STUDIES ON THE SYMBIOTIC ROLE OF ARGININE BIOSYNTHETIC PATHWAY OF TWO GENERA OF RHIZOBIA

Abstract

Nitrogen is an essential nutrient for plants. Its deficiency results in reduced agricultural yields throughout the world. It is the most abundant gaseous element constituting about 78% of the earth's atmosphere but metabolically unavailable directly to higher plants or animals. It is available to some species of microorganisms through biological nitrogen fixation (BNF) in which atmospheric dinitrogen is converted to ammonia by the enzyme nitrogenase. Microorganisms that fix nitrogen are called diazotrophs. Symbiosis between legumes and rhizobia occurs within nodules which are induced mainly on the roots and, in a few cases on the stems. The plant supplies energy rich compounds to the diazotrophs, which in turn reduce atmospheric nitrogen to ammonia. This ammonia is transported from the bacteria to the plant to meet the plant's nutritional nitrogen needs for the synthesis of proteins, enzymes, nucleic acids, and chlorophyll. A total of 415 crosses were performed between the donor strain Escherichia coli WA803 (pGS9) and each of the rhizobial recipient strains, viz., Sinorhizobium meliloti strain Rml021, Rhizobium leguminosarum bv. trifolii strain MTCC905 and R. leguminosarum bv. viciae strain Rlvl. The transconjugants were selected on TY agar medium containing kanamycin (Km) (400 ug/ml) and streptomycin (Sm) (100 ug/ml). A total of 20,750 kanamycin resistant (Kmr) transconjugants were generated. The nutritional requirements of suspected auxotrophs were checked on the basis of their colony forming ability on modified Holliday pools. On the basis of their nutritional requirements all the auxotrophs were classified as follows (the number of auxotrophs isolated have been given in brackets); S. meliloti strain Rml021: arginine (7), methionine (10), leucine (2), tryptophan (1) and histidine (1); R. leguminosarum bv. trifolii MTCC905: arginine (4), glutamine (10), valine (15), phenylalanine (1), glycine (1) and histidine (1); R. leguminosarum bv. viciae Rlvl: arginine (4) and aspartic acid (8). The arginine auxotrophs of S. meliloti Rml021 (IITR1, IITR2, IITR3, IITR4, IITR5, IITR6 and IITR7), R. leguminosarum bv. trifolii MTCC905 (IITR8, IITR9, IITR10 and IITR11) and R. leguminosarum bv. viciae Rlvl (IITR12, IITR13, IITR14 and IITR15) were selected for further studies. The reversion frequencies of the arginine auxotrophs were found to vary from 2.0x 10"9 in S. meliloti IITR 6to 5.7x10"9 in R. leguminosarum bv. viciae IITR13. The biochemical block in each arginine auxotroph was determined by feeding the intermediates of the arginine biosynthetic pathway to the auxotroph and subsequently observing the growth. On the basis of intermediate feeding studies, the arginine auxotrophs were placed in the following three categories: (i) argA/argB/argC/argD/argE mutants: IITR1 and IITR2 ofS. meliloti Rml021; IITR8 of R. leguminosarum bv. trifolii MTCC 905; IITR 12 ofR. leguminosarum bv. viciae Rlvl. These auxotrophs grew on RMM supplemented with ornithine or citrulline. These mutants were also designated as ornithine auxotrophs. (ii) argF/argl mutants: IITR3, IITR4, and IITR5 of S. meliloti Rml021; IITR9 of R. leguminosarum bv. trifolii MTCC905; IITR 13 and IITR 14 of R. leguminosarum bv. viciae Rlvl. These auxotrophs grew on RMM supplemented with citrulline but did not grow on ornithine supplemented RMM. (iii) argH mutants: IITR6 and IITR7 of S. meliloti Rml021; IITR10 and IITR11 of R. leguminosarum bv. trifolii MTCC905; IITR15 of R. leguminosarum bv. viciae Rlvl. These auxotrophs grew on RMM supplemented with arginine but did not grow on citrulline or ornithine supplemented RMM. In order to find out the pleiotropic effects of Tn5 insertions on cell surface characteristics, all the auxotrophs were tested for production of cellulose fibrils, lipopolysaccharides, |$(1—*2) glucans, (3 (1—>3) glucans and succinylated exopolysaccharides. All three parental strains and arginine auxotrophs took up the Congo red dye from the medium, grew in presence of sodium deoxycholate, showed motility on swarming media and fluoresced in presence of calcofluor white under UV light. This indicated the normal production of cellulose fibrils, lipopolysaccharides, P-(l—*2) glucans and succinylated exopolysaccharides in the parental strains and auxotrophs. All the auxotrophs and the parental strains did not take up the aniline blue dye from the medium indicating that p-(l—* 3) glucans were not produced in these auxotrophs and parental strains. The growth of the auxotrophs on RMM supplemented with auxotrophic requirements was same as that of the parental strains on RMM. No change in growth was observed when the glucose in RMMmedium was replaced by any one of the other sugars (sucrose, arabinose, xylose, maltose or mannitol) or dicaboxylic acids (malate, aspartate or glutamate) as a sole carbon source. These findings showed that the Tn5 insertions in these auxotrophs did not affect the cell surface characteristics or the uptake / utilization of sugars and dicarboxylic acids. Symbiotic properties