Enhanced microbial electrosynthesis by using defined co-cultures.

Microbial uptake of free cathodic electrons presents a poorly understood aspect of microbial physiology. Uptake of cathodic electrons is particularly important in microbial electrosynthesis of sustainable fuel and chemical precursors using only CO2 and electricity as carbon, electron and energy source. Typically, large overpotentials (200 to 400 mV) were reported to be required for cathodic electron uptake during electrosynthesis of, for example, methane and acetate, or low electrosynthesis rates were observed. To address these limitations and to explore conceptual alternatives, we studied defined co-cultures metabolizing cathodic electrons. The Fe(0)-corroding strain IS4 was used to catalyze the electron uptake reaction from the cathode forming molecular hydrogen as intermediate, and Methanococcus maripaludis and Acetobacterium woodii were used as model microorganisms for hydrogenotrophic synthesis of methane and acetate, respectively. The IS4-M. maripaludis co-cultures achieved electromethanogenesis rates of 0.1-0.14 µmol cm-2 h-1 at -400 mV vs standard hydrogen electrode and 0.6-0.9 µmol cm-2 h-1 at -500 mV. Co-cultures of strain IS4 and A. woodii formed acetate at rates of 0.21-0.23 µmol cm-2 h-1 at -400 mV and 0.57-0.74 µmol cm-2 h-1 at -500 mV. These data show that defined co-cultures coupling cathodic electron uptake with synthesis reactions via interspecies hydrogen transfer may lay the foundation for an engineering strategy for microbial electrosynthesis.

DEEP BIOSPHERE. Exploring deep microbial life in coal-bearing sediment down to ~2.5 km below the ocean floor.

Microbial life inhabits deeply buried marine sediments, but the extent of this vast ecosystem remains poorly constrained. Here we provide evidence for the existence of microbial communities in ~40° to 60°C sediment associated with lignite coal beds at ~1.5 to 2.5 km below the seafloor in the Pacific Ocean off Japan. Microbial methanogenesis was indicated by the isotopic compositions of methane and carbon dioxide, biomarkers, cultivation data, and gas compositions. Concentrations of indigenous microbial cells below 1.5 km ranged from <10 to ~10(4) cells cm(-3). Peak concentrations occurred in lignite layers, where communities differed markedly from shallower subseafloor communities and instead resembled organotrophic communities in forest soils. This suggests that terrigenous sediments retain indigenous community members tens of millions of years after burial in the seabed.

Potential functional gene diversity involved in methanogenesis and methanogenic community structure in Indian buffalo (Bubalus bubalis) rumen.

Understanding the methanogen community structure and methanogenesis from Bubalus bubalis in India may be beneficial to methane mitigation. Our current understanding of the microbial processes leading to methane production is incomplete, and further advancement in the knowledge of methanogenesis pathways would provide means to manipulate its emission in the future. In the present study, we evaluated the methanogenic community structure in the rumen as well as their potential genes involved in methanogenesis. The taxonomic and metabolic profiles of methanogens were assessed by shotgun sequencing of rumen metagenome by Ion Torrent semiconductor sequencing. The buffalo rumen contained representative genera of all the families of methanogens. Members of Methanobacteriaceae were found to be dominant, followed by Methanosarcinaceae, Methanococcaceae, Methanocorpusculaceae, and Thermococcaceae. A total of 60 methanogenic genera were detected in buffalo rumen. Methanogens related to the genera Methanobrevibacter, Methanosarcina, Methanococcus, Methanocorpusculum, Methanothermobacter, and Methanosphaera were predominant, representing >70 % of total archaeal sequences. The metagenomic dataset indicated the presence of genes involved in the methanogenesis and acetogenesis pathways, and the main functional genes were those of key enzymes in the methanogenesis. Sequences related to CoB--CoM heterodisulfide reductase, methyl coenzyme M reductase, f420-dependent methylenetetrahydromethanopterin reductase, and formylmethanofuran dehydrogenase were predominant in rumen. In addition, methenyltetrahydrofolate cyclohydrolase, methylenetetrahydrofolate dehydrogenase, 5,10-methylenetetrahydrofolate reductase, and acetyl-coenzyme A synthetase were also recovered.

Protein localization analysis of essential genes in prokaryotes.

