Candida glabrata PHO4 Is Necessary and Sufficient for Pho2-Independent Transcription of Phosphate Starvation Genes

Abstract

Comparative genomic analyses of Candida glabrata and Saccharomyces cerevisiae suggest many signal transduction pathways are highly conserved. Focusing on the phosphate signal transduction (PHO) pathway of C. glabrata, we demonstrate that components of the pathway are conserved and confirm the role of CgPHO81, CgPHO80, CgPHO4, and CgMSN5 in the PHO pathway through deletion analysis. Unlike S. cerevisiae, C. glabrata shows little dependence on the transcription factor, Pho2, for induction of phosphate-regulated genes during phosphate limitation. We show that the CgPho4 protein is necessary and sufficient for Pho2-independent gene expression; CgPho4 is capable of driving expression of PHO promoters in S. cerevisiae in the absence of ScPHO2. On the basis of the sequences of PHO4 in the hemiascomycetes and complementation analysis, we suggest that Pho2 dependence is a trait only observed in species closely related to S. cerevisiae. Our data are consistent with trans-regulatory changes in the PHO pathway via the transcription factor Pho4 as opposed to cis-regulatory changes (the promoter).

Novel Acid Phosphatase in Candida glabrata Suggests Selective Pressure and Niche Specialization in the Phosphate Signal Transduction Pathway

“…For all experiments, cells were grown at 30°until logarithmic growth phase (OD 600 $0.5). For phosphate starvation experiments, cells grown to logarithmic phase were harvested by centrifugation, washed three times in media without phosphate, and then transferred to media without phosphate (no-phosphate conditions) or to media with 10 mm KH 2 PO 4 added (high-phosphate conditions) and grown at 30°for 3 hr (Kerwin and Wykoff 2009).…”

“…glabrata mutants were generated using the antibiotic resistance genes KANMX6 or NATMX6 (Kerwin and Wykoff 2009) and homologous recombination to inactivate PMU1, PMU2, or PMU3 in a C. glabrata his3 À background strain (Cormack and Falkow 1999;Hentges et al 2005;Kerwin and Wykoff 2009). Refer to supporting information, Table S1 for a list of primers used to inactivate genes.…”

“…Assays can quantitatively or qualitatively measure the extent of Pho5 activity and serve as a proxy for Pho4 activity. In studying C. glabrata, we observe strong acid phosphatase activity that is regulated by Pho4; however, the sequenced genome does not appear to contain a PHO5 homolog (Kerwin and Wykoff 2009). Through complementation of a Scpho5D strain with a C. glabrata genomic library (Sanglard et al 1999), we identified a three-gene cluster (which we have named CgPMU1, CgPMU2, and CgPMU3) that contains the functional analog of PHO5.…”

“…T HE phosphate signal transduction (PHO) pathway in Candida glabrata is similar to the PHO pathway in Saccharomyces cerevisiae and serves as a model to examine species-specific changes to a signal transduction pathway (Kerwin and Wykoff 2009). C. glabrata is a mammalian pathogen that can cause candidiasis and is relatively closely related to budding yeast, S. cerevisiae (Redondo-Lopez et al 1990;Cormack and Falkow 1999;Domergue et al 2005).…”

“…Thus, identifying differences in the PHO pathway between the two species may shed light on their environmental niche. In both species, the transcription factor Pho4 is critical to the phosphate starvation response, and it appears to be regulated similarly in both species-through phosphorylation by the Pho80/ Pho85/Pho81 complex (Schneider et al 1994;O'Neill et al 1996;Kerwin and Wykoff 2009). However, another transcription factor, Pho2, which is essential for the starvation response in budding yeast, appears to be unimportant in C. glabrata (Barbaric et al 1998;Springer et al 2003;Kerwin and Wykoff 2009).…”