An Integrated Proteomics/Transcriptomics Approach Points to Oxygen as the Main Electron Sink for Methanol Metabolism in Methylotenera mobilis

Abstract

Methylotenera species, unlike their close relatives in the genera Methylophilus, Methylobacillus, and Methylovorus, neither exhibit the activity of methanol dehydrogenase nor possess mxaFI genes encoding this enzyme, yet they are able to grow on methanol. In this work, we integrated a genome-wide proteomics approach, shotgun proteomics, and a genome-wide transcriptomics approach, shotgun transcriptome sequencing (RNA-seq), of Methylotenera mobilis JLW8 to identify genes and enzymes potentially involved in methanol oxidation, with special attention to alternative nitrogen sources, to address the question of whether nitrate could play a role as an electron acceptor in place of oxygen. Both proteomics and transcriptomics identified a limited number of genes and enzymes specifically responding to methanol. This set includes genes involved in oxidative stress response systems, a number of oxidoreductases, including XoxF-type alcohol dehydrogenases, a type II secretion system, and proteins without a predicted function. Nitrate stimulated expression of some genes in assimilatory nitrate reduction and denitrification pathways, while ammonium downregulated some of the nitrogen metabolism genes. However, none of these genes appeared to respond to methanol, which suggests that oxygen may be the main electron sink during growth on methanol. This study identifies initial targets for future focused physiological studies, including mutant analysis, which will provide further details into this novel process.The Methylotenera genus is a recent addition to the family Methylophilaceae (16,17). This genus represents methylotrophs that are not only sufficiently divergent in genomics terms from the formerly described Methylophilaceae but also differ from them by not possessing one of the hallmark enzymes of methylotrophy, methanol dehydrogenase (16,19). However, Methylotenera species are still able, albeit weakly, to grow on methanol (16,17). This capability must be enabled by an alternative enzyme system that remains unidentified. It has been proposed that methanol oxidation by Methylotenera mobilis JLW8 may be linked to (aerobic) denitrification, and XoxF, a protein homologous to the large subunit of methanol dehydrogenase (MxaF), was implicated in this process (18). The more recently described species Methylotenera versatilis 301 possesses the homologs of both XoxF proteins encoded in the genome of M. mobilis JLW8 but does not possess genes for denitrification (19). Thus, two questions remained unanswered with regard to methanol metabolism by Methylotenera: whether, at least in some cases, it is connected to denitrification and whether XoxF enzymes are involved. In order to approach these questions, in this study we integrated shotgun nanoflow scale liquid chromatography-tandem mass spectrometry (LC/MS/MS) proteomics with whole-transcriptome shotgun sequencing (RNA-seq), using M. mobilis JLW8 as a model to evaluate its physiological responses to methanol and nitrate. MATERIALS AND METHODSCultivation and sample preparation for prot...

Insights into Denitrification in Methylotenera mobilis from Denitrification Pathway and Methanol Metabolism Mutants

“…M. mobilis strain JLW8 was routinely cultivated in nitratecontaining MM2 medium supplemented by methylamine, as previously described (13,14). Growth on methanol was tested on plates using solidified MM2 medium or using solidified Hypho (ammonia-containing) medium, or in the respective liquid media, as previously described (14).…”

“…We also mutated a gene predicted to encode a nitric oxide dioxygenase (Nod; mmol_1063) and each of two xoxF genes (mmol_1770 and mmol_2048) that encode homologs of the large subunit of methanol dehydrogenase (11,14,19,20). In addition, a double mutant lacking both XoxF proteins was generated.…”

“…The growth of some of the Methylotenera species is stimulated by nitrate, and it has been proposed that the metabolism of methanol by Methylotenera may be linked to denitrification (13). Denitrification by M. mobilis JLW8 has been previously demonstrated, and putative genes for denitrification have been identified in the genome (13,14). The denitrification pathway is incomplete, due to the lack of nitrous oxide reductase, thus resulting in accumulation of nitrous oxide (13,14).…”

“…Denitrification by M. mobilis JLW8 has been previously demonstrated, and putative genes for denitrification have been identified in the genome (13,14). The denitrification pathway is incomplete, due to the lack of nitrous oxide reductase, thus resulting in accumulation of nitrous oxide (13,14). XoxF enzymes that are homologs of the large subunit of methanol dehydrogenase have been implicated in playing a role in nitrate-dependent growth on methanol (13,14).…”

“…The denitrification pathway is incomplete, due to the lack of nitrous oxide reductase, thus resulting in accumulation of nitrous oxide (13,14). XoxF enzymes that are homologs of the large subunit of methanol dehydrogenase have been implicated in playing a role in nitrate-dependent growth on methanol (13,14). The two goals of the present study were the confirmation of the functions of the genes proposed to be involved in denitrification and investigation of the role of XoxF that is a homolog of the large subunit of methanol dehydrogenase in methanol metabolism and denitrification.…”