Essential genes, those critical for the survival of an organism under certain conditions, play a significant role in pharmaceutics and synthetic biology. Knowledge of protein localization is invaluable for understanding their function as well as the interaction of different proteins. However, systematical examination of essential genes from the aspect of the localizations of proteins they encode has not been explored before. Here, a comprehensive protein localization analysis of essential genes in 27 prokaryotes including 24 bacteria, 2 mycoplasmas and 1 archaeon has been performed. Both statistical analysis of localization information in these genomes and GO (Gene Ontology) terms enriched in the essential genes show that proteins encoded by essential genes are enriched in internal location sites, while exist in cell envelope with a lower proportion compared with non-essential ones. Meanwhile, there are few essential proteins in the external subcellular location sites such as flagellum and fimbrium, and proteins encoded by non-essential genes tend to have diverse localizations. These results would provide further insights into the understanding of fundamental functions needed to support a cellular life and improve gene essentiality prediction by taking the protein localization and enriched GO terms into consideration.

Iron-corroding methanogen isolated from a crude-oil storage tank.

Microbiologically influenced corrosion of steel in anaerobic environments has been attributed to hydrogenotrophic microorganisms. A sludge sample collected from the bottom plate of a crude-oil storage tank was used to inoculate a medium containing iron (Fe(0)) granules, which was then incubated anaerobically at 37 degrees C under an N(2)-CO(2) atmosphere to enrich for microorganisms capable of using iron as the sole source of electrons. A methanogen, designated strain KA1, was isolated from the enrichment culture. An analysis of its 16S rRNA gene sequence revealed that strain KA1 is a Methanococcus maripaludis strain. Strain KA1 produced methane and oxidized iron much faster than did the type strain of M. maripaludis, strain JJ(T), which produced methane at a rate expected from the abiotic H(2) production rate from iron. Scanning electron micrographs of iron coupons that had been immersed in either a KA1 culture, a JJ(T) culture, or an aseptic medium showed that only coupons from the KA1 culture had corroded substantially, and these were covered with crystalline deposits that consisted mainly of FeCO(3).

The electron transfer system of syntrophically grown Desulfovibrio vulgaris.

Interspecies hydrogen transfer between organisms producing and consuming hydrogen promotes the decomposition of organic matter in most anoxic environments. Although syntrophic coupling between hydrogen producers and consumers is a major feature of the carbon cycle, mechanisms for energy recovery at the extremely low free energies of reactions typical of these anaerobic communities have not been established. In this study, comparative transcriptional analysis of a model sulfate-reducing microbe, Desulfovibrio vulgaris Hildenborough, suggested the use of alternative electron transfer systems dependent on growth modality. During syntrophic growth on lactate with a hydrogenotrophic methanogen, numerous genes involved in electron transfer and energy generation were upregulated in D. vulgaris compared with their expression in sulfate-limited monocultures. In particular, genes coding for the putative membrane-bound Coo hydrogenase, two periplasmic hydrogenases (Hyd and Hyn), and the well-characterized high-molecular-weight cytochrome (Hmc) were among the most highly expressed and upregulated genes. Additionally, a predicted operon containing genes involved in lactate transport and oxidation exhibited upregulation, further suggesting an alternative pathway for electrons derived from lactate oxidation during syntrophic growth. Mutations in a subset of genes coding for Coo, Hmc, Hyd, and Hyn impaired or severely limited syntrophic growth but had little effect on growth via sulfate respiration. These results demonstrate that syntrophic growth and sulfate respiration use largely independent energy generation pathways and imply that to understand microbial processes that sustain nutrient cycling, lifestyles not captured in pure culture must be considered.

Metabolic network model of a human oral pathogen.

