TRANSCRIPTIONAL REGULATION OF THE PHOSPHOTRANSACETYLASE-ENCODING AND ACETATE KINASE-ENCODING GENES (PTA AND ACK) FROM METHANOSARCINA-THERMOPHILA

Phosphotransacetylase and acetate kinase catalyze the activation of ac etate to acetyl coenzyme A in the first step of methanogenesis from ac etate in Methanosarcina thermophila. The genes encoding these enzymes (pta and ack) have been cloned and sequenced. They are arranged on the chromosome with pta upstream of ack (M. T. Latimer, and J. G. Ferry, J. Bacteriol. 175:6822-6829, 1993). The activities of phosphotransacet ylase and acetate kinase are at least 8- to 11-fold higher in acetate- growth cells than in cells grown on methanol, monomethylamine, dimethy lamine, or trimethylamine. Northern blot (RNA) analyses demonstrated t hat pta and ack are transcribed as an approximately 2.4-kb polycistron ic message and that the regulation of enzyme synthesis occurs at the m RNA level. Primer extension analyses revealed a transcriptional start site located 27 bp upstream from the translational start of the pta ge ne and 24 bp downstream from a consensus archaeal boxA promoter sequen ce. S1 nuclease protection assays detected transcripts with four diffe rent 3' ends, each of which mapped to the beginning of four consecutiv e direct repeats. Northern blot analysis using an ack-specific probe d etected both the 2.4-kb polycistronic transcript and a smaller 1.4-kb transcript which is the estimated size of monocistronic ack mRNA. A pr imer extension product was detected with an ack-specific primer; the 5 ' end of the product was in the intergenic region between the pta and ack genes but did not follow a consensus archaeal boxA sequence. This result, as well as detection of an additional 1.4-kb mRNA species, sug gests processing of the polycistronic 2.4-kb transcript.