Exploring calcium homeostasis and signalling in Aspergillus nidulans by using a calcium auxotrophy and a calcium-responsive reporter

Calcium is an essential nutrient for most, if not all forms of life. For example, among its roles in mammals, it is essential for muscle contraction, the proper functioning of the nervous system, the ability of cells to divide, fertilisation and the formation of teeth and bones. Among its roles in plants, it is essential for responses to temperature changes and the availability of water and the symbiotic relationship which enables nitrogen gas in the atmosphere to be incorporated into organic molecules needed for growth and the maintenance of life. In bacteria and fungi calcium plays roles in the structure of the organisms and in formation of various forms such as spores. It is also likely to play a role in the ability of bacteria and fungi to infect animals and plants. By learning more about the various roles of calcium in fungi we not only add to basic knowledge about an ubiquitous and important group of organisms but potentially identify ways in which we can prevent fungal growth. For example, if we can identify roles that calcium plays in fungi but does not play in humans then potentially drugs can be found that can treat fungal infections in humans. There is a considerable need for drugs of this sort because very few currently exist and there is a danger because of mutant fungi that these might lose their effectiveness. Similarly, identifying roles that calcium plays in fungi but not in plants can aid in the design of fungicides able to protect plants of agricultural or horticultural importance. It might also point to new ways of preventing spoilage of foods and other commodities by fungi. This proposal involves the use of a soil-borne fungus, Aspergillus nidulans, which is virtually non-pathogenic, to both animals and plants, but which is very easy to manipulate using both traditional genetic methods and modern genetic modification methods. For this work I have obtained two rather unique tools which should prove extremely useful in identifying factors responsible for the distribution of calcium between the environment and the fungus, its distribution within fungal cells and its roles within the fungus. One of these is a combination of mutations which results in a requirement for adding calcium to obtain normal growth of the fungus. Normally, the amount of calcium required for growth is so tiny that calcium contamination of water and the other chemicals required for growth suffices. The behaviour of this calcium-requiring organism is of interest in its own right but, in addition, it can be used to obtain further mutations affecting calcium responses by treating with a DNA damaging agent (mutagen) and seeking derivative organisms which no longer require addition of calcium for growth. The other tool involves a gene made in such a way that a blue colour can be produced in the presence of a certain chemical. The response of this gene, as determined by whether a blue colour is formed and, if so, how intense it is, is influenced by added calcium and by an unfulfilled calcium requirement. By treating an organism containing this gene with a mutagen, we can obtain mutants which differ from their parent in blue colour formation or its intensity in the presence or absence of added calcium. Finally, we will use a system in which a jellyfish gene is inserted into the fungus. The protein made from this gene responds to calcium by emitting blue light. This provides us with a means of following the behaviour of calcium in response to treatments which affect its behaviour in living mutant organisms whose response to calcium is altered as determined by one or both of the above two tools.

In probably all living organisms, calcium ions play a variety of important biological roles. Two almost unique tools for investigating calcium homeostasis and signalling in the ascomycete fungus Aspergillus nidulans have been developed for this work. One of these is a calcium auxotrophy resulting from a combination of regulatory mutations. Although the calcium requirement of this auxotrophy is not absolute, it can be made so by inclusion of a calcium chelator or other cations in the growth medium. The other tool is a reporter gene fusion in which the Escherichia coli lacZ coding region is fused to a calcium-responsive promoter. Expression of this reporter is markedly reduced by inclusion of calcium in the growth medium but significantly increased in calcium auxotrophic trains in the absence of calcium supplementation. I now propose to select extragenic suppressor mutations in a calcium auxotroph containing the reporter gene, having first constructed a non-revertible mutant allele of one of the genes involved in the calcium auxotrophy, and to characterise wild type and mutant versions of the genes containing the extragenic suppressors. In addition, the DNA-binding specificity of the almost certain transcription factor encoded by one of the component genes of the auxotrophy will be determined. A null mutation in the homologue of the Saccharomyces cerevisiae CZ1 (crazy) gene will be constructed and examined for its effect on expression of the calcium-responsive reporter and for its interactions with other mutations affecting calcium homeostasis and signalling. Mutations allowing expression of the reporter gene in the presence of added calcium will be selected and the genes containing these mutations will be characterised. A strain containing two targeted copies of the calcium-responsive reporter gene will be constructed and used to select mutations preventing expression of the reporter gene in medium lacking added calcium. Although the use of two copies of the reporter gene should largely eliminate mutations in the reporter gene itself, other conditions eliciting expression of the reporter gene will be examined in mutants as a means of possibly identifying mutations specifically affecting the calcium response. Genes containing mutations preventing reporter expression will be characterised. Finally, a fungal codon-optimised aequorin-encoding construct developed by Nick Read (Edinburgh) and collaborators will be used to measure cytosolic free calcium concentrations in living hyphae of strains containing a number of the mutations selected in this work. The aequorin luminescence experiments will be done in Nick Read¿s laboratory in Edinburgh and will examine how these mutations affect the response of cytosolic free calcium to mechanical perturbation, hypo-osmotic shock and addition of calcium to the medium. It is anticipated that the mutations obtained in this work will be a valuable resources for biophysicists, cell biologists and biochemists having an interest in fungal calcium homeostasis and signalling.