alternative copper transport system Cus is present instead of Cop and a putative zero-valent sulfur acceptor protein gene appears just before TcDH. Moreover, in contrast to the thiocyanate-oxidizing Thioalkalivibrio species, both genomes of the halophilic SOB contained a gene encoding the enzyme cyanate hydratase. The sulfur-oxidizing pathway in the genome of Thiohalobacter includes a Fcc type of sulfide dehydrogenase, a rDsr complex/AprAB/Sat for oxidation of zero-valent sulfur to sulfate, and an incomplete Sox pathway, lacking SoxCD. The sulfur oxidation pathway reconstructed from the genome of Guyparkeria sp. SCN-R1 was more similar to that of members of the Thiomicrospira-Hydrogenovibrio group, including a Fcc type of sulfide dehydrogenase and a complete Sox complex. One of the outstanding properties of Thiohalobacter is the presence of a Na+-dependent ATP synthase, which is rarely found in aerobic Prokaryotes.Overall, the results showed that, despite an obvious difference in the general sulfur-oxidation pathways, halophilic and haloalkaliphilic SOB belonging to different genera within the Gammaproteobacteria developed a similar unique thiocyanate-degrading mechanism based on the direct oxidative attack on the sulfane atom of thiocyanate.

Genomic analysis of the mesophilic Thermotogae genus Mesotoga reveals phylogeographic structure and genomic determinants of its distinct metabolism.

The genus Mesotoga, the only described mesophilic Thermotogae lineage, is common in mesothermic anaerobic hydrocarbon-rich environments. Besides mesophily, Mesotoga displays lineage-specific phenotypes, such as no or little H2 production and dependence on sulfur-compound reduction, which may influence its ecological role. We used comparative genomics of 18 Mesotoga strains (pairwise 16S rRNA identity >99%) and a transcriptome of M. prima to investigate how life at moderate temperatures affects phylogeography and to interrogate the genomic features of its lineage-specific metabolism. We propose that Mesotoga accomplish H2 oxidation and thiosulfate reduction using a sulfide dehydrogenase and a hydrogenase-complex and that a pyruvate:ferredoxin oxidoreductase acquired from Clostridia is responsible for oxidizing acetate. Phylogenetic analysis revealed three distinct Mesotoga lineages (89.6%-99.9% average nucleotide identity [ANI] within lineages, 79.3%-87.6% ANI between lineages) having different geographic distribution patterns and high levels of intra-lineage recombination but little geneflow between lineages. Including data from metagenomes, phylogeographic patterns suggest that geographical separation historically has been more important for Mesotoga than hyperthermophilic Thermotoga and we hypothesize that distribution of Mesotoga is constrained by their anaerobic lifestyle. Our data also suggest that recent anthropogenic activities and environments (e.g., wastewater treatment, oil exploration) have expanded Mesotoga habitats and dispersal capabilities.

Structure of the flavocytochrome c sulfide dehydrogenase associated with the copper-binding protein CopC from the haloalkaliphilic sulfur-oxidizing bacterium Thioalkalivibrio paradoxusARh 1.

Flavocytochrome c sulfide dehydrogenase from Thioalkalivibrio paradoxus (TpFCC) is a heterodimeric protein consisting of flavin- and monohaem c-binding subunits. TpFCC was co-purified and co-crystallized with the dimeric copper-binding protein TpCopC. The structure of the TpFCC-(TpCopC)2 complex was determined by X-ray diffraction at 2.6 Å resolution. The flavin-binding subunit of TpFCC is structurally similar to those determined previously, and the structure of the haem-binding subunit is similar to that of the N-terminal domain of dihaem FCCs. According to classification based on amino-acid sequence, TpCopC belongs to a high-affinity CopC subfamily characterized by the presence of a conserved His1-Xxx-His3 motif at the N-terminus. Apparently, a unique α-helix which is present in each monomer of TpCopC at the interface with TpFCC plays a key role in complex formation. The structure of the copper-binding site in TpCopC is similar to those in other known CopC structures. His3 is not involved in binding to the copper ion and is 6-7 Å away from this ion. Therefore, the His1-Xxx-His3 motif cannot be considered to be a key factor in the high affinity of CopC for copper(II) ions. It is suggested that the TpFCC-(TpCopC)2 heterotetramer may be a component of a large periplasmic complex that is responsible for thiocyanate metabolism.

