Arman Vinck

Heterogeneity of Aspergillus niger Microcolonies in Liquid Shaken Cultures

ABSTRACT The fungus Aspergillus niger forms (sub)millimeter microcolonies within a liquid shaken culture. Here, we show that such microcolonies are heterogeneous with respect to size and gene expression. Microcolonies of strains expressing green fluorescent protein (GFP) from the promoter of the glucoamlyase gene glaA or the ferulic acid esterase gene faeA were sorted on the basis of diameter and fluorescence using the Complex Object Parametric Analyzer and Sorter (COPAS) technology. Statistical analysis revealed that the liquid shaken culture consisted of two populations of microcolonies that differ by 90 μm in diameter. The population of small microcolonies of strains expressing GFP from the glaA or faeA promoter comprised 39% and 25% of the culture, respectively. Two populations of microcolonies could also be distinguished when the expression of GFP in these strains was analyzed. The population expressing a low level of GFP consisted of 68% and 44% of the culture, respectively. We also show that mRNA accumulation is heterogeneous within microcolonies of A. niger. Central and peripheral parts of the mycelium were isolated with laser microdissection and pressure catapulting (LMPC), and RNA from these samples was used for quantitative PCR analysis. This analysis showed that the RNA content per hypha was about 45 times higher at the periphery than in the center of the microcolony. Our data imply that the protein production of A. niger can be improved in industrial fermentations by reducing the heterogeneity within the culture.

Heterogenic expression of genes encoding secreted proteins at the periphery of Aspergillus niger colonies

Colonization of a substrate by fungi starts with the invasion of exploring hyphae. These hyphae secrete enzymes that degrade the organic material into small molecules that can be taken up by the fungus to serve as nutrients. We previously showed that only part of the exploring hyphae of Aspergillus niger highly express the glucoamylase gene glaA. This was an unexpected finding since all exploring hyphae are exposed to the same environmental conditions. Using GFP as a reporter, we here demonstrate that the acid amylase gene aamA, the α-glucuronidase gene aguA, and the feruloyl esterase gene faeA of A. niger are also subject to heterogenic expression within the exploring mycelium. Coexpression studies using GFP and dTomato as reporters showed that hyphae that highly express one of these genes also highly express the other genes encoding secreted proteins. Moreover, these hyphae also highly express the amylolytic regulatory gene amyR, and the glyceraldehyde-3-phosphate dehydrogenase gene gpdA. In situ hybridization demonstrated that the high expressers are characterized by a high 18S rRNA content. Taken together, it is concluded that two subpopulations of hyphae can be distinguished within the exploring mycelium of A. niger. The experimental data indicate that these subpopulations differ in their transcriptional and translational activity.

Phleomycin Increases Transformation Efficiency and Promotes Single Integrations in Schizophyllum commune

ABSTRACT Phleomycin is mutagenic by introducing double-strand breaks in DNA. The ble gene of Streptoalloteychus hindustanus, which confers resistance to this substance, is widely used as a selection marker for transformation. Schizophyllum commune grows on 25 μg of phleomycin ml−1 after introduction of a resistance cassette based on the ble gene. However, we here report that growth of resistant colonies on this concentration of phleomycin resulted in aberrant colony morphologies. Apparently, phleomycin was mutagenic despite acquired resistance. Therefore, a new selection system was developed based on resistance to the antibiotic nourseothricin. However, the transformation efficiency was tenfold lower than that obtained with phleomycin as a selection agent. This low transformation efficiency could be rescued by addition of a nonselective concentration of phleomycin during protoplast regeneration. This was accompanied by a higher incidence of single-copy integrations and with an increase of expression of key genes involved in double-strand break repair. Taken together, we conclude that the effect of a nonselective concentration of phleomycin strongly resembles the effect of restriction enzyme-mediated integration (REMI) but, unlike REMI, it does not depend on the presence of a target restriction site.

Spatial and Developmental Differentiation of Mannitol Dehydrogenase and Mannitol-1-Phosphate Dehydrogenase in Aspergillus niger

ABSTRACT The presence of a mannitol cycle in fungi has been subject to discussion for many years. Recent studies have found no evidence for the presence of this cycle and its putative role in regenerating NADPH. However, all enzymes of the cycle could be measured in cultures of Aspergillus niger. In this study we have analyzed the localization of two enzymes from the pathway, mannitol dehydrogenase and mannitol-1-phosphate dehydrogenase, and the expression of their encoding genes in nonsporulating and sporulating cultures of A. niger. Northern analysis demonstrated that mpdA was expressed in both sporulating and nonsporulating mycelia, while expression of mtdA was expressed only in sporulating mycelium. More detailed studies using green fluorescent protein and dTomato fused to the promoters of mtdA and mpdA, respectively, demonstrated that expression of mpdA occurs in vegetative hyphae while mtdA expression occurs in conidiospores. Activity assays for MtdA and MpdA confirmed the expression data, indicating that streaming of these proteins is not likely to occur. These results confirm the absence of the putative mannitol cycle in A. niger as two of the enzymes of the cycle are not present in the same part of A. niger colonies. The results also demonstrate the existence of spore-specific genes and enzymes in A. niger.

