A comparative study of glutamate synthesis in free-living Klebsiells R15 and in Klebsiella R15 associated with rhizosphere of rice

Abstract

Klebsiella R15 is a N2 – fixing bacteria isolated from rice root cv. RE 7 in which glutamine synthetase-glutamate synthase (GS-GOGAT) pathway is responsible for ammonium assimilation in combined nitrogen and N2-fixing condition. Since GS is the key enzyme in N-metabolism of Klebsiella R15, comparative study on the GS specific activity and GS protein in free-living and in associative R15 in N2- fixing condition has been conducted. GS activity is measured by transferase assay. It is found that GS specific activity has been increased 7-9 fold in associative bacteria after inoculation 7-8 days. Bacterial GS protein in both conditions have been determined by enzyme linked immunosorbant assay (ELISA) and immunoblot or Western blot analysis, using antibody against purified GS. It is found that GS protein in associative bacteria is 3-5 fold of free-living by ELISA and the intensity of GS protein bands are significantly higher than free-living condition. These results indicate that regulation of GS in associative Klebsiella R15 is at the GS synthesis level. Nitrogenase activity is increased 400-500 fold in associative bacteria on day 6th after inoculation and becomes constant, when GS specific activity of rhizospheric bacteria is increased. At this time, rice root GS specific activity slightly decrease in the presence of bacterial inoculation.It shows that when Klebsiella R15 are associated with rice seedlings, they can fix N2more efficiently and assimilate NH3 should not be directly transferred into rice root. Anti – GS of Klebsiella R15 cross-reacts with GS of E. coli and K. pneumoniae, but does not cross react with rice root GS. Activity staining of native GS polyacrylamide gel electrophoresis immunoblot of denatured GS show that there is no difference in molecular weight of GS complex and their subunit molecular among 3 bacterial strains.