Comparative genomics of citric-acid-producing Aspergillus niger ATCC 1015 versus enzyme-producing CBS 513.88.

The filamentous fungus Aspergillus niger exhibits great diversity in its phenotype. It is found globally, both as marine and terrestrial strains, produces both organic acids and hydrolytic enzymes in high amounts, and some isolates exhibit pathogenicity. Although the genome of an industrial enzyme-producing A. niger strain (CBS 513.88) has already been sequenced, the versatility and diversity of this species compel additional exploration. We therefore undertook whole-genome sequencing of the acidogenic A. niger wild-type strain (ATCC 1015) and produced a genome sequence of very high quality. Only 15 gaps are present in the sequence, and half the telomeric regions have been elucidated. Moreover, sequence information from ATCC 1015 was used to improve the genome sequence of CBS 513.88. Chromosome-level comparisons uncovered several genome rearrangements, deletions, a clear case of strain-specific horizontal gene transfer, and identification of 0.8 Mb of novel sequence. Single nucleotide polymorphisms per kilobase (SNPs/kb) between the two strains were found to be exceptionally high (average: 7.8, maximum: 160 SNPs/kb). High variation within the species was confirmed with exo-metabolite profiling and phylogenetics. Detailed lists of alleles were generated, and genotypic differences were observed to accumulate in metabolic pathways essential to acid production and protein synthesis. A transcriptome analysis supported up-regulation of genes associated with biosynthesis of amino acids that are abundant in glucoamylase A, tRNA-synthases, and protein transporters in the protein producing CBS 513.88 strain. Our results and data sets from this integrative systems biology analysis resulted in a snapshot of fungal evolution and will support further optimization of cell factories based on filamentous fungi.

Phytophthora infestans isolates lacking class I ipiO variants are virulent on Rpi-blb1 potato.

A strategy to control the devastating late blight disease is providing potato cultivars with genes that are effective in resistance to a broad spectrum of Phytophthora infestans isolates. Thus far, most late blight resistance (R) genes that were introgressed in potato were quickly defeated. In contrast, the Rpi-blb1 gene originating from Solanum bulbocastanum has performed as an exclusive broad-spectrum R gene for many years. Recently, the RXLR effector family ipiO was identified to contain Avr-blb1. Monitoring the genetic diversity of the ipiO family in a large set of isolates of P. infestans and related species resulted in 16 ipiO variants in three distinct classes. Class I and class II but not class III ipiO variants induce cell death when coinfiltrated with Rpi-blb1 in Nicotiana benthamiana. Class I is highly diverse and is represented in all analyzed P. infestans isolates except two Mexican P. infestans isolates, and these were found virulent on Rpi-blb1 plants. In its C-terminal domain, IPI-O contains a W motif that is essential for triggering Rpi-blb1-mediated cell death and is under positive selection. This study shows that profiling the variation of Avr-blb1 within a P. infestans population is instrumental for predicting the effectiveness of Rpi-blb1-mediated resistance in potato.

The Phytophthora infestans avirulence gene Avr4 encodes an RXLR-dEER effector.

Resistance in potato against the oomycete Phytophthora infestans is conditioned by resistance (R) genes that are introgressed from wild Solanum spp. into cultivated potato. According to the gene-for-gene model, proteins encoded by R genes recognize race-specific effectors resulting in a hypersensitive response (HR). We isolated P. infestans avirulence gene PiAvr4 using a combined approach of genetic mapping, transcriptional profiling, and bacterial artificial chromosome marker landing. PiAvr4 encodes a 287-amino-acid-protein that belongs to a superfamily of effectors sharing the putative host-cell-targeting motif RXLR-dEER. Transformation of P. infestans race 4 strains with PiAvr4 resulted in transformants that were avirulent on R4 potato plants, demonstrating that PiAvr4 is responsible for eliciting R4-mediated resistance. Moreover, expression of PiAvr4 in R4 plants using PVX agroinfection and agroinfiltration showed that PiAvr4 itself is the effector that elicits HR on R4 but not r0 plants. The presence of the RXLR-dEER motif suggested intracellular recognition of PiAvr4. This was confirmed in agroinfiltration assays but not with PVX agroinfection. Because there was always recognition of PiAvr4 retaining the signal peptide, extracellular recognition cannot be excluded. Deletion of the RXLR-dEER domain neither stimulated nor prevented elicitor activity of PiAvr4. Race 4 strains have frame shift mutations in PiAvr4 that result in truncated peptides; hence, PiAvr4 is apparently not crucial for virulence.

