Generation of Site Specific Mutants of Phosphate Solubilizing Rhizobia for Altered Carbon Source Utilization

Abstract

Two phosphate solubilizing rhizobia were re-characterized for tri-calcium phosphate and rock phosphate solubilization. They were further studied for succinate mediated repression of phosphate solubilization, both qualitatively and quantitatively. For generating rhizobial mutants with constitutive phosphate solubilization phenotype under field conditions, transforming rhizobia with desired plasmid at high efficiency was important. As transformation with rhizobia is not easy, the same was standardized using multiple protocols. A high efficiency transformation protocol for rhizobia has been established with two plasmids, one encoding erythromycin resistance gene and another encoding kanamycin resistance gene. A vector was designed with an attempt to use the generated plasmid as a universal vector for generation of insertional inactivation of desired target for erythromycin and kanamycin sensitive bacteria. Vector encoded for erythromycin and kanamycin resistance gene with lac promoter controlled gfp with temperature sensitive origin of replication to facilitate generation of insertional inactivation mutants using the principle of double homologous recombination. Further, multiple rhizobial and agrobacterial genomes were analysed bioinformatically to determine the putative glucose transporter and putative regulators controlling succinate mediated catabolite repression of phosphate solubilization and preferential organic acid utilization in rhizobia. Primers for such nine shortlisted targets were designed and the amplification was standardized for all the nine genes.