We first evaluated the mutant proteins qualitatively, via growth on plates containing M9 medium plus glycerol or LB medium plus plumbagin (Fig. 1a). As reported previously (Waller et al., 2010), vector-alone controls showed no growth during the assay period on either medium, and the wild-type Ygf Z protein complemented these defects. Of the mutant proteins, the C228A mutant failed to complement on either medium; all other mutants were as

effective as wild type, except ATM signaling pathway the Y229A mutant, which was slightly less effective on LB plus plumbagin. The data for LB medium plus plumbagin confirm and extend those of Lin et al. (2010). To detect more subtle, quantitative effects, growth in M9 plus glycerol was monitored over time in a Bioscreen system. Only the C228A mutant diverged from the wild-type growth curve (Fig. 1b and S1). The poor performance of the C228A mutant could not be attributed merely to low expression of this particular mutant protein, as immunoblot analysis showed its level in growing cells to be at least as high as that of the wild type and all other mutant proteins (Fig. 2). Together, the growth results in both liquid and solid media, and the immunoblot data thus point to C228 as the most crucial residue in the Ygf Z signature motif. To extend these results to the biochemical level, we examined the in vivo activity of MiaB, an Fe/S enzyme that mediates

the methylthiolation of N6-isopentenyladenosine (i6A) in tRNA to 2-methylthio-N6-isopentenyladenosine GSK1120212 molecular weight (ms2i6A). As MiaB activity depends upon Ygf Z activity, the ms2i6A/i6A ratio in tRNA, determined using LC-MS, is a semiquantitative measure of MiaB activity that in effect reports the activity of Ygf Z (Waller et al., 2010). As expected, ΔygfZ cells harbouring

vector alone showed no detectable conversion of i6A to ms2i6A and consequently an ms2i6A/i6A ratio of zero, whereas cells expressing wild-type Ygf Z showed a ratio of 9.1 (Fig. 3). Edoxaban The ratio for a representative mutant with no growth phenotype (G230A) was not significantly different. The ratio of 2.7 for the Y229A mutant was modestly but significantly (P < 0.05) lower than wild type, but the ratio of 0.18 for the C229A mutant was dramatically lower. These results for a biochemical phenotype thus mirror the growth data in showing C228 to be by far the most important single residue for Ygf Z function, with the neighbouring Y229 having a much smaller effect. It is possible that the minor effect of Y229 is because of its influence either on the positioning of C228 for efficient interaction with MiaB, or on the properties of C228, as electrostatic interactions between sulphur-containing residues and aromatic residues are a common structural theme in proteins (Reid et al., 1985; Tauer et al., 2005). The evidence for an absolutely conserved, functionally critical, cysteine residue raises the question of what it does.