Identification of cell wall-associated proteins from Phytophthora ramorum.

The oomycete genus Phytophthora comprises a large group of fungal-like plant pathogens. Two Phytophthora genomes recently have been sequenced; one of them is the genome of Phytophthora ramorum, the causal agent of sudden oak death. During plant infection, extracellular proteins, either soluble secreted proteins or proteins associated with the cell wall, play important roles in the interaction with host plants. Cell walls of P. ramorum contain 1 to 1.5% proteins, the remainder almost exclusively being accounted for by glucan polymers. Here, we present an inventory of cell-wall-associated proteins based on mass spectrometric sequence analysis of tryptic peptides obtained by proteolytic digestion of sodium dodecyl sulfate-treated mycelial cell walls. In total, 17 proteins were identified, all of which are authentic secretory proteins. Functional classification based on homology searches revealed six putative mucins or mucin-like proteins, five putative glycoside hydrolases, two transglutaminases, one annexin-like protein, the elicitin protein RAM5, one protein of unknown function, and one Kazal-type protease inhibitor. We propose that the cell wall proteins thus identified are important for pathogenicity.

Identification of a mitotic recombination hotspot on chromosome III of the asexual fungus Aspergillus niger and its possible correlation with [corrected] elevated basal transcription.

Genetic recombination is an important tool in strain breeding in many organisms. We studied the possibilities of mitotic recombination in strain breeding of the asexual fungus Aspergillus niger. By identifying genes that complemented mapped auxotrophic mutations, the physical map was compared to the genetic map of chromosome III using the genome sequence. In a program to construct a chromosome III-specific marker strain by selecting mitotic crossing-over in diploids, a mitotic recombination hotspot was identified. Analysis of the mitotic recombination hotspot revealed some physical features, elevated basal transcription and a possible correlation with purine stretches.

Amplification generates modular diversity at an avirulence locus in the pathogen Phytophthora.

The destructive late blight pathogen Phytophthora infestans is notorious for its rapid adaptation to circumvent detection mediated by plant resistance (R) genes. We performed comparative genomic hybridization on microarrays (array-CGH) in a near genome-wide survey to identify genome rearrangements related to changes in virulence. Six loci with copy number variation were found, one of which involves an amplification colocalizing with a previously identified locus that confers avirulence in combination with either R gene R3b, R10, or R11. Besides array-CGH, we used three independent approaches to find candidate genes at the Avr3b-Avr10-Avr11 locus: positional cloning, cDNA-AFLP analysis, and Affymetrix array expression profiling. This resulted in one candidate, pi3.4, that encodes a protein of 1956 amino acids with regulatory domains characteristic for transcription factors. Amplification is restricted to the 3' end of the full-length gene but the amplified copies still contain the hallmarks of a regulatory protein. Sequence comparison showed that the amplification may generate modular diversity and assist in the assembly of novel full-length genes via unequal crossing-over. Analyses of P. infestans field isolates revealed that the pi3.4 amplification correlates with avirulence; isolates virulent on R3b, R10, and R11 plants lack the amplified gene cluster. The ancestral state of 3.4 in the Phytophthora lineage is a full-length, single-copy gene. In P. infestans, however, pi3.4 is a dynamic gene that is amplified and has moved to other locations. Modular diversity could be a novel mechanism for pathogens to quickly adapt to changes in the environment.

Selection and characterisation of a xylitol-derepressed Aspergillus niger mutant that is apparently impaired in xylitol transport.

