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The black aspergilli (Aspergillus niger complex) and their role in human, animal, and plant diseases.

SUMMARYThe Aspergillus niger complex, also known as the black aspergilli or section Nigri, comprises a diverse group of filamentous fungi with wide-ranging ecological, industrial, and pathogenic significance. While traditionally associated with food spoilage and industrial fermentation, black aspergilli have emerged as opportunistic pathogens affecting humans, animals, and plants. This review provides a comprehensive synthesis of the taxonomy, ecology, pathogenicity, and antifungal resistance of the A. niger complex. Advances in phylogenetics and whole-genome sequencing have clarified the taxonomy of section Nigri, now comprising six core species in series Nigri. Clinically, A. niger complex is implicated in various conditions, including otomycosis, keratitis, cutaneous infections, onychomycosis, chronic pulmonary aspergillosis, and, less commonly, invasive aspergillosis. In animals, black aspergilli have been isolated from respiratory, cutaneous, and systemic infections, particularly in immunocompromised or stressed hosts. Plant pathogenicity is significant, with A. niger complex contributing to pre- and post-harvest spoilage and producing mycotoxins such as ochratoxin A and oxalic acid. The common finding of elevated minimum inhibitory concentrations (MICs) to triazoles, particularly in both environmental and clinical isolates, raises concern, with underlying mechanisms differing from those characterized in A. fumigatus. Reduced susceptibility is potentially driven by efflux pumps and environmental exposure to azole fungicides. Due to commonly higher MICs, antifungal therapy with itraconazole and isavuconazole may have reduced efficacy, and alternatives such as voriconazole or posaconazole should be considered, guided by susceptibility testing where available. This review emphasizes the need for a One Health approach to managing black aspergilli, integrating surveillance, diagnostics, and targeted interventions across human, veterinary, and agricultural sectors.

Humans↗

Mitochondrial DNA restriction fragment length polymorphisms in field isolates of the Aspergillus niger aggregate.

The mitochondrial DNAs (mtDNAs) and the ribosomal repeat unit (ribosomal DNA, rDNA) of black Aspergillus isolates collected in various parts of the world were examined. Wide-ranging mtDNA variation was observed in natural populations of the Aspergillus niger aggregate. Most isolates were classifiable as A. niger or Aspergillus tubingensis according to their rDNA and mtDNA patterns. The mtDNA variation was distributed unevenly in the populations studied. The mtDNAs of most of the isolates collected in Australia were of the A. tubingensis type, with an unexpectedly high degree of variation, while the rDNA of these isolates exhibited the same A. tubingensis pattern as that of isolates from other locations. Some other local populations displayed very little polymorphism in their mtDNA and rDNA. Hybridization experiments in which cloned A. niger and Aspergillus nidulans mtDNA fragments were used revealed that the two main mtDNA groups corresponding to A. niger and A. tubingensis are more distantly related than concluded earlier. Six of the 13 Brazilian isolates examined exhibited mtDNA and rDNA types different from those of all the other strains and could not be classified into the above species. Classical taxonomic examination of these strains is in progress.

Aspergillus↗

Reverse reaction of Aspergillus niger APC-9319 alpha-galactosidase in a supersaturated substrate solution: production of alpha-linked galactooligosaccharide (alpha-GOS).

The alpha-galactosidase that effectively catalyzes a reverse reaction of galactose, Aspergillus niger APC-9319 alpha-galactosidase, was screened from industrial enzyme preparations for food processing containing alpha-galactosidase activity. Reverse reaction of A. niger APC-9319 alpha-galactosidase was performed using a supersaturated solution (90% galactose [w/v]). A. niger APC-9319 alpha-galactosidase was not inhibited even in high substrate concentration, and effectively catalyzed the reverse reaction. The yield of the reaction product, alpha-linked galactooligosaccharide (alpha-GOS), increased greatly as the initial concentration of galactose increased to 90% (w/v), and was more than 50%. Furthermore, the half life of enzyme activity was about three times as long as that using 60% galactose (w/v). alpha-GOS (1.4 g) was prepared from galactose (3.0 g) by reverse reaction of A. niger APC-9319 alpha-galactosidase. The alpha-GOS contained 58% alpha-galactobiose (alpha-Gal2), 28% alpha-galactotriose, and 14% oligosaccharides larger than alpha-galactotriose. The main component of positional isomers in alpha-Gal2 was alpha-1,6Gal2.

