Transcriptional regulation of the carbon monoxide dehydrogenase gene (cdhA) in Methanosarcina thermophila

Abstract

The mechanisms of gene regulation in the phylogenetic domain Archaea are not yet understood. To examine the expression of a gene encoding a highly regulated catabolic enzyme from the methanogenic archaea, a Methanosarcina thermophila lambda gt11 chromosomal library was probed with antiserum prepared against the 89-kDa subunit of carbon monoxide dehydrogenase, an enzyme which is required for growth and methanogenesis from 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 subunit, and 2 kilobases upstream of cdhA that included an upstream open reading 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 initiation codons for cdhA and ORF1. Each promoter element had sequence similarity to a consensus archaeal promoter sequence. Three discrete mRNA cdhA transcripts of 9.5, 5.6, and 4.8 kilobases and one mRNA ORF1 transcript of < 2 kilobases were identified. All four transcripts were optimally expressed in cells grown with acetate, while growth with the more energetically 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 mRNA transcript were approximately 2 min upon addition of methanol to cells growing exponentially in medium that contained acetate. Results of this study demonstrate that transcription of both cdhA and ORF1 is highly regulated in response to substrate by this methanogenic archaeon.