Ivo Zadra

The siderophore system is essential for viability of Aspergillus nidulans: functional analysis of two genes encoding l-ornithine N 5-monooxygenase (sidA) and a non-ribosomal peptide synthetase (sidC)

The filamentous ascomycete A. nidulans produces two major siderophores: it excretes triacetylfusarinine C to capture iron and contains ferricrocin intracellularly. In this study we report the characterization of two siderophore biosynthetic genes, sidA encoding l‐ornithine N5‐monooxygenase and sidC encoding a non‐ribosomal peptide synthetase respectively. Disruption of sidC eliminated synthesis of ferricrocin and deletion of sidA completely blocked siderophore biosynthesis. Siderophore‐deficient strains were unable to grow, unless the growth medium was supplemented with siderophores, suggesting that the siderophore system is the major iron assimilatory system of A. nidulans during both iron depleted and iron‐replete conditions. Partial restoration of the growth of siderophore‐deficient mutants by high concentrations of Fe2+ (but not Fe3+) indicates the presence of an additional ferrous transport system and the absence of an efficient reductive iron assmilatory system. Uptake studies demonstrated that TAFC‐bound iron is transferred to cellular ferricrocin whereas ferricrocin is stored after uptake. The siderophore‐deficient mutant was able to synthesize ferricrocin from triacetylfusarinine C. Ferricrocin‐deficiency caused an increased intracellular labile iron pool, upregulation of antioxidative enzymes and elevated sensitivity to the redox cycler paraquat. This indicates that the lack of this cellular iron storage compound causes oxidative stress. Moreover, ferricrocin biosynthesis was found to be crucial for efficient conidiation.

The Aspergillus nidulans GATA Factor SREA Is Involved in Regulation of Siderophore Biosynthesis and Control of Iron Uptake

A gene encoding a new GATA factor fromAspergillus nidulans, sreA, was isolated and characterized. SREA displays homology to two fungal regulators of siderophore biosynthesis: about 30% overall identity to SRE fromNeurospora crassa and about 50% identity to URBS1 fromUstilago maydis over a stretch of 200 amino acid residues containing two GATA-type zinc finger motifs and a cysteine-rich region. This putative DNA binding domain, expressed as a fusion protein inEscherichia coli, specifically binds to GATA sequence motifs. Deletion of sreA results in derepression ofl-ornithine-N 5-oxygenase activity and consequently in derepression of the biosynthesis of the hydroxamate siderophore N,N′,N′′-triacetyl fusarinine under sufficient iron supply in A. nidulans. Transcription of sreA is confined to high iron conditions, underscoring the function of SREA as a repressor of siderophore biosynthesis under sufficient iron supply. Nevertheless, overexpression of sreA does not result in repression of siderophore synthesis under low iron conditions, suggesting additional mechanisms involved in this regulatory circuit. Consistent with increased sensitivity to the iron-activated antibiotics phleomycin and streptonigrin, the sreA deletion mutant displays increased accumulation of 59Fe. These results demonstrate that SREA plays a central role in iron uptake in addition to siderophore biosynthesis.

SREA is involved in regulation of siderophore biosynthesis, utilization and uptake in Aspergillus nidulans

Under conditions of low iron availability, most fungi excrete siderophores in order to mobilize extracellular iron. We show that lack of the GATA‐type transcription factor SREA in Aspergillus nidulans not only leads to derepression of siderophore biosynthesis but also to deregulation of siderophore‐bound iron uptake and ornithine esterase expression. Furthermore, SREA deficiency causes increased accumulation of ferricrocin, the siderophore responsible for intracellular iron storage. In sreA deletion strains, extracellular siderophore production is derepressed but still regulated negatively by iron availability, indicating the presence of an additional iron‐regulatory mechanism. In contrast, iron affects ferricrocin accumulation in a positive way, suggesting a protective role for this siderophore in detoxification of intracellular iron excess. The harmfulness of deregulated iron uptake in this mutant is demonstrated by increased expression of genes encoding the antioxidative enzymes catalase CATB and the superoxide dismutases SODA and SODB. It is noteworthy that iron starvation was found to repress catB expression in wild‐type (wt) and SREA‐deficient strains, consistent with catB being subject to SREA‐independent iron regulation. Differential display led to the identification of putative SREA target genes amcA and mirA. The deduced MIRA amino acid sequence displays significant similarity to recently characterized siderophore permeases of Saccharomyces cerevisiae. amcA encodes a putative mitochondrial carrier for the siderophore precursor ornithine, indicating cross‐regulation of siderophore and ornithine metabolism.