Aspergillus niger is known for its biotechnological applications, such as the use of xylanase enzyme for the degradation of hemicellulose. Depending on culture conditions, several polyols may also be accumulated, such as xylitol during D: -xylose oxidation. Also during industrial fermentation of xylose for the production of fuel ethanol by recombinant yeast, xylitol is a by-product. We studied xylitol metabolism by isolating mutants that have impaired xylitol-mediated repression. Genetic and biochemical characterisation revealed that one of these mutants was affected not only in xylitol-mediated carbon repression, but also had impaired xylitol transport.

Aspergillus vadensis, a new species of the group of black Aspergilli.

A strain from the group of black Aspergilli was analysed in detail to determine the species to which it belongs. A detailed analysis of morphology, RFLP patterns and metabolite profiles was carried out. In addition, a phylogenetic tree was constructed for the black Aspergilli using the ITS and the beta-tubulin sequences of the individual strains. The new species differs by its poor growth on glycerol and galacturonate and its unique extrolite profile consisting of aurasperone B, nigragillin, asperazine and kotanins. RFLP analysis using three genes as probes also resulted in a unique pattern. These data indicate that the strain was closely related but not identical to Aspergillus foetidus, Aspergillus niger and Aspergillus tubingensis. It was therefore designated as a novel species and named Aspergillus vadensis.

Oxalic acid production by Aspergillus niger: an oxalate-non-producing mutant produces citric acid at pH 5 and in the presence of manganese.

The external pH appeared to be the main factor governing oxalic acid production by Aspergillus niger. A glucose-oxidase-negative mutant produced substantial amounts of oxalic acid as long as the pH of the culture was 3 or higher. When pH was decreased below 2, no oxalic acid was formed. The activity of oxaloacetate acetylhydrolase (OAH), the enzyme believed to be responsible for oxalate formation in A. niger, correlated with oxalate production. OAH was purified from A. niger and characterized. OAH cleaves oxaloacetate to oxalate and acetate, but A. niger never accumulated any acetate in the culture broth. Since an A. niger acuA mutant, which lacks acetyl-CoA synthase, did produce some acetate, wild-type A. niger is apparently able to catabolize acetate sufficiently fast to prevent its production. An A. niger mutant, prtF28, previously isolated in a screen for strains deficient in extracellular protease expression, was shown here to be oxalate non-producing. The prtF28 mutant lacked OAH, implying that OAH is the only enzyme involved in oxalate production in A. niger. In a traditional citric acid fermentation low pH and absence of Mn2+ are prerequisites. Remarkably, a strain lacking both glucose oxidase (goxC) and OAH (prtF) produced citric acid from sugar substrates in a regular synthetic medium at pH 5 and under these conditions production was completely insensitive to Mn2+.

Disruption of the Aspergillus niger argB gene: a tool for transformation.

We disrupted the Aspergillus niger gene argB, encoding ornithine transcarbamylase. Full characterisation of the argB deletion was performed by Southern blot analysis, growth tests and by means of mitotic recombination, complementation and transformation. The argB locus was found to be physically removed, thus creating an auxotrophic mutation. The latter can be supplemented by addition of arginine into the culture medium. The argB gene and its disruption do not correlate to the argI13 (formerly argB13) allele described. The delta argB is on chromosome I whereas argI13 is on V. In addition, the argI13 mutation can only be complemented by the A. nidulans argB gene, whereas the new argB deletion can be complemented by both the A. niger and A. nidulans argB genes. The delta argB strain has been used to generate several strains in a breeding programme and to study the expression of important genes, such as areA and kexB.

EglC, a new endoglucanase from Aspergillus niger with major activity towards xyloglucan.

