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Growth phase-dependent transcription of the genes that encode the two methyl coenzyme M reductase isoenzymes and N5-methyltetrahydromethanopterin:coenzyme M methyltransferase in Methanobacterium thermoautotrophicum delta H.

ABSTRACT: The genes encoding the two isoenzymes of methyl coenzyme M reductase (MRI and MRII) in Methanobacterium thermoautotrophicum delta H have been cloned and sequenced. The MRI-encoding mcr operon (mcrBDCGA) has been located immediately upstream from the mtr operon (mtrEDCBA) that encodes N5-methyltetrahydromethanopterin:coenzyme M methyltransferase, the enzyme that catalyzes the step preceding the MR-catalyzed reaction in methanogenesis. The MRII-encoding mrt operon (mrtBDGA) has been located between the operon that encodes the methyl viologen-reducing hydrogenase and an open reading frame (designated pyrC) predicted to encode dihydroorotase. Surprisingly, the mrt operon has been found to contain only four genes (mrtBDGA), lacking the equivalent of the mcrC gene that is present in all mcr operons. A protocol that isolates transcripts intact from M. thermoautotrophicum delta H cells has been developed and used, with primer extension and Northern (RNA) blot procedures, to identify the sites of transcription initiation upstream of the mcr, mrt, and mtr operons and to determine the relative numbers of these transcripts in cells at different growth stages. Transcription of the mrt operon was found to occur only at early times in batch cultures and was then replaced by transcription of the mcr operon. Transcripts of the mtr operon were detectable at all times; however, at early times, all mtr transcripts were initiated at the mtr promoter, whereas at later times, during mcr transcription, approximately 3% of mcr transcripts were extended to generate mcr plus mtr transcripts that constituted approximately 20% of all mtr transcripts present.

SUBMITTER: Pihl TD

PROVIDER: S-EPMC196980 | biostudies-other | 1994 Oct

REPOSITORIES: biostudies-other

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Growth phase-dependent transcription of the genes that encode the two methyl coenzyme M reductase isoenzymes and N5-methyltetrahydromethanopterin:coenzyme M methyltransferase in Methanobacterium thermoautotrophicum delta H.

Pihl T D TD Sharma S S Reeve J N JN

Journal of bacteriology 19941001 20

The genes encoding the two isoenzymes of methyl coenzyme M reductase (MRI and MRII) in Methanobacterium thermoautotrophicum delta H have been cloned and sequenced. The MRI-encoding mcr operon (mcrBDCGA) has been located immediately upstream from the mtr operon (mtrEDCBA) that encodes N5-methyltetrahydromethanopterin:coenzyme M methyltransferase, the enzyme that catalyzes the step preceding the MR-catalyzed reaction in methanogenesis. The MRII-encoding mrt operon (mrtBDGA) has been located betwee ...[more]

PMID: 7929010

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Project description:Computer analysis of the archaeal genome databases failed to identify orthologues of all of the bacterial cobamide biosynthetic enzymes. Of particular interest was the lack of an orthologue of the bifunctional nucleoside triphosphate (NTP):5'-deoxyadenosylcobinamide kinase/GTP:adenosylcobinamide-phosphate guanylyltransferase enzyme (CobU in Salmonella enterica). This paper reports the identification of an archaeal gene encoding a new nucleotidyltransferase, which is proposed to be the nonorthologous replacement of the S. enterica cobU gene. The gene encoding this nucleotidyltransferase was identified using comparative genome analysis of the sequenced archaeal genomes. Orthologues of the gene encoding this activity are limited at present to members of the domain Archaea. The corresponding ORF open reading frame from Methanobacterium thermoautotrophicum Delta H (MTH1152; referred to as cobY) was amplified and cloned, and the CobY protein was expressed and purified from Escherichia coli as a hexahistidine-tagged fusion protein. This enzyme had GTP:adenosylcobinamide-phosphate guanylyltransferase activity but did not have the NTP:AdoCbi kinase activity associated with the CobU enzyme of S. enterica. NTP:adenosylcobinamide kinase activity was not detected in M. thermoautotrophicum Delta H cell extract, suggesting that this organism may not have this activity. The cobY gene complemented a cobU mutant of S. enterica grown under anaerobic conditions where growth of the cell depended on de novo adenosylcobalamin biosynthesis. cobY, however, failed to restore adenosylcobalamin biosynthesis in cobU mutants grown under aerobic conditions where de novo synthesis of this coenzyme was blocked, and growth of the cell depended on the assimilation of exogenous cobinamide. These data strongly support the proposal that the relevant cobinamide intermediates during de novo adenosylcobalamin biosynthesis are adenosylcobinamide-phosphate and adenosylcobinamide-GDP, not adenosylcobinamide. Therefore, NTP:adenosylcobinamide kinase activity is not required for de novo cobamide biosynthesis.

Dataset Information

Growth phase-dependent transcription of the genes that encode the two methyl coenzyme M reductase isoenzymes and N5-methyltetrahydromethanopterin:coenzyme M methyltransferase in Methanobacterium thermoautotrophicum delta H.

Publications

Growth phase-dependent transcription of the genes that encode the two methyl coenzyme M reductase isoenzymes and N5-methyltetrahydromethanopterin:coenzyme M methyltransferase in Methanobacterium thermoautotrophicum delta H.

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