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ABSTRACTCandida albicansis an important etiological agent of superficial and life-threatening infections in individuals with compromised immune systems. To date, we know of several overlapping genetic networks that govern virulence attributes in this fungal pathogen. Classical use of deletion mutants has led to the discovery of numerous virulence factors over the years, and genome-wide functional analysis has propelled gene discovery at an even faster pace. Indeed, a number of recent studies using large-scale genetic screens followed by genome-wide functional analysis has allowed for the unbiased discovery of many new genes involved in C. albicansbiology. Here we share our perspectives on the role of these studies in analyzing fundamental aspects of C. albicansvirulence properties.

The putative uridine diphosphate (UDP)-galactose 4-epimerase encoding gene, galE, was isolated from Avibacterium paragallinarum with the use of degenerate primers, colony hybridization and inverse PCR. The data revealed an open reading frame of 1017 bp encoding a protein of 338 amino acids with a molecular weight of 37 kDa and an isoelectric point of 5.5. High sequence homology was obtained with an 87, 91 and 89% sequence identity on protein level towards the galE genes from Actinobacillus pleuropneumoniae, Haemophilus influenza and Pasteurella multocida, respectively. To verify that the cloned galE gene encodes for a UDP-galactose 4-epimeras, this gene was cloned into the pYES- 2 expression vector, followed by transformation in a Saccharomyces cerevisiae gal10 deletion strain. Complementation of the gal10 deletion mutant with the galE gene confirmed that this gene encodes a UDP-galactose 4-epimerase.

Epoxide hydrolase (EH) activity was recently described in yeasts and highly selective hydrolysis of epoxides was observed during whole cell biotransformations. To expand the available molecular data regarding yeast EHs, the EH encoding gene from Rhodosporidium paludigenum(CBS 6565) was isolated, cloned and sequenced. The genomic EH sequence revealed a 1600 bp sequence interrupted by six introns. cDNA sequence analysis revealed an open reading frame of 1236 bp with a deduced polypeptide length of 411 amino acids. The deduced amino acid sequence revealed a relative high degree of sequence homology compared to the amino acid sequence of the EH from Rhodotorula glutinis.

The regulation of endo‐β‐(1,4)‐xylanase production by two different strains of Saccharomyces cerevisiae, each transformed with the XYN2gene from Trichoderma reeseiunder control of the promoter of the alcohol dehydrogenase II (ADH2) gene of S. cerevisiae, was investigated. In batch culture, the rate of xylanase production was severely reduced by the pulse addition of 390 mmol ethanol l−1. Pulses of 190–630 mmol ethanol l−1into aerobic glucose‐limited steady‐state continuous cultures reduced the xylanase activity about five‐fold and showed that ethanol repressed the ADH2promoter, as was evident from Northern blot analyses. Derepression of the ADH2‐regulated xylanase gene occurred at ethanol concentrations below approximately 50 mmol l−1.

The regulation of endo-β-(1,4)-xylanase production by two different strains of Saccharomyces cerevisiae, each transformed with the XYN2gene from Trichoderma reeseiunder control of the promoter of the alcohol dehydrogenase II ( ADH2) gene of S. cerevisiae, was investigated. In batch culture, the rate of xylanase production was severely reduced by the pulse addition of 390 mmol ethanol l −1. Pulses of 190–630 mmol ethanol l −1into aerobic glucose-limited steady-state continuous cultures reduced the xylanase activity about five-fold and showed that ethanol repressed the ADH2promoter, as was evident from Northern blot analyses. Derepression of the ADH2-regulated xylanase gene occurred at ethanol concentrations below approximately 50 mmol l −1.