Cytosolic streaming in vegetative mycelium and aerial structures of Aspergillus niger.

Aspergillus niger forms aerial hyphae and conidiophores after a period of vegetative growth. The hyphae within the mycelium of A. niger are divided by septa. The central pore in these septa allows for cytoplasmic streaming. Here, we studied inter- and intra-compartmental streaming of the reporter protein GFP in A. niger. Expression of the gene encoding nuclear targeted GFP from the gpdA or glaA promoter resulted in strong fluorescence of nuclei within the vegetative hyphae and weak fluorescence in nuclei within the aerial structures. These data and nuclear run on experiments showed that gpdA and glaA are higher expressed in the vegetative mycelium when compared to aerial hyphae, conidiophores and conidia. Notably, gpdA or glaA driven expression of the gene encoding cytosolic GFP resulted in strongly fluorescent vegetative hyphae and aerial structures. Apparently, GFP streams from vegetative hyphae into aerial structures. This was confirmed by monitoring fluorescence of photo-activatable GFP (PA-GFP). In contrast, PA-GFP did not stream from aerial structures to vegetative hyphae. Streaming of PA-GFP within vegetative hyphae or within aerial structures of A. niger occurred at a rate of 10–15 μm s-1. Taken together, these results not only show that GFP streams from the vegetative mycelium to aerial structures but it also indicates that its encoding RNA is not streaming. Absence of RNA streaming would explain why distinct RNA profiles were found in aerial structures and the vegetative mycelium by nuclear run on analysis and micro-array analysis.

Selective transport between heterogeneous hyphal compartments via the plasma membrane lining septal walls of Aspergillus niger

Hyphae of ascomycetes are compartmentalized by septa. The central pore in these septa allows for cytoplasmic streaming. However, many of these pores are closed by Woronin bodies in Aspergillus, which prevents cytoplasmic mixing and thus maintains hyphal heterogeneity. Here, glucose uptake and transport was studied in Aspergillus niger. Glucose uptake was higher in the hyphal population with high transcriptional activity when compared to the population with low transcriptional activity. Glucose was transported from the colony center to the periphery, but not vice versa. This unidirectional flow was similar in the wild-type and the ΔhexA strain that does not form Woronin bodies. This indicated that septal plugging by Woronin bodies does not impact long distance glucose transport. Indeed, the glucose analogue 2-NBDG (2-(N-[7-nitrobenz-2-oxa-1,3-diazol-4-yl]amino)-2-deoxyglucose) translocated to neighboring hyphal compartments despite Woronin body mediated plugging of the septum that separated these compartments. Notably, 2-NBDG accumulated in septal cross walls, indicating that intercompartmental glucose transport is mediated by transporters that reside in the plasma membrane lining the septal cross-wall. The presence of such transporters would thus enable selective transport between heterogeneous compartments.

Mixed colonies of Aspergillus niger and Aspergillus oryzae cooperatively degrading wheat bran

In both natural and man-made environments, microorganisms live in mixed populations, while in laboratory conditions monocultures are mainly used. Microbial interactions are often described as antagonistic, but can also be neutral or cooperative, and are generally associated with a metabolic change of each partner and cause a change in the pattern of produced bioactive molecules. A. niger and A. oryzae are two filamentous fungi widely used in industry to produce various enzymes (e.g. pectinases, amylases) and metabolites (e.g. citric acid). The co-cultivation of these two fungi in wheat bran showed an equal distribution of the two strains forming mixed colonies with a broad range of carbohydrate active enzymes produced. This stable mixed microbial system seems suitable for subsequent commercial processes such as enzyme production. XlnR knock-out strains for both aspergilli were used to study the influence of plant cell wall degrading enzyme production on the fitness of the mixed culture. Microscopic observation correlated with quantitative PCR and proteomic data suggest that the XlnR Knock-out strain benefit from the release of sugars by the wild type strain to support its growth.