FIG. 6.

CBF1 gene deletion is lethal in C. glabrata. (A) Gene replacement of CgCBF1. The 2.2-kb SpeI/HindIII fragment containing the CgCBF1 gene (Fig. 1) was subcloned and is shown as part of pRS-Cg1. For the gene knockout, the CBF1 ORF was replaced by the CgTRP1 gene in a homologous recombination event. Hatched bars (Regions A and B) represent ∼400 bp of homologous sequences on each side of the CgTRP1 gene generated by PCR. Flanking thick lines represent vector sequences. Restriction sites are shown only if relevant. H, HindIII; S, SpeI. (B) Southern blot of genomic DNAs from C. glabrata transformants. Genomic DNAs were extracted from strain CgHTU2001 (lane 1), strain CgHTU2001/random (a transformant that was able to grow after being replated on 5-FOA medium) (lane 2), and the cbf1 deletion strain CgTS1/p112-Cp1 (lane 3) and digested with EcoRI. CgCBF1 was probed with a radioactively labeled PCR fragment generated with primers Cg1/3 and Cg1/4 (Table 2). The labeled band in lane 3 (CgTS1/p112-Cp1) corresponds to the CgCBF1 copy on plasmid p112-Cp1.