Taxonomic distribution, structure/function relationship and metabolic context of the two families of sulfide dehydrogenases: SQR and FCSD

Hydrogen sulfide (H2S) is a versatile molecule with different functions in living organisms: it can work as a metabolite of sulfur and energetic metabolism or as a signaling molecule in higher Eukaryotes. H2S is also highly toxic since it is able to inhibit heme cooper oxygen reductases, preventing oxidative phosphorylation. Due to the fact that it can both inhibit and feed the respiratory chain, the immediate role of H2S on energy metabolism crucially relies on its bioavailability, meaning that studying the central players involved in the H2S homeostasis is key for understanding sulfide metabolism.Two different enzymes with sulfide oxidation activity (sulfide dehydrogenases) are known: flavocytochrome c sulfide dehydrogenase (FCSD), a sulfide:cytochrome c oxidoreductase; and sulfide:quinone oxidoreductase (SQR).In this work we performed a thorough bioinformatic study of SQRs and FCSDs and integrated all published data. We systematized several properties of these proteins: (i) nature of flavin binding, (ii) capping loops and (iii) presence of key amino acid residues. We also propose an update to the SQR classification system and discuss the role of these proteins in sulfur metabolism.

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.

Genomic Analysis of Caldithrix abyssi, the Thermophilic Anaerobic Bacterium of the Novel Bacterial Phylum Calditrichaeota.

The genome of Caldithrix abyssi, the first cultivated representative of a phylum-level bacterial lineage, was sequenced within the framework of Genomic Encyclopedia of Bacteria and Archaea (GEBA) project. The genomic analysis revealed mechanisms allowing this anaerobic bacterium to ferment peptides or to implement nitrate reduction with acetate or molecular hydrogen as electron donors. The genome encoded five different [NiFe]- and [FeFe]-hydrogenases, one of which, group 1 [NiFe]-hydrogenase, is presumably involved in lithoheterotrophic growth, three other produce H2 during fermentation, and one is apparently bidirectional. The ability to reduce nitrate is determined by a nitrate reductase of the Nap family, while nitrite reduction to ammonia is presumably catalyzed by an octaheme cytochrome c nitrite reductase εHao. The genome contained genes of respiratory polysulfide/thiosulfate reductase, however, elemental sulfur and thiosulfate were not used as the electron acceptors for anaerobic respiration with acetate or H2, probably due to the lack of the gene of the maturation protein. Nevertheless, elemental sulfur and thiosulfate stimulated growth on fermentable substrates (peptides), being reduced to sulfide, most probably through the action of the cytoplasmic sulfide dehydrogenase and/or NAD(P)-dependent [NiFe]-hydrogenase (sulfhydrogenase) encoded by the genome. Surprisingly, the genome of this anaerobic microorganism encoded all genes for cytochrome c oxidase, however, its maturation machinery seems to be non-operational due to genomic rearrangements of supplementary genes. Despite the fact that sugars were not among the substrates reported when C. abyssi was first described, our genomic analysis revealed multiple genes of glycoside hydrolases, and some of them were predicted to be secreted. This finding aided in bringing out four carbohydrates that supported the growth of C. abyssi: starch, cellobiose, glucomannan and xyloglucan. The genomic analysis demonstrated the ability of C. abyssi to synthesize nucleotides and most amino acids and vitamins. Finally, the genomic sequence allowed us to perform a phylogenomic analysis, based on 38 protein sequences, which confirmed the deep branching of this lineage and justified the proposal of a novel phylum Calditrichaeota.

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.

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.