GROWTH PHASE-DEPENDENT TRANSCRIPTION OF THE GENES THAT ENCODE THE 2 METHYL COENZYME-M REDUCTASE ISOENZYMES AND N-5-METHYLTETRAHYDROMETHANOPTERIN-COENZYME-M METHYLTRANSFERASE IN METHANOBACTERIUM-THERMOAUTOTROPHICUM DELTA-H

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 mh. operon (mtrEDCBA) that encodes N-5-methgltetrahydromethanopterin:coenzyme M methyltransferase, the enzyme that catalyzes the step preceding the MR-catalyzed reaction in methanogenesis. The MW-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. thermoartfotrophicum 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 mh. operons and to determine the relative numbers of these transcripts in cells at different growth stages. Transcription of the mrf 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 ma operon were detectable at all times; however, at early times, all ma transcripts were initiated at the mtr promoter, whereas at later times, during mcr transcription, similar to 3% of mcr transcripts were extended to generate mcr plus mtr transcripts that constituted similar to 20% of all mtr transcripts present.