Novel acid phosphatase in Candida glabrata suggests selective pressure and niche specialization in the phosphate signal transduction pathway

Abstract

Evolution through natural selection suggests unnecessary genes are lost. We observed that the yeast Candida glabrata lost the gene encoding a phosphate-repressible acid phosphatase (PHO5) present in many yeasts including Saccharomyces cerevisiae. However, C. glabrata still had phosphate starvation-inducible phosphatase activity. Screening a C. glabrata genomic library, we identified CgPMU2, a member of a three-gene family that contains a phosphomutase-like domain. This small-scale gene duplication event could allow for sub- or neofunctionalization. On the basis of phylogenetic and biochemical characterizations, CgPMU2 has neofunctionalized to become a broad range, phosphate starvation-regulated acid phosphatase, which functionally replaces PHO5 in this pathogenic yeast. We determined that CgPmu2, unlike ScPho5, is not able to hydrolyze phytic acid (inositol hexakisphosphate). Phytic acid is present in fruits and seeds where S. cerevisiae grows, but is not abundant in mammalian tissues where C. glabrata grows. We demonstrated that C. glabrata is limited from an environment where phytic acid is the only source of phosphate. Our work suggests that during evolutionary time, the selection for the ancestral PHO5 was lost and that C. glabrata neofunctionalized a weak phosphatase to replace PHO5. Convergent evolution of a phosphate starvation-inducible acid phosphatase in C. glabrata relative to most yeast species provides an example of how small changes in signal transduction pathways can mediate genetic isolation and uncovers a potential speciation gene.

Figure 1.—

Complementation of Scpho5Δ by C. glabrata genomic library. (A) A semiquantitative phosphatase plate assay was performed on wild-type C. glabrata grown in high-phosphate media and in no-phosphate media with the dark color indicating secreted acid phosphatase activity. Five genomic clones conferred phosphatase activity in no-phosphate conditions and repressed phosphatase activity in high-phosphate conditions. (B) The genomic clones with phosphatase activity were further analyzed. Clone A spans nucleotides 740743–747471, clone E 748278–738994, clone Q 747445–735219, and clone T 743041–747455. Clone O ends at nucleotide 747642 (numbering is based on chromosome K sequence NC_006034). Using CgPMU1 primers designed to amplify the ORF, it was determined that clone O contains CgPMU1; however, one end was not refractory to sequencing. The same region of chromosome 11 from S. cerevisiae is below the C. glabrata schematic showing the conserved synteny. The direction of the arrows indicates the direction of transcription.

Figure 2.—

(A) ClustalW alignment of CgPmu proteins with ScPmu1. After alignment, the ALN file was entered into BOXSHADE with a cutoff of identity of 0.7. Solid boxes indicate identity and shaded boxes indicate similarity. (B) Tree of relationships generated from JalView 2.4 using a neighbor joining tree with BLOSSUM62. The sequences used were: Ashbya gossypii AEL304C, Kluyveromyces lactis KLLA0B12628g, Saccharomyces bayanus sbayc559-g5.1, and previously identified protein sequences. This tree is representative of trees generated by other methods as well.

Figure 3.—

Pmu2 is phosphate starvation-inducible acid phosphatase in C. glabrata. (A) Phosphatase plate assay in high- and no-phosphate conditions with only the pmu2Δ strain having a major defect in phosphatase activity. (B) The phosphatase activity (PNPP hydrolysis) of the deletion strains was quantified to determine activity normalized to cell density. Data in this figure and all following figures are expressed as mean ± SEM, n = 3 for each strain unless noted. Generation of new Cgpmu1Δ strain (Figure S1), which did not disrupt CgPMU2 promoter, prevented this effect. (C) Quantitative real-time PCR was performed on the same strains and CgPMU2 was quantified and normalized to the expression level of CgACT1 because the expression of CgACT1 does not change in response to phosphate starvation (Kerwin and Wykoff 2009). Deletion of CgPMU3 appears to lead to increased phosphatase activity and increased levels of CgPMU2 transcript. This 1.5- to 2-fold increase is consistently observed, but was not tractable enough to pursue in this study.

Figure 4.—

CgPMU2 is strongly regulated by phosphate starvation conditions and the transcription factor Pho4. Quantitative real-time PCR was performed on each PMU gene and normalized to the expression level of ACT1. Each primer set was verified to amplify only the indicated gene by examining qPCR of deletion strains (Figure S3). CgPMU1 and CgPMU2 appear regulated by CgPho4, but CgPMU1 to a much lesser extent. The CgPMU3/CgACT1 ratio is ∼0.003 and does not change in response to phosphate condition or CgPHO4 deletion.

Figure 5.—

The CgPMU2 and CgPMU3 ORFs are able to hydrolyze 1-NP or PNPP efficiently. All four ORFs were placed under the control of the phosphate starvation-regulated ScPHO5 promoter and transformed into Cgpmu2Δ strain. Expression of each ORF was verified by RT–qPCR (Figure S4). Phosphatase activity is restored when CgPMU2 and CgPMU3 are expressed as judged by hydrolysis of 1-NP (A) or PNPP (B). Additionally, these ORFs were placed under the control of the CgPMU2 promoter and exhibited similar results (Figure S5). Multiple isolates were subjected to the phosphatase plate assay and these are representative results.

Figure 6.—

Substrate vs. velocity curve with PNPP. Equal amounts of purified enzyme were incubated with various concentrations of PNPP and the amount of PNP formed was monitored at OD₄₀₀. All three exhibit Michaelis–Menten kinetics, and protein from a mock purification exhibited a velocity of ∼0.2 mmol PNP released/min × mg protein regardless of substrate concentration (data not shown).

Figure 7.—

ScPHO5 encodes a phytase that allows C. glabrata to grow with phytic acid as a sole phosphate source. (A) Strains were inoculated at an OD₆₀₀ of 0.001 and grown for 24 hr in SD + 1.2 mm phosphate or 60 hr in SD + 200 μm phytic acid and monitored by measuring OD₆₀₀. The strains at these times points were not dividing rapidly. C. glabrata consistently grows to a higher OD₆₀₀ than S. cerevisiae. Deletion of ScPHO5 has a modest effect on growth in phytic acid (data not shown) because there are other phytases in the S. cerevisiae genome (Olstorpe et al. 2009). (B) A photograph of strains from A, but inoculated at a density of 0.0001 in SD + 200 μm phytic acid and grown for 4 days at 30°.

Figure 8.—

CgPMU2 is required for the growth of C. glabrata in media with organic phosphate as the sole phosphate source. Strains were inoculated at OD₆₀₀ of 0.001 and grown for 20 hr in SD + 0.2 mm phosphate or 72 hr in SD + organic phosphate. GMP, guanosine monophosphate; G2P, glycerol-2-phosphate; P_i, inorganic phosphate. Strains grew much slower in organic phosphate sources and 500 μm G2P was required for measurable growth. This is one representative experiment, but was reproducible.