The microbial community present in the human mouth is engaged in a complex network of diverse metabolic activities. In addition to serving as energy and building-block sources, metabolites are key players in interspecies and host-pathogen interactions. Metabolites are also implicated in triggering the local inflammatory response, which can affect systemic conditions such as atherosclerosis, obesity, and diabetes. While the genome of several oral pathogens has been sequenced, quantitative understanding of the metabolic functions of any oral pathogen at the system level has not been explored yet. Here we pursue the computational construction and analysis of the genome-scale metabolic network of Porphyromonas gingivalis, a gram-negative anaerobe that is endemic in the human population and largely responsible for adult periodontitis. Integrating information from the genome, online databases, and literature screening, we built a stoichiometric model that encompasses 679 metabolic reactions. By using flux balance approaches and automated network visualization, we analyze the growth capacity under amino-acid-rich medium and provide evidence that amino acid preference and cytotoxic by-product secretion rates are suitably reproduced by the model. To provide further insight into the basic metabolic functions of P. gingivalis and suggest potential drug targets, we study systematically how the network responds to any reaction knockout. We focus specifically on the lipopolysaccharide biosynthesis pathway and identify eight putative targets, one of which has been recently verified experimentally. The current model, which is amenable to further experimental testing and refinements, could prove useful in evaluating the oral microbiome dynamics and in the development of novel biomedical applications.

Cloning, expression and purification of DNA-binding protein Mvo10b from Methanococcus voltae.

Mvo10b from the mesophilic archaeon Methanococcus voltae is a member of the Sac10b family which may play an important role in the organization and accessibility of genetic information in Archaea. Since Mvo10b is a DNA-binding protein as the other member in the Sac10b family, to obtain a recombinant Mvo10b requires an efficient and inexpensive expression and purification system for producing the protein free of nucleic acid contamination. Previously, the hyperthermophilic archaeal Ssh10b of the Sac10b family was successfully purified. However, the protocol adopted to purify Ssh10b is not appropriate for purifying the mesophilic Mvo10b. This study describes the successful expression and purification of the recombinant Mvo10b. The expression of recombinant Mvo10b was carried out in Escherichia coli, and the target protein was expressed in the soluble form. The protein was purified by polyethyleneimine (PEI) precipitation followed by nickel ion metal affinity chromatography. The purity of Mvo10b was checked to insure being free of nucleic acid contamination. The final protein yield is about 30mg/l of LB culture. The ensemble of NMR and far-UV CD data shows that the purified Mvo10b has abundant regular secondary structures and is correctly folded, which may have similar 3D structure as its hyperthermophilic counterpart [P62A]Ssh10b. The developed protocol has potential application in the production of the other thermophilic and mesophilic proteins in the Sac10b family.

Methanococcus aeolicus sp. nov., a mesophilic, methanogenic archaeon from shallow and deep marine sediments.

Three strains of CO(2)-reducing methanogens were isolated from marine sediments. Strain PL-15/H(P) was isolated from marine sediments of the Lipari Islands, near Sicily and the other two strains, Nankai-2 and Nankai-3(T), were isolated from deep marine sediments of the Nankai Trough, about 50 km from the coast of Japan. Analysis of the cellular proteins and 16S rRNA gene sequences indicated that these three strains represented a single novel species that formed a deep branch of the mesophilic methanococci. Phylogenetic analysis indicated that the three strains were most closely related to Methanothermococcus okinawensis (95 % 16S rRNA gene sequence similarity). However, strains PL-15/H(P), Nankai-2 and Nankai-3(T) grew at temperatures that were more similar to those of recognized species within the genus Methanococcus. Strain Nankai-3(T) grew fastest at 46 degrees C. Results of physiological and biochemical tests allowed the genotypic and phenotypic differentiation of strains PL-15/H(P), Nankai-2 and Nankai-3(T) from closely related species. The name Methanococcus aeolicus sp. nov. is proposed, with strain Nankai-3(T) (=OCM 812(T)=DSM 17508(T)) as the type strain.

Identification and characterization of a L-tyrosine decarboxylase in Methanocaldococcus jannaschii.

Methanofuran is the first coenzyme in the methanogenic pathway used by the archaeon Methanocaldococcus jannaschii, as well as other methanogens, to reduce CO2 to methane. The details of the pathway for the biosynthesis of methanofuran and the responsible genes have yet to be established. A clear structural element in all known methanofurans is tyramine, likely produced by the decarboxylation of L-tyrosine. We show here that the mfnA gene at M. jannaschii locus MJ0050 encodes a thermostable pyridoxal phosphate-dependent L-tyrosine decarboxylase that specifically produces tyramine. Homologs of this gene are widely distributed among euryarchaea but are not specifically related to known bacterial or plant tyrosine decarboxylases.

Methanocaldococcus indicus sp. nov., a novel hyperthermophilic methanogen isolated from the Central Indian Ridge.

