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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM032183-08
Application #
3280782
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1983-07-01
Project End
1989-07-31
Budget Start
1987-08-01
Budget End
1988-07-31
Support Year
8
Fiscal Year
1987
Total Cost
Indirect Cost
Name
University of South Carolina at Columbia
Department
Type
Schools of Arts and Sciences
DUNS #
111310249
City
Columbia
State
SC
Country
United States
Zip Code
29208
Hu, C Z; Yoch, D C (1990) Complementation of a pleiotropic Nif-Gln regulatory mutant of Rhodospirillum rubrum by a previously unrecognized Azotobacter vinelandii regulatory locus. Arch Microbiol 154:528-35
Yoch, D C; Li, J D; Hu, C Z et al. (1988) Ammonia switch-off of nitrogenase from Rhodobacter sphaeroides and Methylosinus trichosporium: no evidence for Fe protein modification. Arch Microbiol 150:1-5
Gandy, E L; Yoch, D C (1988) Relationship between nitrogen-fixing sulfate reducers and fermenters in salt marsh sediments and roots of Spartina alterniflora. Appl Environ Microbiol 54:2031-6
Li, J D; Hu, C Z; Yoch, D C (1987) Changes in amino acid and nucleotide pools of Rhodospirillum rubrum during switch-off of nitrogenase activity initiated by NH4+ or darkness. J Bacteriol 169:231-7
Whiting, G J; Gandy, E L; Yoch, D C (1986) Tight coupling of root-associated nitrogen fixation and plant photosynthesis in the salt marsh grass Spartina alterniflora and carbon dioxide enhancement of nitrogenase activity. Appl Environ Microbiol 52:108-13
Yoch, D C; Whiting, G J (1986) Evidence for NH4+ switch-off regulation of nitrogenase activity by bacteria in salt marsh sediments and roots of the grass Spartina alterniflora. Appl Environ Microbiol 51:143-9
Schultz, J E; Gotto, J W; Weaver, P F et al. (1985) Regulation of nitrogen fixation in Rhodospirillum rubrum grown under dark, fermentative conditions. J Bacteriol 162:1322-4
Gotto, J W; Yoch, D C (1985) Regulation of nitrogenase activity by covalent modification in Chromatium vinosum. Arch Microbiol 141:40-3