Aspergillus niger↗

Purification and partial characterization of an extracellular ribonuclease from a mutant of Aspergillus niger.

A new extracellular ribonuclease (RNase) from a mutant of Aspergillus niger, named A. niger SA-13-20 RNase, was purified to homogeneity by (NH4)2SO4 fractionation (50-85%), DEAE-cellulose anion-exchange chromatography, ultrafiltration and Sephacryl HR-200 chromatography. The enzyme was purified up to 54.4-fold with a final yield of 24.5%. There were differences in the molecular weight, pI value and some physico-chemical properties between A. niger SA-13-20 RNase and that from the parent strain. The enzyme is monomeric and its molecular weight and isoelectric point were 40.1 kDa and 5.3, respectively. The N-terminal amino acid sequence of A. niger SA-13-20 RNase was TIDTYSSDSP. The optimum pH, temperature and buffer concentration for the enzymatic reaction were 3.5, 65 degrees C, and 0.175 M, respectively. Metal ions, such as K+, NH4+, Mg2+, and Ca2+ at the concentration of 1.0 mM had a slight activation effect on the enzyme activity and (NH4)2SO4 activated the enzyme significantly. The enzyme was stable at pH lower than 8.5 and was easy to inactivate in strong alkali solution.

Aspergillus niger↗

Semi-solid-state fermentation of Eicchornia crassipes biomass as lignocellulosic biopolymer for cellulase and 3-glucosidase production by cocultivation of Aspergillus niger RK3 and Trichoderma reesei MTCC164.

An aquatic weed biomass, Eicchornia crassipes, present in abundance and leading to a threatening level of water pollution was used as substrate for cellulase and beta-glucosidase production using wild-type strain Aspergillus niger RK3 that was isolated from decomposing substrate. Alkali treatment of the biomass (10%) resulted in a 60-66% increase in endoglucanase, exoglucanase, and beta-glucosidase production by the A. niger RK3 strain in semi-solid-state fermentation. Similarly, the alkali-treated biomass led to a 45-54% increase in endo- and exoglucanase and a higher (98%) increase in beta-glucosidase production by Trichoderma reesei MTCC164 under similar conditions. However, the cocultivation of A. niger RK3 and T. reesei MTCC164 at a ratio of 3:1 showed a 20-24% increase in endo- and exoglucanase activities and about a 13% increase in the beta-glucosidase activity over the maximum enzymatic activities observed under single culture conditions. Multistep physical (ultraviolet) and chemical (N-methyl-N'-nitrosoguanidine, sodium azide, colchicine) mutagenesis of the A. niger RK3 strain resulted in a highly cellulolytic mutant, UNSC-442, having an increase of 136, 138, and 96% in endoglucanase, exoglucanase, and beta-glucosidase, activity, respectively. The cocultivation of mutant UNSC-442 along with T. reesei MTCC164 (at a ratio of 3:1) showed a further 10-11% increase in endo- and exoglucanase activities and a 29% increase in beta-glucosidase activity in semi-solid-state fermentation.

Aspergillus niger↗

Genetic analysis in the asexual fungus Aspergillus niger.

The genetics of A. niger has been developed since 1980. An overview is presented of the advances in developing methods and collecting data. Important tools have been a) the application of essentially different methods to isolate mutants, b) the adaptation to A. niger ofA. nidulans methodology for analysis of the parasexual cycle, c) the choice of marker genes, and in some cases the artificial introduction of such genes, to select homozygous segregants arising from mitotic recombination. With the use of parasexual recombination, a genetic linkage map of A. niger has been established. In total, 110 nuclear and 1 cytoplasmic (mitochondrial) markers are available. The application of A. niger genetics in applied research is illustrated by examples.

Aspergillus niger↗

[Cloning and sequences comparison of promoters from Aspergillus niger].

