Genetic basis for metabolism of methylated sulfur compounds in Methanosarcina species

Abstract

Methanosarcina acetivorans uses a variety of methylated sulfur compounds as carbon and energy sources. Previous studies implicated the mtsD, mtsF, and mtsH genes in catabolism of dimethylsulfide, but the genes required for use of other methylsulfides have yet to be established. Here, we show that a four-gene locus, designated mtpCAP-msrH, is specifically required for growth on methylmercaptopropionate (MMPA). The mtpC, mtpA, and mtpP genes encode a putative corrinoid protein, a coenzyme M (CoM) methyltransferase, and a major facilitator superfamily (MFS) transporter, respectively, while msrH encodes a putative transcriptional regulator. Mutants lacking mtpC or mtpA display a severe growth defect in MMPA medium but are unimpaired during growth on other substrates. The mtpCAP genes comprise a transcriptional unit that is highly and specifically upregulated during growth on MMPA, whereas msrH is monocistronic and constitutively expressed. Mutants lacking msrH fail to transcribe mtpCAP and grow poorly in MMPA medium, consistent with the assignment of its product as a transcriptional activator. The mtpCAP-msrH locus is conserved in numerous marine methanogens, including eight Methanosarcina species that we showed are capable of growth on MMPA. Mutants lacking the mtsD, mtsF, and mtsH genes display a 30% reduction in growth yield when grown on MMPA, suggesting that these genes play an auxiliary role in MMPA catabolism. A quadruple ΔmtpCAP ΔmtsD ΔmtsF ΔmtsH mutant strain was incapable of growth on MMPA. Reanalysis of mtsD, mtsF, and mtsH mutants suggests that the preferred substrate for MtsD is dimethylsulfide, while the preferred substrate for MtsF is methanethiol.

Importance: Methylated sulfur compounds play pivotal roles in the global sulfur and carbon cycles and contribute to global temperature homeostasis. Although the degradation of these molecules by aerobic bacteria has been well studied, relatively little is known regarding their fate in anaerobic ecosystems. In this study, we identify the genetic basis for metabolism of methylmercaptopropionate, dimethylsulfide, and methanethiol by strictly anaerobic methanogens of the genus Methanosarcina. These data will aid the development of predictive sulfur cycle models and enable molecular ecological approaches for the study of methylated sulfur metabolism in anaerobic ecosystems.

FIG 1

Methanosarcina enzymes involved in C-1 metabolism. (A) Schematic of the three-subunit, substrate-specific MT1/MT2 system for activation of methanol and methylamines found in all Methanosarcina species. The designations for specific MT1/MT2 enzymes and their cognate substrates are shown. (B) Schematic of the biochemically characterized two-subunit methylsulfide MT1/MT2 system from M. barkeri. (C) Schematic of the putative function of a single-subunit DMS MT1/MT2 system, based on genetic analysis of the mtsD, mtsF, and mtsH genes of M. acetivorans (19). (D) Schematic of the putative function of an H₄MPT:CoM methyltransferase system encoded by the mtsD, mtsF, and mtsH genes of M. acetivorans, based on biochemical analysis of the MtsF (CmtA) protein (21).

FIG 2

Growth of M. acetivorans strains in MMPA medium. The indicated mutants were grown in HS medium with 20 mM MMPA. The strains used were WWM82 (parental strain), WWM830 (ΔmtpC), WWM898 (complemented ΔmtpC), WWM831 (ΔmtpA), WWM899 (complemented ΔmtpA), WWM832 (ΔmtpP), WWM833 (ΔmsrH), WWM900 (complemented ΔmsrH), WWM829 (ΔmtpCAP), WWM901 (complemented ΔmtpCAP), WWM816 (ΔmtsDFH), and WWM897 (ΔmtpCAP ΔmtsDFH). Error bars represent standard deviations of the results from triplicate cultures. OD₆₀₀, optical density at 600 nm.

FIG 3

RNA-seq read coverage of M. acetivorans grown on MMPA. (Top) mRNA read coverage of the entire chromosome; (middle) mRNA read coverage of the mtpCAP-msrH locus; (bottom) mRNA read coverage of the mtpA-mtpP intergenic region. Note that the coverage between genes never drops below the coverage within the genes, suggesting that mtpCAP are cotranscribed.

FIG 4

Relative mRNA abundances of the ΔmsrH mutant versus the parental strain during growth on TMA plus MMPA. Volcano plot shows the fold differences in transcript abundance between WWM833 (ΔmsrH) and WWM82 (parental strain), based on the EDGE test plotted against statistical significance. Only the three genes indicated show significant differences between the two strains.

FIG 5

Putative roles for MtpCA and MtsD/-F/-H in methylsulfide metabolism. (Top) Schematic of a proposed bifunctional MT1/MT2 enzyme, in which MtpA catalyzes transfer of the methyl group from MMPA to the corrinoid protein MtpC and subsequent methyl transfer from methyl-MtpC to CoM. (Bottom) Schematic of a proposal for broad substrate specificity in the MtsD/-F/-H proteins. These putative multifunctional enzymes would allow bypass of the normal energy-conserving/-dependent Mtr H₄MPT:CoM methyltransferase while providing a mechanism to introduce methyl groups from methylsulfides into both the reductive and the oxidative branch of the methylotrophic pathway of methanogenesis. Note that the direction of methyl transfer between CH₃-H₄MPT and CH₃-CoM determines whether it is exergonic or endergonic and, thus, whether sodium ions are extruded or consumed.