The genes mvhDGA, which encode the subunit polypeptides of the methyl viologen-reducing hydrogenase in Methanobacterium thermoautotrophicum strain AH, have been cloned and sequenced. These genes, together with a fourth open reading frame designated mvhB, are tightly linked and appear to form an operon that is transcribed starting 42 base pairs upstream of mvhD. The organization and sequences ofthe mvhG and mvhA genes indicate a common evolutionary ancestry with genes encoding the small and large subunits of hydrogenases in eubacterial species. The product of the mvhB gene is predicted to contain six tandomly repeated bacterial-ferredoxinlike domains and, therefore, is predicted to be a polyferredoxin that could contain as many as 48 iron atoms in 12 Fe4S4 clusters.Methanobacterium thermoautotrophicum reduces CO2 to CH4 using H2 as the reductant. Therefore, hydrogenase activity is essential for methanogenesis in this species, and two hydrogenases have been purified and characterized from extracts of M. thermoautotrophicum (1-3). In this report we describe the organization and structure of the clustered genes (mvhDGA) that encode subunits ofthe hydrogenase that does not reduce cofactor F420, the enzyme conventionally designated as the methyl viologen-reducing hydrogenase (MV hydrogenase desired recombinant clones by their ability to direct the synthesis of antigens in Escherichia coli that bound rabbit antibodies raised against the a subunit of the F420-reducing hydrogenase, purified as previously described from M. thermoautotrophicum AH (2). DNA prepared from positive clones was subcloned into pUC8 (11) and sequenced (Fig. 1). § § Determination of Amino Acid Sequences. The aminoterminal sequences of the subunit polypeptides of MVhydrogenase purified from M. thermoautotrophicum AH and separated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis were determined by using an Applied Biosystems 470A gas-phase protein sequencer. The subunits did not have N-terminal methionyl residues. The N-terminal amino acid sequence of the a subunit was found to be '40% identical to the N-terminal sequence of the a subunit of the F420-reducing hydrogenase (2). This conservation of amino acid residues presumably accounts for the immunological cross-reactivity of the two polypeptides.RNA Preparation, Primer Extension, and RNA Sequencing. Total cellular RNA was prepared from lysates of M. thermoautotrophicum strain AH. 32P-labeled oligonucleotide primers were synthesized and hybridized to the M. thermoautotrophicum AH RNA, and the hybrid molecules were used in primer extension procedures to determine the 5' end of the mvh transcript (9) and to sequence the transcript of the mvhG gene (12) in the region of the cloned TGA codon (see Results).Primer-Directed Amplification and Sequencing of M. thermoautotrophicum AH Genomic DNA. Oligonucleotide primers (24 mers) were synthesized complementary to the sequences located 42 bp 5' and 42 bp 3' from the position at which the TGA codon had been detected in the cloned mvhG gen...

Transcription initiation of the hisA gene in vivo in the archaebacterium Methanococcus vannielii, as determined by nuclease S1 and primer extension analyses, occurs 73 base pairs (bp) upstream of the translation initiation site. Binding of M. vannielii RNA polymerase protects 43 bp of DNA, from 35 bp upstream (-35) to 8 bp downstream (+8) of the hisA mRNA initiation site, from digestion by DNase I and exonuclease III. An A + T rich region, with a sequence which conforms to the consensus sequence for promoters of stable RNA-encoding genes in methanogens, is found at the same location (-25) upstream of the polypeptide-encoding hisA gene. It appears therefore that a TATA-like sequence is also an element of promoters which direct transcription of polypeptide-encoding genes in this archaebacterium.