An Aspergillus niger (A. niger) genomic library was constructed in a promoter-trap vector, which contains a Hygromycin B (Hy) phosphotransferase-encoding gene (hph) and screened for DNA fragments with promoter activity by applying the sib selection procedure. A functional promoter PX27 was identified. Both DNA strands of this fragment were sequenced and showed no significant homology to the sequence already in the database. Comparison of the sequences of all known promoters from A. niger revealed a consensus CTTCTC, as a novel motif of the A. niger promoters.

Aspergillus niger↗

[No solution for neonatal mortality in sub-saharan Africa? Evaluation and perspectives in the urban environment of Niamey, Niger].

Niger has one of the highest mortality rates of infants (222/1000) and children under five years old (318/1000), with 15% of them suffering from malnutrition. Yet, neonatal mortality was not considered as the top priority of public health in Niger, where 85 to 90% of the deliveries succeed without any medical care and 70% of the population live more than 10 kilometers from the nearest medical center. Also, in the African countries which have adopted expensive neonatal care centers following the occidental model, the lethality rate is high and maintenance is difficult. Thus, alternative strategies should be considered to reduce the neonatal mortality, according to the local possibilities. This was carried out in Niamey, the capital of Niger. A retrospective study of the activities in the pediatric and obstetric wards was carried out from 1985 to 1992. This was associated with a descriptive prospective survey of a sample of 149 pregnant women followed from the first prenatal consultation to the end of the neonatal period. Results showed that possibilities exist in Niamey to reduce neonatal mortality. The concentration of medical personnel was high compared to the rest of the country, and the health infrastructure was diversified. However, the knowledge of neonatal care was lacking. Use of prenatal care was high and deliveries at home without medical assistance concerned only 14.3% of the total births. Mortality observed in the obstetrical ward (6.7/1000) corresponded to less than a quarter of the estimated neonatal mortality (28.6/1000). Neonatal mortality in the pediatric ward was high (43.8%), predominantly on the first day of admittance (45% of the deaths), especially for the low birth weights (under 2,500 g) (62.4%). These figures underline the necessity to improve the care of the newborns and to link prenatal prevention, obstetrical care and pediatrics. The prospective survey showed that although the ratio of prenatal visits per woman was high (3.8), the quality of the care was inadequate. Correct newborn care was rare and no examination could detect or prevent complications during the short stay of less than 24 hours in the obstetrical ward. To lower the neonatal mortality, service could be improved concerning the material conditions of prenatal consultations, reorientation of prenatal consultations towards detection, correct treatment of the risk factors of neonatal mortality, obstetrical screening and care, and training of the midwives. The adoption of inexpensive measures was suggested, including the training of pediatric nurses in each maternity ward, screening and treatment of newborns at risk in small units integrated within the obstetrical ward, and the requirement of a consultation before the traditional feast of giving names, which occurs on the seventh day of life in Niger. These measures were considered as priorities before considering construction of expensive neonatal centers and assume the participation of the public health personnel and policy makers. Some of these suggestions are now being implemented.

Health Priorities↗

Aspergillus niger I-1472 and Pycnoporus cinnabarinus MUCL39533, selected for the biotransformation of ferulic acid to vanillin, are also able to produce cell wall polysaccharide-degrading enzymes and feruloyl esterases.

The filamentous fungal strains Aspergillus niger I-1472 and Pycnoporus cinnabarinus MUCL39533, previously selected for the bioconversion of ferulic acid to vanillic acid and vanillin respectively, were grown on sugar beet pulp. A large spectrum of polysaccharide-degrading enzymes was produced by A. niger and very few levels of feruloyl esterases were found. In contrast, P. cinnabarinus culture filtrate contained low amount of polysaccharide-degrading enzymes and no feruloyl esterases. In order to enhance feruloyl esterases in A. niger cultures, feruloylated oligosaccharide-rich fractions were prepared from sugar beet pulp or cereal bran and used as carbon sources. Number of polysaccharide-degrading enzymes were induced. Feruloyl esterases were much higher in maize bran-based medium than in sugar beet pulp-based medium, demonstrating the ability of carbon sources originating from maize to induce the synthesis of feruloyl esterases. Thus, A. niger I-1472 could be interesting to release ferulic acid from sugar beet pulp or maize bran.

