Genome sequence and comparative analysis of the solvent-producing bacterium Clostridium acetobutylicum.

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.

Phylogenetic analysis of 18 thermophilic Methanobacterium isolates supports the proposals to create a new genus, Methanothermobacter gen. nov., and to reclassify several isolates in three species, Methanothermobacter thermautotrophicus comb. nov., Methanothermobacter wolfeii comb. nov., and Methanothermobacter marburgensis sp. nov.

Using a combination of 16S rRNA analysis and antigenic fingerprinting consisting of new and published data, the phylogenetic position of 18 thermophilic isolates currently classified as Methanobacterium species was reinvestigated. The results were verified by independent methods, including, where applicable, plasmid and phage typing. Comparative analysis of 16S rRNA data for 30 strains belonging to the order Methanobacteriales strongly suggested that mesophilic and thermophilic Methanobacterium isolates are distantly related and should be assigned to separate genera. For the thermophilic strains the genus Methanothermobacter was initially proposed by Boone, Whitman and Rouvière. Furthermore, the results support a reclassification of 15 isolates in three species within the proposed genus: (i) Methanothermobacter thermautotrophicus comb. nov., containing eight isolates, six of which are able to utilize formate (type strain deltaHT); (ii) Methanothermobacter wolfeii comb. nov., containing four formate-utilizing isolates (type strain DSM 2970T); (iii) Methanothermobacter marburgensis sp. nov., containing three obligately autotrophic isolates (type strain MarburgT). Of the nine isolates formerly referred to as Methanobacterium thermoformicicum, six were reclassified as Methanothermobacter thermautotrophicus and three as Methanothermobacter wolfeii.

Hydrogen regulation of growth, growth yields, and methane gene transcription in Methanobacterium thermoautotrophicum deltaH.

Changes in growth rate, methanogenesis, growth yield (Y(CH4)), and methane gene transcription have been correlated with changes in the supply of H2 to Methanobacterium thermoautotrophicum deltaH cells growing on H2 plus CO2 in fed-batch cultures. Under conditions of excess H2, 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 H2-dependent N5,N10-methenyltetrahydromethanopterin (methenyl-H4MPT) reductase (MTH) and methyl coenzyme M reductase II (MRII), respectively. Reducing the H2 supply, by decreasing the percentage of H2 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 H2-limited growth conditions, cultures grew with a continuously increasing Y(CH4) and the mtd and mcr genes that encode the reduced coenzyme F420-dependent N5,N10-methenyl-H4MPT 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 H2 supply could be reversed by restoring a high H2 supply. Methane production continued, but at a low and constant rate, and only mcr transcripts could be detected when the H2 supply was reduced to a level insufficient for growth. ftsA transcripts, which encode coenzyme F390 synthetase, were most abundant in cells growing with high H2 availability, consistent with coenzyme F390 synthesis signaling a high exogenous supply of reductant.

Growth- and substrate-dependent transcription of the formate dehydrogenase (fdhCAB) operon in Methanobacterium thermoformicicum Z-245.

The formate dehydrogenase-encoding fdhCAB operon and flanking genes have been cloned and sequenced from Methanobacterium thermoformicicum Z-245. fdh transcription was shown to be initiated 21 bp upstream from fdhC, although most fdh transcripts terminated or were processed between fdhC and fdhA. The resulting fdhC, fdhAB, and fdhCAB transcripts were present at all growth stages in cells growing on formate but were barely detectable during early exponential growth on H2 plus CO2. The levels of the fdh transcripts did, however, increase dramatically in cells growing on H2 plus CO2, coincident with the decrease in the growth rate and the onset of constant methanogenesis that occurred when culture densities reached an optical density at 600 nm of approximately 0.5. The mth transcript that encodes the H2-dependent methenyl-H4 MPT reductase (MTH) and the frh and mvh transcripts that encode the coenzyme F420-reducing (FRH) and nonreducing (MVH) hydrogenases, respectively, were also present in cells growing on formate, consistent with the synthesis of three hydrogenases, MTH, FRH, and MVH, in the absence of exogenously supplied H2. Reducing the H2 supply to M. thermoformicicum cells growing on H2 plus CO2 reduced the growth rate and CH4 production but increased frh and fdh transcription and also increased transcription of the mtd, mer, and mcr genes that encode enzymes that catalyze steps 4, 5, and 7, respectively, in the pathway of CO2 reduction to CH4. Reducing the H2 supply to a level insufficient for growth resulted in the disappearance of all methane gene transcripts except the mcr transcript, which increased. Regions flanking the fdhCAB operon in M. thermoformicicum Z-245 were used as probes to clone the homologous region from the Methanobacterium thermoautotrophicum deltaH genome. Sequencing revealed the presence of very similar genes except that the genome of M. thermoautotrophicum, a methanogen incapable of growth on formate, lacked the fdhCAB operon.

