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

Abstract

The methanogenic archaeon Methanosarcina mazei strain Gö1 uses versatile carbon sources and is able to fix molecular nitrogen with methanol as carbon and energy sources. Here, we demonstrate that when growing on trimethylamine (TMA), nitrogen fixation does not occur, indicating that ammonium released during TMA degradation is sufficient to serve as a nitrogen source and represses nif gene induction. We further report on the transcriptional regulation of soluble methyltransferases, which catalyze the initial step of methylamine consumption by methanogenesis, in response to different carbon and nitrogen sources. Unexpectedly, we obtained conclusive evidence that transcription of the mtmB 2 C 2 operon, encoding a monomethylamine (MMA) methyltransferase and its corresponding corrinoid protein, is highly increased under nitrogen limitation when methanol serves as a carbon source. In contrast, transcription of the homologous mtmB 1 C 1 operon is not affected by the nitrogen source but appears to be increased when TMA is the sole carbon and energy source. In general, transcription of operons encoding dimethylamine (DMA) and TMA methyltransferases and methylcobalamine:coenzyme M methyltransferases is not regulated in response to the nitrogen source. However, in all cases transcription of one of the homologous operons or genes is increased by TMA or its degradation products DMA and MMA.The archaeon Methanosarcina mazei strain Gö1 belongs to the methylotrophic methanogens of the order Methanosarcinales. Those species have the most versatile substrate spectrum within the methanogenic archaea and are able to grow on H 2 plus CO 2 , acetate, or methylotrophic substrates such as methanol or methylamines as sole carbon and energy sources (10, 21). We recently showed that M. mazei is able to use molecular nitrogen as sole nitrogen source when growing on methanol and characterized a single nitrogen fixation (nif) gene cluster encoding a molybdenum-containing nitrogenase (6). For M. barkeri it has been demonstrated that degradation of each trimethylamine (TMA), dimethylamine (DMA), and monomethylamine (MMA) leads to the release of ammonium into the medium (13). However, whether M. mazei fixes nitrogen when growing on methylamines or directly uses the ammonium generated by the disproportion of methylamines has never been studied.The first step of the disproportion of methylamines is catalyzed by soluble methyltransferases, which transfer the methyl group to a cognate corrinoid protein whereby each different methylamine requires its specific methyltransferase and corresponding corrinoid protein (2,7,8). 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 po...

The appearance of pyrrolysine in tRNA His guanylyltransferase by neutral evolution

“…This could result in loss of read-through or down regulation of Pyl-containing proteins. We could show that, in agreement with previous experiments (7,8), no difference in read-through efficiency on methanol or TMA is observed (Fig. 3A, lanes 1 and 2), indicating that incorporation of Pyl into proteins is independent of growth substrate and perhaps entirely unregulated.…”

“…The importance of Pyl in these enzymes initially suggested that the availability of Pyl-tRNA Pyl could also be regulated in a similar fashion. Quantitative PCR data revealed constitutive expression of tRNA Pyl in growth on trimethylamine (TMA) or on methanol in Methanosarcina mazei (8); likewise a monomethylamine methyltransferase (MtmB1) expression construct could be overexpressed in M. acetivorans during growth on methanol (7). Nevertheless, the fact that Pyl is found in the active sites of the methylamine methyltransferases and that all Pyl-decoding organisms share the Pyl operon and at least one methylamine methyltransferase has led to the belief that Pyl was selectively retained during evolution to support growth on methylamines (9).…”

The Ether-Cleaving Methyltransferase System of the Strict Anaerobe Acetobacterium dehalogenans : Analysis and Expression of the Encoding Genes

“…Recent reports have concentrated on the characteristics of the archaeal MTs, their cofactors (22,31), the protein structure (20), or the occurrence of isozymes (8,16,41,42) as well as their differential transcription (23,61). Similar to the case for the O-demethylases of A. dehalogenans, a corrinoid AE, designated either Map or Ram (3,9), was required for the methanogenic MT reactions.…”

Carbon source-dependent expansion of the genetic code in bacteria

“…Pyl is expressed independently of the carbon source in Methanosarcina mazei (16) and that Pyl-containing proteins were recovered from M. acetivorans cells grown on methanol (15). These studies indicate that the expression of particular Pyl-containing proteins is regulated in archaeal organisms, but Pyl-tRNA Pyl formation and encoding of UAG as Pyl is constitutive and has not been shown to be regulated during the lifetime of the cell.…”