Journal Article↗

Evidence for a non-phosphorylated route of galactose breakdown in cell-free extracts of Aspergillus niger.

Aspergillus niger could utilize D-galactose as sole source of carbon. Cell-free extracts of D-galactose-grown mycelia were able to catalyze the oxidation of D-galactose to D-galactonic acid-gamma-lactone (GalA-gamma-lact) in the presence of NAD, followed by the appearance of 2-keto-3-deoxy-D-galactonate (KDGal), pyruvate and glyceraldehyde. From 10 &mgr;moles only 6.6 &mgr;moles of GalA-gamma-lact were disappeared after 60 min of reaction indicating the presence of GalA-gamma-lactonase. Identification of GalA-gamma-lact was achieved by ascending paper chromatography. KDGal, pyruvate and glyceraldehyde were also chromatographically identified in the reaction mixture containing D-galactonate which suggests that D-galactonate is degraded into pyruvate and glyceraldehyde via the intermediate formation of KDGal. Such reactions are supposed to be catalyzed by an inducible D-galactonate dehydratase and a constitutive KDGal aldolase. The amount of KDGal, pyruvate and glyceraldehyde were found to be almost equivalent and the equilibrium of the reaction being toward the formation of KDGal. The apparent equilibrium constant (K(eq)) was calculated and found to be 0.5 x 10(-3) M. Results also proved the reversibility of the reaction catalyzed by KDGal aldolase of A. niger. In the light of the findings obtained from the degradation of D-galactose by cell-free extracts of A. niger grown on D-galactose and D-galactonate a nonphosphorolytic pathway was suggested to be operative for the degradation of D-galactose in extracts of A. niger.

Journal Article↗

Structures and expression patterns of two tropinone reductase genes from Hyoscyamus niger.

In the biosynthesis of tropane alkaloids, two tropinone reductases (TRs) catalyze reduction of tropinone to different stereoisomers, tropine and pseudotropine. Two TRs from Hyoscyamus niger have 64% of identical amino acids and hence a common evolutionary origin. In this study, genomic clones of TRs were isolated from H. niger. Their sequence comparison showed that although they have the same exon/intron organization, sequence similarity was restricted to the coding regions. In H. niger transgenic hairy roots, both TR promoters activated transcription of the reporter genes in endodermis and pericycle of the roots. A quantitative reporter assay and a nuclear run-on experiment indicated that the two genes are transcribed at a similar rate. The results indicate that although different activity levels have been observed for the TR enzymes in the H. niger root, the TR genes per se conserve similar tissue-specific expression pattern and transcriptional rate.

Alcohol Oxidoreductases↗

Diagnostic value of conidia associated with pulmonary oxalosis: evidence of an Aspergillus niger infection.

Bronchoalveolar lavage (BAL) material is commonly received in cytopathology for the exclusion of microorganisms. When crystalline material suggestive of calcium oxalate is present in the specimen, a search for fungal elements should be undertaken. Aspergillus niger is the hyaline mold associated with the presence of oxalate crystals. Commonly fragments of hyphae and occasionally entire conidiophores may be present in BAL specimens from patients with aspergillosis. We report a case of a patient with saprophytic colonization of a bullous/cavitary lesion. The BAL consisted of abundant acute inflammation, crystalline material suggestive of oxalate, and darkly pigmented conidia. Although an extensive search was undertaken, hyphal fragments could not be found. The suspicion of an A. niger infection was confirmed by culture. We believe that even in the absence of hyphal fragments, darkly pigmented, occasionally rough-walled conidia are sufficient evidence to be highly suspicious of an A. niger infection in patients with pulmonary oxalosis.

Aspergillosis↗

Metabolism of citric acid production by Aspergillus niger: model definition, steady-state analysis and constrained optimization of citric acid production rate.