An autotrophic, hyperthermophilic methanogen, strain SL43(T), was isolated from a deep-sea hydrothermal chimney sample collected on the Central Indian Ridge at a depth of 2420 m. The coccoid, surface-layer-carrying, Gram-negative-staining cells were heavily flagellated and exhibited a slight tumbling motility. The temperature range for growth at pH 6.5 was 50-86 degrees C, with optimum growth at 85 degrees C. The optimum pH for growth was 6.6 and the optimum NaCl concentration for growth was 30 g l(-1). The novel isolate used H(2) and CO(2) as the only substrates for growth and produced methane. Selenium and yeast extract stimulated growth significantly. In the presence of CO(2) and H(2), the organism reduced elemental sulfur to hydrogen sulfide. Growth was inhibited by chloramphenicol and rifampicin, but not by ampicillin, kanamycin, penicillin or streptomycin. The G+C content of the genomic DNA was 30.7 mol%. On the basis of 16S rRNA gene sequence analysis, this organism was most closely related to Methanocaldococcus infernus ME(T) (3.2 % distance). Its phylogenetic distinctiveness was confirmed by RFLP analysis of the 16S rDNA, a reliable tool for differentiating hyperthermophilic methanococci. On the basis of phylogenetic and physiological characteristics, it is proposed that strain SL43(T) (=DSM 15027(T)=JCM 11886(T)) be designated as the type strain of a novel species, Methanocaldococcus indicus sp. nov.

Characterization of Methanococcus voltaei strain P2F9701a: a new methanogen isolated from estuarine environment.

A new methanogen, designated as strain P2F9701a (= OCM 745), was isolated from a water sample of estuarine environment in Elrin Shi, Taiwan. Cells of strain P2F9701a were motile coccus (0.7 approximately 1.1 micron) with tufts of flagella. Gas vacuoles were observed, and the protein cell wall was composed of S-layer protein subunit with Mr of 74,700. Cells catabolized formate and H2+CO2 to produce methane, but not acetate, methanol, and trimethylamine. Strain P2F9701a grew in the range of 30-42 degrees C, with optimal growth temperature at 37 degrees C, but did not grow below 28 degrees C or above 42 degrees C. This estuarine isolate P2F9701a tolerated well the NaCl concentration between 0.02 and 1.03 m, and the optimal salt for growth was 0.17 m. Although phylogenetic analytic results indicated that P2F9701a belongs to the mesophilic, hydrogenotrophic marine methanogen of Methanococcus voltaei, the occurrence of gas vacuoles, tufts of flagella, eury-halotolerant and steno-thermotolerant characters of strain P2F9701a are different from mesophilic Methanococcus spp. that had been reported.

Methanococcus vulcanius sp. nov., a novel hyperthermophilic methanogen isolated from East Pacific Rise, and identification of Methanococcus sp. DSM 4213T as Methanococcus fervens sp. nov.

An autotrophic, hyperthermophilic methanogen (M7T) was isolated from a deep-sea hydrothermal chimney sample collected on the East Pacific Rise at a depth of 2600 m. The coccoid-shaped cells are flagellated and exhibit a slight tumbling motility. The temperature range for growth at pH 6.5 was 49-89 degrees C, with optimum growth at 80 degrees C. The optimum pH for growth was 6.5, and the optimum NaCl concentration for growth was around 25 g l-1. The new isolate used H2 and CO2 as the only substrates for growth and methane production. Tungsten, selenium and yeast extract stimulated growth significantly. In the presence of CO2 and H2, the organism reduced elemental sulphur to hydrogen sulphide. Growth was inhibited by chloramphenicol and rifampicin, but not by ampicillin, kanamycin, penicillin and streptomycin. The G + C content of the genomic DNA was 31 mol%. As determined by 16S rDNA gene sequence analysis, this organism was closely related to Methanococcus jannaschii strain JAL-1T. However, despite the high percentage of similarity between their 16S rDNA sequences (97.1%), the DNA-DNA hybridization levels between these strains were less than 5%. On the basis of these observations and physiological traits, it is proposed that this organism should be placed in a new species, Methanococcus vulcanius. The type strain is M7T (= DSM 12094T). During the course of this study, the 16S rDNA sequence analysis placed Methanococcus sp. strain AG86T (= DSM 4213T) as a close relative of M. jannaschii strain JAL-1T. However, the weak level of DNA-DNA hybridization with this strain (< 10%) allowed the proposal that strain AG86T also constitutes a new species, Methanococcus fervens.