A novel gene, eglC, encoding an endoglucanase, was cloned from Aspergillus niger. Transcription of eglC is regulated by XlnR, a transcriptional activator that controls the degradation of polysaccharides in plant cell walls. EglC is an 858-amino-acid protein and contains a conserved C-terminal cellulose-binding domain. EglC can be classified in glycoside hydrolase family 74. No homology to any of the endoglucanases from Trichoderma reesei was found. In the plant cell wall xyloglucan is closely linked to cellulose fibrils. We hypothesize that the EglC cellulose-binding domain anchors the enzyme to the cellulose chains while it is cleaving the xyloglucan backbone. By this action it may contribute to the degradation of the plant cell wall structure together with other enzymes, including hemicellulases and cellulases. EglC is most active towards xyloglucan and therefore is functionally different from the other two endoglucanases from A. niger, EglA and EglB, which exhibit the greatest activity towards beta-glucan. Although the mode of action of EglC is not known, this enzyme represents a new enzyme function involved in plant cell wall polysaccharide degradation by A. niger.

Isolation of a fluffy mutant of Aspergillus niger from chemostat culture and its potential use as a morphologically stable host for protein production.

Chemostat cultivation of Aspergillus niger and other filamentous fungi is often hindered by the spontaneous appearance of morphologic mutants. Using the Variomixing bioreactor and applying different chemostat conditions we tried to optimize morphologic stability in both ammonium- and glucose-limited cultures. In most cultivations mutants with fluffy (aconidial) morphology became dominant. From an ammonium-limited culture, a fluffy mutant was isolated and genetically characterized using the parasexual cycle. The mutant contained a single morphological mutation, causing an increased colony radial growth rate. The fluffy mutant was subjected to transformation and finally conidiospores from a forced heterokaryon were shown to be a proper inoculum for fluffy strain cultivation.

Isolation and characterization of two specific regulatory Aspergillus niger mutants shows antagonistic regulation of arabinan and xylan metabolism.

This paper describes two Aspergillus niger mutants (araA and araB) specifically disturbed in the regulation of the arabinanase system in response to the presence of L-arabinose. Expression of the three known L-arabinose-induced arabinanolytic genes, abfA, abfB and abnA, was substantially decreased or absent in the araA and araB strains compared to the wild-type when incubated in the presence of L-arabinose or L-arabitol. In addition, the intracellular activities of L-arabitol dehydrogenase and L-arabinose reductase, involved in L-arabinose catabolism, were decreased in the araA and araB strains. Finally, the data show that the gene encoding D-xylulose kinase, xkiA, is also under control of the arabinanolytic regulatory system. L-Arabitol, most likely the true inducer of the arabinanolytic and L-arabinose catabolic genes, accumulated to a high intracellular concentration in the araA and araB mutants. This indicates that the decrease of expression of the arabinanolytic genes was not due to lack of inducer accumulation. Therefore, it is proposed that the araA and araB mutations are localized in positive-acting components of the regulatory system involved in the expression of the arabinanase-encoding genes and the genes encoding the L-arabinose catabolic pathway.

Isolation of Aspergillus niger creA mutants and effects of the mutations on expression of arabinases and L-arabinose catabolic enzymes.

Aspergillus niger mutants relieved of carbon repression were isolated from an areA parental strain by selection of colonies that exhibited improved growth on a combination of 4-aminobutanoic acid (GABA) and D-glucose. In addition to derepression of the utilization of GABA as a nitrogen source in the presence of D-glucose, three of the four mutants also showed derepression of L-alanine and L-proline utilization. Transformation of the mutants with the A. niger creA gene, encoding the repressor protein CREA, re-established the areA phenotype on GABA/D-glucose, identifying the mutations as creAd. The creA gene mapped on chromosome IV by linkage analysis and contour-clamped homogeneous electric field hybridization. The creA mutants obtained were used to study the involvement of CREA in repression by D-glucose of arabinases and L-arabinose catabolism in A. niger. In wild-type A. niger, alpha-L-arabinofuranosidase A, alpha-L-arabinofuranosidase B, endo-arabinase, L-arabinose reductase and L-arabitol dehydrogenase were induced on L-arabinose, but addition of D-glucose prevented this induction. Repression was relieved to varying degrees in the creA mutants, showing that biosynthesis of arabinases and L-arabinose catabolic enzymes is under control of CREA.

