Analysis of a Stress-Inducible DNA-Binding Protein in a Cyanobacterium

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9634049 Bullerjahn This project is an investigation of a mechanism by which the cyanobacterium Synechococcus sp. strain PCC 7942 can adapt to oxidative stress. The gene dpsA, encoding a stress-induced DNA-binding protein has been cloned and sequenced. Previous work has shown that the DpsA protein accumulates under conditions of nutrient stress as well during the stationary phase of the growth curve. The protein forms extremely stable hexameric complexes that bind DNA; studies of the evolutionarily divergent Escherichia coli gene and its product indicate the DpsA protein serves to protect the chromosome from oxidative stress during long-term nutrient depletion and exposure to peroxide. However, a major difference between the E. coli and Synechococcus polypeptides is the presence of a heme-binding domain in the C-terminal half of the Synechococcus DpsA protein. It is suggested that the function of heme is to consume excess peroxide by a catalase mechanism. There is potentially a higher demand for detoxifying reactive species of oxygen in oxygen-producing phototrophs such as cyanobacteria. Mutants defective in the production of DpsA will be examined with respect to their ability to survive conditions of photooxidative damage and peroxide treatment. Truncated and chimeric forms of dpsA will also be introduced into dpsA null mutants of Synechococcus in order to test whether the presence of the heme-binding domain plays an important role in survival. RNA blots and promoter fusions will be used to determine the environmental conditions contributing to dpsA transcription. Immunocytochemical methods will be used to attempt to determine the location of the DpsA/DNA complex in the cells during adaptation to stationary phase. This study may help determine whether there is a distinct structural region of the nucleoid that is preferentially protected by the DpsA protein complex. Lastly, the yeast two-hybrid system will be employed to screen for possible protein-protein interactions that might be involved in regulating assembly and turnover of the stable DpsA complex. Overall, this work will help reveal mechanisms involved in global stress responses in cyanobacteria, and other phototrophs. %%% This study is concerned with how the cyanobacterium Synechococcus sp. strain PCC 7942 can adapt to conditions which cause oxidative damage to cell components. The gene dpsA and the protein it encodes DpsA will be studied. DpsA helps protect the cells against oxidative damage by an unusual mechanism. This proposal will focus on how light and other environmental conditions control production of DpsA. These studies will provide new information about how photosynthetic organisms respond to oxidative stress and change. ***