ROLE OF ARGININE BIOSYNTHETIC PATHWAY OF Sinorhizohium meliloti IN SYMBIOSIS

Abstract

Biological nitrogen fixation, via the legume-rhizobium symbiosis, is a renewable and natural source of nitrogen for plants. Rhizobia enter into symbiotic relationship with legumes and induce the formation of root nodules where the atmospheric nitrogen is fixed in the form of ammonia by the changed form of rhizobial bacteria called bacteroids. A number of genes and nutritional conditions in the developing bacteroids are supposed to affect nitrogen fixation. Earlier reports have shown that some metabolites of the biosynthetic pathways of some amino acids, nucleotide bases and vitamins have a role in symbiosis. This work was taken up with the aim to study the role of arginine biosynthetic pathway of Sinorhizobium meliloti in symbiosis. S. meliloti Rmd201, a streptomycin resistant derivative of the strain AK631, was conjugated with Escherichia coli WA803 (pGS9). Kanamycin resistant transconjugants of S. meliloti were selected by plating the mating mixture on complete medium (Tryptone yeast extract) agar plates containing kanamycin (400 ixg/ml) and streptomycin (100 ug/ml). A total of 7,650 Tn5- induced kanamycin resistant transconjugants were obtained from 128 crosses. These transconjugants were screened for auxotrophs by streaking on Rhizobium minimal medium (RMM). The nutritional requirements of the auxotrophs were determined on RMM supplemented with Holliday pools. Following auxotrophs (the number of each type is given in brackets) were obtained: arginine (15), methionine (3), tryptophan (2), cysteine (7), uracil (3) and adenine (2). The 15 arginine-requiring auxotrophs (AK3, AK4, AK5, AK6, AK7, AK8, AK9, AK10, AK11, AK12, AK13, AK14, AK15, AK16, AK25) obtained were used for further studies. The frequency of spontaneous reversion to prototrophy was determined for each auxotroph by plating a known number of mutant cells on RMM. The reversion frequencies varied from 1.0 x10"9 for AK5 to 3.0 x 10"9for AK15. When transposon Tn5-encoded kanamycin resistance marker of each arginine auxotroph was transferred to S. meliloti ZB201 strain, all kanamycin resistant transconjugants (150 in each case) were found to be arginine auxotrophs. One hundred per cent co-transfer of Tn5 and auxotrophy indicated complete linkage of transposon Tn5 insertion to auxotrophy. The biochemical block in each arginine-requiring auxotroph was determined by streaking it on the minimal medium (RMM) supplemented with different intermediates, viz. ornithine, citrulline and arginosuccinic acid, of the arginine biosynthetic pathway. On the basis of intermediate feeding arginine auxotrophic mutants were classified into following four categories: ( i ) argA/argB/argC/argD/argE mutants (AK3, AK4 and AK5) which grew on RMM supplemented with ornithine, citrulline or arginosuccinic acid; these mutants were also designated as ornithine auxotrophs, (ii) argF/argl mutants (AK6, AK7, AK8, AK10, AK11, AK13, AK14, AK15, AK16 and AK25 ) which did not grow on RMM supplemented with ornithine but grew on RMM supplemented with citrulline or arginosuccinic acid, ( iii ) argG mutant (AK12) which did not grow on RMM supplemented with ornithine or citrulline but grew on RMM supplemented with arginosuccinic acid and ( iv ) argH mutant (AK9) which did not grow on RMM supplemented with ornithine, citrulline or arginosuccinic acid. All the mutants, like the parental strain Rmd201, showed growth in presence of sodium deoxycholate (DOC), took up congo red dye, fluoresced in presence of calcoflour white under the UV light and showed motility on swarm plates, indicating the normal production of lipopolysaccharides, cellulose fibrils, succinylated exopolysaccharides and P(1^2) glucans, respectively. These auxotrophs did not show binding with aniline blue dye which indicated that p(1^3) glucans, like the parental strain, were not produced. These results showed that the Tn5 insertions had no pleiotropic effects on cell surface characteristics. No change in growth of arginine auxotrophs was observed when glucose in RMM medium was replaced by any one of the other sugars (sucrose, arabinose, xylose, maltose, lactose, galactose, mannitol or fructose) or dicarboxylic acids (malic acid, succinic acid, aspartic acid, glutamic acid or fumaric acid) as a sole carbon source. These findings showed that the uptake/utilization of sugars and dicarboxylic acids was not affected by Tn5 insertions in the arginine auxotrophs. The symbiotic properties of arginine auxotrophs were determined by