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. 2014 Jul 7;111(29):10520–10525. doi: 10.1073/pnas.1411267111

Fig. 1.

Fig. 1.

SepCysE is an essential factor for tRNA-dependent Cys biosynthesis. (A) The direct and indirect pathways for Cys-tRNACys formation. The direct pathway acylates tRNACys with Cys by CysRS. The indirect pathway acylates tRNACys with Sep by SepRS (encoded by sepS), and then Sep is converted to Cys by SepCysS (encoded by pscS) with a sulfur donor. (B) Pull-down of His6-tagged SepCysS expressed in M. maripaludis. Proteins purified from 200 mL cells transformed with the empty vector (left lane) or with the vector for His6-SepCysS expression (right lane) were separated by SDS/PAGE, in-gel tryptic digested, and analyzed by liquid chromatography-tandem mass spectrometry analysis. The locus tag of identified proteins (>60% coverage) only present in the right lane are labeled in bold. (C) Growth of the M. maripaludis strains in the defined medium (McNA) with 1 mM l-Cys. (D) Growth of the M. maripaludis strains in McNA without Cys. scsE−, ΔsepS/ΔscsE mutant with SepRS expressed from a vector; sepS+/scsE+, ΔsepS/ΔscsE mutant with both SepRS and SepCysE expressed from a vector; sepS−, ΔsepS/ΔscsE mutant with SepCysE expressed from a vector; sepS−/scsE−, ΔsepS/ΔscsE double mutant. Data are mean ± SDs from three replicate cultures.