STUDIES ON THE ROLE OF PYRIMIDINE BIOSYNTHETIC PATHWAY OF Rhizohium meliloti IN SYMBIOSIS

Abstract

The present work was undertaken to determine the symbiotic role of pyrimidine biosynthetic pathway in Rhizohium meliloti (presently called Sinorhizobium meliloti). The primary objectives of this work were isolation, characterization and symbiotic studies of pyrimidine auxotrophs of S. meliloti. Random transposon Tn5 mutagenesis was employed to generate pyrimidine auxotrophs of S. meliloti strain Rmd201 which is a Smr derivative of strain AK631. Conjugations between donor E. coli WA803 (pGS9) and recipient S. meliloti Rmd201 yielded 7,350 Kmr transconjugants from 147 crosses. Screening of these transconjugants resulted in isolation of 37 auxotrophs. Streaking of these aaxotrophs on nutritional pools yielded twelve uracil, two uracil + arginine and two arginine auxotrophs. Six uracil and three uracil + arginine auxotrophs isolated by other researchers in this lab were also included in this study. Intermediate feeding and intermediate accumulation studies were conducted for biochemical characterization of pyrimidine auxotrophs. On the basis of these studies, uracil + arginine and uracil auxotrophs, were divided into three groups: (i) car mutants (NV1, NV15, RH33. RH37, RH47): Grew on minimal medium supplemented with uracil and arginine; also grew on carbamoyl phosphate supplemented minimal medium, (ii) pyrC mutants (NV18. NV21. NV32. VK12. VK19. VK43, RH7. RH9): Grew on orotic acid or dihydroorotic acid supplemented minimal medium. (iii) pyrE/pyrF mutants (NV6. NV9. NV12, NV19, NV23. NV26, NV33, NV34. NV37. RH36): Did not grow on minimal medium supplemented with any of the pyrimidine biosynthetic intermediates (used in the study); accumulated orotic acid in liquid minimal medium. Arginine aaxotrophs grew on ornithine or citrulline supplemented minimal medium indicating that the positions of biochemical block in these auxotrophs were before ornithine. The uracil, uracil + arginine and arginine auxotrophs were similar to the parental strain w.r.t. cell surface molecules (lipopolysaccharides, cellulose fibrils, succinylated exopolysaccharides and [3-glucans), utilization of carbon sources, salt and acid tolerances, change in pH of the medium and growth patterns indicating that the symbiotic defects of these auxotrophs were not caused by a change in any of the above characteristics. The linkage of Tn5 insertion to auxotrophy in each auxotroph (uracil/uracil + arginine/arginine) was determined by mobilizing the Tn5-containing genomic fragment from the aaxotroph into the S. meliloti strain ZB555 (Cys", Phe", Rf, Smr) with the help of genome mobilizing plasmid pJB3JI and subsequently checking for donor's auxotrophy(ies) in Kmr transconjugants. All Kmr transconjugants thus obtained showed respective donor's auxotrophy confirming the linkage of Tn5 insertion to auxotrophy. This also proved that no other Tn5 insertion occurred in the genome of this auxotroph. Transconjugants ofS. meliloti strain ZB555 carrying kanamycin resistance and respective auxotrophy when inoculated on alfalfa plants, showed the symbiotic defect like the donor auxotroph. The revertant of each auxotroph showed normal symbiosis, like the parental strain Rmd201, with alfalfa plants. These results showed that a single Tn5 insertion in each auxotroph was responsible for auxotrophy and symbiotic defect. Genetic mapping of Tn5 insertion in each of the uracil and uracil + arginine aaxotrophs was performed using plasmid pJB3JI mediated mapping method. These mutations were mapped in 41.7% region of chromosome between cys46 and purl5/168 loci. Precise mapping was not possible due to unavailability of mapping strains for complete chromosomal region. Symbiotic properties of uracil, uracil + arginine and arginine auxotrophs were determined by inoculating them on alfalfa (Medicago sativa cv. T9) plants grown aseptically on nitrogen-free slants. All these auxotrophs induced white nodules, and mean plant heights and dry weights of these plants did not differ significantly from those of the uninoculated plants, indicating the inability of these auxotrophs to fix nitrogen. The car, pyrC and arg mutants induced spherical/irregular nodules whereas the nodules induced by the pyrE/pyrF mutants were cylindrical like the parental strain induced nodules. This showed that the extent of nodule development was related to the position of mutation in the pyrimidine biosynthetic pathway. The symbiotic defects of uracil and uracil + arginine auxotrophs were not restored on addition of uracil or its intermediates (and arginine in case of uracil + arginine aaxotrophs) to the plant nutrient medium. This may be due to the failure of the aaxotrophs located in nodules to utilize these compounds from the medium. The symbiotic defects of arginine auxotrophs were restored on supplementation of arginine, citrulline or ornithine to plant nutrient medium. The methylene blue stained root portions (1cm long) of alfalfa plants inoculated with the auxotrophs were observed for root hair curling and infection thread formation. All auxotrophs induced root hair curling and resulted in infection thread formation. in Six weeks old nodules induced by uracil and uracil + arginine auxotrophs were fixed and embedded in epoxy araldite resin. Semithin and ultrathin sections of these nodules were examined under light and electron microscopes. In the nodules induced by car mutants lysis of rhizobial bacteria occurred immediately after their release into nodule cells from the infection threads. The defect in these nodules appears to be at the stage of bacterial release. The release of rhizobial bacteria into nodule cells occurred normally in the nodules induced by the pyrC mutants but the transformation of the released bacteria into bacteroids was not complete. Hence in these nodules the block occurred during the development of nitrogen fixation zone. In the nodules induced by the pyrE/pyrF mutants the rhizobial release into plant cells was normal and in most of the rhizobial cells transformation from bacterial to bacteroid stage was almost complete but the nitrogen fixation zones of these nodules were not fully developed like those of the parental strain induced nodules. Since the nodules induced by pyrC mutants showed advanced structural features over those of the nodules induced by car mutants, carbamoyl phosphate/carbamoyl phosphate synthetase/carbamoyl aspartate may be involved in symbiosis. The nodules induced by pyrE/pyrF mutants were also structurally more advanced than the nodules formed by pyrC mutants, indicating that dihydroorotic acid/dihydroorotase/orotic acid may also have symbiotic function(s). The symbiotic defects of nodules induced by pyrE/pyrF mutants may be due to unavailability of orotidine monophosphate or lack of orotate phosphoribosyltransferase/orotidine monophosphate decarboxylase activity. Hence an undiminished metabolic flow through pyrimidine biosynthetic pathway in S. meliloti appears to be essential for the effective nodule development on alfalfa plants.