Supplementation of Sulfide or Acetate and 2-Mercaptoethane Sulfonate Restores Growth of the Methanosarcina acetivorans Δ hdrABC Deletion Mutant during Methylotrophic Methanogenesis

Abstract

Methanogenic archaea are important organisms in the global carbon cycle that grow by producing methane gas. Methanosarcina acetivorans is a methanogenic archaeum that can grow using methylated compounds, carbon monoxide, or acetate and produces renewable methane as a byproduct. However, there is limited knowledge of how combinations of substrates may affect metabolic fluxes in methanogens. Previous studies have shown that heterodisulfide reductase, the terminal oxidase in the electron transport system, is an essential enzyme in all methanogens. Deletion of genes encoding the nonessential methylotrophic heterodisulfide reductase enzyme (HdrABC) results in slower growth rate but increased metabolic efficiency. We hypothesized that increased sulfide, supplementation of mercaptoethanesulfonate (coenzyme M, CoM-SH), or acetate would metabolically alleviate the effect of the ΔhdrABC mutation. Increased sulfide improved growth of the mutant as expected; however, supplementation of both CoM-SH and acetate together were necessary to reduce the effect of the ΔhdrABC mutation. Supplementation of CoM-SH or acetate alone did not improve growth. These results support our model for the role of HdrABC in methanogenesis and suggest M.acetivorans is more efficient at conserving energy when supplemented with acetate. Our study suggests decreased Hdr enzyme activity can be overcome by nutritional supplementation with sulfide or coenzyme M and acetate, which are abundant in anaerobic environments.

Keywords: Methanosarcina; archaea; heterodisulfide reductase; methane; methanogenesis.

Figure 1

Model for the effect of HdrABC deletion on methylotrophic methanogenesis. When ΔhdrABC is deleted, the terminal oxidase reaction that regenerates CoM-SH and CoB-SH cofactors is slowed. As a result, methane formation rate decreases as free CoM-SH and CoB-SH are depleted while CH₃-CoM and CoM-S-S-CoB accumulates. In addition, CH₃-Cbl is susceptible to H₂S in the medium, resulting in formation of CH₃-SH (orange) and (CH₃)₂S, which triggers expression of methylsulfide methyltransferase enzymes. To regenerate CoM-SH, cells up-regulate genes for CoM-SH and CoB-SH biosynthesis (bold) and CoM:methyltransferases (thick blue arrow). The oxidative branch of methylotrophic methanogenesis pathway is shown in blue arrows. CoB-SH, coenzyme B thiol; CoM-SH, Coenzyme M thiol; CoM-S-S-CoB, coenzyme M-coenzyme B heterodisulfide; Fd, ferredoxin; Fd_red, reduced ferredoxin; H₂S, hydrogen sulfide (green); H₄MPT, tetrahydromethanopterin; MFR, methanofuran; MPh, methanophenazine; MPhH₂, reduced methanophenazine. Enzymes involved in the Wolfe Cycle: (a) formyl-methanofuran dehydrogenase (Fmd), (b) formyl-methanofuran:H₄MPT formyl transferase (Ftr), (c) methenyl-H₄MPT cyclohydrolase (Mch), (d) F₄₂₀-dependent methylene-H₄MPT dehydrogenase (Mtd), (e) F₄₂₀-dependent methylene-H₄MPT reductase (Mer), (f) methyl-H₄MPT:coenzyme M methyltransferase (Mtr), (g) methyl-coenzyme M reductase (Mcr), (h) heterodisulfide reductase HdrABC, (i) ferredoxin:methanophenazine oxidoreductase Rnf, (j) Proton-translocating methanophenazine:heterodisulfide reductase (HdrED), (k) Sodium–proton antiporter (MrpA), (l) proton-pumping F₄₂₀H₂: methanophenazine reductase (Fpo). Figure adapted from [3].

Figure 2

Effect of sulfur source on growth of the ΔhdrABC mutant on methanol. (a) doubling times in hours for the parent (blue) and ΔhdrABC mutant (orange) strains in low sulfide culture medium with sodium sulfide concentrations of 0.025 Mm–0.4 mM. Error bars showing standard deviation may be obscured by the symbols. The linear trendline and Pearson R² coefficients are shown indicating no relationship between the doubling time and sulfide concentration for either strain. (b) growth of parent (blue) and ΔhdrABC mutant (orange) strains over time in culture medium in which sodium sulfide has been omitted. Averages were calculated from a minimum of three independent biological replicates per treatment. Error bars have been omitted for clarity. OD, optical density at 600 nm.

Figure 3

Effect of acetate supplementation on growth of the ΔhdrABC mutant. (a) growth curves for the parent (blue) on methanol as energy source (closed circles) and methanol with acetate as energy sources (open circles). (b) growth curves for the ΔhdrABC mutant (orange) on methanol as energy source (closed circles) and methanol with acetate as energy sources (open circles). Averages were calculated from a minimum of three independent biological replicates per treatment. Error bars have been omitted for clarity. OD, optical density at 600 nm.

Figure 4

Effect of CoM-SH and acetate supplementation on growth of the ΔhdrABC mutant. (a) growth of parent (red) and ΔhdrABC mutant (black) strains on methanol as energy source (closed circles) with 1 mM CoM-SH supplementation. (b) growth of parent (red) and ΔhdrABC mutant (black) strains on methanol plus acetate (open circles) with 1 mM CoM-SH supplementation. Averages were calculated from a minimum of three independent biological replicates per treatment. Error bars have been omitted for clarity. OD, optical density at 600 nm.

Figure 5

In silico analysis of the electron transport chain of M. acetivorans. (a) represents the metabolic network of iST807 which was generated using fluxer [11]. (b) represents the relative reaction flux of hdrABC compared to hdrED when acetate is the carbon source. (c) represents the relative reaction flux of hdrABC compared to hdrED when methanol is the carbon source. (d) represents the reaction flux distribution for $10\frac{mmol}{gDW.hr}$ of acetate uptake (red numbers) and for $10\frac{mmol}{gDW.hr}$ of methanol uptake (green numbers).

Figure 6

Model for complementation of ΔhdrA1B1C1 function by supplementation with CoM-SH, acetate, and sulfide. Abbreviations and enzymes are as indicated in Figure 1. Exogenously supplied CoM-SH is indicated in bold. Pink arrows indicate acetotrophic methanogenesis pathway. Note several steps are bi-directional between methylotrophic and acetotrophic pathways. Figure adapted from [3].