9507490 Curtis The cyanobacteria are a very ancient and diverse group of photosynthetic bacteria which have a wide distribution in freshwater, marine, and terrestrial environments. They are unique among photosynthetic prokaryotes in their ability to perform an oxygen-evolving, or oxygenic, form of photosynthesis characteristic of plant chloroplasts. A subset of the cyanobacteria can respond to nitrogen limitations in the environment by the production of fixed nitrogen. Nitrogen fixation is catalyzed by the oxygen-sensitive enzyme nitrogenase and requires large amounts of energy in the form of ATP and reductant. In cyanobacteria, this energy requirement is met through photosynthesis, and this presents a dilemma as the oxygen byproduct of photosynthesis inhibits nitrogenase. In Anabaena sp. strain PCC 7120 (Anabaena 7120), this problem is circumvented by spatial separation of nitrogen fixation and photosynthesis. In the presence of fixed nitrogen, Anabaena 7120 grows as filaments of uniform photosynthetic vegetative cells. Starvation for fixed nitrogen triggers the initiation of a differentiation pathway that culminates in the production of a new cell specialized for nitrogen fixation. These terminally differentiated cells, heterocysts, develop from vegetative cells at regular intervals along the filament. During the thirty to forty hours required for development, the cells destined to become heterocysts undergo a series of biochemical and morphological alterations that provide an environment favorable for nitrogen fixation. Though the genetic mechanisms governing heterocyst development are largely unknown, there is considerable precedent in other bacterial systems for control at the level of gene transcription. As in other developmental systems, heterocyst differentiation involves the sequential activation and repression of a large number of gene sets. Although the regulation of gene expression during heterocyst development undoubtedly occurs at many levels, transcriptional control is believed to play a major role. The broad goal of this project is to extend our knowledge of how genes are regulated in Anabaena 7120 with an emphasis on transcriptional control. The work is focused on three genes, rbcL, gnd, and AD239, that fall into three classes with regard to regulation of transcript levels during vegetative growth and heterocyst development. The objectives are to understand how these genes are regulated: 1) The rbcL gene, which encodes a enzyme of carbon fixation is abundantly expressed in vegetative cells and turned off heterocysts. Experiments are proposed to identify cis elements that regulate rbcL expression in vegetative and heterocyst cells, and to begin identification of trans-acting factors that interact with these cis elements. 2) The gnd gene, which encodes an enzyme of carbon metabolism, is expressed at low levels during vegetative growth, and up-regulated during heterocyst development. Experiments address in which cell types the multiple gnd promoters are utilized, the relative contribution of each promoters to gnd expression, and the location of cis elements required for up-regulation during development. 3) The AD239 gene, of unknown identity, is not expressed in vegetative cells, and is induced by nitrogen starvation. Experiments address when, and in which cell types AD239 is expressed, whether AD239 is required for heterocyst development, and the location of cis elements required for expression of the gene after nitrogen starvation. %%% This research will help us understand how photosynthesizing cells can fix nitrogen from their environment. This process is essential to plant life, and requires the addition of fertilizers if the cells cannot do it themselves. ***
