GENETIC AND BIOCHEMICAL STUDIES ON BIOSYNTHESIS OF SOME AMINO ACIDS IN RHIZOBIUM

Abstract

The present work was taken up with an aim to focus on the influence of biosynthetic pathways of amino acids of Rhizobium meliioti on symbiotic nitrogen fixation. The objectives taken were generation of amino acid auxotrophs with defective symbiotic properties, their characterization and finding out the role of the intermediates of the affected biosynthetic pathways in normal symbiosis. To generate auxotrophs, random transposon mutagenesis of R. meliioti strain Rmd201 (Strr derivative of AK631) was carried out with the help of transposon Tn5 delivery vector pGS9. Conjugations between E. coli strain WA803 (Met", Thi") harbouring the suicide plasmid pGS9 and R. meliioti Rmd201 yielded Tn5 induced kanamycin resistant transconjugants. Six thousand transconjugants were collected from 45 crosses, purified and screened on minimal medium for auxotrophs. The nutritional requirements of auxotrophs which failed to grow on minimal medium were determined on minimal medium supplemented with nutritional pools. Out of thirty auxotrophs obtained, ten aromatic amino acid auxotrophs - six tryptophan (VK1, H4, H6, VK15, VK28 and VK30), one phenylalanine (H38), one tyrosine (Rl) and two aro auxotrophs requiring all three amino acids, viz., tryptophan, tyrosine and phenylalanine (VK18 and H5)- were chosen for further studies. Biochemical characterization of tryptophan auxotrophs included intermediate feeding and intermediate accumulation studies, and cross-feeding assays. Based on the results of these experiments tryptophan auxotrophs were placed into three categories as follows : (i) trpE mutants (H4, VK1 & H6) which grew on anthranilic acid and did (0 not accumulate any of the intermediates of the tryptophan biosynthetic pathway. (ii) VK15, that grew on indole and accumulated anthranilic acid. (iii) Tryptophan synthase mutants (VK28 &VK30) which grew only on minimal medium supplemented with tryptophan and accumulated anthranilic acid and indole glycerol phosphate. The three trp mutants, viz., VK15, VK 28 and VK30 cross-fed the trpE mutants and VK28 & VK30 cross-fed VK15. Symbiotic properties of aromatic amino acid auxotrophs were determined by inoculating them on alfalfa seedlings {Medicago satiua cv. T9) grown aseptically on nitrogen free agar slants. All auxotrophs induced nodulation but the mean shoot dry weight of the plants inoculated by these auxotrophs (except tyrosine auxotroph and tryptophan synthase mutants) were significantly less than the shoot dry weight of the parental strain inoculated plants indicating reduced nitrogen fixation abilities of these mutant strains. trpE, ctro and phe mutants showed almost no nitrogen fixation (Fix"), whereas VK15 was partially effective. Early events in the nodulation process like root hair curling and infection thread formation were followed by observing the methylene blue stained root portions of alfalfa plants inoculated with the mutant strains. All mutants induced root hair curling and infection thread formation. Nodules from six week old plants were fixed, post fixed and embedded in araldite epoxy resin. Semithin and ultrathin sections of the nodules were observed under light and transmission electron microscopes, respectively. Nodules elicited by Rmd201 contained four well formed zones.- meristematic (ii) region, infection zone, nitrogen fixation zone and senescence zone. TEM studies showed all stages of bacteroid development in these nodules. Tyrosine and tryptophan synthase mutants which were Fix+ formed nodules whose histology resembled that of parental strain induced nodules in all aspects. trpE and aro mutants formed nodules with unusual defects. The meristematic zone in these nodules was normal but the infection zone was very extensive and occupied most part of the nodule. The nitrogen fixing region was restricted to few layers at the base of the nodule. TEM studies revealed normal bacteroid development only in the nitrogen fixing zone. The bacteroids in the extended infection zone and in the zone between infection and nitrogen fixing zone showed no tendency towards maturation. The cytoplasm of these bacteroids was homogeneous unlike the bacteroids from the same zones of nodules induced by Rmd201 where heterogeneous cytoplasm was exhibited by the bacteroids. VK15, which is a slightly leaky mutant, exhibited novel symbiotic defects. The plants inoculated with this strain were stunted but did not appear chlorotic presumably because traces of nitrogen fixation occurred. The nodules were elongate, cylindrical and slightly pinkish. Electron microscopic studies showed all stages of bacteroid development. phe mutant induced nodules with normal meristematic and infection zones but the zone after infection region was very extensive. The cells in this region were studded with many amyloplasts. The nitrogen fixation zone was not observed. Bacterial release did occur in these nodules but soon after their release they degenerated. The linkage of auxotrophy to Tn5 insertion was tested by transferring (iii) the Tn5 inserted portion of the genome of the auxotrophs into another R. meliioti strain ZB555 with the help of plasmid pJB3JI. All Kmr transconjugants obtained showed the respective donor's auxotrophy. This confirmed that auxotrophy was due to Tn5 insertion. This also proved that no other independent Tn5 insertion occurred elsewhere in the genome of these auxotrophs. The position of Tn5 insertion in VK15 was located using plasmid pJB3JI mediated mapping method. The Tn5 insertion in this mutant mapped at the trpl5 locus which has been reported earlier. In order to find out the pleiotropic effects of Tn5 insertions, all auxotrophs were tested for cellulose fibrils, p-(l->2) glucans, acidic exopolysaccharides and lipopolysaccharides production and in every case their production seemed normal. Dicarboxylic acids (malate, succinate, etc.) were effectively utilized by these auxotrophs confirming that the symbiotic lesions were not due to dct' phenotype. From the results it is obvious that a normal flow of metabolites through aromatic amino acid biosynthetic pathways is required for successful R. melilotialfalfa symbiosis. Tryptophan and tyrosine but not phenylalanine seem to be available to the bacteria in planta. Anthranilic acid appear to be required for proper nodule development. The fact that aro mutants resembled the trpE mutants rather than the phe mutant in their symbiotic defectiveness confirms this. Though anthranilic acid is required for symbiosis, a subsequent intermediate(s) in the tryptophan pathway also appear to influence nitrogen fixation process.