Phylogeny of Methanopyrus kandleri based on methyl coenzyme M reductase operons.

The mcrBDCGA operon that encodes methyl coenzyme M reductase (MR) in the hyperthermophile Methanopyrus kandleri was cloned and sequenced. The results of a phylogenetic analysis of the nine MR sequences now available support the position that M. kandleri is a separate methanogen lineage. As in other methanogens, the M. kandleri mcr operon is located immediately upstream of the mtrE gene, the promoter-proximal gene in an operon that encodes the N5-methyltetrahydromethanopterin:coenzyme M methyltransferase that catalyzes the step preceding the MR-catalyzed reaction in methanogenesis. In contrast to other methanogens and hyperthermophilic members of the Archaea, CG dinucleotides and CG-containing codons occur frequently in M. kandleri DNA. The MR subunit-encoding genes are preceded by sequences consistent with ribosome binding sites, indicating that mRNA-rRNA base pairing can still direct translation initiation in cells growing at temperatures above 100 degrees C.

Isolation and characterization of a copper-resistant methanogen from a copper-mining soil sample.

A copper-resistant methanogen for which the CuSO4 MICs were approximately 2- to 36-fold higher than those for other methanogens tested was isolated from a copper-mining area in the upper peninsula of Michigan. The rod-shaped methanogen used H2-CO2 or formate, but not acetate or methanol, as a growth substrate. Standing incubation with H2-CO2 medium resulted in a mat-like surface growth, dependent on the presence of hydrogen. The presence of 1 mM cupric salt resulted in longer filamentous and intertwined cells. Antigenic fingerprinting, 16S rRNA gene analysis, morphology, and substrate use suggest that the new isolate is a novel strain of Methanobacterium bryantii that is able to use formate.

Cloning, sequencing, and growth phase-dependent transcription of the coenzyme F420-dependent N5,N10-methylenetetrahydromethanopterin reductase-encoding genes from Methanobacterium thermoautotrophicum delta H and Methanopyrus kandleri.

The mer genes, which encode the coenzyme F420-dependent N5,N10-methylenetetrahydromethanopterin reductases (CH2 = H4MPT reductases), and their flanking regions have been cloned from Methanobacterium thermoautotrophicum delta H and Methanopyrus kandleri and sequenced. The mer genes have DNA sequences that are 57% identical and encode polypeptides with amino acid sequences that are 57% identical and 71% similar, with calculated molecular masses of 33.6 and 37.5 kDa, respectively. In M. thermoautotrophicum, mer transcription has been shown to initiate 10 bp upstream from the ATG translation initiating codon and to generate a monocistronic transcript approximately 1 kb in length. This transcript was synthesized at all stages of M. thermoautotrophicum delta H growth in batch cultures but was found to increase in abundance from the earliest stages of exponential growth, reaching a maximum level at the mid-exponential growth phase. For comparison, transcription of the ftr gene from M. thermoautotrophicum delta H that encodes the formylmethanofuran:tetrahydromethanopterin formyltransferase (A. A. DiMarco, K. A. Sment, J. Konisky, and R. S. Wolfe, J. Biol. Chem. 265:472-476, 1990) was included in this study. The ftr transcript was found similarly to be monocistronic and to be approximately 1 kb in length, but, in contrast to the mer transcript, the ftr transcript was present at maximum levels at both the early and the mid-exponential growth stages.

Characterization of a 45-kDa flavoprotein and evidence for a rubredoxin, two proteins that could participate in electron transport from H2 to CO2 in methanogenesis in Methanobacterium thermoautotrophicum.

