In the photosynthetic bacterium Rhodospirillum rubrum nitrogenase activity is inhibited when fixed nitrogen compounds (like NH+) are added to an N2 fixing culture. The inhibition of nitrogenase was long known to be due the covalent modification of one of the subunits of the Fe protein by a nucleotide factor; this nucleotide has been recently identified (by others) to be ADP-ribose. The mechanism responsible for the NH+ activation of ADP-ribosyltransferase remains unknown. It is the goal of this proposal to understand this NH4+-induced switch-off of nitrogenase activity in photosynthetic bacteria. Mutants defective in their ability to inactivate nitrogenase will be isolated. Cells will be mutagenized by nitrosoguanidine and Tn5 mutagenesis. In the analysis of these mutants we will determine what part of the inactivation process NH4+, glutamine, phenazine methosulfate and darkness have in common (both the oxidant and darkness also lead to Fe protein modification). While these metabolic, chemical and environmental factors all initiate inactivation, analysis of mutants will allow us sort out those mechanistic steps which all have in common and those steps which differ. Inactivation mutants will also be tested to determine if the Fe protein activating enzyme is still functional; this is to determine if the inactivating enzyme and the activating enzyme are the same or different enzymes. Cell extracts will be examined after initiation of nitrogenase switch-off in vivo by NH4+ to determine if there are significant changes in pool sizes of nucleotides and amino acids. Changes in a nucleotide or amino acid may serve as a regulatory signal for this process. Pool changes initiated by NH4+ will be compared to those caused by PMS and darkness. Conditions will be established to inactivate Fe protein in vitro. These experiments will be designed on the assumption that NAD is the donor molecule for the ADP-ribosyltransferase. A prelude to these experiments will be to assay R. rubrum extracts for NAD glycohydrolyase activity which is a side reaction of ADP-ribosyltransferase, the activity of physiological interest. Finally nifH (the Fe protein structural gene) from Klebsiella Anabaena and Rhizobium (organisms that do not switch-off their nitrogenases) will be introduced into nif- mutants of R. rubrum on plasmid vectors to determine if these Fe proteins can be regulated by the switch-off regulatory system of R. rubrum.
