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Isolation of a chitin synthase gene ( CHS 1) from Candida albicans by expression in Saccharomyces cerevisiae
Author(s) -
AuYoung J.,
Robbins P. W.
Publication year - 1990
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.1990.tb00587.x
Subject(s) - chitin synthase , saccharomyces cerevisiae , biology , candida albicans , biochemistry , chitin , corpus albicans , heterologous expression , yeast , mutant , microbiology and biotechnology , gene , recombinant dna , chitosan
Summary Chitin synthase activity was studied in yeast and hyphal forms of Candida albicans. pH‐activity profiles showed that yeast and hyphae contain a protease‐dependent activity that has an optimum at pH 6.8. In addition, there is an activity that is not activated by proteolysis in vitro and which shows a peak at pH 8.0. This suggests there are two distinct chitin syntheses in C albicans. A gene for chitin synthase from C. albicans (CHS1) was cloned by heterologous expression in a Saccharomyces cerevisiae chs 1 mutant. Proof that the cloned chitin synthase is a C. albicans membrane‐bound zymogen capable of chitin biosynthesis in vitro was based on several criteria, (i) the CHS 1 gene complemented the S. cerevisiae chs 1 mutation and encoded enzymatic activity which was stimulated by partial proteolysis; (ii) the enzyme catalyses incorporation of [ 14 C]‐GlcNAc from the substrate, UDP[U‐ 14 C]‐GlcNAc, into alkali‐insoluble chitin; (iii) Southern analysis showed hybridization of a C. albicans CHS1 probe only with C. albicans DNA and not with S. cerevisiae DNA; (iv) pH profiles of the cloned enzyme showed an optimum at pH 6.8. This overlaps with the pH‐activity profiles for chitin synthase measured in yeast and hyphal forms of C. albicans. Thus, CHS 1 encodes only part of the chitin synthase activity in C. albicans. A gene for a second chitin synthase in C. albicans with a pH optimum at 6.0 is proposed. DNA sequencing revealed an open reading frame of 2328 nucleotides which predicts a polypeptide of M r 88281 with 776 amino acids. The alignment of derived amino acid sequences revealed that the CHS 1 gene from C. albicans (canCHS 1) is homologous (37% amino acid identity) to the CHS 1 gene from S. cerevisiae (sacCHS 1).