Isolation of a chitin synthase gene (CHS1) from Candida albicans by expression in Saccharomyces cerevisiae.
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 synthases in C. albicans. A gene for chitin synthase from C. albicans (CHS1) was cloned by heterologous expression in a Saccharomyces cerevisiae chs1 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 CHS1 gene complemented the S. cerevisiae chs1 mutation and encoded enzymatic activity which was stimulated by partial proteolysis; (ii) the enzyme catalyses incorporation of [14C]-GlcNAc from the substrate, UDP[U-14C]-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, CHS1 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 8.0 is proposed. DNA sequencing revealed an open reading frame of 2328 nucleotides which predicts a polypeptide of Mr 88,281 with 776 amino acids. The alignment of derived amino acid sequences revealed that the CHS1 gene from C. albicans (canCHS1) is homologous (37% amino acid identity) to the CHS1 gene from S. cerevisiae (sacCHS1).
Au-Young
J
J
Center for Cancer Research, Massachusetts Institute of Technology, Cambridge 02139.
Robbins
P W
PW
D000595:Amino Acid Sequence; D001483:Base Sequence; D015139:Blotting, Southern; D002176:Candida albicans / Q000201:enzymology*; D002686:Chitin / Q000032:analysis; D002687:Chitin Synthase / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D005816:Genetic Complementation Test; D005964:Glucosyltransferases / Q000235:genetics*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D012689:Sequence Homology, Nucleic Acid
D011994:Recombinant Proteins; D002686:Chitin; D005964:Glucosyltransferases; D002687:Chitin Synthase