Genome analyses of the carboxydotrophic sulfate-reducers Desulfotomaculum nigrificans and Desulfotomaculum carboxydivorans and reclassification of Desulfotomaculum caboxydivorans as a later synonym of Desulfotomaculum nigrificans.

Desulfotomaculum nigrificans and D. carboxydivorans are moderately thermophilic members of the polyphyletic spore-forming genus Desulfotomaculum in the family Peptococcaceae. They are phylogenetically very closely related and belong to 'subgroup a' of the Desulfotomaculum cluster 1. D. nigrificans and D. carboxydivorans have a similar growth substrate spectrum; they can grow with glucose and fructose as electron donors in the presence of sulfate. Additionally, both species are able to ferment fructose, although fermentation of glucose is only reported for D. carboxydivorans. D. nigrificans is able to grow with 20% carbon monoxide (CO) coupled to sulfate reduction, while D. carboxydivorans can grow at 100% CO with and without sulfate. Hydrogen is produced during growth with CO by D. carboxydivorans. Here we present a summary of the features of D. nigrificans and D. carboxydivorans together with the description of the complete genome sequencing and annotation of both strains. Moreover, we compared the genomes of both strains to reveal their differences. This comparison led us to propose a reclassification of D. carboxydivorans as a later heterotypic synonym of D. nigrificans.

Genome analysis of Desulfotomaculum gibsoniae strain Groll(T) a highly versatile Gram-positive sulfate-reducing bacterium.

Desulfotomaculum gibsoniae is a mesophilic member of the polyphyletic spore-forming genus Desulfotomaculum within the family Peptococcaceae. This bacterium was isolated from a freshwater ditch and is of interest because it can grow with a large variety of organic substrates, in particular several aromatic compounds, short-chain and medium-chain fatty acids, which are degraded completely to carbon dioxide coupled to the reduction of sulfate. It can grow autotrophically with H2 + CO2 and sulfate and slowly acetogenically with H2 + CO2, formate or methoxylated aromatic compounds in the absence of sulfate. It does not require any vitamins for growth. Here, we describe the features of D. gibsoniae strain Groll(T) together with the genome sequence and annotation. The chromosome has 4,855,529 bp organized in one circular contig and is the largest genome of all sequenced Desulfotomaculum spp. to date. A total of 4,666 candidate protein-encoding genes and 96 RNA genes were identified. Genes of the acetyl-CoA pathway, possibly involved in heterotrophic growth and in CO2 fixation during autotrophic growth, are present. The genome contains a large set of genes for the anaerobic transformation and degradation of aromatic compounds, which are lacking in the other sequenced Desulfotomaculum genomes.

A genomic view on syntrophic versus non-syntrophic lifestyle in anaerobic fatty acid degrading communities.

In sulfate-reducing and methanogenic environments complex biopolymers are hydrolyzed and degraded by fermentative micro-organisms that produce hydrogen, carbon dioxide and short chain fatty acids. Degradation of short chain fatty acids can be coupled to methanogenesis or to sulfate-reduction. Here we study from a genome perspective why some of these micro-organisms are able to grow in syntrophy with methanogens and others are not. Bacterial strains were selected based on genome availability and upon their ability to grow on short chain fatty acids alone or in syntrophic association with methanogens. Systematic functional domain profiling allowed us to shed light on this fundamental and ecologically important question. Extra-cytoplasmic formate dehydrogenases (InterPro domain number; IPR006443), including their maturation protein FdhE (IPR024064 and IPR006452) is a typical difference between syntrophic and non-syntrophic butyrate and propionate degraders. Furthermore, two domains with a currently unknown function seem to be associated with the ability of syntrophic growth. One is putatively involved in capsule or biofilm production (IPR019079) and a second in cell division, shape-determination or sporulation (IPR018365). The sulfate-reducing bacteria Desulfobacterium autotrophicum HRM2, Desulfomonile tiedjei and Desulfosporosinus meridiei were never tested for syntrophic growth, but all crucial domains were found in their genomes, which suggests their possible ability to grow in syntrophic association with methanogens. In addition, profiling domains involved in electron transfer mechanisms revealed the important role of the Rnf-complex and the formate transporter in syntrophy, and indicate that DUF224 may have a role in electron transfer in bacteria other than Syntrophomonas wolfei as well. This article is a part of a Special Issue entitled: 18th European Bioenergetics Conference (Biochim. Biophys. Acta, Volume 1837, Issue 7, July 2014).

Genome analysis of Desulfotomaculum kuznetsovii strain 17(T) reveals a physiological similarity with Pelotomaculum thermopropionicum strain SI(T).

