[Subtractive gene cloning and gene-disruption for elucidation of pseudohyphal formation in Candida tropicalis]

Abstract

The dimorphic transition from yeast to pseudohyphae in the petroleum-assimilating yeast Candida tropicalis occurs following the addition of ethanol to glucose semi-defined medium. Subtractive gene cloning was performed on the cDNA from the yeast-growing control culture and on that from the ethanol-supplemented one (the ethanol culture). A homologue of Schizosaccharomyces pombe nmt1+ or Saccharomyces cerevisiae THI5 was isolated from the cDNA fraction as a preferentially expressed gene for the ethanol culture. This homologue was tentatively called Ctnmt1+, since exogenous thiamine repressed its expression in C. tropicalis growth media. The ethanol culture showed a biphasic pattern of growth phases and the expression of Ctnmt1+ occurred at the first growth phase. The supplementation of thiamine to the ethanol culture at the first phase was followed by repression of Ctnmt1+ expression and also delay of pseudohyphal growth: filamentous growth was inhibited and chains of yeast cells were formed. A Ctnmt1+ disruptant of this organism did not show thiamine auxotrophy and produced pseudohyphal filaments even in the control culture. The supplementation of oxythiamine, an analog of thiamine, to the control culture was followed by the appearance of pseudohyphal filaments, indicating the participation of thiamine during the process of pseudohyphal growth in this organism.