Heterogeneity in Microbial Micro-colonies

Abstract

Filamentous microbes play an important role in nutrient recycling by degrading dead organic material. Moreover, they can be used as cell factories for the production of pharmaceuticals and enzymes and metabolites. In my Thesis I studied the fungal cell factories Aspergillus niger and Aspergillus oryzae and bacterial cell factories of the genus Streptomyces. Filamentous microbes form colonies by means of an interconnected network of hyphae. Colonies formed in industrial bioreactors are ≤ 1mm and are dubbed micro-colonies. I studied whether all micro-colonies are the same or whether distinct populations of colonies can be distinguished. Statistical analysis following flowcytometry showed that liquid cultures of A. niger consisted of two populations of micro-colonies that differed in size and expression. It is known that micro-colony size depends on the surface properties of spores. Therefore I tested the ΔfwnA, ΔolvA and ΔbrnA strains that are affected in the biosynthesis of the pigment that resides at the surface of spores. These mutants formed larger and more homogenous micro-colonies than the control. Micro-colonies of ΔfwnA were even normally distributed. Additional analysis showed that conidia morphology and biophysical properties of the ΔbrnA and the ΔfwnA strains were similar as the control. However, conidia of ΔolvA were larger, more negatively charged, highly hydrophilic, and rodlets were almost completely absent. Two populations of micro-colonies could also be shown in liquid cultures of Streptomyces coelicolor, Streptomyces lividans, Streptomyces scabies, and Streptomyces griseus. Heterogeneity in A. niger is not only observed between micro-colonies but also within these colonies. The amount of RNA per individual hypha was 45 times higher at the periphery. Moreover, expression of genes was shown to be heterogeneous between neighboring hyphae. In A. oryzae it was shown that 40 % of the first three septa of hyphae at the periphery are closed. The plugging state of these septa did neither depend on the plugging state of neighboring septa nor on environmental conditions. Septa were always closed when a Woronin body was localized at the septal pore and were open when it was absent. This process is dynamic, implying that septal closure is reversible. Septal plugging explains hyphal heterogeneity between neighboring hyphae. Expression of glaA and aguA was normally distributed in neighboring hyphae of a strain lacking Woronin bodies, while it was heterogeneous in the wild-type background. The fact that microbial micro-colonies are heterogeneous has implications how analyses should be interpreted. By using the whole culture, an average is determined and may not reflect the composition or activity of individual populations. Therefore, individual populations should be studied to understand mechanisms underlying biological processes