Complete genome sequence of Syntrophothermus lipocalidus type strain (TGB-C1).
ABSTRACT: Syntrophothermus lipocalidus Sekiguchi et al. 2000 is the type species of the genus Syntrophothermus. The species is of interest because of its strictly anaerobic lifestyle, its participation in the primary step of the degradation of organic maters, and for releasing products which serve as substrates for other microorganisms. It also contributes significantly to maintain a regular pH in its environment by removing the fatty acids through ?-oxidation. The strain is able to metabolize isobutyrate and butyrate, which are the substrate and the product of degradation of the substrate, respectively. This is the first complete genome sequence of a member of the genus Syntrophothermus and the second in the family Syntrophomonadaceae. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 2,405,559 bp long genome with its 2,385 protein-coding and 55 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project.
Project description:The syntrophic degradation of branched-chain fatty acids (BCFAs) such as 2-methylbutyrate and isobutyrate is an essential step in the production of methane from proteins/amino acids in anaerobic ecosystems. While a few syntrophic BCFA-degrading bacteria have been isolated, their metabolic pathways in BCFA and short-chain fatty acid (SCFA) degradation as well as energy conservation systems remain unclear. In an attempt to identify these pathways, we herein performed comparative genomics of three syntrophic bacteria: 2-methylbutyrate-degrading "Syntrophomonas wolfei subsp. methylbutyratica" strain JCM 14075(T) (=4J5(T)), isobutyrate-degrading Syntrophothermus lipocalidus strain TGB-C1(T), and non-BCFA-metabolizing S. wolfei subsp. wolfei strain Göttingen(T). We demonstrated that 4J5 and TGB-C1 both encode multiple genes/gene clusters involved in ?-oxidation, as observed in the Göttingen genome, which has multiple copies of genes associated with butyrate degradation. The 4J5 genome possesses phylogenetically distinct ?-oxidation genes, which may be involved in 2-methylbutyrate degradation. In addition, these Syntrophomonadaceae strains harbor various hydrogen/formate generation systems (i.e., electron-bifurcating hydrogenase, formate dehydrogenase, and membrane-bound hydrogenase) and energy-conserving electron transport systems, including electron transfer flavoprotein (ETF)-linked acyl-CoA dehydrogenase, ETF-linked iron-sulfur binding reductase, ETF dehydrogenase (FixABCX), and flavin oxidoreductase-heterodisulfide reductase (Flox-Hdr). Unexpectedly, the TGB-C1 genome encodes a nitrogenase complex, which may function as an alternative H2 generation mechanism. These results suggest that the BCFA-degrading syntrophic strains 4J5 and TGB-C1 possess specific ?-oxidation-related enzymes for BCFA oxidation as well as appropriate energy conservation systems to perform thermodynamically unfavorable syntrophic metabolism.
Project description:To understand the physiological basis of methanogenic archaea living on interspecies H(2) transfer, the protein expression of a hydrogenotrophic methanogen, Methanothermobacter thermautotrophicus strain ?H, was investigated in both pure culture and syntrophic coculture with an anaerobic butyrate oxidizer Syntrophothermus lipocalidus strain TGB-C1 as an H(2) supplier. Comparative proteomic analysis showed that global protein expression of methanogen cells in the model coculture was substantially different from that of pure cultured cells. In brief, in syntrophic coculture, although methanogenesis-driven energy generation appeared to be maintained by shifting the pathway to the alternative methyl coenzyme M reductase isozyme I and cofactor F(420)-dependent process, the machinery proteins involved in carbon fixation, amino acid synthesis, and RNA/DNA metabolisms tended to be down-regulated, indicating restrained cell growth rather than vigorous proliferation. In addition, our proteome analysis revealed that ? subunits of proteasome were differentially acetylated between the two culture conditions. Since the relevant modification has been suspected to regulate proteolytic activity of the proteasome, the global protein turnover rate could be controlled under syntrophic growth conditions. To our knowledge, the present study is the first report on N-acetylation of proteasome subunits in methanogenic archaea. These results clearly indicated that physiological adaptation of hydrogenotrophic methanogens to syntrophic growth is more complicated than that of hitherto proposed.
Project description:Small-subunit rRNA sequences were obtained for two saturated fatty acid-beta-oxidizing syntrophic bacteria, Syntrophomonas sapovorans and Syntrophomonas wolfei LYB, and sequence analysis confirmed their classification as members of the family Syntrophomonadaceae. S. wolfei LYB was closely related to S. wolfei subsp. wolfei, but S. sapovorans did not cluster with the other members of the genus Syntrophomonas. Five oligonucleotide probes targeting the small-subunit rRNA of different groups within the family Syntrophomonadaceae, which contains all currently known saturated fatty acid-beta-oxidizing syntrophic bacteria, were developed and characterized. The probes were designed to be specific at the family, genus, and species levels and were characterized by temperature-of-dissociation and specificity studies. To demonstrate the usefulness of the probes for the detection and quantification of saturated fatty acid-beta-oxidizing syntrophic bacteria in methanogenic environments, the microbial community structure of a sample from a full-scale biogas plant was determined. Hybridization results with probes for syntrophic bacteria and methanogens were compared to specific methanogenic activities and microbial numbers determined with most-probable-number estimates. Most of the methanogenic rRNA was comprised of Methanomicrobiales rRNA, suggesting that members of this order served as the main hydrogen-utilizing microorganisms. Between 0.2 and 1% of the rRNA was attributed to the Syntrophomonadaceae, of which the majority was accounted for by the genus Syntrophomonas.