The DNA sequences of a region that includes the hisA gene of two related methanogenic archaebacteria, Methanococcus Methanogens are members of the archaebacteria, a group of prokaryotic organisms exhibiting several properties that are distinct from those seen in either eubacteria or eukaryotes (1, 2). Therefore, it was somewhat surprising when it was found that DNA cloned from several methanogenic species could complement auxotrophic mutations in Escherichia coli, Bacillus subtilis (3-6), and Salmonella typhimurium (unpublished results). In the case of two methanogens of the genus Methanococcus (vannielii and voltae), it was possible to clone methanogen DNA that complemented the hisA mutation of E. coli (4, 6). Since the hisA-complementing activity was efficiently expressed regardless of the orientation of the cloned DNA fragment within the vector, it seemed likely that the methanogen DNA harbored sequences that were recognized as signals for both transcription and translation. That methanococcal DNA would contain signals for initiation ofan early step in translation in E. coli was expected because these methanococci have the sequence A-U-C-A-C-C-U-C-C at the 3' end of their 16S rRNA (1), which is similar to that found at the 3' end of E. coli 16S rRNA (7). This sequence is believed to be important for initial binding of ribosomes to mRNA molecules (8).Since both M. voltae and M. vannielii have an overall base composition of --70% A+T (1), they should statistically contain those high A+T-rich sequences that often function as promoters in prokaryotes. Whether similar A+T-rich sequences are used as promoters for recognition by methanogen DNA-dependent RNA polymerases is currently unknown. The subunit composition of RNA polymerases from archaebacteria more closely resembles eukaryotic RNA polymerase II than eubacterial RNA polymerase (9), and thus archaebacterial promoters may well be eukaryotic rather than prokaryotic in their sequence organization.To better understand the molecular basis for expression of the hisA-complementing methanogen genes in E. coli and to provide basic information for evaluating possible mechanisms of gene expression in methanogens, we have sequenced the hisA-complementing genes ofboth M. voltae and M. vannielfi. The sequences obtained indicate evolutionary divergence of archaebacterial structural genes, support the prediction ofribosomal binding sites, and demonstrate repetitive elements within the intergenic regions. MATERIALS AND METHODSBacterial Strains and Phage. E. coli K-12 strain X760, which is a hisA auxotroph (4), was used to determine if plasmid derivatives carried hisA-complementing DNA.Enzymes and Chemicals. Restriction enzymes were purchased from Bethesda Research Laboratories, BoehringerMannheim, or New England Biolabs and were used in accordance with the supplier's directions. Klenow DNA polymerase, T4 DNA ligase, and proteinase K were purchased from Boehringer Mannheim. Deoxy and dideoxynucleotide triphosphates were purchased from Pharmacia and P-L Biochemicals. ...

A 2.7 kilobase pair (Kb) fragment of DNA, which complements mutations in the hisI locus of Escherichia coli, has been cloned and sequenced from the genome of the methanogenic archaebacterium Methanococcus vannielii. The cloned DNA directs the synthesis of three polypeptides, with molecular weights of 71,000, 29,000 and 15,600 in minicells of E. coli. Subcloning and mutagenesis demonstrates that hisI complementation results from the activity of the 15,600 molecular weight polypeptide. The primary structure of this archaebacterial gene and its gene product have been compared with the functionally equivalent gene and protein from the eubacterium E. coli (hisI) (Chiariotti et al. 1986) and from the eucaryote Saccharomyces cerevisiae (his4A) (Donahue et al. 1982). The DNA sequences of the archaebacterial and eubacterial genes are 40% homologous, the archaebacterial and eucaryotic DNA sequences are 47% homologous and, as previously reported (Bruni et al. 1986) the eubacterial and eucaryotic DNA sequences are 45% homologous. In E. coli the hisI locus is part of a bifunctional gene (hisI/E) within the single his operon. In S. cerevisiae the his4A locus is part of a multifunctional gene (his4) which encodes a protein with at least four enzymatic activities. The his genes of S. cerevisiae do not form an operon and are not physically linked. The M. vannielii hisI gene does not appear to be part of a multifunctional DNA sequence and, although it does appear to be within an operon, the open reading frames (ORFs) 5' and 3' to the M. vannielii hisI gene are not related to any published his sequences.(ABSTRACT TRUNCATED AT 250 WORDS)

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