The genome sequence of the solvent-producing bacterium Clostridium acetobutylicum ATCC 824 has been determined by the shotgun approach. The genome consists of a 3.94-Mb chromosome and a 192-kb megaplasmid that contains the majority of genes responsible for solvent production. Comparison of C. acetobutylicum to Bacillus subtilis reveals significant local conservation of gene order, which has not been seen in comparisons of other genomes with similar, or, in some cases closer, phylogenetic proximity. This conservation allows the prediction of many previously undetected operons in both bacteria. However, the C. acetobutylicum genome also contains a significant number of predicted operons that are shared with distantly related bacteria and archaea but not with B. subtilis. Phylogenetic analysis is compatible with the dissemination of such operons by horizontal transfer. The enzymes of the solventogenesis pathway and of the cellulosome of C. acetobutylicum comprise a new set of metabolic capacities not previously represented in the collection of complete genomes. These enzymes show a complex pattern of evolutionary affinities, emphasizing the role of lateral gene exchange in the evolution of the unique metabolic profile of the bacterium. Many of the sporulation genes identified in B. subtilis are missing in C. acetobutylicum, which suggests major differences in the sporulation process. Thus, comparative analysis reveals both significant conservation of the genome organization and pronounced differences in many systems that reflect unique adaptive strategies of the two gram-positive bacteria.

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Changes in growth rate, methanogenesis, growth yield (Y CH4 ), and methane gene transcription have been correlated with changes in the supply of H 2 to Methanobacterium thermoautotrophicum ⌬H cells growing on H 2 plus CO 2 in fed-batch cultures. Under conditions of excess H 2 , biomass and methanogenesis increased exponentially and in parallel, resulting in cultures with a constant Y CH4 and transcription of the mth and mrt genes that encode the H 2 -dependent N 5 ,N 10 -methenyltetrahydromethanopterin (methenyl-H 4 MPT) reductase (MTH) and methyl coenzyme M reductase II (MRII), respectively. Reducing the H 2 supply, by decreasing the percentage of H 2 in the input gas mixture or by reducing the mixing speed of the fermentor impeller, decreased the growth rate and resulted in lower and constant rates of methanogenesis. Under such H 2 -limited growth conditions, cultures grew with a continuously increasing Y CH4 and the mtd and mcr genes that encode the reduced coenzyme F 420 -dependent N 5 ,N 10 -methenyl-H 4 MPT reductase (MTD) and methyl coenzyme M reductase I (MRI), respectively, were transcribed. Changes in the kinetics of growth, methanogenesis, and methane gene transcription directed by reducing the H 2 supply could be reversed by restoring a high H 2 supply. Methane production continued, but at a low and constant rate, and only mcr transcripts could be detected when the H 2 supply was reduced to a level insufficient for growth. ftsA transcripts, which encode coenzyme F 390 synthetase, were most abundant in cells growing with high H 2 availability, consistent with coenzyme F 390 synthesis signaling a high exogenous supply of reductant.Most methanogens can grow and generate energy in a mineral salts medium with CO 2 and H 2 as the sole carbon and energy sources (14,41), and this is the only known lifestyle for Methanobacterium thermoautotrophicum ⌬H. There are seven steps in the energy-generating H 2 -dependent pathway of CO 2 reduction to CH 4 (7, 33) and the enzymes that catalyze these reactions have been characterized, in detail, from strains of M. thermoautotrophicum (2,8,10,13,27,42). The methane genes that encode these enzymes have been cloned and sequenced (3,8,10,13,18,21,22,24,32,35,36), leading to the discovery of two [Ni,Fe]-hydrogenases, designated the methyl viologenreducing hydrogenase (MVH) and the coenzyme F 420 -reducing hydrogenase (FRH) (1, 25); two formylmethanofuran dehydrogenases, one containing tungsten and one containing molybdenum (2, 13); two N 5 ,N 10 -methenyltetrahydromethanopterin (methenyl-H 4 MPT) reductases, designated MTH and MTD (18,22,42); and two methyl coenzyme M reductases, designated MRI and MRII (4,24,27). These enzymes facilitate the uptake of the reductant H 2 and catalyze steps 1, 4, and 7, respectively, in the reduction of CO 2 to CH 4 (7, 33). This complexity presumably provides M. thermoautotrophicum cells with the ability to adjust to changes in the availability of the substrates and of the metals employed as enzyme cofactors in methanogenesis.In 1980, Schönh...

Two regions of the Methanobacterium thermoautotrophicum genome containing genes that encode enzymes involved in methanogenesis (methane genes) have been cloned and sequenced to determine the extent of methane gene clustering and conservation. One region from the M. thermoautotrophicum strains ⌬H and Winter, extending ϳ13.5 kb upstream from the adjacent mvhDGAB and mrtBDGA operons that encode the methyl-viologen-reducing hydrogenase (MVH) and the methyl coenzyme M reductase II (MRII), respectively, was sequenced, and 76% sequence identity and very similar gene organizations were demonstrated. Five closely linked open reading frames were located immediately upstream of the mvh operon and were designated flpECBDA. The flpCBD genes encode amino acid sequences that are 31, 47, and 65% identical to the primary sequences of the ␣ and ␤ subunits of formate dehydrogenase and the ␦ subunit of MVH, respectively. Located immediately upstream of the flp genes was the mth gene, which encodes the H 2 -dependent methylene-tetrahydromethanopterin dehydrogenase (MTH). In contrast to this mth-flp-mvh-mrt cluster of methane genes, a separate ϳ5.4-kb genomic fragment cloned from M. thermoautotrophicum ⌬H contained only one methane gene, the mtd gene, which encodes the 8-hydroxy-5-deazaflavin (H 2 F 420 )-dependent methylene-tetrahydromethanopterin dehydrogenase (MTD). Northern (RNA) blot experiments demonstrated that mth was transcribed only at early growth stages in fermentor-grown cultures of M. thermoautotrophicum ⌬H, whereas mtd was transcribed at later growth stages and in the stationary phase. Very similar transcription patterns have been observed by T. D. Pihl, S. Sharma, and J. N. Reeve (J. Bacteriol. 176:6384-6391, 1994) for the MRI-and MRII-encoding operons, mrtBDGA and mcrBDCGA, in M. thermoautotrophicum ⌬H, suggesting coordinated regulation of methane gene expression. In contrast to the growth phase-dependent transcription of the mth/mrt and mtd/mcr genes, transcription of the mvhDGAB and frhADGB operons, which encode the two (NiFe) hydrogenases in M. thermoautotrophicum ⌬H, was found to occur at all growth stages. Seven enzymatic steps in the H 2 -dependent reduction of CO 2 to CH 4 by strains of the thermophilic archaeon Methanobacterium thermoautotrophicum have been characterized. During this process, CO 2 is progressively reduced through the formyl, methenyl, methylene, and methyl levels and transferred from methanofuran to tetrahydromethanopterin (H 4 MPT) to coenzyme M (CoM). In the final reaction, methyl-CoM is reduced and methane is released. Characterization of the enzymes involved in this pathway has led to the discovery of isoenzymes and of functionally equivalent enzymes (for a review see reference 35). The first step in methanogenesis is catalyzed by either a tungsten-or molybdenum-containing formyl-methanofuran dehydrogenase (29). The fourth reaction, the reduction of ,N 10 -methylene-H 4 MPT, can be catalyzed by either an H 2 -dependent (cofactor F 420 -independent) or F 420 -dependent methylene-H 4 MPT de...