Methanobacterium thermoautotrophicum strains contain a flavoprotein (flavoprotein A) that copurifies with the H2:heterodisulfide oxidoreductase complex. In this study, we report the iron-dependent synthesis and biochemical properties of flavoprotein A, cloning and sequencing of the flavoprotein-A-encoding gene (fpaA) and the co-transcription of fpaA with two downstream open reading frames, one of which (rdxA) appears to encode a rubredoxin. Native flavoprotein A has been shown to be a homodimer of a 45-kDa polypeptide that contains 1.3 mol FMN/45-kDa subunit but no iron or acid-labile sulfur. Catalytic amounts of the H2:heterodisulfide oxidoreductase complex or of the F420-reducing hydrogenase reduced flavoprotein A with H2, at specific rates of 0.3-0.4 U/mg enzyme, generating up to 70% flavin semiquinone before reduction to the flavin hydroquinone was observed. This intermediate accumulation of the semiquinone species had a kinetic rather than a thermodynamic basis, because the semiquinone form of flavoprotein A, generated by photoreduction, disproportionated quantitatively to the quinone and hydroquinone species. The midpoint potential of the quinone/hydroquinone couple was estimated to be 230 +/- 15 mV, at pH 7.6, versus the normal hydrogen electrode. Quantitation of Western blots demonstrated that flavoprotein A constituted approximately 1.5% of the soluble protein in cells grown in an iron-sufficient medium but that this increased to about 6% of the cellular protein when the iron the medium was depleted. The increase in the flavoprotein A content of cells grown under iron-limiting conditions was mirrored by a decrease in the content of the iron-rich polyferredoxin that also copurified with the H2:heterodisulfide oxidoreductase complex. The fpaA gene, cloned and sequenced from M. thermoautotrophicum strain delta H, encodes 404 amino acids in a sequence that has a C-terminal domain (approximately 130 amino acid residues) with features consistent with a flavodoxin structure. The remainder of flavoprotein A has sequences that are also predicted to be present in the N-terminal region of the orf14 gene product, which also appears to be an enlarged flavodoxin, encoded in the nif region of Rhodobacter capsulatus. Immediately downstream from fpaA, two open reading frames designated orfX and rdxA, have been located and shown by Northern-blot analyses to be co-transcribed with fpaA, although approximately 50% of fpaA-orfX-rdxA transcripts terminated or were cleaved within rdxA. Primer extension studies revealed that transcription of this transcriptional unit (the fpa operon) was initiated 32 nucleotides upstream of fpaA, at a site 25 nucleotides downstream from a sequence consistent with an archaeal TATA-box promoter element.(ABSTRACT TRUNCATED AT 400 WORDS)

Organization and growth phase-dependent transcription of methane genes in two regions of the Methanobacterium thermoautotrophicum genome.

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 delta H and Winter, extending approximately 13.5 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 alpha and beta subunits of formate dehydrogenase and the delta subunit of MVH, respectively. Located immediately upstream of the flp genes was the mth gene, which encodes the H2-dependent methylene-tetrahydromethanopterin dehydrogenase (MTH). In contrast to this mth-flp-mvh-mrt cluster of methane genes, a separate approximately 5.4-kb genomic fragment cloned from M. thermoautotrophicum delta H contained only one methane gene, the mtd gene, which encodes the 8-hydroxy-5-deazaflavin (H2F420)-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 delta 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, im M. thermoautotrophicum deltaH, 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 deltaH, was found to occur at all growth stages.

Distribution and characterization of plasmid-related sequences in the chromosomal DNA of different thermophilic Methanobacterium strains.

The genomes of several thermophilic members of the genus Methanobacterium were analyzed for homology to the related restriction-modification plasmids pFV1 and pFZ1 from M. thermoformicicum strains THF and Z-245, respectively. Two plasmid regions, designated FR-I and FR-II, could be identified with chromosomal counterparts in six Methanobacterium strains. Multiple copies of the pFV1-specific element FR-I were detected in the M. thermoformicicum strains CSM3, FF1, FF3 and M. thermoautotrophicum delta H. Sequence analysis showed that one FR-I element had been integrated in almost identical sequence contexts into the chromosomes of the strains CSM3 and delta H. Comparison of the FR-I elements from these strains with that from pFV1 revealed that they consisted of two subfragments, boxI (1118 bp) and boxII (383 bp), the order of which is variable. Each subfragment was identical on the sequence level with the corresponding plasmid-borne element and was flanked by terminal direct repeats with the consensus sequence A(A/T)ATTT. These results suggest that FR-I represents a mobile element. FR-II was located on both plasmids pFV1 and pFZ1, and on the chromosome of M. thermoformicicum strains THF, CSM3 and HN4. Comparison of the nucleotide sequences of the two plasmid FR-II copies and that from the chromosome of strain CSM3 showed that the FR-II segments were approximately 2.5-3.0 kb in size and contained large open reading frames (ORFs) that may encode highly related proteins with an as yet unknown function.

