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.

Fig. 1.

Log₂-normalized protein abundances. (a) Methanol plus nitrate versus methylamine. Shown in red are select proteins detected at higher abundances under methylamine growth conditions (Mmol_1913, putative metal transporter component; Mmol_1575, methylamine dehydrogenase large subunit). Shown in green are select proteins detected at higher abundances in methanol under nitrate growth conditions (Mmol_1770, XoxF; Mmol_2290, nitrogen regulatory protein PII; Mmol_0995, PQQ biosynthesis protein C; Mmol_2227, alkyl hydroxyperoxide reductase; Mmol_0150, catalase/peroxidase; Mmol_0228, peroxiredoxin). (b) Methanol plus nitrate versus methanol plus nitrate plus ammonium. Shown in blue are proteins detected at higher abundances in methanol with nitrate (Mmol_2289, conserved hypothetical protein; Mmol_1110, DNA-binding protein; Mmol_1063, NO dioxygenase).

Fig. 2.

Expression of nitrate metabolism genes and abundances of nitrate metabolism enzymes in M. mobilis. Solid arrows represent protein abundances and dashed lines represent transcription levels (the widths of the arrows correlate with levels of transcription/protein abundance). Green reflects induction during growth/transition to methanol in the presence of nitrate. Yellow reflects no significant change in transcription/protein abundance. Red asterisks denote regulation by nitrogen source (nitrate or ammonium; see text). Gray indicates lack of detection. Mmol identifiers correspond to respective enzymes/subunits.

Fig. 3.

Comparison of protein and transcript abundances and protein and transcript fold changes. (a and b) Protein and transcript abundances are shown for methylamine (a) and methanol plus nitrate (b). Correlations of proteomics and transcriptomics results for panels a and b are 0.64 and 0.5, respectively. (c and d) Comparisons of fold changes are shown for methanol plus nitrate versus methylamine (c) and methanol plus nitrate versus methanol plus ammonium (transcriptomics) or methanol plus nitrate plus ammonium (proteomics) (d). The dots were made semitransparent in order to provide an indication of density.