Mating-type distribution within a phylogenetic tree, heterokaryon compatibility, and subsequent diploid formation were studied in 24 Aspergillus niger sensu stricto strains. The genomes of the 24... Show moreMating-type distribution within a phylogenetic tree, heterokaryon compatibility, and subsequent diploid formation were studied in 24 Aspergillus niger sensu stricto strains. The genomes of the 24 strains were sequenced and analyzed revealing an average of 6.1 ± 2.0 variants/kb between Aspergillus niger sensu stricto strains. The genome sequences were used together with available genome data to generate a phylogenetic tree revealing 3 distinct clades within Aspergillus niger sensu stricto. The phylogenetic tree revealed that both MAT1-1 and MAT1-2 mating types were present in each of the 3 clades. The phylogenetic differences were used to select for strains to analyze heterokaryon compatibility. Conidial color markers (fwnA and brnA) and auxotrophic markers (pyrG and nicB) were introduced via CRISPR/Cas9-based genome editing in a selection of strains. Twenty-three parasexual crosses using 11 different strains were performed. Only a single parasexual cross between genetically highly similar strains resulted in a successful formation of heterokaryotic mycelium and subsequent diploid formation, indicating widespread heterokaryon incompatibility as well as multiple active heterokaryon incompatibility systems between Aspergillus niger sensu stricto strains. The 2 vegetatively compatible strains were of 2 different mating types and a stable diploid was isolated from this heterokaryon. Sclerotium formation was induced on agar media containing Triton X-100; however, the sclerotia remained sterile and no ascospores were observed. Nevertheless, this is the first report of a diploid Aspergillus niger sensu stricto strain with 2 different mating types, which offers the unique possibility to screen for conditions that might lead to ascospore formation in A. niger.Show less
Brule, T. van den; Punt, M.; Seekles, S.J.; Segers, F.J.J.; Houbraken, J.; Hazeleger, W.C.; ... ; Besten, H.M.W. den 2022
Microbial species are inherently variable, which is reflected in intraspecies genotypic and phenotypic differences. Strain-to-strain variation gives rise to variability in stress resistance and... Show moreMicrobial species are inherently variable, which is reflected in intraspecies genotypic and phenotypic differences. Strain-to-strain variation gives rise to variability in stress resistance and plays a crucial role in food safety and food quality. Here, strain variability in heat resistance of asexual spores (conidia) of the fungal species Aspergillus niger, Penicillium roqueforti and Paecilomyces variotii was quantified and compared to bacterial variability found in the literature. After heat treatment, a 5.4- to 8.6-fold difference in inactivation rate was found between individual strains within each species, while the strain variability of the three fungal species was not statistically different. We evaluated whether the degree of intraspecies variability is uniform, not only within the fungal kingdom, but also amongst different bacterial species. Comparison with three spore-forming bacteria and two non-spore-forming bacteria revealed that the variability of the different species was indeed in the same order of magnitude, which hints to a microbial signature of variation that exceeds kingdom boundaries. Show less
Seekles, S.J.; Dam, J. van; Arentshorst, M.; Ram, A.F.J. 2022
Weak acids, such as sorbic acid, are used as chemical food preservatives by the industry. Fungiovercome this weak-acid stress by inducing cellular responses mediated by transcription factors. In... Show moreWeak acids, such as sorbic acid, are used as chemical food preservatives by the industry. Fungiovercome this weak-acid stress by inducing cellular responses mediated by transcription factors. In ourresearch, a large-scale sorbic acid resistance screening was performed on 100 A. niger sensu stricto strains isolated fromvarious sources to study strain variability in sorbic acid resistance. Theminimal inhibitory concentration of undissociated (MICu) sorbic acid at pH = 4 in the MEB of the A. niger strains varies between 4.0 mMand 7.0 mM, with the average out of 100 strains being 4.8 0.8 mM, when scored after 28 days. MICu valueswere roughly 1mMlowerwhen tested in commercial ice tea. Genome sequencingof the most sorbic-acid-sensitive strain among the isolates revealed a premature stop codon inside thesorbic acid response regulator encoding gene sdrA. Repairing this missense mutation increased thesorbic acid resistance, showing that the sorbic-acid-sensitive phenotype of this strain is caused by theloss of SdrA function. To identify additional transcription factors involved in weak-acid resistance,a transcription factor knock-out library consisting of 240 A. niger deletion strains was screened. Thescreen identified a novel transcription factor,WarB, which contributes to the resistance against a broadrange of weak acids, including sorbic acid. The roles of SdrA,WarA andWarB in weak-acid resistance,including sorbic acid, were compared by creating single, double and the triple knock-out strains. Allthree transcription factors were found to have an additive effect on the sorbic acid stress response. Show less