Modular organization of related Archaeal plasmids encoding different restriction-modification systems in Methanobacterium thermoformicicum.

Nucleotide sequence comparison of the related 13513-bp plasmid pFV1 and the 11014-bp plasmid pFZ1 from the thermophilic archaeon Methanobacterium thermoformicicum THF and Z-245, respectively, revealed a homologous, approximately 8.2 kb backbone structure that is interrupted by plasmid-specific elements. Various highly conserved palindromic structures and an ORF that could code for a NTP-binding protein were identified within the backbone structure and may be involved in plasmid maintenance and replication. Each plasmid contains at comparable locations a module which specifies components of different restriction-modification (R/M) systems. The R/M module of pFV1 contained, in addition to the genes of the GGCC-recognizing R/M system MthTI, an ORF which may be involved in repair of G-T mismatches generated by deamination of m5C at high temperatures.

Identification of the CTAG-recognizing restriction-modification systems MthZI and MthFI from Methanobacterium thermoformicicum and characterization of the plasmid-encoded mthZIM gene.

Two CTAG-recognizing restriction and modification (R/M) systems, designated MthZI and MthFI, were identified in the thermophilic archaeon Methanobacterium thermoformicicum strains Z-245 and FTF, respectively. Further analysis revealed that the methyltransferase (MTase) genes are plasmid-located in both strains. The plasmid pFZ1-encoded mthZIM gene of strain Z-245 was further characterized by subcloning and expression studies in Escherichia coli followed by nucleotide sequence analysis. The mthZIM gene is 1065 bp in size and may code for a protein of 355 amino acids (M(r) 42,476 Da). The deduced amino acid sequence of the M.MthZI enzyme shares substantial similarity with four distinct regions from several m4C- and m6A-MTases, and contains the TSPPY motif that is so far only found in m4C-MTases. Partially overlapping with the mthZIM gene and in reverse orientation, an additional ORF was identified with a size of 606 bp potentially coding for a protein of 202 amino acids (M(r) 23.710 Da). This ORF is suggested to encode the corresponding endonuclease R.MthZI.

Characterization of the archaeal, plasmid-encoded type II restriction-modification system MthTI from Methanobacterium thermoformicicum THF: homology to the bacterial NgoPII system from Neisseria gonorrhoeae.

A restriction-modification system, designated MthTI, was localized on plasmid pFV1 from the thermophilic archaeon Methanobacterium thermoformicicum THF. The MthTI system is a new member of the family of GGCC-recognizing restriction-modification systems. Functional expression of the archaeal MthTI genes was obtained in Escherichia coli. The mthTIR and mthTIM genes are 843 and 990 bp in size and code for proteins of 281 (32,102 Da) and 330 (37,360 Da) amino acids, respectively. The deduced amino acid sequence of M.MthTI showed high similarity with that of the isospecific methyltransferases M.NgoPII and M.HaeIII. In addition, extensive sequence similarity on the amino acid level was observed for the endonucleases R.MthTI and R.NgoPII. Moreover, the endonuclease and methyltransferase genes of the thermophilic MthTI system and those of the Neisseria gonorrhoeae NgoPII system show identical organizations and high (54.5%) nucleotide identity. This finding suggests horizontal transfer of restriction-modification systems between members of the domains Bacteria and Archaea.

DNA relatedness among some thermophilic members of the genus Methanobacterium: emendation of the species Methanobacterium thermoautotrophicum and rejection of Methanobacterium thermoformicicum as a synonym of Methanobacterium thermoautotrophicum.

DNA reassociation was used to determine levels of relatedness among four thermophilic Methanobacterium strains that are able to use formate and between these organisms and two representative strains of Methanobacterium thermoautotrophicum, strain delta HT (= DSM 1053T = ATCC 29096T) (T = type strain) and strain Marburg (= DSM 2133). Three homology groups were delineated, and these groups coincided with the clusters identified by antigenic fingerprinting. The first group, which had levels of cross hybridization that ranged from 73 to 99%, included M. thermoautotrophicum delta HT, Methanobacterium thermoformicicum Z-245, Methanobacterium sp. strain THF, and Methanobacterium sp. strain FTF. The second and third groups were each represented by only one strain, Methanobacterium sp. strain CB-12 and M. thermoautotrophicum Marburg, respectively (cross-hybridization levels, 13 to 30 and 29 to 33%, respectively). Our results indicate that the name M. thermoformicicum should be rejected as it is a synonym of M. thermoautotrophicum. The taxonomic positions of strains Marburg and CB-12 need further investigation.