The deep-subsurface sulfate reducer Desulfotomaculum kuznetsovii employs two methanol-degrading pathways

Abstract

Methanol is generally metabolized through a pathway initiated by a cobalamine-containing methanol methyltransferase by anaerobic methylotrophs (such as methanogens and acetogens), or through oxidation to formaldehyde using a methanol dehydrogenase by aerobes. Methanol is an important substrate in deep-subsurface environments, where thermophilic sulfate-reducing bacteria of the genus Desulfotomaculum have key roles. Here, we study the methanol metabolism of Desulfotomaculum kuznetsovii strain 17^T, isolated from a 3000-m deep geothermal water reservoir. We use proteomics to analyze cells grown with methanol and sulfate in the presence and absence of cobalt and vitamin B12. The results indicate the presence of two methanol-degrading pathways in D. kuznetsovii, a cobalt-dependent methanol methyltransferase and a cobalt-independent methanol dehydrogenase, which is further confirmed by stable isotope fractionation. This is the first report of a microorganism utilizing two distinct methanol conversion pathways. We hypothesize that this gives D. kuznetsovii a competitive advantage in its natural environment.

Fig. 1

Comparative proteomics results. a Volcano plot with comparison of cells grown on 20 mM methanol with and without supplementation of cobalt and vitamin B12. Data are from four independent replicates (Supplementary Data 1). b Identification of the predicted function of proteins depicted in the volcano plot and corresponding label-free quantification (LFQ) values for proteins quantified in cells grown with different electron donors (20 mM ethanol, 20 mM ethanol, 20 mM methanol with and without supplementation of cobalt, and vitamin B12)

Fig. 2

Hypothesized methanol metabolism pathways in D. kuznetsovii. Methanol is oxidized to CO₂ by an alcohol dehydrogenase (ADH), aldehyde ferredoxin oxidoreductase (AFO), and a formate dehydrogenase (FDH). When cobalt is present in the environment a second concurrent methanol-oxidizing pathway is induced and part of the methanol is methylated to methyl-tetrahydrofolate (CH₃-THF). Subsequently, CH₃-THF is oxidized to CO₂ generating the same amount of electrons. Locus tag numbers are indicated for boxed enzymes

Fig. 3

Neighbor-joining tree based on MtaB amino acid sequences. The sequences were obtained from a BLASTp analysis, using MtaB of D. kuznetsovii as the query sequence. MtaB of D. kuznetsovii is printed in bold. Closed circles represent bootstrap values of 75% or higher. Scale bar represents 10% sequence difference

Fig. 4

Neighbor-joining tree based on ADH amino acid sequences. The sequences were obtained from a BLASTp analysis, using ADHs of D. kuznetsovii as query sequences. ADHs of D. kuznetsovii are printed in bold and an arrow points at the methanol-oxidizing ADH. Closed circles represent bootstrap values of 75% or higher. Scale bar represents 10% sequence difference

Fig. 5

Stable carbon isotope fractionation analysis of D. kuznetsovii. a Percentage of methanol degraded in time. b SCIF analysis data, presented as the delta ¹³C fractionation values of methanol set out against the methanol degradation. Open symbols are controls without bacteria