Epsilon Open Archive

Abstract

In nature filamentous fungi and bacteria compete for space and nutrients. Both kinds of organisms have evolved mechanisms such as synthesis of antibiotic secondary metabolites to defeat other microbes. Studies of synthesis of antibiotics and microbial ecology in general have important applications in both agriculture and medicine. This thesis deals with molecular responses in the filamentous fungus Aspergillus nidulans to inhibitors of V-ATPases, bafilomycin and concanamycin. These antibiotics are produced by various species within the bacterial genus Streptomyces. The main function of V-ATPases in fungi is to keep the vacuoles acidified. Inhibition of V-ATPases leads to fungal hyperbranching and extremely reduced radial growth. Changes at the molecular level were observed when the fungus was treated with the antibiotics. Using mRNA differential display, five genes with changed expression after treatment were identified. A proteomic approach was used to screen for affected proteins, and 20 proteins displayed changed abundance after antibiotic treatment. Five of these were successfully identified. Most of these gene products were previously unknown, but one could be directly linked to disrupted V-ATPases. The function of several others could not, at this point, be directly related to inhibited V-ATPases. In this thesis, two genes were further characterised. The first of them, vmaA, encodes a major subunit of the V-ATPase. Disruption of vmaA confirmed that the V-ATPase is the main target for bafilomycin and concanamycin in A. nidulans. This mutant strain promises to be a useful tool in further studies of the identified gene products. The most extensively studied gene in this work is phiA. This gene was identified by mRNA differential display, and was up-regulated by bafilomycin. Surprisingly, phiA was found to be essential for normal asexual development. This is intriguing, and several hypotheses can be formulated, of which the most likely is that the induced phiA expression after inhibited V-ATPases is due to secondary effects in the fungus, i.e. caused by triggering growth arrest. In this thesis, several molecular methods, e.g. differential display, proteomics, and immunohisto-chemistry, have been used successfully to study interactions between bacteria and filamentous fungi.