In an attempt to provide a rational basis for the optimization of citric acid production by A. niger, we developed a mathematical model of the metabolism of this filamentous fungus when in conditions of citric acid accumulation. The present model is based in a previous one, but extended with the inclusion of new metabolic processes and updated with currently available kinetic data. Among the different alternatives to represent the system behavior we have chosen the S-system representation within power-law formalism. This type of representation allows us to verify not only the ability of the model to exhibit a stable steady state of the integrated system but also the robustness and quality of the representation. The model analysis is shown to be self-consistent, with a stable steady state, and in good agreement with experimental evidence. Moreover, the model representation is sufficiently robust, as indicated by sensitivity and steady-state and dynamic analyses. From the steady-state results we concluded that the range of accuracy of the S-system representation is wide enough to model realistic deviations from the nominal steady state. The dynamic analysis indicated a reasonable response time, which provided further indication that the model is adequate. The extensive assessment of the reliability and quality of the model put us in a position to address questions of optimization of the system with respect to increased citrate production. We carried out the constrained optimization of A. niger metabolism with the goal of predicting an enzyme activity profile yielding the maximum rate of citrate production, while, at the same time, keeping all enzyme activities within predetermined, physiologically acceptable ranges. The optimization is based on a method described and tested elsewhere that utilizes the fact that the S-system representation of a metabolic system becomes linear at steady state, which allows application of linear programming techniques. Our results show that: (i) while the present profile of enzyme activities in A. niger at idiophase steady state yields high rates of citric acid production, it still leaves room for changes and suggests possible optimization of the activity profile to over five times the basal rate synthesis; (ii) when the total enzyme concentration is allowed to double its basal value, the citric acid production rate can be increased by more than 12-fold, and even larger values can be attained if the total enzyme concentration is allowed to increase even more (up to 50-fold when the total enzyme concentration may rise up to 10-fold the basal value); and (iii) the systematic search of the best combination of subsets of enzymes shows that, under all conditions assayed, a minimum of 13 enzymes need be modified if significant increases in citric acid are to be obtained. This implies that improvements by single enzyme modulation are unlikely, which is in agreement with the findings of some investigators in this and other fields.

Aspergillus niger↗

Optimization of catalase biosynthesis in submerged cultures of Aspergillus niger mutant.

The effect of some medium components, viscous substances and metabolic inhibitors, on catalase production by mutant Aspergillus niger has been studied in shake culture. Altering the composition of the basal medium, particularly substituting NaNO3 for KNO3, and peptone for yeast extract brought an increase in extra- and intracellular catalase activity by 1.5- and 3-fold, respectively. The addition of 2.0-6.0 mg sodium alginate or pectin/ml as viscous additive to the medium, containing glucose as carbon source, increased the medium viscosity and catalase production in shake culture by about 2.8- to 3.0-fold. The highest yield of extracellular catalase activity of A. niger was obtained in the presence of sodium orthovanadate and Triton X-100, which improved the activity of this enzyme by about 1.5-2.2-fold. A significant increase in intracellular catalase activity was observed in the presence of hematin, Tween 80 and sodium orthovanadate (1.7-, 1.6- and 1.4-fold respectively). The time course of growth and enzyme production by A. niger in the optimized medium is also reported.

Alginates↗

Impact of biological factors on binding media identification in art objects: identification of animal glue in the presence of Aspergillus niger.

The materials and especially organic materials used for creation of art objects can be utilized by various microorganisms for their growth and facilitate the microbial colonization of the object. An understanding of the chemical alterations in artefacts caused by the presence of microorganisms can be crucial for correct identification of the materials initially used for the artefact creation--nowadays an important step in restoration and/or art-historical investigation of the art object. The present article describes a model experiment in which we investigated the possible chemical alterations in animal glue films used as substrate for growth of the fungus Aspergillus niger. The sterilized animal glue solution was poured into Petri dishes, inoculated with Aspergillus niger, and subsequently incubated at 15 degrees C for 0, 7, 9, 14, and 28 days. After interruption of incubation, the content of the Petri dish was analyzed for amino acid composition by the GC-MS based method. It was found that the growth of Aspergillus niger on animal glue films did not cause significant changes in the amino acid composition of the film and had no impact on animal glue identification.

Adhesives↗

Purification and characterization of benzoate-para-hydroxylase, a cytochrome P450 (CYP53A1), from Aspergillus niger.

