This project supports research on the regulation of nitrogen metabolism by ADP-ribosylation of nitrogenase and other proteins in the photosynthetic bacterium Rhodospirillum rubrum. The system is unique in that the first two enzymes of the nitrogen assimilatory pathway are regulated by separate covalent modification cycles: dinitrogenase reductase is reversibly ADP-ribosylated and glutamine synthetase is reversibly adenylylated. Recently, it was discovered that R. rubrum glutamine synthetase can also be ADP-ribosylated in vitro and this suggests a mechanism by which the two cascades can be coordinated in vivo. In addition to the dinitrogenase reductase component of the nitrogenase complex, the R. rubrum ADP-ribosyl transferase will ADP-ribosylate dinitrogenase reductases from other organisms. Biochemical studies have shown that these alternate targets have distinctly different requirements for modification. The genes for these alternate targets will be transfered to R. rubrum so that a biochemical analysis of their properties as targets in vivo can be made. Results from these studies will delimit the requirements for ADP- ribosylation of dinitrogenase reductase in vivo.%%% Organisms must adapt their metabolism to respond to their environment. Microorganisms, in particular, are subject to rapid and divergent changes in their environment. Rhodospirillum rubrum is remarkable in its ability grow under drastically different conditions photosynthetically, fermentatively and by respiration. It is capable of using both very simple carbon sources, such as carbon dioxide and carbon monoxide, and a variety of complex carbon sources. It is capable of assimilating nitrogen in the form of ammonia nitrogen and also possesses the ability to control metabolism by reversible covalent modification of certain enzymes. Dinitrogenase reductase of the nitrogenase enzyme system is reversibly modified by adenosine diphosphoribose, glutamine synthetase is regulated by adenylylation and perhaps by ADP- ribosylation, and it is likely that other metabolic pathways are regulated by these mechanisms. Thus, the study of nitrogen assimilation in R. rubrum offers the opportunity to study the interaction of separate regulatory cascades which must respond to some of the same stimuli. This research will provide for an understanding of the relationship of these cascades to each other and their integration into metabolism in the cell.***//
