Differential regulation of the three methanol methyltransferase isozymes in Methanosarcina acetivorans C2A

Abstract

Genetic analysis of the three methanol-specific methyltransferase 1 operons (mtaCB1, mtaCB2, and mtaCB3) in Methanosarcina acetivorans led to the suggestion that each of them has a discrete function during growth on methanol, which might be reflected in differential gene regulation (Pritchett and Metcalf, Mol. Microbiol. 56:1183-1194, 2005). To test this suggestion, reporter gene fusions were constructed for each of the three operons, and their expression was examined under various growth conditions. Expression of the mtaCB1 and mtaCB2 fusions was 100-fold and 575-fold higher, respectively, in methanol-grown cells than in trimethylamine (TMA)-grown cells. The mtaCB3 fusion was expressed at low levels on methanol, TMA, and dimethylamine but was significantly upregulated on monomethylamine and acetate. When TMA- or acetate-grown cultures were shifted to methanol, the mtaCB1 fusion was expressed most highly during exponential phase, whereas the mtaCB2 fusion, although strongly induced prior to mtaCB1 expression, did not reach full expression levels until stationary phase. The mtaCB3 fusion was transiently expressed prior to entry into exponential phase during a TMA-to-methanol substrate shift experiment. When acetate-grown cells were shifted to medium containing both TMA and methanol, TMA utilization commenced prior to utilization of methanol; however, these two substrates were consumed simultaneously later in growth. Under these conditions expression of the mtaCB2 and mtaCB3 fusions was delayed, suggesting that methylamines may repress their expression.

FIG. 1.

Simplified scheme for methanogenesis from methanol. Methanol (CH₃OH) is converted to methyl coenzyme M (2-mercaptoethanesulfonic acid) (CoM) by the concerted effort of three proteins. Methyltransferase 1 (MT1) is comprised of methanol:5-hydroxy-benzimidazolyl-cobamide methyltransferase, MtaB, and the corrinoid-binding protein, MtaC. The product of the MT1 reaction, methyl-MtaC is the substrate for a second methyltransferase (MT2 or MtaA), which transfers the methyl group from the corrinoid protein to coenzyme M. Methyl coenzyme M (CH₃-CoM) is then disproportionated, with one molecule oxidized to CO₂ to provide the electrons required for the reduction of three CH₃-CoM molecules to methane (not all steps are shown).

FIG. 2.

PmtaC::uidA expression during a substrate shift from trimethylamine to methanol. Cultures adapted to TMA (50 mM) were switched to medium containing methanol (125 mM). The OD₄₂₀ indicates growth, and the methanol concentration was determined by gas chromatography. The β-glucuronidase activity indicates expression from the specific promoters. (A) PmtaC1::uidA; (B) PmtaC2::uidA; (C) PmtaC3::uidA. The y axes indicate log OD₄₂₀ (red), methanol concentration (dark blue), and β-glucuronidase activity (light blue). The β-glucuronidase activities of the three strains were very different, and thus the scales used for specific activity are not identical. Triplicate experiments were performed for each fusion strain. Equivalent results were obtained for all replicates; a representative curve is shown for each experiment.

FIG. 3.

PmtaC::uidA expression during a substrate shift from acetate to methanol. Cultures adapted to acetate (120 mM) were switched to medium containing methanol (125 mM). The OD₄₂₀ indicates growth, and the methanol concentration was determined by gas chromatography. The β-glucuronidase activity indicates expression from the specific promoters. (A) PmtaC1::uidA; (B) PmtaC2::uidA; (C) PmtaC3::uidA. The y axes indicate log OD₄₂₀ (red), methanol concentration (dark blue), and β-glucuronidase activity (light blue). The β-glucuronidase activities of the three strains were very different, and thus the scales used for specific activity are not identical. Duplicate experiments were performed for each fusion strain. Equivalent results were obtained for all replicates; a representative curve is shown for each experiment.

FIG. 4.

PmtaC::uidA expression during a substrate shift from acetate to methanol and trimethylamine. Cultures adapted to acetate (120 mM) were switched to medium containing both methanol (125 mM) and TMA (50 mM). The OD₄₂₀ indicates growth, and the methanol, TMA, DMA, and MMA concentrations were determined by gas chromatography. The β-glucuronidase specific activity indicates expression from the specific promoters. (A) PmtaC1::uidA; (B) PmtaC2::uidA; (C) PmtaC3::uidA. The y axes indicate log OD₄₂₀ (red), methanol concentration (dark blue), and β-glucuronidase activity. The β-glucuronidase activities of the strains were very different, and thus the scales used for specific activity are not the same. Duplicate experiments were performed for each fusion strain. Equivalent results were obtained for both replicates; a representative curve is shown for each experiment.