Micro-organisms have developed systems to adapt to sudden changes in the environment. Here we describe the response of the yeastSaccharomyces cerevisiae to osmotic stress. A drop in the water activity (aw) of the medium following the addition of NaCl led to an immediate shrinkage of the cells. During the 2 h following the osmotic shock the cells partially restored their cell volume. This process depended on active protein synthesis. During the recovery period the cells accumulated glycerol intracellularly as a compatible solute and very little glycerol was leaking out of the cell. We have investigated in more detail the enzymes of glycerol metabolism and found that only the cytoplasmic glycerol-3-phosphate dehydrogenase was strongly induced. The level of induction was dependent on the yeast strain used and the degree of osmotic stress. The synthesis of cytoplasmic glycerol-3-phosphate dehydrogenase is also regulated by glucose repression. Using mutants defective in glucose repression (hxk2?), or derepression (snf1?), and with invertase as a marker enzyme, we show that glucose repression and the osmotic-stress response system regulate glycerol-3-phosphate dehydrogenase synthesis independently. We infer that specific control mechanisms sense the osmotic situation of the cell and induce responses such as the production and retention of glycerol.

The NAD‐dependent glycerol‐3‐phosphate dehydrogenase (glycerol‐3‐phosphate:NAD??? oxidoreductase; EC 1.1.1.8; G3P DHG) was purified 178‐fold to homogeneity from Saceharomyces cerevisiuestrain H44‐3D by affinity‐ and ion‐exchange chromatography, SDS‐PAGE indicited that the enzyme had a molecular mass of approximately 42,000 (± 1.000) whereas a molecular mass of 68,000 was observed using gel filtration, implying that the enzyme may exist as a dimer, The pH optimum for the reduction or dihydroxyacetone phosphate (DHAP) was 7.6 and the enzyme had a pt of 7.4. NADPH will not subititute for NADH as coenzyme in the reduction of DHAP. The oxidation of glycerol‐3‐phosphate (G3P) occurs at 3% of the rate of DHAP reduction at pH 7.0. Apparent Kmvalues obtained were 0.023 and 0.54 mM ror NADH and DHAP, respectively, NAD, fructose‐1,6‐bisphosphate (FBP), ATP and ADP inhibited G3P DHG activity, K1values obtained for NAD with NADH as variable substrate and FBP with DHAP as variable substrate were 0.93 and 4.8 mM, respectively.

The NAD-dependent glycerol-3-phosphate dehydrogenase (glycerol-3-phosphate:NAD??? oxidoreductase; EC 1.1.1.8; G3P DHG) was purified 178-fold to homogeneity from Saceharomyces cerevisiuestrain H44-3D by affinity- and ion-exchange chromatography, SDS-PAGE indicited that the enzyme had a molecular mass of approximately 42,000 (± 1.000) whereas a molecular mass of 68,000 was observed using gel filtration, implying that the enzyme may exist as a dimer, The pH optimum for the reduction or dihydroxyacetone phosphate (DHAP) was 7.6 and the enzyme had a pt of 7.4. NADPH will not subititute for NADH as coenzyme in the reduction of DHAP. The oxidation of glycerol-3-phosphate (G3P) occurs at 3% of the rate of DHAP reduction at pH 7.0. Apparent Kmvalues obtained were 0.023 and 0.54 mM ror NADH and DHAP, respectively, NAD, fructose-1,6-bisphosphate (FBP), ATP and ADP inhibited G3P DHG activity, K1values obtained for NAD with NADH as variable substrate and FBP with DHAP as variable substrate were 0.93 and 4.8 mM, respectively.

The regulation of endo-ß-(1,4)-xylanase production by two different strains of Saccharomyces cerevisiae, each transformed with the XYN2 gene from Trichoderma reesei under control of the promoter of the alcohol dehydrogenase II (ADH2) gene of S. cerevisiae, was investigated. In batch culture, the rate of xylanase production was severely reduced by the pulse addition of 390 mmol ethanol l-1. Pulses of 190–630 mmol ethanol l-1 into aerobic glucose-limited steady-state continuous cultures reduced the xylanase activity about five-fold and showed that ethanol repressed the ADH2 promoter, as was evident from Northern blot analyses. Derepression of the ADH2-regulated xylanase gene occurred at ethanol concentrations below approximately 50 mmol l-1.