inoculating alfalfa {Medicago sativa cv. T9 and cv. A2) seedlings grown aseptically on nitrogen free agar media slants with these auxotrophs. The parental strain Rmd201 and the arginine mutants in the later part of the pathway (after ornithine) induced cylindrical, pink nodules on both primary and lateral roots of plants of both alfalfa cultivars, T9 and A2. These results indicated that the nitrogen fixing efficiencies of these mutants were similar to that of the parental strain. The nodules induced by ornithine auxotrophs and AK10, an argF/argl mutant, were spherical/irregular and white; these nodules were located mostly on the lateral roots. These results indicated that these auxotrophs did not fix nitrogen. Normal symbiosis, like that of the parental strain, was observed when alfalfa plants were inoculated with the spontaneous revertants of ornithine auxotrophs. The reisolation studies showed 100% occupancy of nodules by the arginine auxotrophs. Light microscopic studies of the longitudinal section of the nodule induced by the parental strain Rmd201 showed a central tissue surrounded by several peripheral tissues. The central tissue was differentiated into five zones, viz., meristematic zone, infection zone, interzone between infection and nitrogen fixation zones, nitrogen fixation zone and senescence zone. The internal structural features of the nodules induced by arginine auxotrophs argF/argl (AK11), argG (AK12) and argH (AK9) were similar to those of the parental strain induced nodules. Distinct peripheral and central tissues like those in the parental strain induced nodules were seen in the nodules of the ornithine auxotrophs AK3 and AK4. The central tissues of these nodules, like that of the parental strain induced nodules, were differentiated into five zones; however, the infection and the senescence zones were larger than those of Rmd201 nodules. The nitrogen fixation zone in the nodules of ornithine auxotrophs was identified on the basis of its visual resemblance to the nitrogen fixation zone of the Rmd201 induced nodules. In comparison to the parental strain induced nodule, few nodule cells were infected with rhizobia in the interzone and the so-called nitrogen fixation zone. TEM studies of the ultrathin section of the nodule induced by the parental strain Rmd201 showed the presence of poly-, -hydroxy butyrate (PHB) granules in the rhizobial bacteria in infection threads and in freshly released rhizobia in nodule cells. Peribacteroid membrane (pbm) surrounded each freshly released bacterial cell. Some bacteroids in the senescence zone had broken pbm. The cytoplasm of the rhizobial bacteria in the infection zone was electron dense whereas the cytoplasm of the bacteroids in the interzone and nitrogen fixation zone was heterogeneous in the sense that it contained electron dense and electron transparent regions. The cytoplasm of the bacteroids in the senescence zone was electron transparent. Most of the bacteroids in the nitrogen fixation zone were elongated. All stages of the infection of plant cells by rhizobial bacteria and the subsequent bacteroidal development in the nodules induced by the ornithine auxotrophs were similar to those seen in the parental strain induced nodule except that the bacteroids in the so-called nitrogen fixation zone of ornithine mutants were mostly spherical or oval. TEM studies revealed that the internal structures of the nodules induced by argF/argl, argG and argH mutants were similar to the internal structure of the nodule induced by the parental strain Rmd201. The induction of fully effective nodules by arginine auxotrophs, each having a biochemical block in one of the last three steps (i.e. after ornithine) of the arginine biosynthetic pathway (with the exception of one mutant AK10), on two cultivars of alfalfa plants showed that alfalfa host plant is able to provide the required quantity of arginine to S. meliloti bacteria during symbiosis. AK10 mutant, apart from having a Tn5 insertion in the argF gene, appears to have another mutation in one of its symbiotic genes. The studies on the ornithine requiring arginine auxotrophs indicate that ornithine or an ornithine-derived factor is required for the normal development of nitrogen fixation zone and complete transformation of rhizobial bacteria into bacteroids during the symbiosis of S. meliloti with alfalfa plants. Normal symbiotic activity of the prototrophs of ornithine auxotrophs indicated that a single Tn5 insertion in each of these auxotrophs was responsible for auxotrophy and the symbiotic defect.