Identification of the mcrD gene product and its association with component C of methyl coenzyme M reductase in Methanococcus vannielii

Abstract

A mcrD-lacZ gene fusion has been constructed and expressed under lacP control in Escherichia coli. Antibodies raised against the product of this gene fusion have been used in Western blotting (immunoblotting) to demonstrate the gene product of mcrD (gpmcrD) in Methanococcus vannielii. The a, j, and -y subunit polypeptides of component C of methyl coenzyme M reductase (MR) were coprecipitated with gpmcrD when bound by antibodies raised either against MR or against gpmcrD-lacZ. This association of MR and gpmcrD did not withstand polyacrylamide gel electrophoresis under nondenaturing conditions. Component C of methyl coenzyme M reductase (MR) catalyzes the terminal reaction in methanogenesis, releasing methane (2,8,9,11,19). MR, which constitutes approximately 10% of the cellular protein, has been purified from several methanogens and has been shown in every case to contain three different polypeptide subunits which form a holoenzyme with the organization a232.Y2 (2,8,9). The genes encoding the a, ,B, and -y subunits, mcrA, mcrB, and mcrG, respectively, have been cloned and sequenced from five different methanogens (3,4,7,12,22) and have been found in every case to be part of a cluster of tightly linked, cotranscribed genes, arranged mcrBDCGA and designated the mcr operon. All extant mcr operons appear to have evolved from a common ancestor (12,22,23). The extent of divergence of the mcr sequences correlates well with current methanogen phylogenetics as determined by differences in ribosomal RNA sequences (11). The mcrD and mcrC genes are conserved and found at the same location in all five sequenced mcr operons (12, 22), arguing for important and conserved functions for the gene products of mcrD (gpmcrD) and mcrC (gpmcrC), respectively. These polypeptides do not, however, copurify with the MR holoenzyme, so their functions, indeed their existence, in methanogens has remained a matter of speculation. The mcrD gene of Methanococcus vannielii was expressed when cloned in Escherichia coli (7), and by using antibodies raised against the product of a mcrD-lacZ fusion (gpmcrD-lacZ)

mRNAs in the methanogenic archaeon Methanococcus vannielii: numbers, half‐lives and processing

“…The mcrC and mcrD gene products (gpmcrCand gpmcrD) are present in much smaller amounts in methanogens than are gpmcrA, gpmcrB and gpmcrG, i.e. the a, p and y subunits of MR (Eliermann ef ai, 1988(Eliermann ef ai, , 1989Sherf and Reeve. 1990;Stroup and Reeve.…”

“…The mcrC and mcrD gene products (gpmcrCand gpmcrD) have been identified in M. vannielii but are present in very much reduced amounts relative to the r, p, and y subunits of MR {QpmcrA, gpmcrB and gpmcrG. respectively) and their functions remain unknown (Sherf and Reeve, 1990;Stroup and Reeve, 1993a,b). Different-strength RBSs and/or processing of a primary mcrBDCGA transcript into shorter mRNAs, with different half-lives, have been proposed to explain how very large amounts of the y.. \\.…”

“…The two transcripts of M. vannielii housekeeping genes that were chosen for study encode proteins with very etal.. 1987;Sherf and Reeve, 1990). Sites of transcription initiation (p) and termination (t).…”

Methanogenesis: genes, genomes, and who's on first?

“…The M. jannaschii genome contains an mcrBDCGA operon, and mcrBDCGA operons have been cloned from M. thermoautotrophicum Marburg and ⌬H, M. fervidus, M. kandleri, M. barkeri, M. vannielii, and M. voltae and sequenced (7,8,16,52,67,75,123). Despite their conservation and investigation (89,98,99), the functions of MCRC and MCRD have not been determined.…”

Regulated gene expression in methanogens

“…Despite the fact that both the mcrD and the mcrC gene product have been identified in Me'. cannielii, the function of rncrD and mcrC is presently not known [25,26].…”