RUI: Continued Investigation of the Ambient pH Signal Transduction Pathway of Aspergillus Nidulans

Eckerd College, Saint Petersburg FL

Investigators

Abstract

Cells of microorganisms as well as human cells encounter changes in environmental pH. Responses of cells to altered environmental pH may involve changes in gene expression. A system for the regulation of gene expression by environmental pH was first identified in the filamentous Ascomycete fungus Aspergillus nidulans. Components of this regulatory system include the pacC gene-encoded zinc-finger transcription factor and the products of the palA, B, C, F, H and I genes, which lead to activation of the PacC transcription factor at alkaline pH. This system ensures that extracellular enzymes are produced under conditions of pH where they can function: acid phosphatase under acid conditions and alkaline phosphatase under alkaline conditions, for example. The pH regulatory system is also important for alkaline adaptation (survival in alkaline environments) and for conidial development in A. nidulans. The aims of this research are to discover new components of this regulatory system and to further define the roles of known components. Research on the A. nidulans pH signalling pathway has contributed to the larger field of cell signalling. For example, the palB gene product was the first example of a calpain, calcium-activated cysteine protease in a microorganism and in a specific signalling pathway. The study of pH regulation in the model organism A. nidulans has also helped the understanding of pH regulation in fungi of medical and economic importance. The study of pH regulation is most advanced in A. nidulans. This fact and the availability of well- developed genetics tools and resources make A. nidulans one of the best systems for the study of this important eukaryotic regulatory pathway. In this research, a recently-developed gene library will be used to identify high copy number suppressors of A. nidulans strains with mutations in the pH regulatory pathway. With this library, the expression of A. nidulans genomic DNA inserts is under control of the inducible niiA (nitrite reductase) promoter. As the plasmid used to construct the library also has the AMA1 sequence, which allows extrachromosomal maintenance of plasmids, isolation of plasmids from Aspergillus transformants is straightforward. Significant preliminary results have already been achieved in this study. For example, 166 putative copy number suppressors of a pacC loss-of-function mutation have been obtained. An assay for palB protease activity will also be developed and work will be completed to determine the cellular location of the palI gene product, which may function as the pH-sensing protein. For this study, a palI-green fluorescent protein gene fusion construct has been made. In addition to the contribution to scientific knowledge of the proposed research, the project will make an important contribution to education. As undergraduate students will participate in this research, it will provide valuable training for future scientists and educators.