GENETIC AND SYMBIOTIC STUDIES ON PURINE AUXOTROPHS OF Sinorhizobium meliloti

Abstract

Sinorhizobium meliloti Rmd201 (Nod+,Fix+,Strr) strain, a derivative of the wild type strain Rm41, was subjected to random Tn5 mutagenesis with the help of suicide plasmid pGS9, carrying transposon Tn5. Two thousand and two hundred kanamycin resistant transconjugants were obtained from crosses between E. coli WA803 (pGS9) and S. meliloti strain Rmd201. These transconjugants were streaked on minimal medium and twelve auxotrophs were identified on the basis of their inability to grow in this medium. Streaking of these auxotrophs on nutritional pools yielded two adenine auxotrophic mutants. Two adenine+thiamine and eight adenine auxotrophs isolated by other research workers were also included in further studies. The positions of blocks in the purine biosynthetic pathway for these auxotrophs were determined by intermediate feeding and cross feeding studies and based on these results the purine auxotrophs were divided into four groups as follows: Group (I) Purine auxotrophs, NV2 and NV24, which require both adenine and thiamine for their growth and each of these, has a block in the adenine-thiamine pathway before amino imidazole riboside (AIR). Group (II) Purine auxotrophs, NV10, NV28, and AL7 that require amino imidazole carboxamide riboside (AICAR) for their growth and each of these have a block in the adenine pathway before AICAR. Group (III) Purine auxotrophs, AL3, VK37, VK38, VK40 and RH13 which require inosine or adenine for their growth and each of these has a block in the adenine pathway before inosine monophosphate (IMP) and group (IV) Purine auxotrophs, VK27 and RH19, which require adenine for their growth and each of these, has a block in the pathway before adenosine monophosphate (AMP).

In order to find out the pleiotropic effects of Tn5 insertions, all purine auxotrophs were tested for production of exopolysaccharides, lipopolysaccharides, cellulose fibrils, (3 (1-2) glucans and utilization of dicarboxylic acids. AH these auxotrophs were found to utilize dicarboxylic acids as a sole carbon source and produce exopolysaccharides, lipopolysaccharides and cellulose fibrils as the parental strain Rmd201, whereas, these mutants showed less motility in swarm medium than the parental strain which related to production of (3 (1-2) glucans. The symbiotic properties of different purine auxotrophs along with the parental strain 5. meliloti Rmd201 were studied by inoculating them on alfalfa (Medicago sativa) seedlings grown on nitrogen free agar slants. The nodules induced by the parental strain Rmd201 were of cylindrical shape and pink in color. The mutants induced-nodules were round /irregular in shape and white in color. The mean shoot dry weights and mean shoot high of the plants inoculated with purine auxotrophs did not differ significantly from those of uninoculated alfalfa plants. These indicate the ineffectiveness of these mutants in symbiotic nitrogen fixation. The ineffectiveness of these mutants in nitrogen fixation were also reflected by comparing total nitrogen content of plant inoculated with parental strain and that inculated with purine auxotrophs. All purine auxotrophs were found to induce root hair deformation and infection thread formation. The addition of adenine and other intermediates to plant growth medium did not restore effectiveness in these mutants. For histological studies, the nodules from six weeks old plants were cut, fixed and embedded in araldite epoxy resin. Thin and ultrathin sections of nodules were cut and examined under light and transmission electron microscopes, respectively. The nodules induced by the parental strain Rmd201 had four zones: meristematic zone,

infection zone, nitrogen fixation zone and senescence zone. Cells of meristematic zone were found to be typically small and isodiametric in shape. This region was devoid of rhizobia. A network of infection threads was seen in the infection zone. Poly-(3- hydroxybuterate (phb) inclusions were found in the bacterial cytoplasm. In the nitrogen fixation zone of nodule, the mature bacteroids were elongated. The younger bacteroids exhibited a relatively homogeneous cytoplasm. In the distal part of the nitrogen fixation zone, the bacteroids had proliferated to the extent that the most of the host cell cytoplasm was occupied by elongated bacteroids. The organelles of host cell and bacteroids were found at the peripheral position around the centrally located vacuole. In mature bacteroids, which were present in the proximal part of nitrogen fixation zone, the cytoplasm was heterogeneous due to condensation of nucleic acid material. The transition from the nitrogen fixation zone to senescence zone occurred abruptly. Light microscopic studies of the nodules induced by purine auxotrophs did not show significant differences among themselves. The longitudinal section of whole nodule in each case showed the lack of distinct zones like the parental strain induced nodules; most of the cortical cells were devoid of bacteroids, while a few contained amyloplasts. Infection threads and many peripheral vascular bundles were commonly seen in the nodule sections. At ultrastructural level, the nodules induced by group I auxotrophic mutant NV2 showed infection threads containing bacteria in lytic condition. The released bacteroids started degrading at this stage. The nodules induced by group II (AL7) and group III (VK38) auxotrophs showed a large number of uninvaded cells with starch granules. Some of the cortical cells contained bacteroids with heterogeneous cytoplasm and normal shape, while some others contained bacteroids with heterogeneous (iii) cytoplasm and were abnormal in shape. It seems that the lysis of bacteroids occurred immediately after their release. In the nodules induced by group IV auxotroph VK 27 bacteroidal development appeared to be relatively more advance as compared to that in the nodules induced by the purine auxotrophs of groups I, II and III. These results showed that a normal flow of metabolites through purine biosynthetic pathway is required for successful S. meloliti-a\Mfa symbiosis. Some intermediates and enzymes of purine biosynthetic pathway may have a role in bacteroidal development and maturation.