Regulation of Two Clusters of Nitrogenase Genes of the Cyanobacterium Anabaena

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Thiel 9723754 Cyanobacteria of the genus Anabaena have evolved specialized cells called heterocysts that function exclusively for nitrogen fixation. Some strains, notably Anabaena variabilis, have multiple genes that encode different nitrogenases. There are three clusters of genes in A. variabilis that encode three nitrogenases: a V-dependent nitrogenase (vnf) and two molybdenum-dependent nitrogenases, one that functions under aerobic conditions (nil1) and one that functions only under strictly anaerobic conditions (nif2). These genes are tightly regulated; however, very little is known about their regulation except that it is probably not via the (54- NifA system described in other nitrogen-fixing bacteria. Thus, an understanding of nitrogenase gene regulation in A. variabilis can broaden our understanding of mechanisms of procaryotic gene regulation. The goal of this research is to identify the cis elements and trans-acting factors that function in the environmental control of the nif1 and vnf genes that function under aerobic conditions in heterocysts of A. variabilis. The objectives are to 1 ) identify and characterize a putative repressor made in Mo-grown cells that controls V-nitrogenase gene expression, 2) identify the DNA-binding site for the putative repressor and determine the nucleotides that are important for repression, 3) identify and characterize a putative repressor made in ammonium-grown cells that controls nif1 and vnf expression in the presence of fixed nitrogen, and 4) identify the DNA-binding site for this regulatory protein. Nitrogen fixation, the conversion of atmospheric nitrogen gas to ammonia by the enzyme nitrogenase, is found exclusively in bacteria, including many cyanobacteria. Nitrogen fixation sustains the growth of not only these bacteria, but also of many plants that receive nitrogen by associations with nitrogen-fixing bacteria. In all organisms nitrogen fixation is tightly regulated. Very little is known about nitrogen regulation in many agriculturally impo rtant nitrogen-fixing bacteria, including the cyanobacteria. The primary objective of this research project is to use the tools of molecular genetics to understand how the cyanobacterium, Anabaena variabilis, controls two sets of genes, nif1 and vnf, that allow these cells to fix nitrogen under diverse environmental conditions.