Analysis of the sltA (stzA) gene and its orthologues in Aspergillus nidulansand other filamentous fungi

Abstract

Generation and phenotypic analyses of stzA gene deletion strains of Aspergillus nidulans implies that stzA is allelic to sltA, with the encoded transcription factor regulating tolerance to cations, DNA-damaging agents and high arginine concentrations. The similar severe sensitivity of a sltA1 mutant (GO281) and stzA deletion mutants to these stresses indicated that the premature termination codon in sltA1 represents a total loss-of-function mutation. It was also verified that StzA has no regulatory role in the utilisation of carbon sources. Findings were supported by phenotypic analyses of recombinant progeny resulting from sexual crosses between sltA1 and sltA+ strains. Bioinformatic analysis of genes involved in the osmotic stress response revealed that their promoters were significantly enriched for StzA binding site motifs compared to those of the control group, indicating that StzA may regulate many of these genes that comprise the High Osmolarity Glycerol (HOG) pathway. Although this pathway is activated by fludioxonil, stzA deletants and stzA+ strains showed similar sensitivities to this fungicide. Phenotypic analyses indicate that StzA does not regulate tolerance to sources of oxidative stress, non-ionic osmotic stress or components of the Cell Wall Integrity (CWI) pathway. A. nidulans StzA appears to have no role in cellulase or xylanase expression as revealed by the results of a dinitrosalicylic acid (DNS) assay. Trichoderma reesei ace1 deletant and wild-type strains showed similar sensitivities to cations, DNA-damaging agents, arginine, neomycin, acidic and alkaline pH. These results confirm that A. nidulans StzA and T. reesei Ace1 regulate the distinct phenotypes of abiotic stress tolerance and cellulase and xylanase expression, respectively, despite these two proteins sharing 58% overall amino acid similarity. All twenty-nine StzA orthologues identified are restricted to filamentous ascomycetes of the Pezizomycotina subphylum and may therefore represent specific and novel antifungal drug targets. The C-termini of StzA proteins are highly variable in both length and sequence, with no apparent conservations in amino acids or predicted secondary structure. This region is considered most likely to influence the divergent functions of StzA proteins. Conservations of individual residues in the N-termini correspond to conserved secondary structure (alpha helices) among StzA proteins, implying shared functions for StzA proteins in this region. Regulators of two major nitrogen metabolic pathways (CpcA and AreA) may regulate stzA expression. Statistically significant putative CpcA binding sites were positionally conserved in 26 out of 29 stzA orthologue promoters, indicating an interaction between stzA and CpcA, a transcription factor that mediates the cross pathway control of amino acid biosynthesis. REALALE sequences, likely to be of retrotransposon origin, containing putative overlapping binding sites for StzA and AreA, were found in eleven stzA promoters of the Eurotiomycetes class, indicating an interaction between stzA and the global nitrogen metabolite repressor AreA. Intriguingly, REALALE-containing promoters identified across the genome of A. nidulans were significantly enriched for StzA binding site motifs when compared to a control group of genes. Hence, REALALE may have regulatory significance that extends to other A. nidulans genes.