9604270 Meeks The overall goal of this project is to characterize transcriptional and posttranslational regulation of glucose-6-phosphate dehydrogenase (G6PD) expression in the nitrogen fixing, facultatively heterotrophic cyanobacterium Nostoc punctiforme. G6PD is the initial enzyme of the oxidative pentose phosphate pathway. Previous work in this laboratory established that G6PD is essential for nitrogen fixation and dark growth of N. punctiforme, that transcription of G6PD increases during growth in the absence of ammonium and presence of N2, and that G6PD catalytic activity is dependent on the product of a cotranscribed gene termed opcA. The specific objectives are to: 1. Identify the mechanisms of regulation of transcription of G6PD with the respect to the nitrogen responsive operator/promoter region P1 and P5. Promoter activity in the 5' regions of P1 and P5 will be verified by in vivo (reporter gene expression) and in vitro (footprinting and runoff transcription assay) experiments. The regulatory 5' regions of P1 and P5 will be used in mobility shift assays to identify and isolate DNA binding proteins that function as activators or repressors. 2. Determine the role of OpcA in G6PD catalytic activity. Assays of specific activity, and activity stains and immunodetection in native protein gels will be used to examine the catalytic and aggregation states of G6PD, dependent on OpcA, in extracts from wild-type and opcA- mutant strains. This work will provide information about transcription in an ecologically significant organism. The overall goal of this project is to identify the biochemical mechanisms involved in expression of the enzyme glucose-6-phosphate dehydrogenase (G6PD) in the cyanobacterium (blue-green alga) Nostoc punctiforme. Previous work in the laboratory established that G6PD is essential for nitrogen fixation in this, and related, organisms. Photosynthetic, nitrogen fixing cyanobacteria are the most nutritionally independent organisms on earth and have an es sentially ubiquitous distribution in habitats exposed to light; thus, they are important for maintaining stable environments might be used in bioremediation of certain polluted habitats. DNA sequences that regulate the synthesis of enzymes such as G6PD could be used to construct biodegrading metabolic pathways in cyanobacteria. There are two specific objectives in this project: identification of DNA sequence important in regulation of enzyme synthesis, and of protein-protein interactions in enzyme activity. Biochemical assays monitoring protein-DNA interactions will be used to identify the important DNA sequences and regulated steps in the synthesis of G6PD. The role of an associated protein in the formation of more active highly aggregated states of G6PD will be determined by concurrently measuring enzyme activity and molecular size (aggregated state). This project will provide new information about an ecologically significant group of organisms.
