STUDIES ON ISOLEUCINE, VALINE AND LEUCINE AUXOTROPHS OF Sinorhizobium meliloti

Abstract

The present work was undertakento study the symbiotic role of isoleucine, valine, and leucine biosynthetic pathways of Sinorhizobhim meliloti. Random transposon Tn5 mutagenesis was employed to generate isoleucine, valine, and leucine auxotrophs of S. meliloti strain Rmd201 which is a streptomycin resistant derivative of strain AK631. Matings between me donor strain E. coli WA803 carrying suicide plasmid pGS9 and recipient strain S. meliloti Rmd201 yielded 5,450 kanamycin resistant transconjugants from 122 crosses. Thirty transconjugants out ofthese were found to be auxotrophs. Based on the growth patterns of these auxotrophs on nutritional pools, four isoleucine + valine and one leucine auxotrophs were identified. Six isoleucine + valine and two leucine auxotrophs isolated by other researchers in this lab were also included in this study. Intermediate feeding, intermediate accumulation and cross-feeding studies were done to identify the mutated gene in each auxotroph. On the basis of these studies, isoleucine +valine auxotrophs were classified into following categories: Category I: ihB / ilvG mutant (VK4) which grew on Rhizobium minimal medium (RMM) supplemented with a-acetolactate and isoleucine, and accumulated pyruvic acid. Category II: ihC mutants (RH1, RH30, VK5, NV29, SY2 and PS7) which did not grow on RMM supplemented with a-acetolactate and isoleucine, and were cross-fed by RH3, RH18 and VK44. Category III: itvD mutants (RH3, RH18 and VK44) which grew on RMM supplemented with a-keto-pVmethyrvalerate andvaline and were not cross-fed by any other isoleucine+ valine auxotroph. The leucine auxotrophs were placed in the following categories: Category I: leuC / leuD mutant (RS3) which did not grow on RMM supplemented with a-isopropylmalate and was cross-fed by RH14 and SY1 leucine auxotrophs. Category II: leuB mutants (RH14 and SY1) which grew on RMM supplemented with aketoisocaproate and cross-fed RS3 leucine auxotroph. The isoleucine +valine, and leucine auxotrophs were similar to the parental strain w.r.t. cell surface molecules (lipopolysaccharides, cellulose fibrils, succinylated exopolysaccharides and fS-glucans), and utilization of dicarboxylic acids and sugars indicating mat the symbiotic defects of these auxotrophs were not caused by a change in any of the above characteristics. 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 ZB555 (Cys", Phe", Rf, Sin1). Kanamycin resistance transconjugants were selected and tested for the presence of the donor's auxotrophic markers). All kanamycin resistant transconjugants showed donor's auxotrophy. This result confirmed 100% linkage of auxotrophy to Tn5 insertion as well as ruled out the possibility of occurrence ofother independent Tn5 insertions in each mutant Plasmid-mediated mapping method was used to locate the position of transposon Tn5 in each isoleucine + valine auxotroph. The donor strain of each auxotroph, constructed by the introduction of plasmid pJB3H, was mated with four S. meliloti recipient strams ZB555, ZB556, ZB557 and ZB205, the markers of which cover most of the chromosome. In all crosses, selection was made for the transfer of kanamycin resistance marker which is encoded by Tn5. The co-transfer percentages of the u kanamycin resistance marker with the unselected markers were determined by streaking the transconjugants on appropriate plates. Kanamycin resistance showed 24, 9 and 5% co-transfer values with ade-15 marker in isoleucine + valine auxotrophs RHl8, RHl and VK4, respectiverywhen S. melilotiZB205 strain was used as a recipient. Symbiotic properties of isoleucine + valine, and leucine auxotrophs were determined by inoculating mem on alfalfa (Medicago sativa cv. T9) seedlings grown asepticalry on nitrogen-free agar slants. The ihB I ilvG and ilvC mutants did not induce nodules and the Nod* phenotype was not restored by the supplementation of plant nutrient medium with isoleucine and valine, or a-keto-0-methyrvalerate and valine. All HvD mutants induced nitrogen fixing nodules on alfalfa plants and nitrogen fixing efficiencies of these mutants were similar to the nitrogen fixing efficiency of the parental strain Rmd201. All leucine auxotrophs of S. meliloti were Nod+ Fix and the symbiotic defect wasnot restored by supplementation of the plant nutrient medium with leucine or a-ketoisocaproate. The ihB / ihG auxotroph VK4 did not induce root hair curling and infection thread formation. The ihC auxotrophs (RHl, RH30, VK5, NV29, SY2 and PS7) were capable of curling root hair but no infection threads in root hairs were found. The ihD auxotrophs (RH3,- RHl8 and VK44) and all leucine auxotrophs resulted in root hair curlingand infection thread formation. Histological studies revealed that the nodules induced by the leuB mutants were structurally more advanced than the leuC I leuD mutant induced nodules. In the former case rhizobial bacteria were released from the infection thread into plant cells whereas in the latter case the rhizobia did not come out of the infection threads. Histological in structure of the nodule induced by leuB mutant RHl4 was improved to some extent by the supplementation ofthe plant nutrientmediumwith leucine. The above results indicated that the expression of ihB, ihG and ihC genes of S. meliloti is required for induction of nodules onalfalfa plants. The genes leuC I leuD and leuB ofS. meliloti appear to be required for normal nodule development The leuC I leuD gene may be involved in the release of rhizobia from infection threads.