The Trimethylamine Methyltransferase Gene and Multiple Dimethylamine Methyltransferase Genes of Methanosarcina barkeri Contain In-Frame and Read-Through Amber Codons

Abstract

Three different methyltransferases initiate methanogenesis from trimethylamine (TMA), dimethylamine (DMA) or monomethylamine (MMA) by methylating different cognate corrinoid proteins that are subsequently used to methylate coenzyme M (CoM). Here, genes encoding the DMA and TMA methyltransferases are characterized for the first time. A single copy of mttB, the TMA methyltransferase gene, was cotranscribed with a copy of the DMA methyltransferase gene, mtbB1. However, two other nearly identical copies of mtbB1, designated mtbB2 and mtbB3, were also found in the genome. A 6.8-kb transcript was detected with probes to mttB and mtbB1, as well as to mtbC and mttC, encoding the cognate corrinoid proteins for DMA:CoM and TMA:CoM methyl transfer, respectively, and with probes to mttP, encoding a putative membrane protein which might function as a methylamine permease. These results indicate that these genes, found on the chromosome in the order mtbC, mttB, mttC, mttP, and mtbB1, form a single transcriptional unit. A transcriptional start site was detected 303 or 304 bp upstream of the translational start of mtbC. The MMA, DMA, and TMA methyltransferases are not homologs; however, like the MMA methyltransferase gene, the genes encoding the DMA and TMA methyltransferases each contain a single in-frame amber codon. Each of the three DMA methyltransferase gene copies from Methanosarcina barkeri contained an amber codon at the same position, followed by a downstream UAA or UGA codon. The C-terminal residues of DMA methyltransferase purified from TMA-grown cells matched the residues predicted for the gene products of mtbB1, mtbB2, or mtbB3 if termination occurred at the UAA or UGA codon rather than the in-frame amber codon. The mttB gene from Methanosarcina thermophila contained a UAG codon at the same position as the M. barkeri mttB gene. The UAG codon is also present in mttB transcripts. Thus, the genes encoding the three types of methyltransferases that initiate methanogenesis from methylamine contain in-frame amber codons that are suppressed during expression of the characterized methyltransferases.

Effects of Nitrogen and Carbon Sources on Transcription of Soluble Methyltransferases in Methanosarcina mazei Strain Gö1

“…mazei mtbC 1 , mttB 1 , mttC 1 , mttP, and mtbB 1 (MM 1687 to MM 1694) have been shown to form a transcriptional unit (K. Veit and R. A. Schmitz, unpublished), which is in accordance to the genomic organization in M. barkeri (15). Thus, in the following experiments the mttB 1 gene was exemplarily investigated for the regulation of the complete operon encoding DMA and TMA methyltransferase 1, their corresponding corrinoid proteins, and a potential TMA permease.…”

“…This analysis showed that the observed changes in expression of the mtmBC operons during lag-or stationary-phase growth compared to exponential- Transcription of pyl genes does not appear to be affected by nitrogen limitation and the presence of TMA. All methylamine methyltransferase transcripts possess an internal amber codon (UAG) (15), which encodes the recently discovered amino acid pyrrolysine (12,19). Thus, synthesis of functional methylamine methyltransferases is dependent on the expression of the pyl genes, whose gene products are responsible for the synthesis of pyrrolysyl-tRNA Pyl .…”

“…The indicated open reading frame numbers were obtained from GenBank, of the National Center for Biotechnology (4). Genes are designated according to Paul et al (15). The dotted lines indicate the in-frame amber codon of methylamine methyltransferases.…”

“…Within this nomenclature, B describes the substrate specific methyltransferase, C the corrinoid binding polypeptide, and A the CoM-methylating protein. For M. barkeri multiple and nearly identical copies of the operons encoding DMA and MMA methyltransferases and their respective corrinoid proteins have been identified (15,19). Analyzing the genome sequence of M. mazei, seven operons coding for methylamine methyltransferases and their corresponding corrinoid proteins and three genes coding for methylcobalamine:CoM methyltransferases have been identified (4), an overview of which is depicted in Fig.…”

“…The demethylation of the cognate corrinoid proteins is finally catalyzed by a methylcobalamine:coenzyme M (CoM) methyltransferase (9) resulting in methyl-CoM, the precursor of methane. The genes encoding these enzymes were named according to Krzycki and coworkers (15), where the final letter designates the polypeptide function. Within this nomenclature, B describes the substrate specific methyltransferase, C the corrinoid binding polypeptide, and A the CoM-methylating protein.…”