The Candida albicans 14‐3‐3 gene, BMH1, is essential for growth

Abstract

The 14-3-3 proteins are a family of conserved small acidic proteins that have been implicated in playing major roles in a wide variety of signalling cascades. In Saccharomyces cerevisiae, the 14-3-3 genes (BMH1 and BMH2) are essential for normal pseudohyphal induction and normal bud cell development. The Bmh proteins function in the cAMPdependent RAS/MAPK and rapamycin-sensitive signalling cascades. Deletion of only one BMH gene demonstrates no phenotypic differences under normal growth conditions. Strains deleted of both BMH1 and BMH2 are either non-viable or demonstrate sensitivity to environmental stresses. In Schizosaccharomyces pombe, the BMH homologues (RAD24 and RAD25) are essential for cell cycle control after DNA damage and deletion of both genes renders the cell inviable. The 14-3-3 gene in Candida albicans (BMH1) was identified using a novel adherence assay and differential display RT-PCR. Unlike other yeasts, C. albicans has only one 14-3-3 gene (BMH1). It was not possible to construct double knockouts by routine methods. These results suggested that the C. albicans BMH1 gene is essential. The essentiality of C. albicans BMH1 was confirmed by a PCR disruption technique. The C. albicans bmh1D/BMH1 heterozygotes exhibit growth and morphogenetic defects. Therefore, the BMH1 gene in C. albicans (Accession No. AF038154) is an excellent candidate to improve our understanding of the coordinate regulation of cell cycle and morphogenesis.

Mutant alleles of the essential 14-3-3 gene in Candida albicans distinguish between growth and filamentation

“…The heterozygote strains BH1 and BA1 were constructed by transforming BWP17 with the PCR product bmh1 : HIS1 or bmh1 : ARG4 as described for BUM1 (Cognetti et al, 2002). The PCR products were derived by amplifying the HIS1 gene or ARG4 gene from pGEM-HIS1 or pRS-ARGDSpeI, respectively, with primers Bmh511FL and Bmh1300RL (Table 2).…”

“…pKJ3 and pLUX were digested with KpnI and SacI and ligated to give plasmid pLB. Site-directed mutagenesis was performed using the QuikChange site-directed mutagenesis kit (Stratagene) following the manufacturer's directions and as performed previously (Cognetti et al, 2002). pLB was mutagenized with primers tsmutF and tsmutR to introduce the S188P temperature-sensitive (Ts) mutation to give plasmid pLBTs, primers K51EF and K51ER to give plasmid pLBKE, and primers 14mutF and 14mutR to introduce L14QA15QE16R to give pLBdim.…”

“…The 14-3-3 gene in C. albicans, BMH1, is essential (Cognetti et al, 2002) and thus it is not possible to identify its function using conventional knock-out strategies. Therefore, conditional mutants were constructed.…”

“…Therefore, conditional mutants were constructed. A first attempt was to place the remaining BMH1 allele in a heterozygote strain under the control of the MET3 promoter (Care et al, 1999;Cognetti et al, 2002). However, all transformants retained an additional wild-type copy, even those that contained the desired integration (Cognetti et al, 2002).…”

“…In C. albicans, there is only one 14-3-3 protein and it is required for vegetative growth and optimal filamentation (Cognetti et al, 2002). The ability of C. albicans to adapt is a key step in its pathogenesis and involves careful regulation of multiple cellular processes.…”