A new black Aspergillus species, A. vadensis, is a promising host for homologous and heterologous protein production.

A new species of the group of black aspergilli, Aspergillus vadensis, was analyzed for its potential as a host for homologous and heterologous protein production. Unlike the other black aspergilli, this strain does not acidify the culture medium when nitrate is the nitrogen source and only produces very low levels of extracellular proteases, mainly serine metalloproteases. The stability of A. tubingensis feruloyl esterase A (FaeA) was compared upon production in wild-type A. vadensis, A. tubingensis, and an A. niger strain in which the three main protease-encoding genes were disrupted. The production of FaeA in A. vadensis resulted in larger amounts of intact protein than production in A. tubingensis and was similar to production in an A. niger protease disruptant, confirming in vivo the low proteolytic activity of A. vadensis. The protoplast formation and transformation efficiencies of A. vadensis were much higher than those of A. niger. These characteristics make A. vadensis a very promising candidate for homologous, and possibly heterologous, protein production.

The abfB gene encoding the major alpha-L-arabinofuranosidase of Aspergillus nidulans: nucleotide sequence, regulation and construction of a disrupted strain.

Using a DNA fragment containing the Aspergillus niger abfB gene as a probe, the homologous Aspergillus nidulans gene, designated abfB, has been cloned from a genomic library containing size-selected HindIII fragments. The nucleotide sequence of the A. nidulans abfB gene shows strong homology with the A. niger abfB, Trichoderma reesei abf-1 and Trichoderma koningii alpha-L-arabinofuranosidase/beta-xylosidase genes. Regulation of abfB expression has been investigated in cultures induced with L-arabitol. The accumulation of abfB mRNA, total alpha-L-arabinofuranosidase activity and AbfB protein levels have been determined in a wild-type A. nidulans strain as well as in different mutant strains. These strains are affected either in their response to ambient pH (paIA1 and pacC(c)14 mutants), carbon catabolite repression (creA(d)4 mutant), the ability to utilize L-arabitol as a carbon source (araA1 mutant) or a combination of both latter genotypes (araA1 creA(d)4). The results obtained indicate that the expression of the A. nidulans abfB gene was higher at acidic pHs and was superinduced in this double mutant. Furthermore, disruption of the abfB gene demonstrated that in A. nidulans AbfB is the major p-nitrophenyl alpha-L-arabinofuranoside-hydrolysing activity but at least one minor activity is expressed, which is involved in the release of L-arabinose from polysaccharides.

Cyclic AMP-dependent protein kinase is involved in morphogenesis of Aspergillus niger.

The cAMP signal transduction pathway controls many processes in fungi. The pkaR gene, encoding the regulatory subunit (PKA-R) of cAMP-dependent protein kinase (PKA), was cloned from the industrially important filamentous fungus Aspergillus niger. To investigate the involvement of PKA in morphology of A. niger, a set of transformants which overexpressed pkaR or pkaC (encoding the catalytic subunit of PKA) either individually or simultaneously was prepared as well as mutants in which pkaR and/or pkaC were disrupted. Strains overexpressing pkaR or both pkaC and pkaR could not be distinguished from the wild-type, suggesting that regulation of PKA activity is normal in these strains. Absence of PKA activity resulted in a two- to threefold reduction in colony diameter on plates. The most severe phenotype was observed in the absence of PKA-R, i.e., very small colonies on plates, absence of sporulation and complete loss of growth polarity during submerged growth. Suppressor mutations easily developed in the DeltapkaR mutant and one of these mutants appeared to lack PKA-C activity. These data suggest that cAMP-dependent protein phosphorylation in A. niger regulates growth polarity and formation of conidiospores.

Onset of carbon catabolite repression in Aspergillus nidulans. Parallel involvement of hexokinase and glucokinase in sugar signaling.

