Transcriptional regulation of the phosphotransacetylase-encoding and acetate kinase-encoding genes (pta and ack) from Methanosarcina thermophila

Abstract

Phosphotransacetylase and acetate kinase catalyze the activation of acetate to acetyl coenzyme A in the first step of methanogenesis from acetate 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 phosphotransacetylase and acetate kinase are at least 8- to 11-fold higher in acetate-grown cells than in cells grown on methanol, monomethylamine, dimethylamine, or trimethylamine. Northern blot (RNA) analyses demonstrated that pta and ack are transcribed as an approximately 2.4-kb polycistronic message and that the regulation of enzyme synthesis occurs at the mRNA level. Primer extension analyses revealed a transcriptional start site located 27 bp upstream from the translational start of the pta gene and 24 bp downstream from a consensus archaeal boxA promoter sequence. S1 nuclease protection assays detected transcripts with four different 3' ends, each of which mapped to the beginning of four consecutive direct repeats. Northern blot analysis using an ack-specific probe detected both the 2.4-kb polycistronic transcript and a smaller 1.4-kb transcript which is the estimated size of monocistronic ack mRNA. A primer 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, suggests processing of the polycistronic 2.4-kb transcript.