Two Components of a velvet-Like Complex Control Hyphal Morphogenesis, Conidiophore Development, and Penicillin Biosynthesis in Penicillium chrysogenum

ABSTRACT Penicillium chrysogenum is the industrial producer of the antibiotic penicillin, whose biosynthetic regulation is barely understood. Here, we provide a functional analysis of two major homologues of the velvet complex in P. chrysogenum, which we have named P. chrysogenumvelA (PcvelA) and PclaeA. Data from array analysis using a ΔPcvelA deletion strain indicate a significant role of PcVelA on the expression of biosynthesis and developmental genes, including PclaeA. Northern hybridization and high-performance liquid chromatography quantifications of penicillin titers clearly show that both PcVelA and PcLaeA play a major role in penicillin biosynthesis in a producer strain that underwent several rounds of UV mutagenesis during a strain improvement program. Both regulators are further involved in different developmental processes. While PcvelA deletion leads to light-independent conidial formation, dichotomous branching of hyphae, and pellet formation in shaking cultures, a ΔPclaeA strain shows a severe impairment in conidiophore formation under both light and dark conditions. Bimolecular fluorescence complementation assays provide evidence for a velvet-like complex in P. chrysogenum, with structurally conserved components that have distinct developmental roles, illustrating the functional plasticity of these regulators in genera other than Aspergillus.

xylP promoter-based expression system and its use for antisense downregulation of the Penicillium chrysogenum nitrogen regulator NRE.

ABSTRACT A highly inducible fungal promoter derived from thePenicillium chrysogenum endoxylanase (xylP) gene is described. Northern analysis and the use of a β-glucuronidase (uidA) reporter gene strategy showed thatxylP expression is transcriptionally regulated. Xylan and xylose are efficient inducers, whereas glucose strongly represses the promoter activity. Comparison of the same expression construct as a single copy at the niaD locus in P. chrysogenumand at the argB locus in Aspergillus nidulansdemonstrated that the xylP promoter is regulated similarly in these two species but that the level of expression is about 80 times higher in the Aspergillus species. The xylPpromoter was found to be 65-fold more efficient than the isopenicillin-N-synthetase (pcbC) promoter inPenicillium and 23-fold more efficient than the nitrate reductase (niaD) promoter in Aspergillus under induced conditions. Furthermore, the xylP promoter was used for controllable antisense RNA synthesis of the nre-encoded putative major nitrogen regulator of P. chrysogenum. This approach led to inducible downregulation of the steady-state mRNA level of nre and consequently to transcriptional repression of the genes responsible for nitrate assimilation. In addition, transcription of nreB, which encodes a negative-acting nitrogen regulatory GATA factor of Penicillium, was found to be subject to regulation by NRE. Our data are the first direct evidence that nre indeed encodes an activator in the nitrogen regulatory circuit in Penicillium and indicate that cross regulation of the controlling factors occurs.

Sexual reproduction and mating-type–mediated strain development in the penicillin-producing fungus Penicillium chrysogenum

Penicillium chrysogenum is a filamentous fungus of major medical and historical importance, being the original and present-day industrial source of the antibiotic penicillin. The species has been considered asexual for more than 100 y, and despite concerted efforts, it has not been possible to induce sexual reproduction, which has prevented sexual crosses being used for strain improvement. However, using knowledge of mating-type (MAT) gene organization, we now describe conditions under which a sexual cycle can be induced leading to production of meiotic ascospores. Evidence of recombination was obtained using both molecular and phenotypic markers. The identified heterothallic sexual cycle was used for strain development purposes, generating offspring with novel combinations of traits relevant to penicillin production. Furthermore, the MAT1-1–1 mating-type gene, known primarily for a role in governing sexual identity, was also found to control transcription of a wide range of genes with biotechnological relevance including those regulating penicillin production, hyphal morphology, and conidial formation. These discoveries of a sexual cycle and MAT gene function are likely to be of broad relevance for manipulation of other asexual fungi of economic importance.

