Methanol-Dependent Gene Expression Demonstrates that Methyl-Coenzyme M Reductase Is Essential in Methanosarcina acetivorans C2A and Allows Isolation of Mutants with Defects in Regulation of the Methanol Utilization Pathway

Abstract

Methanosarcina acetivorans C2A is able to convert several substrates to methane via at least four distinct methanogenic pathways. A common step in each of these pathways is the reduction of methyl-coenzyme M (CoM) to methane catalyzed by methyl-CoM reductase (MCR). Because this enzyme is used in each of the known pathways, the mcrBDCGA operon, which encodes MCR, is expected to be essential. To validate this prediction, a system for conditional gene inactivation was developed. A heterologous copy of the mcrBDCGA operon was placed under the control of the highly regulated mtaC1 promoter, which directs the expression of genes involved in methanol utilization, and recombined onto the M. acetivorans chromosome. This allowed for disruption of the endogenous mcr operon in the presence of methanol. Because the PmtaC1 promoter is transcribed only during growth on methanol, mcrBDCGA was rendered methanol dependent and the strain was unable to grow in trimethylamine media, strongly suggesting that mcrBDCGA is essential. Upon prolonged incubation, suppressed mutants which expressed mcrBDCGA constitutively could be selected. Expression analysis of PmtaC1::uidA gene fusions in several isolated suppressed mutants suggests that they carry transactive mutations leading to deregulation of all genes under control of this promoter. Subsequently, proteome analysis of one such suppressed mutant revealed that all known proteins derived from mtaC1 promoterdependent expression were constitutively expressed in this mutant. This genetic system can therefore be employed for the testing of essential genes and for the identification of genes under a common regulatory mechanism by making regulatory mutations phenotypically selectable.

The evolutionary history of Cys-tRNA Cys formation

“…To obtain a clearer picture of the phylogenetic distribution of SepRS͞ SepCysS and the methanogenesis genes, directed environmental sequencing efforts are called for, and genetic experiments in Methanosarcina acetivorans, e.g., ref. 51, which contains both the archaeal and bacterial routes for cysteine coding and biosynthesis, will help in understanding why this organism has retained the archaeal pathway and shed light on the reason for the apparent functional redundancy. Directed by hypotheses generated from the evolutionary data, such work will lead to a deeper understanding of these archaea and the connection between information processing and metabolism in cellular evolution.…”

Differential Regulation of the Three Methanol Methyltransferase Isozymes in Methanosarcina acetivorans C2A

“…The uidA gene of E. coli encodes the easily assayable enzyme ␤-glucuronidase, which has previously been used as a reporter gene in Methanosarcina and other methanoarchaea (2,12,23). We constructed uidA reporter gene fusions to the promoter regions of the mtaCB1 (23), mtaCB2, and mtaCB3 operons.…”

“…To our knowledge, the level of regulation measured in our mtaCB1 and mtaCB2 fusions is unprecedented in the archaeal domain. We have recently shown that it is possible to select constitutive mutants by fusing the mtaC1 promoter to an essential gene (23), suggesting that genetic approaches should be fruitful in unraveling this mystery.…”

“…The method used for preparation of cell extracts and the ␤-glucuronidase assay method were methods described previously (23).…”

Regulation of putative methyl‐sulphide methyltransferases in Methanosarcina acetivorans C2A

“…Cultures were not pre-adapted for b-glucuronidase activity measurements on DMS in order to minimize handling of this highly toxic (to humans) chemical. The preparation of cell extracts and b-glucuronidase assay method were as previously described (Rother et al, 2005), using a Varian Cary 50 UV-Vis spectrophotometer equipped with an 18 multicell holder was used for all assays.…”