STUDIES ON AUXOTROPHIC MUTANTS OF SULFUR-CONTAINING AMINO ACIDS OF Sinorhizobium meliloti

Abstract

The present work was taken up to focus on the influence of the biosynthetic pathways of sulfur-containing amino acids of Sinorhizobium meliloti on the symbiotic characteristics of this bacterium. S. meliloti strain Rmd201, a streptomycin resistant derivative of the strain AK631, was mutagenized with transposon Tn5 that codes for resistance to kanamycin. The suicide vector pGS9 carrying Tn5 was introduced into S. meliloti from Escherichia coli strain WA803(pGS9) by conjugation. Kanamycin resistant (Km') transconjugants were selected by plating the mating mixture on tryptone yeast extract (TY) agar medium containing kanamycin (400 |Jg/ml) and streptomycin (100 (Jg/ml). Three thousand Tn5 derivatives (Km1) of S. meliloti strain Rmd201 were obtained and out of these derivatives 12 did not grow on Rhizobium minimal medium (RMM). These were considered to be auxotrophs and purified for single colonies on TY agar medium containing kanamycin and streptomycin. On the basis of growth on RMM supplemented with modified Holliday pools, the auxotrophs were classified as follows (the number of each kind is given in brackets): adenine (2), adenine + thiamine (1), cysteine (2), methionine (1), uracil (2), tryptophan (1) and tryptophan + tyrosine + phenylalanine (1). The nutritional requirement(s) of two auxotrophs could not be determined. Three sulfur amino acid auxotrophs isolated during this study and 24 such auxotrophs generated previously in this lab were used for further studies. Based on the results of cross feeding and intermediate feeding studies sulfur amino acid auxotrophs were placed in the following categories: Category I: Cysteine auxotrophs (BA4, BA7, VK6, VK10, VK13, VK17, VK20, VK32, VK33, NV8, NV11, NV15, NV16, NV17, NV20, NV25, NV30 and NV55); grew on RMM supplemented with sodium sulfide but not sodium sulfite and hence these auxotrophs were sulfite reductase {cysIlcysJIcysG) mutants. Further studies showed that these auxotrophic mutants were cysl/cysj mutants. Category II: metAlmetZ methionine auxotrophs (BA8, VK21, VK31, NV22, NV35, and NV36); grew on RMM supplemented with cystathionine orhomocysteine. Category HI: metE methionine auxotrophs (VK29 and VK36); did not grow on RMM supplemented with cystathionine or homocysteine but grew on RMM supplemented with cyanocobalamin (vitamin B12). Category IV: metF methionine auxotroph (VK39); did not grow on RMM supplemented with cystathionine, homocysteine or cyanocobalamin. All cysl/cysj mutants were able to grow on RMM agar medium supplemented with methionine, homocysteine or cystathionine. The methionine auxotrophs did not show any growth in the liquid RMM medium supplemented with cysteine. For confirming the linkage of Tn5 insertion to auxotrophy in each auxotroph, the plasmid pJB3JI was used to mobilize the Tn5 containing chromosomal segment into S meliloti recipient strain ZB557 (Phe\ Leu", Rf, Smr). Kmr transconjugants were selected and tested for the presence of the donor's auxotrophic marker. All Kmr transconjugants obtained showed donor's auxotrophy. This result confirmed 100% linkage of auxotrophy to Tn5 insertion as well as ruled out the possibility of occurrence of other independent Tn5 insertions in each mutant. The cysteine and methionine auxotrophs were similar to the parental strain with respect to the production of cell surface molecules (lipopolysaccharides, cellulose fibrils, succinylated exopolysaccharides and 3-glucans) and utilization of dicarboxylic acids and sugars indicating that the Tn5 insertions in these mutants did not result in any change in the cell surface molecules or carbon source utilization properties and the symbiotic defects of methionine auxotrophs were not caused by a change in any of the above characteristics. Symbiotic properties of sulfur-containing amino acid auxotrophs were determined by inoculating alfalfa (Medicago sativa) seedlings grown aseptically on nitrogen free agar slants with these auxotrophs. The parental strain Rmd201 and all its cysteine auxotrophs induced pink nodules. The dry weights of the plants inoculated with the cysteine auxotrophs did not differ significantly from that of the plants inoculated with the parental strain indicating that the nitrogen fixing abilities of the cysteine auxotrophs were similar to that of the parental strain. All methionine auxotrophs formed white nodules which did not fix nitrogen. Delayed nodulation and reduction in nodule number per plant were observed in case of methionine auxotrophs. Normal symbiosis was restored on addition of methionine to plant growth medium or when alfalfa plants were inoculated with methionine revertants. Nodules from six weeks old alfalfa plants were fixed and embedded in araldite embedding medium. Semithin and ultrathin sections were obtained and observed under light and transmission electron microscopes, respectively. The parental strain Rmd201 induced nodule had the following distinct zones, viz., apical meristem, infection, inter. nitrogen fixation and senescence, in the central tissue. A large number of bacteroids were arranged around a centrally located large vacuole in each infected nodule cell. All stages of bacteroidal development were visible under transmission electron microscope. Histological studies of the metA/metZ auxotrophs revealed that the nitrogen fixation zone in the nodule induced by each of these mutants was not fully developed like that of the nodule induced by the parental strain Rmd201. Some differences were found in the histological structures of the nodules induced by metE and metFmutants.