Regulation ofnif gene expression in Bradyrhizobium japonicum

Bradyrhizobium japonicum USDA 110 derivative L2-110 (L2) bacteroids contained slightly reduced levels of nifH and nifDK mRNA compared to the nitrogen-fixing USDA 110 derivative MN-110 (MN). However, compared to MN, L2 nitrogenase components were reduced three-fold indicating a posttranscriptional mutation(s) which affected L2 nitrogenase levels. L2 nitrogenase activity was reconstituted when functional dinitrogenase reductase was added to L2 crude bacteroid extracts but was not reconstituted by addition of functional dinitrogenase suggesting an inactive L2 dinitrogenase reductase. Sequencing of the nifH gene from L2 and MN revealed both to be identical. Collectively, these data indicated that L2 lacked nitrogenase activity at the posttranslational level nifH gene expression. {dollar}sp{lcub}31{rcub}{dollar}P NMR showed L2 nodules had severely reduced ATP/ADP ratios suggesting ADP would inhibit nitrogenase activity in L2 bacteroids at the level of dinitrogenase reductase. L2 appears to be deficient in establishing or maintaining energy status resulting in the lack of symbiotic nitrogenase activity.; Examination of in vivo protein phosphorylation for L2 and MN grown under aerobic, hyperosmolar, and nitrogen-fixing ex planta conditions showed clear differences in {dollar}sp{lcub}32{rcub}{dollar}P-labeling for three proteins. The three intensely {dollar}sp{lcub}32{rcub}{dollar}P-labeled proteins had molecular weights of 55, 60, and 97 kDa. The phosphate linkage for the 60 kDa protein was acid, base, and phosphatase stable suggesting nucleotidylation of this protein. The 60 kDa protein showed increased {dollar}sp{lcub}32{rcub}{dollar}P-labeling under hyperosmolar stress (i.e. glutamate accumulation) and ex planta conditions (i.e. NH{dollar}sb4sp+{dollar} accumulation) and was more intensely labeled in MN vs L2 ex planta cultures. Together, these data suggest the 60 kDa protein may be glutamine synthetase I.; In vivo {dollar}sp{lcub}32{rcub}{dollar}P-labeling and biotinylation of ex planta proteins using N-hydroxysuccinimide esters of biotin followed by analyses of streptavidin precipitated proteins indicated the 55 and 60 may be closely associated with the inner membrane whereas the 97 kDa protein may have a cell wall location. In vivo protein biotinylation was characterized using Escherichia coli as a model system. Biotinylation of different E. coli strains baring an inducible cytosolic or inner membrane protein marker resulted in biotinylation of the inner membrane marker but not the cytosolic marker.