of all 15 arginine auxotrophs were determined by inoculating them, along with parental strains, onto their respective host plants, namely, alfalfa {Medicago sativa L.), clover (Trifolium alexandrium L.) and lentil (Lens culinaris Medik.). The S. meliloti Rml021 strain and its argF/argl (IITR3, IITR4 and IITR5) and argH (IITR6 and IITR7) auxotrophs induced cylindrical and pink nodules on both primary and lateral roots of plants of each of the 10 cultivars of alfalfa. The nodules induced by argA/argB/argC/argD/argE auxotrophs (IITR1 and IITR2) were of irregular shape and light pink colour and these were located on both primary and lateral roots ofall the 10 cultivars of alfalfa. The data on mean dry plant weight, total nitrogen content per plant and acetylene reduction activity indicated that the nitrogen fixing efficiencies of the argF/argl and argH mutants were significantly less than nitrogen fixing efficiencies of the parental strain with 8 cultivars of alfalfa; with the remaining 2 cultivars the differences were nonsignificant. However, the nitrogen fixing efficiencies of argF/argl and argH mutants were significantly more than the nitrogen fixing efficiencies of argA/argB/argC/argD/argE mutants on all the cultivars of alfalfa. The dry plant weight and total nitrogen content per plant of argA/argB/argC/argD/argE mutants were very similar to those of the uninoculated plants showing that these strains fixed no or very little nitrogen. Similar results were obtained with the arginine auxotrophs of R. leguminosarum bv. trifolii MTCC905 (IITR10 and IITR11) and R. leguminosarum bv. viciae Rlvl (IITR15) on 4 and 6 cultivars of clover and lentil respectively. Normal symbiosis, like that of the parental strains, was observed when the host plants were inoculated with the spontaneous revertants of ornithine auxotrophs. Reisolation of bacteria from the nodules induced by the arginine auxotrophic mutants showed 100% occupancy of nodules by these mutants. Light microscopic studies of the nodules induced by each of the parental strains S. meliloti Rml021, R. leguminosarum bv. trifolii MTCC905 and R. leguminosarum bv. viciae Rlvl revealed that each nodule had a central tissue surrounded by several peripheral tissues. The central tissue consisted of five zones, viz., meristematic zone, infection zone, interzone, nitrogen fixation zone and senescence zone. Small, uninfected cells and infection threads were seen in the meristematic zone. Nitrogen fixation zone was quite large and most of the cells in this zone contained differentiated rhizobia called bacteroids. Lysed nodule cells were present in the senescence zone. The internal structure of the nodules induced by the argF/argl and argH mutants of S. meliloti Rml021, R. leguminosarum bv. trifolii MTCC 905 and R. leguminosarum bv. viciae Rlvl appeared almost similar to the internal structure of the nodules induced by the parental strains. The nodules induced by the ornithine auxotrophs ofS. meliloti Rml021 (IITR1 and IITR2), R. leguminosarum bv. trifolii MTCC905 (IITR8) and R. leguminosarum bv. viciae Rlvl (IITR12) had distinct peripheral and central tissues. The central tissue in each of these nodules was differentiated into five zones as the nodules induced by the parental strains, however the nitrogen fixation zone was poorly developed and the senescence zone was quite extensive. Transmission electron microscopic (TEM) studies of the ultrathin sections of the nodules induced by the parental strains S. meliloti Rml021, R. leguminosarum bv. trifolii MTCC905 and R. leguminosarum bv. viciae Rlvl revealed the presence of the poly-(3- hydroxybutyrate (Phb) granules in the rhizobial bacteria in the infection threads. The bacteroids were surrounded by a membrane called peribacteroid membrane (Pbm). In the senescence zone some bacteroids had broken Pbm. The cytoplasm of the rhizobial bacteria was electron dense where as the cytoplasm of the bacteroids in the interzone and nitrogen fixation zone contained both electron dense and electron transparent regions. The bacteroids in the nitrogen fixation zone were mostly elongated. The ultrastructure of the nodule induced by the argF/argl (IITR3, IITR4, IITR5, IITR9, IITR13 and IITR14) and argH (IITR6, IITR7, IITR10, IITR11, and IITR15) auxotrophs was almost similar to that of the nodules induced by the parental strains. The TEM studies of the ultrathin sections induced by the ornithine auxotrophs showed the rupturing of Pbm in many bacteroids in the nitrogen fixation zone and bacteroidal cytoplasm had more electron transparent regions in comparison to that of the parental strains. This work indicated that ornithine or an intermediate of ornithine biosynthesis may be required for the transformation of the rhizobial bacteria into bacterioids and normal development of the nitrogen fixation zone during the symbiosis of S. meliloti Rml021, R. leguminosarum bv. trifolii MTCC 905 and R. leguminosarum bv. viciae Rlvl with alfalfa, clover and lentil plants, respectively. Some cultivars of alfalfa and lentil appear to provide the required amount of arginine to the rhizobia in the nodules.