The SSK2 MAPKKK of Candida albicans is required for oxidant adaptation in vitro

The Ssk2p (MAPKKK) of Candida albicans was deleted and functions assigned based on phenotyping studies. SSK2 deletion was first attempted using the UAU1 disruption method. All transformants lacking one copy of SSK2 appeared to be triploids, suggesting that the SSK2 is essential for the organism. To verify this observation, a strain was constructed in which one allele was deleted using the SAT1 flipper disruption method. The second allele was then placed under control of the on/off tetracycline-regulatable (TetR) promoter. The transcription of SSK2 was measured by reverse transcriptase-PCR and although the promoter was somewhat leaky, transcript was significantly reduced in an ssk2/TetR-SSK2 transformant (AT2) in the presence of doxycycline. Strains AT1 and AT2 constructed using the SAT1 flipper and TetR promoter method, respectively, were studied phenotypically in different growth media to determine the role of Ssk2p in morphogenesis. The mutants were also compared under on/off conditions in the presence of 1.5 M NaCl and various types of oxidants. Strain AT2 demonstrated resistance to 1.5 M NaCl in the absence of doxycycline but was inhibited by 8 mM hydrogen peroxide.