ORGANIZATION AND GROWTH PHASE-DEPENDENT TRANSCRIPTION OF METHANE GENES IN 2 REGIONS OF THE METHANOBACTERIUM-THERMOAUTOTROPHICUM GENOME

Two regions of the Methanobacterium thermoautotrophicum genome contain ing genes that encode enzymes involved in methanogenesis (methane gene s) have been cloned and sequenced to determine the extent of methane g ene clustering and conservation, One region from the M. thermoautotrop hicum strains Delta H and Winter, extending similar to 13.5 kb upstrea m from the adjacent mvhDGAB and mrtBDGA operons that encode the methyl -viologen-reducing hydrogenase (MVH) and the methyl coenzyme M reducta se II (MRII), respectively, was sequenced, and 76% sequence identity a nd very similar gene organizations were demonstrated, Five closely lin ked open reading frames were located immediately upstream of the mvh o peron 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 H-2-dependent methylene-tet yahydromethanopterin dehydrogenase (MTH). In contrast to this mth-JEp- mvh-mrt duster of methane genes, a separate similar to 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 (M TD), Northern (RNA) blot experiments demonstrated that mth was transcr ibed only at early growth stages in fermenter-grown cultures of M. the rmoautotrophicum Delta H, whereas mtd was transcribed at later growth stages and in the stationary phase, Very similar transcription pattern s have been observed by T. D. Pihl, S. Sharma, and J. N. Reeve (J. Bac teriol, 176:6384-6391, 1994) for the MRI- and MRII-encoding operons, m rtBDGA and mcrBDCGA, in M. thermoautotrophicum Delta H, suggesting coo rdinated regulation of methane gene expression, In contrast to the gro wth phase-dependent transcription of the mth/mrt and mtd/mcr genes, tr anscription of the mvhDGAB and frhADGB operons, which encode the two ( NiFe) hydrogenases in M. thermoautotrophicum Delta H, was found to Occ ur at all growth stages.