GENETIC AND SYMBIOTIC STUDIES ON AROMATIC AMINO ACID AUXOTROPHS OF Sinorhizobium meliloti

Abstract

Random Tn5 insertion mutants of Sinorhizobium meliloti strain Rmd201 were isolated by transposon Tn5 mutagenesis. Conjugation between donor Escherichia coli strain WA803, harbouring the Tn5 on its suicide plasmid pGS9, and recipient S. meliloti Rmd201 resulted in the generation of five thousand kanamycin resistant transconjugants from 85 crosses. Screening these transconjugants yielded in the isolation of twenty two auxotrophs, unable to grow on Rhizobium minimal medium (RMM). Streaking these auxotrophs on nutritional pools resulted in the determination of four auxotrophs of aromatic amino acid biosynthetic pathways (two of tryptophan, one of tyrosine and one of phenylalanine). Two auxotrophs requiring all three aromatic amino acids, viz., tryptophan, tyrosine and phenylalanine, and three tryptophan auxotrophs of S. meliloti Rmd201, isolated by other researchers in the lab were also included in this study. Based on the location of metabolic block in the aromatic amino acid biosynthetic pathways, these auxotrophs were classified into four groups: i. aro mutants (NV3 and BA2): Grew on RMM supplemented with all three aromatic amino acids, ii. trp mutants which were further classified into three subgroups : a. trpE(G) mutants (FN2 and FN3): Grew on RMM supplemented with anthranilic acid, indole or tryptophan and did not accumulate anthranilic acid and indolesglycerol phosphate in RMM. b. trpD, trpF, or trpC mutants (NV7 and NV31): Grew on indole or tryptophan supplemented RMM and accumulated anthranilic acid in RMM. c. trpB mutant (BA6): Grew only on tryptophan supplemented RMM and accumulated anthranilic acid and indole-3-glycerol phosphate in RMM. iii. tyrA mutant (FN4): Grew only on tyrosine supplemented RMM. iv. pheA mutant (FN9): Grew only on phenylalanine supplemented RMM. Similar to the parental strain Rmd201, all aromatic amino acid auxotrophs were able to infect the root hairs and form nodules on the roots of alfalfa (Medicago sativa cv. T9) plants (Nod+). However, the nodules induced by aro, trpE(G) and pheA mutants were symbiotically ineffective (Fix"). The plants nodulated by these mutants were weak, stunted and became chlorotic six weeks after inoculation which showed the inability of these auxotrophs to fix nitrogen. The remaining Tn5 insertion mutants, represented by trpD, trpF or trpC, trpB and tyrA mutants elicited fully effective nodules on the roots of alfalfa plants. These plants were healthy, green (indicating that nitrogen was being fixed) and resembled the parental strain Rmd201 inoculated plants in all respects. The symbiotic ability of trpE(G) mutants was restored on supplementation of plant growth medium with anthranilic acid (upto 10 fig/ml). However, supplementation of plant growth medium with indole (upto 5 ug/ml) or tryptophan (upto 20 ug/ml) could not restore the symbiotic ability of trpE(G) mutants. At 2.5 ug/ml supplementation of anthranilic acid, only partial restoration was observed, whereas complete restoration took place at 5, 7.5 and 10 ug/ml supplementation of anthranilic acid. The trpE(G) mutants, supplemented with 2.5 ug/ml of anthranilic acid, formed slightly pink nodules on alfalfa roots. The mean height and dry shoot weight of these plants differed significantly from those of the parental strain Rmd201 inoculated plants and with the uninoculated controls. However, the trpE(G) mutants inoculated plants, supplemented with 5-10 ug/ml of anthranilic acid, resembled those of the parental strain Rmd201 inoculated plants in all aspects. Atomic absorption spectrometer analysis of iron uptake by auxotrophs revealed that the trpE(G) mutants, in grown on minimal medium having iron (supplemented with minimal nutritional requirements of the auxotrophs), took up less amount of iron than anthranilic acidproducing mutants. These findings suggest that anthranilic acid has a role in symbiotic process. The symbiotic functions of pheA and aro mutants were not restored by addition of phenylalanine (upto 30 u.g/ml) and aromatic amino acids (upto 5 fig/ml), respectively, to the plant growth medium. Light microscopic observations of nodules induced by the parental strain Rmd201 showed normal developmental stages of the distinct zones, viz., distal meristematic, infection, amyloplast-rich inter, nitrogen-fixing and proximal senescence zones. Transmission electron microscopy of these zones revealed all stages of bacteroid development. The microscopic observations of nodules elicited by aro and trpE(G) mutants exhibited striking similarities. Each of these nodules showed extensive infection zone which occupied most part of the middle nodule tissues, while the nitrogen-fixing region was poorly developed and restricted to a few layers just beneath the infection zone. The bacteroids in these zones did not show functional maturation. The senescence zone occupied almost one third of the nodules and contained deteriorating bacteroids. Histology of nodules formed by trpE(G) mutants, supplemented with 2.5 fig/ml of anthranilic acid, showed that the rhizobial release into the nodule cells was normal and in most of the rhizobial cells differentiation into bacteroidal state was almost complete. Nitrogen-fixing zones of these nodules were not fully developed and contained bacteroids in a degenerating condition. However, in the nodules induced by the same mutants, supplemented with 5, 7.5 or 10 u,g/ml of anthranilic acid, the nitrogen-fixing zones were fully developed. IV Light and electron microscopy of nodules elicited by pheA mutant exhibited no distinctive cellular zones. The release of rhizobial bacteria into nodule cells was normal, but the differentiation of released bacteria into bacteroids was not complete and they were observed to be in a degenerating condition after their release. Microscopy of trpD, trpF or trpC, trpB and tyrA mutant-nodules showed normal developmental stages. The internal histological features of these nodules at structural and ultrastructural levels resembled those of the nodules induced by Rmd201 strain in all respects. The aromatic amino acid auxotrophs were similar to the parental strain Rmd201 with respect to the production of cell surface carbohydrate molecules (pglucans, cellulose fibrils, lipopolysaccharides and succinylated exopolysaccharides), utilization of carbon sources (C4-dicarboxylic acids and sugars) and production of cytochrome coxidase indicating that the symbiotic defects of aro, trpE(G) and pheA mutants were not caused by a change in any of the above mentioned characteristics. The defective symbiosis of these auxotrophs could be merely due to the loss of biosynthetic gene functions through Tn5 insertion into aro, trpE(G) and pheA genes. Hence, the functions of aro, trpE(G) and pheA genes involved in aromatic amino acid biosynthetic pathways of S. meliloti are required for an effective nodule development and optimal symbiotic nitrogen fixation. However, the informations regarding the role they play in symbiosis are not well established. The restoration of the symbiotic ability of trpE(G) mutants with exogenous supplementation of anthranilic acid confirmed its role in symbiosis, most probably by facilitating iron uptake which plays significant role in nitrogen fixation.