GENETIC AND BIOCHEMICAL STUDIES ON BIOSYNTHESIS OF AMINO ACIDS, NUCLEOTIDE BASES AND VITAMINS IN RHIZOBIUM

Abstract

The Rhizobium meliloti strain Rmd 201 (Nod+ Fix+ Smr), a derivative of wild type strain Rm 41, was subjected to random transposon Tn5 mutagenesis with the help of transposon introducing plasmid pGS9 carrying Tn5. Six thousand transconjugants obtained at a frequency of 3xl0"5 from the crosses between the E. coli strain WA 803 {met, thi) harbouring suicide plasmid pGS9 and Rmd 201 were collected, purified and screened for auxotrophs based on their inability to grow on minimal medium. The screening of Tn5 derivatives yielded 29 auxotrophs, the frequency of isolation of auxotrophs being 0.48%. The nature of auxotrophy of each mutant was then determined on nutritional pools. Symbiotic properties of all auxotrophs were tested and are as follows : Nod" (isoleucine + valine), Nod+, Fix+ (methionine and cysteine/methionine), Nod+, Fix" (uracil, uracil + arginine and adenine). When the revertants were similarly examined, they resembled the parental type strain Rmd 201, indicating that changes in the symbiotic behaviour of the above strains could be attributed to their auxotrophic mutations (Table 6). In all the above plant experiments, the bacteria were isolated from the nodules to confirm that nodules were colonized by the same strain with which the plant was inoculated. From a collection of 29 auxotrophs 9 pyrimidine auxotrophs were selected for further detailed studies. The position of block in the pyrimidine biosynthetic pathway in these auxotrophs was determined by intermediate feeding and intermediate (ii) accumulation studies. Based on the results the pyrimidine auxotrophs were divided into three groups as follows: I Carbamoyl phosphate synthetase mutants which require carbamoyl phosphate for their growth (H33, H37 and H47). II The mutants which have blocks in the pathway between carbamoyl phosphate and orotic acid (VE12, VE19, VE43, H7 and H9). III The mutant that does not grow on orotic acid but grows on uracil supplementation, evidently having a block in the pathway after orotic acid (H36). Symbiotic properties of pyrimidine auxotrophs were investigated by inoculating them on alfalfa (Medicago sativa) seedlings grown aseptically on nitrogen free agar slants. The symbiotic properties of auxotrophs in the second group except H7 (VE12, VE19, VE43 and H9) could not be determined as the nodules were always occupied by their prototrophic revertants. The carbamoyl phosphate mutants (H33, H37 and H47), the mutant H7 which has a block between carbamoyl phosphate and orotic acid and the mutant H36 that has a block in its pyrimidine biosynthetic pathway after orotic acid were nod+ and fix", i.e., they formed nodules which did not fix nitrogen. In order to test the authenticity of the nodules formed by these auxotrophs, root hair curling and infection thread formation were observed under light microscope. All auxotrophs were found to induce root hair curling and infection thread formation. For histological studies the nodules from six weeks old plants were fixed and embedded in araldite epoxy resin. Semithin and ultrathin sections

were obtained and observed under light microscope and transmission electron microscope, respectively. Alfalfa nodules induced by the parental strain Rmd 201 were elongate, cylindrical and had distinct typical meristem, infection, nitrogen fixation and senescent zones. The bacteria in the infection thread and just after release into the host cell were approximately 1.0 to 1.5 urn long. At this stage poly-p-hydroxybutyrate inclusions were found in the bacterial cytoplasm. In the nitrogen fixation zone of the nodule the mature bacteroids increased significantly in size becoming 3 to 8 urn long. In addition, they proliferated to the extent that most of the host cytoplasm was occupied by elongated bacteroids. The younger bacteroids exhibited a relatively homogeneous cytoplasm and were found in the distal part of nitrogen fixation zone. In the mature bacteroids, which were present in more proximal part of the nitrogen fixation zone, the cytoplasm exhibited greater heterogeneity because of the condensation of nucleic acid material. The nodules induced by strain H33 were ineffective and distinguishable in morphology from those induced by wild type. These nodules were usually white, spherical and found on secondary roots. The longitudinal section of the nodule did not show distinct zones. Most of the cells in the nodule were without bacteroids. Extensive branching of infection threads was found. At the ultrastructural level the occasional release of bacteria into plant cell cytoplasm was seen. The released bacteria showed homogeneous cytoplasm. Mature bacteroids with features as described above were not seen in the plant cells. It seems that transformation of young bacteroids to mature bacteroids did not occur in this case.

Nodules elicited by H7 were spherical or irregular in shape and white in colour. Light microscopic studies revealed absence of distinct zones. Ultrastructural studies showed extensive branching of infection threads and occasional release of bacteria into plant cell cytoplasm was observed. The released bacteria exhibited a relatively homogenous cytoplasm. The bacteroidal development did not occur beyond first stage. The pyrimidine auxotroph H36 formed nodules that exhibited distinct zones viz. meristematic zone, infection zone, poorly developed nitrogenfixing zone and senescent zone. Electron microscopic studies revealed that in the infection zone electron dense homogeneous young bacteroids with phb granules were seen. In the nitrogen-fixing zone bacteroids were elongated and properly organised. Bacteroids in this region showed partially heterogeneous cytoplasm which were lacking proper condensation of nucleic acid materials. It seems that transformation from young bacteroids to mature bacteroids did not occur fully. Moreover some cells showed this bacteroidal cytoplasm in electron transparent condition with ruptured peribacteroidal membrane. These bacteroids looks like lysing in this stage. For confirming the linkage of auxotrophy to Tn5 insertion, the plasmid pJB3JI was used to mobilize the Tn5 containing chromosomal segments of pyrimidine auxotrophs into another R.meliloti strain ZB 555 (Cys, Phe, Rifr, Sm'). All Kmr transconjugants obtained showed donor's auxotrophy. This confirmed the linkage of auxotrophy to Tn5 insertion as well as ruled out the possibility of occurrence of other independent Tn5 insertions in these mutants. Since the plasmid pJB3JI used in this study showed poor genome

mobilizing ability, precise mapping was not possible but the possibility of occurrence of pyr loci in 2/3 of the chromosome could be ruled out. In order to find out the pleiotropic effects of Tn5 insertions all auxo trophs were tested for the production of exopolysaccharides, lipopolysaccharides, cellulose fibrils, 0-1 -> 2 glucans and utilization of dicarboxylic acids (malate, aspartate, succinate, etc.). Like the wild type all auxotrophs utilised dicarboxylic acids as carbon source and produced the above mentioned cell surface molecules. From the above results it seems that intermediates of the pyrimidine biosynthetic pathway have a role in bacteroid development as blocks at different steps of the pathway resulted in bacteroids blocked at different stages of development.