Homologous recombination in the antibiotic producer Penicillium chrysogenum: strain ΔPcku70 shows up-regulation of genes from the HOG pathway

In Penicillium chrysogenum, the industrial producer of the β-lactam antibiotic penicillin, generating gene replacements for functional analyses is very inefficient. Here, we constructed a recipient strain that allows efficient disruption of any target gene via homologous recombination. Following isolation of the Pcku70 (syn. hdfA) gene encoding a conserved eukaryotic DNA-binding protein involved in non-homologous end joining (NHEJ), a Pcku70 knockout strain was constructed using a novel nourseothricin-resistance cassette as selectable marker. In detailed physiological tests, strain ΔPcku70 showed no significant reduction in vegetative growth due to increased sensitivity to different mutagenic substances. Importantly, deletion of the Pcku70 gene had no effect on penicillin biosynthesis. However, strain ΔPcku70 exhibits higher sensitivity to osmotic stress than the parent strain. This correlated well with comparative data from microarray analyses: Genes related to the stress response are significantly up-regulated in the Pcku70 deletion mutant. To demonstrate the applicability of strain ΔPcku70, three genes related to β-lactam antibiotic biosynthesis were efficiently disrupted, indicating that this strain shows a low frequency of NHEJ, thus promoting efficient homologous recombination. Furthermore, we discuss strategies to reactivate Pcku70 in strains successfully used for gene disruptions.

Overexpression of nreB, a New GATA Factor-encoding Gene of Penicillium chrysogenum, Leads to Repression of the Nitrate Assimilatory Gene Cluster

To investigate the mechanism of nitrogen metabolite repression in the biotechnologically important fungusPenicillium chrysogenum a polymerase chain reaction approach was employed to identify transcription factors involved in this regulatory circuit, leading to the isolation of a new gene (nreB) encoding a 298 amino acid protein. Despite a low overall amino acid sequence identity of approximately 30%, it shares several features with Dal80p/Uga43p and Gzf3p/Nil2p, both repressors in nitrogen metabolism in Saccharomyces cerevisiae. All three proteins contain an N-terminal GATA-type zinc finger motif, displaying 86% amino acid sequence identity, and a putative leucine zipper motif in the C terminus. Northern blot analysis revealed the presence of twonreB transcripts, 1.8 and 1.5 kilobases in length, that differ in polyadenylation sites. The steady state level of both transcripts is subject to nitrogen metabolite repression. The putative DNA binding domain of NREB, expressed as a fusion protein inEscherichia coli, binds in vitro to GATA sites of its own 5′-upstream region as well as in the promoter of the nitrate assimilation gene cluster. Consistent with a role in the regulation of nitrogen metabolism, overexpression of nreB leads to repression of nitrate assimilatory genes. Hence, the simple view of nitrogen regulation by four GATA factors in yeast, but only one key regulator in filamentous ascomycetes seems no longer valid.

Iron starvation leads to increased expression of Cu/Zn-superoxide dismutase in Aspergillus

In a search for iron‐regulated proteins of Aspergillus nidulans and Aspergillus fumigatus a 16‐kDa protein was identified which is about 5‐fold upregulated during iron starvation in both species and which can be approximately 500‐fold enriched by simple one‐step chromatography on Amberlite XAD‐16 resin. N‐terminal protein sequence analysis and cloning of the respective A. nidulans cDNA identified this protein as a Cu/Zn‐superoxide dismutase (SODA). Northern analysis revealed that upregulation of sodA expression occurs at the level of transcript accumulation. This seems to be a specific low iron response and not a general starvation answer since sodA transcript levels do not respond to carbon or nitrogen starvation. In contrast, copper depletion leads to transcriptional downregulation of sodA. Furthermore, sodA expression was found still to be subject to iron regulation in an A. nidulans mutant lacking SREA, a regulator of iron homeostasis, indicating that sodA expression is regulated by an SREA‐independent mechanism. The data presented suggest that SODA plays a protective role under iron deplete conditions.

The Aspergillus nidulans GATA transcription factor gene areB encodes at least three proteins and features three classes of mutation

In Aspergillus nidulans, the principal transcription factor regulating nitrogen metabolism, AREA, belongs to the GATA family of DNA‐binding proteins. In seeking additional GATA factors, we have cloned areB, which was originally identified via a genetic screen for suppressors of areA loss‐of‐function mutations. Based on our analysis, areB is predicted to encode at least three distinct protein products. These arise from the use of two promoters, differential splicing and translation initiating at AUG and non‐AUG start codons. All the putative products include a GATA domain and a putative Leu zipper. These regions show strong sequence similarity to regulatory proteins from Saccharomyces cerevisiae (Dal80p and Gzf3p), Penicillium chrysogenum (NREB) and Neurospora crassa (ASD4). We have characterized three classes of mutation in areB; the first are loss‐of‐function mutations that terminate the polypeptides within or before the GATA domain. The second class truncates the GATA factor either within or upstream of the putative Leu zipper but retains the GATA domain. The third class fuses novel gene sequences to areB with the potential to produce putative chimeric polypeptides. These novel gene fusions transform the putative negative‐acting transcription factor into an activator that can partially replace areA.