The role of hexose phosphorylating enzymes in the signaling of carbon catabolite repression was investigated in the filamentous fungus Aspergillus nidulans. A d-fructose non-utilizing, hexokinase-deficient (hxkA1, formerly designated frA1) strain was utilized to obtain new mutants lacking either glucokinase (glkA4) or both hexose kinases (hxkA1/glkA4). d-Glucose and d-fructose phosphorylation is completely abolished in the double mutant, which consequently cannot grow on either sugar. The glucokinase single mutant exhibits no nutritional deficiencies. Three repressible diagnostic systems, ethanol utilization (alcA and alcR genes), xylan degradation (xlnA), and acetate catabolism (facA), were analyzed in these hexose kinase mutants at the transcript level. Transcriptional repression by d-glucose is fully retained in the two single kinase mutants, whereas the hexokinase mutant is partially derepressed for d-fructose. Thus, hexokinase A and glucokinase A compensate each other for carbon catabolite repression by d-glucose in the single mutants. In contrast, both d-glucose and d-fructose repression are severely impaired for all three diagnostic systems in the double mutant. Unlike the situation in Saccharomyces cerevisiae, the hexose phosphorylating enzymes play parallel roles in glucose repression in A. nidulans.

Glycerol dehydrogenase, encoded by gldB is essential for osmotolerance in Aspergillus nidulans.

We have characterized the Aspergillus nidulans gldB gene encoding a NADP+-dependent glycerol dehydrogenase. A basal expression level was observed for gldB, which increased significantly under conditions of hyper-osmotic shock (1 M NaCl). Growth of strains in which gldB was disrupted was severely reduced on plates containing 1% glucose and 1 M NaCl, but these strains were able to grow on plates containing 1 M NaCl and 1% glycerol, arabitol, mannitol or erythritol. Uptake of these polyols compensated for the inability of the gldB disruptants to produce glycerol. Presence of 1% glucose in these plates prevented growth restoration by all the polyols tested with the exemption of glycerol, indicating that uptake of mannitol, arabitol and erythritol is subject to glucose repression, whereas uptake of glycerol is significantly less or not repressed. No intracellular glycerol dehydrogenase activity could be detected in the gldB disruption strains. Intracellular glycerol levels in these strains were strongly decreased compared to wild type, whereas intracellular mannitol, erythritol and arabitol levels were increased. Conidia of the gldB disruption strain did not accumulate glycerol upon germination in glucose media with or without 1 M NaCl and germ tube emergence was significantly delayed in this strain in the presence of 1 M NaCl in comparison to the wild type. These data indicate that gldB is essential for osmotolerance in A. nidulans and that the pathways for glycerol biosynthesis under osmotic stress differ between yeast and filamentous fungi.

Mannitol is required for stress tolerance in Aspergillus niger conidiospores.

D-Mannitol is the predominant carbon compound in conidiospores of the filamentous fungus Aspergillus niger and makes up 10 to 15% of the dry weight. A number of physiological functions have been ascribed to mannitol, including serving as a reserve carbon source, as an antioxidant, and to store reducing power. In this study, we cloned and characterized the A. niger mpdA gene, which encodes mannitol 1-phosphate dehydrogenase (MPD), the first enzyme in the mannitol biosynthesis pathway. The mpdA promoter contains putative binding sites for the development-specific transcription factors BRLA and ABAA. Furthermore, increased expression of mpdA in sporulating mycelium suggests that mannitol biosynthesis is, to a certain extent, developmentally regulated in A. niger. Inactivation of mpdA abolished mannitol biosynthesis in growing mycelium and reduced the mannitol level in conidiospores to 30% that in the wild type, indicating that MPD and mannitol 1-phosphate phosphatase form the major metabolic pathway for mannitol biosynthesis in A. niger. The viability of spores after prolonged storage and germination kinetics were normal in an mpdA null mutant, indicating that mannitol does not play an essential role as a reserve carbon source in A. niger conidia. However, conidiospores of a DeltampdA strain were extremely sensitive to a variety of stress conditions, including high temperature, oxidative stress and, to a lesser extent, freezing and lyophilization. Since mannitol supplied in the medium during sporulation repaired this deficiency, mannitol appears to be essential for the protection of A. niger spores against cell damage under these stress conditions.