TRANSCRIPTIONAL REGULATION OF THE CARBON-MONOXIDE DEHYDROGENASE GENE (CDHA) IN METHANOSARCINA-THERMOPHILA

The mechanisms of gene regulation in the phylogenetic domain Archaea a re not yet understood. To examine the expression of a gene encoding a highly regulated catabolic enzyme from the methanogenic archaea, a Met hanosarcina thermophila lambdagt11 chromosomal library was probed with antiserum prepared against the 89-kDa subunit of carbon monoxide dehy drogenase, an enzyme which is required for growth and methanogenesis f rom acetate. A 2.3-kilobase DNA fragment was isolated that encoded 300 bases of the 5'-end of cdhA, the gene which encodes the 89-kDa subuni t, and 2 kilobases upstream of cdhA that included an upstream open rea ding frame (ORF1). Primer extension analyses determined that cdhA and ORF1 each had a single transcriptional initiation site located 370 and 9 nucleotides, respectively, 5' of the putative translation initiatio n codons for cdhA and ORF1. Each promoter element had sequence similar ity to a consensus archaeal promoter sequence. Three discrete mRNA cdh A transcripts of 9.5, 5.6, and 4.8 kilobases and one mRNA ORF1 transcr ipt of <2 kilobases were identified. All four transcripts were optimal ly expressed in cells grown with acetate, while growth with the more e nergetically favorable substrates methanol and trimethylamine caused a significant reduction in levels of the cdhA and ORF1 mRNA's. The half -lives of the 5' ends of the three cdhA transcripts and entire ORF1 mR NA transcript were approximately 2 min upon addition of methanol to ce lls growing exponentially in medium that contained acetate. Results of this study demonstrate that transcription of both cdhA and ORF1 is hi ghly regulated in response to substrate by this methanogenic archaeon.