Differentiation in colonies of Aspergillus niger

Abstract

The aim of this Thesis was to study genome-wide gene expression in colonies of A. niger and to relate this to nutrient availability, growth, and secretion. Maltose and xylose were chosen as carbon sources for this study. These compounds result in different sets of extracellular enzymes. In Chapter 2 a novel method is described, the ring plate system, enabling simultaneous detection of protein secretion and environmental conditions (e.g. pH and nutrient concentration) in different parts of the colony. Colonies are grown on a porous polycarbonate membrane that is placed on a plate with 6 ring-shaped wells filled with liquid medium. Pulse labelling with radioactive amino acids showed that secretion of proteins occurs mainly at the periphery of the colony. Chapter 3 describes genome wide expression of 5 concentrical zones (i.e. from centre to periphery) of colonies grown in a thin agarose layer in between two polycarbonate membranes (sandwiched colonies) that had been placed on a nutrient agar medium containing xylose or maltose as carbon source. Protein secretion and growth mainly occurred in the outer zone of colonies. This correlated with carbon source availability but nitrate, sulphate and phosphate were not limited under the colony. Expression in each zone of the colony was strongly affected by the nature of the carbon source. However, the position within the colony also had a strong impact. The finding that expression profiles at the periphery of the colony changes in time, despite the fact that nutrient availability within this zone did not change in time, supports the hypothesis that developmental processes independently from the carbon source affect gene expression in a colony. Taken together, the results show that colonies are highly differentiated despite the fact that the cytoplasm is a continuous system. Expression profiles of A. niger were also determined in liquid shaken cultures and compared to the sandwiched culture. To this end, liquid shaken cultures were grown until the nutrient conditions mimicked those observed at the periphery of the sandwiched colony. Genome wide analysis of the mRNA obtained from the liquid culture revealed two components within the expression profile; one component shows similarity to expression at the periphery of sandwiched colonies, the other to that within the central zones. The sandwiched colony thus seems to be a good model system to study expression in liquid shaken culture. Sandwiched colonies were transferred to a fresh medium for 24 h to address whether the centre of colonies can be reactivated to secrete proteins (Chapter 4). Indeed, secretion was observed in two central zones of the colony after colonies had been transferred. Suprisingly, protein secretion in the central zones did not correlate with growth as determined by the incorporation of N-acetylglucosamine. After transfer, two zones remained inactive in secretion. These zones were shown to sporulate after the upper polycarbonate membrane was removed. Apparently, these zones were programmed to sporulate and not to secrete. These findings also demonstrate the differentiation within the colony. Transfer of colonies to fresh xylose medium also enabled us to establish whether the effect of the spatial position within the colony on global expression can be solely attributed to the availability of the carbon source (Chapter 4). Interestingly, genome wide expression showed that the availability of the carbon source had a limited effect on the spatial expression in the colony. Most variation is apparently caused by non-carbon source related developmental processes. Chapter 5 describes strategies to improve protein production by involving a larger part of the colony in the process of secretion. 6-day-old wild-type colonies only produced laccase in the centre of the colony. Increasing the part of the mycelium secreting laccase was also accomplished by transforming A. niger with a construct encompassing the laccase gene lccA under regulation of the glaA or the gpdA promoter. Transformants not only showed laccase activity in the centre but also in middle zones of the colony. However, the spatial laccase activity did not correlate with the expression of the glaA and gpdA promoters. Results indicate that young colonies are not able to produce laccase (at least lccA). Furthermore, LccA has affinity for the cell wall resulting in a slow release into the culture medium. Results are summarized and discussed in Chapter 6.