Project description:Members of Methanocellales are widespread in paddy field soils and play the key role in methane production. These methanogens feature largely in these organisms’ adaptation to low H2 and syntrophic growth with anaerobic fatty acid oxidizers. The adaptive mechanisms, however, remain unknown. In the present study, we determined the transcripts of 21 genes involved in the key steps of methanogenesis and acetate assimilation of Methanocella conradii HZ254, a strain recently isolated from paddy field soil. M. conradii was grown in monoculture and syntrophically with Pelotomaculum thermopropionicum (a propionate syntroph) or Syntrophothermus lipocalidus (a butyrate syntroph). Comparison of the relative transcript abundances showed that three hydrogenase-encoding genes and all methanogenesis-related genes tested were upregulated in cocultures relative to monoculture. The genes encoding formylmethanofuran dehydrogenase (Fwd), heterodisulfide reductase (Hdr), and the membrane-bound energy-converting hydrogenase (Ech) were the most upregulated among the evaluated genes. The expression of the formate dehydrogenase (Fdh)-encoding gene also was significantly upregulated. In contrast, an acetate assimilation gene was downregulated in cocultures. The genes coding for Fwd, Hdr, and the D subunit of F420-nonreducing hydrogenase (Mvh) form a large predicted transcription unit; therefore, the Mvh/Hdr/Fwd complex, capable of mediating the electron bifurcation and connecting the first and last steps of methanogenesis, was predicted to be formed in M. conradii. We propose that Methanocella methanogens cope with low H2 and syntrophic growth by (i) stabilizing the Mvh/Hdr/Fwd complex and (ii) activating formatedependent methanogenesis.
Project description:Syntrophy is a thermodynamically required mutualistic cooperation between fatty acid-oxidizing bacteria and methanogens that plays the important role in organic decomposition and methanogenesis in anoxic environments. In this study, three experiments were conducted to evaluate the cell-to-cell interaction in a thermophilic coculture consisting of <i>Syntrophothermus lipocalidus</i> and <i>Methanocella conradii</i> and a mesophilic coculture consisting of <i>Syntrophomonas wolfei</i> and <i>Methanococcus maripaludis</i>. First, syntrophs and methanogens were inoculated at different initial cell ratios to evaluate the growth synchronization. The quantitative PCR analysis revealed that the organism with a lower relative abundance at the beginning always grew faster, and the cell ratio converged over time to relative constant values in both the thermophilic and mesophilic cocultures. Next, intermittent ultrasound and constant shaking treatments were used to evaluate the influence of physical disturbance on microbial aggregation in the mesophilic coculture. The fluorescence <i>in situ</i> hybridization and scanning electron microscopy revealed that the tendency of syntrophic aggregation was not affected by the physical disturbances, although the activity was slightly depressed. <i>Syntrophomonas</i> dominated in the initial microbial aggregates, which, however, did not grow until <i>Methanococcus</i> was attached and increased to a significant extent, indicating the local growth synchronization during the formation and maturation of syntrophic aggregates. Last, microfluidic experiments revealed that whether or not <i>Syntrophomonas</i> or <i>Methanococcus</i> was loaded first, the second organism preferred moving to the place where the first organism was located, suggesting the cell-to-cell attraction between <i>Syntrophomonas</i> and <i>Methanococcus</i>. Collectively, our study demonstrated the growth synchronization and cell-to-cell attraction between the butyrate-oxidizing bacteria and methanogens for optimizing the syntrophic cooperation.
Project description:An ecogenomic analysis of the methanogenic microbial community in a laboratory-scale up-flow anaerobic sludge blanket (UASB) reactor treating soy sauce-processing wastewater revealed a synergistic metabolic network. Granular sludge samples were collected from the UASB reactor operated under psychrophilic (20°C) conditions with a COD removal rate >75%. A 16S rRNA gene amplicon sequencing-based microbial community analysis classified the major microbial taxa as Methanothrix, Methanobacterium, Pelotomaculaceae, Syntrophomonadaceae, Solidesulfovibrio, and members of the phyla Synergistota and Bacteroidota. Draft genomes of dominant microbial populations were recovered by metagenomic shotgun sequencing. Metagenomic- and metatranscriptomic-assisted metabolic reconstructions indicated that Synergistota- and Bacteroidota-related organisms play major roles in the degradation of amino acids. A metagenomic bin of the uncultured Bacteroidales 4484-276 clade encodes genes for proteins that may function in the catabolism of phenylalanine and tyrosine under microaerobic conditions. Syntrophomonadaceae and Pelotomaculaceae oxidize fatty acid byproducts presumably derived from the degradation of amino acids in syntrophic association with aceticlastic and hydrogenotrophic methanogen populations. Solidesulfovibrio organisms are responsible for the reduction of sulfite and may support the activity of hydrogenotrophic methanogens and other microbial populations by providing hydrogen and ammonia using nitrogen fixation-related proteins. Overall, functionally diverse anaerobic organisms unite to form a metabolic network that performs the complete degradation of amino acids in the psychrophilic methanogenic microbiota.