Desulfotomaculum kuznetsovii is a moderately thermophilic member of the polyphyletic spore-forming genus Desulfotomaculum in the family Peptococcaceae. This species is of interest because it originates from deep subsurface thermal mineral water at a depth of about 3,000 m. D. kuznetsovii is a rather versatile bacterium as it can grow with a large variety of organic substrates, including short-chain and long-chain fatty acids, which are degraded completely to carbon dioxide coupled to the reduction of sulfate. It can grow methylotrophically with methanol and sulfate and autotrophically with H2 + CO2 and sulfate. For growth it does not require any vitamins. Here, we describe the features of D. kuznetsovii together with the genome sequence and annotation. The chromosome has 3,601,386 bp organized in one contig. A total of 3,567 candidate protein-encoding genes and 58 RNA genes were identified. Genes of the acetyl-CoA pathway, possibly involved in heterotrophic growth with acetate and methanol, and in CO2 fixation during autotrophic growth are present. Genomic comparison revealed that D. kuznetsovii shows a high similarity with Pelotomaculum thermopropionicum. Genes involved in propionate metabolism of these two strains show a strong similarity. However, main differences are found in genes involved in the electron acceptor metabolism.

Complete genome sequence of Syntrophobacter fumaroxidans strain (MPOB(T)).

Syntrophobacter fumaroxidans strain MPOB(T) is the best-studied species of the genus Syntrophobacter. The species is of interest because of its anaerobic syntrophic lifestyle, its involvement in the conversion of propionate to acetate, H2 and CO2 during the overall degradation of organic matter, and its release of products that serve as substrates for other microorganisms. The strain is able to ferment fumarate in pure culture to CO2 and succinate, and is also able to grow as a sulfate reducer with propionate as an electron donor. This is the first complete genome sequence of a member of the genus Syntrophobacter and a member genus in the family Syntrophobacteraceae. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 4,990,251 bp long genome with its 4,098 protein-coding and 81 RNA genes is a part of the Microbial Genome Program (MGP) and the Genomes to Life (GTL) Program project.

Transcription of fdh and hyd in Syntrophobacter spp. and Methanospirillum spp. as a diagnostic tool for monitoring anaerobic sludge deprived of molybdenum, tungsten and selenium.

Formate dehydrogenases and hydrogenases contain molybdenum or tungsten and/or selenium. These enzymes are crucial for interspecies formate and hydrogen transfer between propionate degrading Syntrophobacter spp. and methanogenic Methanospirillum spp. Here we used reverse transcription of total RNA followed by quantitative PCR (RT-qPCR) with specific primers to get insight into interspecies formate and hydrogen transfer. Transcriptional regulation of formate dehydrogenases and hydrogenases in Syntrophobacter and Methanospirillum spp. in a propionate-fed up-flow anaerobic sludge bed (UASB) reactor was examined. In both microorganisms formate dehydrogenase and hydrogenase coding genes (fdh and hyd respectively) were transcribed simultaneously. During 249 days in which molybdenum, tungsten and selenium were not supplied to the reactor feed, the microbial activity and transcription of fdh and hyd in Syntrophobacter spp. decreased. Transcription of fdh and hyd in Methanospirillum spp. did not decrease, but transcription of fdh increased when after 249 days molybdenum, tungsten and selenium were supplied to the reactor feed. The developed RT-qPCR is a technique that can give rapid information about active processes in methanogenic granular sludge and may contribute to predict metal limitation and failure in UASB reactors.

Growth- and substrate-dependent transcription of formate dehydrogenase and hydrogenase coding genes in Syntrophobacter fumaroxidans and Methanospirillum hungatei.

Transcription of genes coding for formate dehydrogenases (fdh genes) and hydrogenases (hyd genes) in Syntrophobacter fumaroxidans and Methanospirillum hungatei was studied following growth under different conditions. Under all conditions tested, all fdh and hyd genes were transcribed. However, transcription levels of the individual genes varied depending on the substrate and growth conditions. Our results strongly suggest that in syntrophically grown S. fumaroxidans cells, the [FeFe]-hydrogenase (encoded by Sfum_844-46), FDH1 (Sfum_2703-06) and Hox (Sfum_2713-16) may confurcate electrons from NADH and ferredoxin to protons and carbon dioxide to produce hydrogen and formate, respectively. Based on bioinformatic analysis, a membrane-integrated energy-converting [NiFe]-hydrogenase (Mhun_1741-46) of M. hungatei might be involved in the energy-dependent reduction of CO(2) to formylmethanofuran. The best candidates for F(420)-dependent N(5),N(10)-methyl-H(4) MPT and N(5),N(10),-methylene-H(4)MPT reduction are the cytoplasmic [NiFe]-hydrogenase and FDH1. 16S rRNA ratios indicate that in one of the triplicate co-cultures of S. fumaroxidans and M. hungatei, less energy was available for S. fumaroxidans. This led to enhanced transcription of genes coding for the Rnf-complex (Sfum_2694-99) and of several fdh and hyd genes. The Rnf-complex probably reoxidized NADH with ferredoxin reduction, followed by ferredoxin oxidation by the induced formate dehydrogenases and hydrogenases.

