Methanosarcina acetivorans contains a functional ISC system for iron-sulfur cluster biogenesis

Abstract

Background: The production of methane by methanogens is dependent on numerous iron-sulfur (Fe-S) cluster proteins; yet, the machinery involved in Fe-S cluster biogenesis in methanogens remains largely unknown. Methanogen genomes encode uncharacterized homologs of the core components of the ISC (IscS and IscU) and SUF (SufBC) Fe-S cluster biogenesis systems found in bacteria and eukaryotes. Methanosarcina acetivorans contains three iscSU and two sufCB gene clusters. Here, we report genetic and biochemical characterization of M. acetivorans iscSU2.

Results: Purified IscS2 exhibited pyridoxal 5'- phosphate-dependent release of sulfur from L-cysteine. Incubation of purified IscU2 with IscS2, cysteine, and iron (Fe²⁺) resulted in the formation of [4Fe-4S] clusters in IscU2. IscU2 transferred a [4Fe-4S] cluster to purified M. acetivorans apo-aconitase. IscU2 also restored the aconitase activity in air-exposed M. acetivorans cell lysate. These biochemical results demonstrate that IscS2 is a cysteine desulfurase and that IscU2 is a Fe-S cluster scaffold. M. acetivorans strain DJL60 deleted of iscSU2 was generated to ascertain the in vivo importance of IscSU2. Strain DJL60 had Fe-S cluster content and growth similar to the parent strain but lower cysteine desulfurase activity. Strain DJL60 also had lower intracellular persulfide content compared to the parent strain when cysteine was an exogenous sulfur source, linking IscSU2 to sulfur metabolism.

Conclusions: This study establishes that M. acetivorans contains functional IscS and IscU, the core components of the ISC Fe-S cluster biogenesis system and provides the first evidence that ISC operates in methanogens.

Fig. 1

Purified IscS2 binds PLP and is a homodimer. a SDS-PAGE analysis of purified IscS2 (cropped image of original). Lane 1, MW marker; lane 2, IscS2 (2.5 μg). b UV-visible spectra of 20 μM IscS2 (dotted line) or IscS2^PLP (solid line) in 50 mM Tris pH 7.2, 150 mM NaCl. c Size-exclusion chromatography of IscS2. IscS2 (3.3 mg loaded) was analyzed by size-exclusion chromatography with 50 mM Tris pH 8.0, 150 mM NaCl, 2 mM DTT, 10% glycerol. The molecular weight of IscS2 was calculated from a standard curve (inset). The square represents the V_e/V_o of IscS2 with a calculated molecular weight of 84 kDa, consistent with homodimer (87 kDa)

Fig. 2

Fe-S cluster reconstitution of purified IscU2. a SDS-PAGE analysis of purified IscU2 (cropped image of original). Lane 1, MW marker; lane 2, IscU2 (2.4 μg). b Anaerobic UV-visible spectra of 20 μM IscU2 (dotted line), IscU2^S-FeS (black line) and IscU2^C-FeS (gray line) in 50 mM Tris pH 7.2, 150 mM NaCl. c Anaerobic size exclusion chromatography of 14.8 mg of IscU2 (gray line) and 15.6 mg of IscU2^C-FeS (black line) in 50 mM Tris pH 8.0, 150 mM NaCl, 2 mM DTT, 10% glycerol. The molecular weight of IscU2 and IscU2^C-FeS were calculated with a standard curve (inset). The calculated MW of IscU2 (solid square symbol) was 12 kDa and the calculated MW of IscU2^C-FeS (open square symbol) was 26 kDa

Fig. 3

EPR spectra of IscU2. a Expanded view, trace A: IscU2^C-FeS (100 μM) reduced with dithionite, trace B: IscU2^S-FeS (100 μM) reduced with dithionite, trace C: IscU2^S-FeS (100 μM) as such. Numbers shown represent g values. b Detailed view, trace A: IscU2^C-FeS reduced with dithionite, trace B: IscU2^S-FeS reduced with dithionite

Fig. 4

Reconstitution of apo-AcnA activity by [4Fe-4S]-IscU2. Apo-AcnA (4 μM) was incubated with IscU2^S-FeS (40 μM) or iron (Fe²⁺) and sulfide (S²⁻) (80 μM each) and aconitase activity was measured over time

Fig. 5

PCR confirmation of iscSU2 deletion in M. acetivorans strain DJL60. a Schematic showing replacement of iscSU2 with pac-hpt in strain DJL60 and predicted PCR products are indicated by P1–4. b Gel image of products of PCR reactions P1–4 with WWM73 and DJL60 genomic DNA

Fig. 6

Growth of M. acetivorans strains WWM73 and DJL60 with different sulfur sources. Each strain was grown in HS_DTT medium containing 125 mM methanol supplemented with 3 mM cysteine (Cys) and/or 3 mM sodium sulfide (Na₂S). Growth was monitored by the optical density at 600 nm (OD₆₀₀). Data points are the mean of n = 3 with error bars ± STD.

Fig. 7

Cysteine desulfurase activity, Fe-S cluster, and persulfide levels in cell lysates of strains WWM73 and DJL60. Cysteine desulfurase activity (a), Fe-S cluster content (b) and persulfide content (c) were measured as described in methods. Asterisks indicate significant difference between WWM73 and DJL60 lysate, as determined by t-test (p < 0.02 for A, p < 0.01 for C)