Rapid identification of hyperthermophilic methanococci isolated from deep-sea hydrothermal vents.

16S rDNAs amplified by PCR from 22 hyperthermophilic methanococci isolated from deep-sea hydrothermal vents were compared with those of the six type strains of the genus Methanococcus by RFLP analysis. Restriction fragments obtained with Haelli enabied four of the type species to be distinguished. Restrictions with HhaI, BstUI and MspI were necessary to differentiate Methanococcus jannaschii and Methanococcus fervens. The results indicate that the 16S rDNA PCR-RFLP method provides a rapid and reliable tool for the identification of newly isolated hyperthermophilic Methanococcus spp.

Methanococcus infernus sp. nov., a novel hyperthermophilic lithotrophic methanogen isolated from a deep-sea hydrothermal vent.

An autotrophic, extremely thermophilic methanogen (ME(T)) was isolated from a deep-sea hydrothermal chimney sample collected on the Mid-Atlantic Ridge at a depth of 3000 m. The heavily flagellated cells are motile and coccoid shaped. The new strain growths between 55 and 91 degrees C, with an optimum growth temperature at 85 degree C. The optimum pH for growth is 6.5, and the optimum sea salt concentration for growth is around 25 g l-1. The organism uses H2 and CO2 as the only substrate for growth and methane production. Tungsten, selenium and yeast extract stimulate growth significantly. In the presence of CO2 and H2, the organism reduces elemental sulphur to hydrogen sulphide. The G+C content of the genomic DNA is 33 mol%. As determined by 16S gene sequence analysis, this organism is closely related to Methanococcus jannaschii strain JAL-1T. However, no significant homology was observed between them with DNA-DNA hybridization. It is proposed that this organism should be placed in a new species, Methanococcus infernus. The type strain is ME(T) (= DSM 11812T).

Sequence of archaeal Methanococcus jannaschii alpha-amylase contains features of families 13 and 57 of glycosyl hydrolases: a trace of their common ancestor?

Two sequentially different, seemingly unrelated alpha-amylase families exist, known as family-13 and family-57 glycosyl hydrolases. Despite the common enzyme activity, it has as yet been impossible to detect any sequence similarity between the two families. The detailed analysis of the recently determined sequence of the alpha-amylase from methanogenic archaeon Methanococcus jannaschii using the sensitive Hydrophobic Cluster Analysis method revealed that this alpha-amylase contains features of both families of alpha-amylases. Thus the M. jannaschii alpha-amylase is similar to the Pyrococcus furiosus alpha-amylase from family 57 while it obviously contains most of the sequence fingerprints characteristic for alpha-amylase family 13. Importantly, a glutamic acid residue equivalent with the family-13 catalytic glutamate positioned in the beta 5-strand segment was identified in members of family 57. The results presented in this report indicate that the two families, 13 and 57, are either the products of a very distant common ancestor or have evolved from each other, although at present they can represent two different alpha-amylase families with evolved different catalytic mechanisms, catalytic machinery and folds.

[The composition and distribution of some kinds of anaerobic microorganisms in Yinqion basin].

Under the strictly anaerobic conditions, the population of Sulfate-reducing bacteria, fermentative bacteria and methanogenic bacteria of serial samples got from erect sections of different Sedimentary of Yingqiong basin (a typical marine sedimentary environment) were measured by MPN method. The morphology of different kinds of bacteria and the metabolic types of methanogen and methanogenic activity were observed. The relation between population of bacteria and some indexes were compared. The results show that SRB present in all of the samples. The distribution of SRB and fermentative bacteria have no interrelation with the depth of samples but SRB has interrelation with the SO(4)2- concentration, and fermentative bacteria has negative interrelation with the contents of organic matter. Two kinds of methanogen present in all of the samples. They belong to Methanobacterium and Methanococcus, respectively. Their types of nutriment are H2/CO2.

Genomes: Methanococcus jannaschii and the golden fleece.

The total genome sequence of the archaeon Methanococcus jannaschii completes the trilogy of genomes for the three domains of life-Archaea, Bacteria and Eucarya. It will have far-reaching consequences for evolutionary studies and for the way in which experimental biology is performed.