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A new genus and species of a nonmotile gram-negative rod, Syntrophobacter wolinii , is the first bacterium described which degrades propionate only in coculture with an H 2 -using organism and in the absence of light or exogenous electron acceptors such as O 2 , sulfate, or nitrate. It was isolated from methanogenic enrichments from an anaerobic municipal sewage digestor, using anaerobic roll tubes containing a medium with propionate as the energy source in association with an H 2 -using, sulfate-reducing Desulfovibrio sp. which cannot utilize fatty acids other than formate. S. wolinii produced acetate and, presumably, CO 2 and H 2 (or formate) from propionate. In media without sulfate and with Methanospirillum hungatei , a methanogen that uses only H 2 -CO 2 or formate as an energy source, acetate, methane, and, presumably, CO 2 were produced from propionate and only small amounts of Desulfovibrio sp. were present. Isolation in coculture with the methanogen was not successful. S. wolinii does not use other saturated fatty acids as energy sources.

A strain of anaerobic, syntrophic, propionate-oxidizing bacteria, strain LYPT (= OCM 661T; T = type strain), was isolated and proposed as representative of a new genus and new species, Smithella propionica gen. nov., sp. nov. The strain was enriched from an anaerobic digestor and isolated. Initial isolation was as a monoxen i c prop i on at e-d eg ra d i ng co-cu It u re con t a i n i ng Methanospirillum hungateii JF-lT as an H, -and formate-using partner. Later, an axenic culture was obtained by using crotonate as the catabolic substrate. The previously described propionate-degrading syntrophs of the genus Syntrophobacter also grow in co-culture with methanogens such as Methanospirillum hungateii, forming acetate, CO, and methane from propionate. However, Smithella propionica differs by producing less methane and more acetate; in addition, it forms small amounts of butyrate. Smithella propionica and Syntrophobacter wolinii grew within similar ranges of pH, temperature and salinity, but they differed significantly in substrate ranges and catabolic products. Unlike Syntrophobacter wolinii, Smithella propionica grew axenically on crotonate, although very slowly. Co-cultures of Smithella propionica grew on propionate, and grew slowly on crotonate or butyrate. Syntrophobacter wolinii and Syntrophobacter pfennigii grow on propionate plus sulfate, whereas Smithella propionica did not. Comparisons of 165 rDNA genes indicated that Smithella propionica is most closely related to Syntrophus, and is more distantly related to Syntrophobacter.

We generated draft genome sequences for two cold-adapted Archaea, Methanogenium frigidum and Methanococcoides burtonii, to identify genotypic characteristics that distinguish them from Archaea with a higher optimal growth temperature (OGT). Comparative genomics revealed trends in amino acid and tRNA composition, and structural features of proteins. Proteins from the cold-adapted Archaea are characterized by a higher content of noncharged polar amino acids, particularly Gln and Thr and a lower content of hydrophobic amino acids, particularly Leu. Sequence data from nine methanogen genomes (OGT 15°-98°C) were used to generate 1111 modeled protein structures. Analysis of the models from the cold-adapted Archaea showed a strong tendency in the solvent-accessible area for more Gln, Thr, and hydrophobic residues and fewer charged residues. A cold shock domain (CSD) protein (CspA homolog) was identified in M. frigidum, two hypothetical proteins with CSD-folds in M. burtonii, and a unique winged helix DNA-binding domain protein in M. burtonii. This suggests that these types of nucleic acid binding proteins have a critical role in cold-adapted Archaea. Structural analysis of tRNA sequences from the Archaea indicated that GC content is the major factor influencing tRNA stability in hyperthermophiles, but not in the psychrophiles, mesophiles or moderate thermophiles. Below an OGT of 60°C, the GC content in tRNA was largely unchanged, indicating that any requirement for flexibility of tRNA in psychrophiles is mediated by other means. This is the first time that comparisons have been performed with genome data from Archaea spanning the growth temperature extremes from psychrophiles to hyperthermophiles.

We calculated the potential H2 and formate diffusion between microbes and found that at H2 concentrations commonly found in nature, H2 could not diffuse rapidly enough to dispersed methanogenic cells to account for the rate of methane synthesis but formate could. Our calculations were based on individual organisms dispersed in the medium, as supported by microscopic observations of butyrate-degrading cocultures. We isolated an axenic culture of Syntrophomonas wolfei and cultivated it on butyrate in syntrophic coculture with Methanobacterium formicicum; during growth the H2 concentration was 63 nM (10.6 Pa). S. wolfei contained formate dehydrogenase activity (as does M. formicicum), which would allow interspecies formate transfer in that coculture. Thus, interspecies formate transfer may be the predominant mechanism of syntrophy. Our diffusion calculations also indicated that H2 concentration at the cell surface of H2-consuming organisms was low but increased to approximately the bulk-fluid concentration at a distance of about 10 ,um from the surface. Thus, routine estimation of kinetic parameters would greatly overestimate the Km for H2 or formate.

Bacillus infernus sp. nov. was isolated from ca. 2,700 m below the land surface in the Taylorsville Triassic Basin in Virginia. B. infernus was a strict anaerobe that grew on formate or lactate with Fe(III), MnO,, trimethylamine oxide, or nitrate (reduced to nitrite) as an electron acceptor, and it also grew fermentatively on glucose. Type strain TH-23 and five reference strains were gram-positive rods that were thermophilic (growth occurred at 61"C), halotolerant (good growth occurred in the presence of Na+ concentrations up to 0.6 M), and very slightly alkaliphilic (good growth occurred at pH 7.3 to 7.8). A phylogenetic analysis of its 16s rRNA indicated that B. infernus should be classified as a new species of the genus Bacilius. B. infernus is the only strictly anaerobic species in the genus Bacillus.

Representatives of the family Methanosarcinuceae were analyzed phylogenetically by comparing partial sequences of their methyl-coenzyme M reductase (mcrI) genes. A 490-bp fragment from the A subunit of the gene was selected, amplified by the PCR, cloned, and sequenced for each of 25 strains belonging to the Metlzanosarcinuceae. The sequences obtained were aligned with the corresponding portions of five previously published sequences, and all of the sequences were compared to determine phylogenetic distances by Fitch distance matrix methods. We prepared analogous trees based on 16s rRNA sequences; these trees corresponded closely to the mcrI trees, although the mcrI sequences of pairs of organisms had 3.01 f 0.541 times more changes than the respective pairs of 16s rRNA sequences, suggesting that the mcrI fragment evolved about three times more rapidly than the 16s rRNA gene. The qualitative similarity of the mcrI and 16s rRNA trees suggests that transfer of genetic information between dissimilar organisms has not significantly affected these sequences, although we found inconsistencies between some mcrI distances that we measured and previously published DNA reassociation data. It is unlikely that multiple mcrI isogenes were present in the organisms that we examined, because we found no major discrepancies in multiple determinations of mcrI sequences from the same organism. Our primers for the PCR also match analogous sites in the previously published rncrII sequences, but all of the sequences that we obtained from members of the Methanosarcinaceae were more closely related to mcrI sequences than to mcrII sequences, suggesting that members of the Methanosarcinuceae do not have distinct mcrII genes,

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