Human fungal pathogens accelerate into the genomics era.

A report on the Euroconference on Fungal Virulence Factors and Disease: 'Human Fungal Pathogens', Seefeld, Austria, 8-13 September 2001.

NRG1 represses yeast-hypha morphogenesis and hypha-specific gene expression in Candida albicans.

We have characterized CaNrg1 from Candida albicans, the major fungal pathogen in humans. CaNrg1 contains a zinc finger domain that is conserved in transcriptional regulators from fungi to humans. It is most closely related to ScNrg1, which represses transcription in a Tup1-dependent fashion in Saccharomyces cerevisiae. Inactivation of CaNrg1 in C.albicans causes filamentous and invasive growth, derepresses hypha-specific genes, increases sensitivity to some stresses and attenuates virulence. A tup1 mutant displays similar phenotypes. However, unlike tup1 cells, nrg1 cells can form normal hyphae, generate chlamydospores at normal rates and grow at 42 degrees C. Transcript profiling of 2002 C.albicans genes reveals that CaNrg1 represses a subset of CaTup1-regulated genes, which includes known hypha-specific genes and other virulence factors. Most of these genes contain an Nrg1 response element (NRE) in their promoter. CaNrg1 interacts specifically with an NRE in vitro. Also, deletion of two NREs from the ALS8 promoter releases it from Nrg1-mediated repression. Hence, CaNrg1 is a transcriptional repressor that appears to target CaTup1 to a distinct set of virulence-related functions, including yeast-hypha morphogenesis.

The essential and ancillary role of glutathione in Saccharomyces cerevisiae analysed using a grande gsh1 disruptant strain.

A grande gsh1 disruptant mutant of Saccharomyces cerevisiae was generated by crossing a petite disruptant to a wild-type grande strain. This strain was relatively stable, but generated petites at an elevated frequency, illustrating the ancillary role of glutathione (GSH) in the maintenance of the genetic integrity of the mitochondrial genome. The availability of the grande gsh1 deletant enabled an evaluation of the role of GSH in the cellular response to hydrogen peroxide independent of the effects of a petite mutation. The mutant strain was more sensitive to hydrogen peroxide than the wild-type strain but was still capable of producing an adaptive stress response to this compound. GSH was found to be essential for growth and sporulation of the yeast, but the intracellular level needed to support growth was at least two orders of magnitude less than that normally present in wild-type cells. This surprising result indicates that there is an essential role for GSH but only very low amounts are needed for growth. This result was also found in anaerobic conditions, thus this essential function does not involve protection from oxidative stress. Suppressors of the gsh1 deletion mutation were isolated by ethylmethanesulfonate mutagenesis. These were the result of a single recessive mutation (sgr1, suppressor for glutathione requirement) that relieved the requirement for GSH for growth on minimal medium but did not affect the sensitivity to H(2)O(2) stress. Interestingly, the gsh1 sgr1 mutant generated petites at a lower rate than the gsh1 mutant. Thus, it is suggested that the essential role of GSH is involved in the maintenance of the mitochondrial genome.

Regulation of gene expression during meiosis in Saccharomyces cerevisiae: SPR3 is controlled by both ABFI and a new sporulation control element.

The SPR3 gene encodes a sporulation-specific homolog of the yeast Cdc3/10/11/12 family of bud neck filament proteins. It is expressed specifically during meiosis and sporulation in Saccharomyces cerevisiae. Analysis of the sporulation-specific regulation of SPR3 has shown that it is strongly activated under sporulating conditions but shows low levels of expression under nonsporulating conditions. A palindromic sequence located near the TATA box is essential to the developmental regulation of this gene and is the only element directly activating SPR3 at the right time during sporulation. Within the palindrome is a 9-bp sequence, gNCRCAAA(A/T) (midsporulation element [MSE]), found in the known control regions of three other sporulation genes. A previously identified ABFI element is also needed for activation. The MSE has been shown to activate a heterologous promoter (CYC1) in a sporulation-specific manner. Related sequences, including an association of MSE and ABFI elements, have been found upstream of other genes activated during the middle stage of S. cerevisiae sporulation. One group of these may be involved in spore coat formation or maturation.

Characterization of the ugatA gene of Ustilago maydis, isolated by homology to the gatA gene of Aspergillus nidulans.

A gene encoding a putative GABA aminotransferase (ugatA) was isolated from the basidiomycete Ustilago maydis via heterologous hybridization to the GABA aminotransferase gene (gatA) of Aspergillus nidulans . The derived amino-acid sequence of ugatA shows strong identity throughout the protein to the GABA aminotransferase enzymes from A. nidulans and Saccharomyces cerevisiae. Northern analysis in U. maydis indicated that the ugatA transcript is inducible by the omega-amino acids GABA and beta-alanine, and is not subject to nitrogen catabolite repression. With the use of ugatA promoter-lacZ fusion constructs, it was demonstrated that the removal of sequences located approximately 250 bp 5' to the translational start site of ugatA (including multiple copies of a 7-bp direct repeat) resulted in the loss of induction by omega-amino acids. While the ugatA gene under the control of the A. nidulans gatA promoter was able to fully complement a gatA- phenotype in A. nidulans, the full-length ugatA gene was not, suggesting a lack of expression from the U. maydis promoter in A. nidulans. A U. maydis strain with a gene disruption at the ugatA locus showed decreased growth on beta-alanine as a sole nitrogen source, but was able to grow on GABA as a sole nitrogen source, indicating an alternative pathway for the utilization of GABA in U. maydis.