Coenzyme F420-dependent sulfite reductase-enabled sulfite detoxification and use of sulfite as a sole sulfur source by Methanococcus maripaludis

Abstract

Coenzyme F(420)-dependent sulfite reductase (Fsr) of Methanocaldococcus jannaschii, a sulfite-tolerant methanogen, was expressed with activity in Methanococcus maripaludis, a sulfite-sensitive methanogen. The recombinant organism reduced sulfite to sulfide and grew with sulfite as the sole sulfur source, indicating that Fsr is a sulfite detoxification and assimilation enzyme for methanogens and that M. maripaludis synthesizes siroheme.

FIG. 1.

Growth and methanogenesis in cultures of M. maripaludis strains with sulfide or sulfite as a sole sulfur source. (A) pEFJ9, an expression vector for fsr based on pMEV2.1.1, an E. coli-M. maripaludis shuttle vector (12). P_hmvA, Methanococcus voltae histone promoter; neo, neomycin resistance gene; bla, β-lactamase gene; Eco ori, origin for vector replication in E. coli; ORFless 1 and ORFless 2, elements that contain multiple potential stem-loop structures and direct and inverted repeats, lack open reading frames (ORFs), and are necessary for replication in M. maripaludis (19). (B) Growth, represented by the optical density of the culture at 600 nm. Sulfur sources used were 2 mM sulfide (filled symbols) and 2 mM sulfite (open symbols). M. m., M. maripaludis. The period of incubation without shaking is marked accordingly. Other growth conditions are as described in Materials and Methods. (C) Methanogenesis and sulfide production in an M. maripaludis(pEFJ9) culture with sulfite (final concentration, 2 mM) as the sole sulfur source. The headspace and culture volumes were 370 and 150 ml, respectively. Other details are the same as those described for panel B.

FIG. 2.

Effect of sulfite addition on growth and methanogenesis in cultures of M. maripaludis strains initiated with sulfide as the sulfur source. (A) Growth, as represented by the optical density of the culture at 600 nm. The cultures were initiated in a medium with sulfide as the sole sulfur source. Two of these cultures (filled symbols) received sodium sulfite (final concentration, 2 mM) at the times shown by arrows. The period of unstirred incubation is marked. Other growth conditions are as described in Materials and Methods. (B) Methanogenesis, as represented by the concentration of methane in the headspace gas (volume, 370 ml). Other details are the same as those described for panel A.

FIG. 3.

Expression of recombinant Fsr in Methanococcus maripaludis (Mm) and native Fsr in Methanocaldococcus jannaschii (Mj). The study involved sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of cell lysates of the following: M. jannaschii grown on sulfide and sulfite (lanes A and B, respectively), molecular mass standards (lane C), M. maripaludis(pEFJ9) grown on sulfite (lane D), and M. maripaludis(pMEV2.1.1) grown on sulfide (lane E). The concentrations of both sulfide and sulfite were 2 mM. The arrow points to the location of the Fsr polypeptide. The molecular masses (in kDa) for the standards (as in lane C) are shown to the right of the gel.