FIG. 7.

Expression of vipD shows low levels of toxicity for yeast. (A) Either wild-type VipD or VipD containing mutations in the putative phospholipase A active site can be toxic to the yeast S. cerevisiae. Constructs containing the indicated vipD alleles under the control of the galactose-regulated promoter (pGAL) were integrated at the URA3 locus in S. cerevisiae. The resulting strains, along with a strain containing pGAL but not vipD, were grown in rich medium to stationary phase. Tenfold serial dilutions were spotted onto rich medium containing either dextrose (which represses expression of vipD) or galactose (which induces expression of vipD) and incubated for 3 days at 30°C. For each vipD-expressing construct, an isolate displaying wild-type growth on galactose medium (rows 1) and one with a slow-growth phenotype (rows 2) are shown. (B) vipD-expressing strains that are slow growing on galactose medium produce higher levels of VipD. Equivalent amounts of the strains grown on galactose medium in (A) were lysed in sample buffer, separated by SDS-PAGE, and analyzed by Western blotting using an anti-VipD antibody.