Benzoate-para-hydroxylase (CYP51A or BpH) and NADPH:cytochrome P450 reductase from the filamentous fungus Aspergillus niger were purified to apparent homogeneity, using an overproducing A. niger strain. This is the first membrane-bound fungal cytochrome P450 to be isolated and characterized. Combining BpH with NADPH:cytochrome P450 oxidoreductase in the presence of the phospholipid dilauryl phosphatidylcholine restored the BpH activity, although to only a minor extent. Spectral analysis of BpH showed characteristic spectra for a cytochrome P450. Substrate binding studies with purified BpH as a function of temperature and as a function of pH were performed. Temperature-dependent studies, at pH 8.0, showed that the simplified spin equilibrium model originally proposed for camphor binding to cytochrome P450cam (M. T. Fisher and S. G. Sligar, 1987, Biochemistry 26, 4797-4803) also applies to the benzoate-BpH system. Two equilibrium constants were determined, K(1) for substrate binding without a spin change and K(2) for the spin change of the benzoate-BpH complex. pH-dependent binding studies showed that both K(1) and K(2) increase with pH, indicative of a higher affinity. As K(1) decreases more strongly with pH than K(2), we suggest that benzoate first binds to a binding site on the outside of the protein in a pH-dependent way, followed by transfer to the inside of the protein causing a spin change at the heme iron. The strong pH dependence of K(1) could be the result of the need to break salt bridges at the binding site on the outside of the protein. pH-dependent kinetic studies with microsomes showed that the apparent K(M) values followed the trend observed for benzoate binding to purified BpH, while k(cat) values were virtually constant between pH 6.6 and 8.0 and decreased above pH 8, probably due to loss of productive interaction between BpH and NADPH:cytochrome P450 oxidoreductase. Research into the substrate specificity of BpH showed that BpH can only use benzoic acid and some of its derivatives. Monosubstitution on the phenyl ring is allowed but only at certain positions with specific, not too large groups. Substitution always leads to a lower affinity of the substrate. With one exception, all substrates were converted to their 4-hydroxy derivative. The exception, 3-methoxybenzoate, was demethylated to yield 3-hydroxybenzoate only. The restricted number of substrates and the specificity in catalysis suggest that BpH is not a general-purpose hydroxylase but that its role is confined to benzoate hydroxylation in the beta-ketoadipate pathway of A. niger.

Aspergillus niger↗

Organic acid production by Aspergillus niger in recycling culture analyzed by capillary electrophoresis.

Wild-type Aspergillus niger N402 and glucoamylase++ overproducing transformant A. niger N402[pAB6-10]B1 have grown in maltodextrin- and xylose-limited recycling culture at pH 4.5 on mineral medium. The only products formed were organic acids and proteins, among which glucoamylase. The production of organic acids by the fungus has been analyzed qualitatively and quantitatively using capillary electrophoresis. The only organic acids produced in these cultures were substantial amounts of citric acid. This is the first demonstration of abundant oxalic acid production and a very low citric acid production by submerged cultures of A. niger. In the maltodextrin-limited culture the oxalic acid production rate increased during the first 80 h of cultivation and decreased after that time. In xylose-limited recycling culture the oxalic acid production rate always increased in time and highest values were found in the last samples taken from the culture after about 140 h of cultivation. Oxalic acid production rates were highest by the wild-type strain grown on xylose as carbon source, i.e., when the lowest glucoamylase production rates were observed. A clear negative correlation was found between the oxalic acid production rate and the respiration quotient (RQ). An increase in the oxygen consumption rate, due to the production of strongly oxidized oxalic acid, caused the RQ to be lowest at those stages of recycling cultivation when highest oxalic acid production rates were observed.

Aspergillus niger↗

Identification and cloning of a second phytase gene (phyB) from Aspergillus niger (ficuum).

An Aspergillus niger (ficuum) genomic DNA lambda EMBL3 library was probed with a 354-bp DNA fragment obtained by polymerase chain reaction of A. niger DNA with oligonucleotides based on partial amino acid sequence of a pH 2.5 optimum acid phosphatase. A clone containing a 1605 bp segment (phyB) encoding the 479 amino acid enzyme was isolated and found to contain four exons. Global alignment revealed 23.5% homology to Aspergillus niger phytase (PhyA); four regions of extensive homology were identified. Some of these regions may contain catalytic sites for phosphatase function.

6-Phytase↗