Genetics and physiology of respiration in Aspergillus nidulans : a thesis presented in partial fulfilment of the requirements for the degree of Doctor in Philosophy in Molecular Genetics at Massey University, Palmerston North, New Zealand
Obligate aerobes such as Aspergillus nidulans primarily use the classical respiratory pathway for ATP production. However, the use of alternative energy-producing processes in A. nidulans was first speculated upon when cytochrome c-deficient strains were observed to be viable upon fermentable carbon sources. It was therefore postulated that the cycA- strains of A. nidulans may use fermentation and an alternative respiratory pathway to compensate for the non-functioning cytochrome c-dependent pathway. Characterisation of the A. nidulans cytochrome c-deficient strains was carried out. The growth parameters for strain A68 were consistent with other cytochrome c mutants; the strain grew more slowly than the corresponding wildtype strain on fermentable carbon sources, and produced higher levels of ethanol. Spectral analysis confirmed the lack of detectable levels of cytochrome c in the cycA- strains, and decreased levels of cytochrome c oxidase, consistent with the non-functioning cytochrome c-dependent respiratory pathway. The presence of a hemoglobin-like molecule in the cycA- and cycA+ strains was determined by CO binding assays. Inhibitor studies determined the presence of an alternative respiratory pathway in cycA- and cycA+ strains of A. nidulans. An active cytochrome c-dependent pathway was found to be present in the cycA+ strains, yet absent from the cycA- strains. Results also suggested the presence of a putative third terminal oxidase in the cycA- and cycA+ strains. Increased levels of b-type heme observed in the redox spectra of the cycA- strains were suggested to be associated with the putative third terminal oxidase. Therefore, the cycA- strains of A. nidulans appear to use fermentation and the alternative respiratory pathway to compensate for the non-functioning cytochrome c-dependent pathway. The putative hemoglobin molecule identified in these strains may also function as a terminal oxidase, in addition to the putative third terminal oxidase. PCR amplification with degenerate primers was carried out to confirm the presence of an AOX gene in cycA+ and cycA- strains of A. nidulans. The product of the AOX gene is likely to function as a terminal oxidase in the alternative pathway. A comparison of fungal and plant AOX protein sequences was cauied out. A conserved cysteine residue which has been implicated in dimer formation and pyruvate regulation was found to be absent from the fungal sequences. A preliminary expression study of the A. nidulans AOX gene was carried out by RT-PCR. The putative regulatory elements identified within the A. nidulans AOX gene promoter are also located within the promoters of other respiratory-related genes (eg. A. nidulans cycA) and genes involved in reducing the formation of reactive oxygen species (ROS) (eg. A. nidulans catA-C and sod1). This implies similar mechanisms of regulation, which may be controlled in a coordinated manner. A functional analysis of the A. nidulans cycA gene promoter was carried out to identify important regulatory elements. Reporter constructs containing cycA-lacZ, fusion genes were transformed into A. nidulans. Although integrated at the argB locus, the constructs had very low levels of lacZ expression (with the exception of the positive control). A number of parameters were investigated, and a 'promoter switch' experiment commenced, however the cause of the faulty cycA-lacZ expression system was not determined. Functional expression of the A. nidulans cycA gene promoter in yeast was also attempted, as regulatory mechanisms controlling cytochrome c expression are believed to be analogous in A. nidulans and Saccharomyces cerevisiae. Reporter constructs containing cycA-lacZ fusion genes were transformed into S. cerevisiae, with resulting low β-galactosidase activity, similar to the results in A. nidulans. The wildtype strain of A. nidulans from which the cycA gene promoter fragment was amplified was shown by spectral analysis to have low levels of cytochrome c, in comparison to other wildtype strains of A. nidulans. Therefore, it is possible that the low levels of lacZ expression from the cycA-lacZ fusion genes may be representative of the level of cycA expression in that strain. However, low levels of lacZ expression were also observed for the S. cerevisiae positive control, indicating that the expression system was not working properly. Therefore, the expression of the A. nidulans cycA gene promoter in S. cerevisiae could not be assessed in this study.