Disruption analysis of metallothionein-encoding genes in Candida glabrata.
Candida glabrata harbors multiple genes encoding metallothionein (MT). We have disrupted MT-IIa, an amplified locus, and MT-IIb, a single-copy gene, to determine the roles of various MT genes in CuSO4 resistance in C. glabrata. The concentration of CuSO4 required to inhibit the growth by 50% (IC50) of a C. glabrata strain harboring an amplified MT-IIa locus and a single-copy MT-IIb and MT-I genes was 7 mM in a synthetic complete medium. The IC50 decreased to approx. 1 mM when the amplified MT-IIa locus was deleted. The disruption of the MT-IIb gene decreased the IC50 further to 0.1 mM. The CuSO4 resistance in a strain lacking both of the MT-II genes was attributable to MT-I; no evidence was found for the production of (gamma EC)nG isopeptides. The comparison of the nucleotide sequence of MT-IIb to that of MT-IIa revealed the same coding sequence with differences in the 5' region. However, substantial differences were found in the 3' region. MT-IIb was expressed since we were able to purify the protein from the strain that had an intact MT-IIb gene, but a deleted MT-IIa gene. In addition, CuSO4 resistance was provided by MT-IIb. Northern analysis of the total RNA from varied C. glabrata strains indicated no significant changes in the expression of MT-I in the presence or absence of the MT-II genes.
Mehra
R K
RK
Department of Medicine, University of Utah Medical Center, Salt Lake City 84132.
Thorvaldsen
J L
JL
Macreadie
I G
IG
Winge
D R
DR
D000595:Amino Acid Sequence; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D002175:Candida / Q000187:drug effects / Q000235:genetics*; D003300:Copper / Q000494:pharmacology*; D019327:Copper Sulfate; D004252:DNA Mutational Analysis; D004352:Drug Resistance, Microbial / Q000235:genetics; D008668:Metallothionein / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000235:genetics
D003300:Copper; D008668:Metallothionein; D019327:Copper Sulfate