Project description:We investigated long-chain fatty acid (LCFA)-degrading anaerobic microbes by enrichment, isolation, and RNA-based stable isotope probing (SIP). Primary enrichment cultures were made with each of four LCFA substrates (palmitate, stearate, oleate, or linoleate, as the sole energy source) at 55 degrees C or 37 degrees C with two sources of anaerobic granular sludge as the inoculum. After several transfers, we obtained seven stable enrichment cultures in which LCFAs were converted to methane. The bacterial populations in these cultures were then subjected to 16S rRNA gene-based cloning, in situ hybridization, and RNA-SIP. In five of seven enrichment cultures, the predominant bacteria were affiliated with the family Syntrophomonadaceae. The other two enrichment cultures contained different bacterial populations in which the majority of members belonged to the phylum Firmicutes and the class Deltaproteobacteria. After several attempts to isolate these dominant bacteria, strain MPA, belonging to the family Syntrophomonadaceae, and strain TOL, affiliated with the phylum Firmicutes, were successfully isolated. Strain MPA converts palmitate to acetate and methane in syntrophic association with Methanospirillum hungatei. Even though strain TOL assimilated [(13)C]palmitate in the original enrichment culture, strain TOL has not shown the ability to degrade LCFAs after isolation. These results suggest that microbes involved in the degradation of LCFAs under methanogenic conditions might not belong only to the family Syntrophomonadaceae, as most anaerobic LCFA-degrading microbes do, but may also be found in phylogenetically diverse bacterial groups.
Project description:Long-chain fatty acid (LCFA) degradation is a key step in methanogenic treatment of wastes/wastewaters containing high concentrations of lipids. However, despite the importance of LCFA-degrading bacteria, their natural diversity is little explored due to the limited availability of isolate information and the lack of appropriate molecular markers. We therefore investigated these microbes by using RNA-based stable isotope probing. We incubated four methanogenic sludges (mesophilic sludges MP and MBF and thermophilic sludges TP and JET) with (13)C-labeled palmitate (1 mM) as a substrate. After 8 to 19 days of incubation, we could detect (13)C-labeled bacterial rRNA. A density-resolved terminal restriction fragment length polymorphism fingerprinting analysis showed distinct bacterial populations in (13)C-labeled and unlabeled rRNA fractions. The bacterial populations in the (13)C-labeled rRNA fractions were identified by cloning and sequencing of reverse-transcribed 16S rRNA. Diverse phylogenetic bacterial sequences were retrieved, including those of members of the family Syntrophaceae, clone cluster MST belonging to the class Deltaproteobacteria, Clostridium clusters III and IV, phylum Bacteroidetes, phylum Spirochaetes, and family Syntrophomonadaceae. Although Syntrophomonadaceae species are considered to be the major fatty acid-degrading syntrophic microorganisms under methanogenic conditions, they were detected in only two of the clone libraries. These results suggest that phylogenetically diverse bacterial groups were active in situ in the degradation of LCFA under methanogenic conditions.
Project description:Anaerobic long-chain fatty acid (LCFA)-degrading bacteria were identified by combining selective enrichment studies with molecular approaches. Two distinct enrichment cultures growing on unsaturated and saturated LCFAs were obtained by successive transfers in medium containing oleate and palmitate, respectively, as the sole carbon and energy sources. Changes in the microbial composition during enrichment were analyzed by denaturing gradient gel electrophoresis (DGGE) profiling of PCR-amplified 16S rRNA gene fragments. Prominent DGGE bands of the enrichment cultures were identified by 16S rRNA gene sequencing. A significant part of the retrieved 16S rRNA gene sequences was most similar to those of uncultured bacteria. Bacteria corresponding to predominant DGGE bands in oleate and palmitate enrichment cultures clustered with fatty acid-oxidizing bacteria within Syntrophomonadaceae and Syntrophobacteraceae families. A low methane yield, corresponding to 9 to 18% of the theoretical value, was observed in the oleate enrichment, and acetate, produced according to the expected stoichiometry, was not further converted to methane. In the palmitate enrichment culture, the acetate produced was completely mineralized and a methane yield of 48 to 70% was achieved from palmitate degradation. Furthermore, the oleate enrichment culture was able to use palmitate without detectable changes in the DGGE profile. However, the palmitate-specialized consortia degraded oleate only after a lag phase of 3 months, after which the DGGE profile had changed. Two predominant bands appeared, and sequence analysis showed affiliation with the Syntrophomonas genus. These bands were also present in the oleate enrichment culture, suggesting that these bacteria are directly involved in oleate degradation, emphasizing possible differences between the degradation of unsaturated and saturated LCFAs.