Syntrophic butyrate and propionate oxidation processes: from genomes to reaction mechanisms.

In anoxic environments such as swamps, rice fields and sludge digestors, syntrophic microbial communities are important for decomposition of organic matter to CO2 and CH4 . The most difficult step is the fermentative degradation of short-chain fatty acids such as propionate and butyrate. Conversion of these metabolites to acetate, CO2 , formate and hydrogen is endergonic under standard conditions and occurs only if methanogens keep the concentrations of these intermediate products low. Butyrate and propionate degradation pathways include oxidation steps of comparably high redox potential, i.e. oxidation of butyryl-CoA to crotonyl-CoA and of succinate to fumarate, respectively, that require investment of energy to release the electrons as hydrogen or formate. Although investigated for several decades, the biochemistry of these reactions is still not completely understood. Genome analysis of the butyrate-oxidizing Syntrophomonas wolfei and Syntrophus aciditrophicus and of the propionate-oxidizing Syntrophobacter fumaroxidans and Pelotomaculum thermopropionicum reveals the presence of energy-transforming protein complexes. Recent studies indicated that S. wolfei uses electron-transferring flavoproteins coupled to a menaquinone loop to drive butyryl-CoA oxidation, and that S. fumaroxidans uses a periplasmic formate dehydrogenase, cytochrome b:quinone oxidoreductases, a menaquinone loop and a cytoplasmic fumarate reductase to drive energy-dependent succinate oxidation. Furthermore, we propose that homologues of the Thermotoga maritima bifurcating [FeFe]-hydrogenase are involved in NADH oxidation by S. wolfei and S. fumaroxidans to form hydrogen.

Structural insight into substrate binding and catalysis of a novel 2-keto-3-deoxy-D-arabinonate dehydratase illustrates common mechanistic features of the FAH superfamily.

The archaeon Sulfolobus solfataricus converts d-arabinose to 2-oxoglutarate by an enzyme set consisting of two dehydrogenases and two dehydratases. The third step of the pathway is catalyzed by a novel 2-keto-3-deoxy-D-arabinonate dehydratase (KdaD). In this study, the crystal structure of the enzyme has been solved to 2.1 A resolution. The enzyme forms an oval-shaped ring of four subunits, each consisting of an N-terminal domain with a four-stranded beta-sheet flanked by two alpha-helices, and a C-terminal catalytic domain with a fumarylacetoacetate hydrolase (FAH) fold. Crystal structures of complexes of the enzyme with magnesium or calcium ions and either a substrate analog 2-oxobutyrate, or the aldehyde enzyme product 2,5-dioxopentanoate revealed that the divalent metal ion in the active site is coordinated octahedrally by three conserved carboxylate residues, a water molecule, and both the carboxylate and the oxo groups of the substrate molecule. An enzymatic mechanism for base-catalyzed dehydration is proposed on the basis of the binding mode of the substrate to the metal ion, which suggests that the enzyme enhances the acidity of the protons alpha to the carbonyl group, facilitating their abstraction by glutamate 114. A comprehensive structural comparison of members of the FAH superfamily is presented and their evolution is discussed, providing a basis for functional investigations of this largely unexplored protein superfamily.

Identification of the missing links in prokaryotic pentose oxidation pathways: evidence for enzyme recruitment.

The pentose metabolism of Archaea is largely unknown. Here, we have employed an integrated genomics approach including DNA microarray and proteomics analyses to elucidate the catabolic pathway for D-arabinose in Sulfolobus solfataricus. During growth on this sugar, a small set of genes appeared to be differentially expressed compared with growth on D-glucose. These genes were heterologously overexpressed in Escherichia coli, and the recombinant proteins were purified and biochemically studied. This showed that D-arabinose is oxidized to 2-oxoglutarate by the consecutive action of a number of previously uncharacterized enzymes, including a D-arabinose dehydrogenase, a D-arabinonate dehydratase, a novel 2-keto-3-deoxy-D-arabinonate dehydratase, and a 2,5-dioxopentanoate dehydrogenase. Promoter analysis of these genes revealed a palindromic sequence upstream of the TATA box, which is likely to be involved in their concerted transcriptional control. Integration of the obtained biochemical data with genomic context analysis strongly suggests the occurrence of pentose oxidation pathways in both Archaea and Bacteria, and predicts the involvement of additional enzyme components. Moreover, it revealed striking genetic similarities between the catabolic pathways for pentoses, hexaric acids, and hydroxyproline degradation, which support the theory of metabolic pathway genesis by enzyme recruitment.