Generation and Characterization of Random Mutants of Phosphate Solubilizing Rhizobia

Abstract

Nitrogen (N) fixing bacteria plays a very important role in biogeochemical cycle in the soil, which results in enhanced plant growth. Rhizobia are present in root nodules which enrich the N pool of the soil. Rhizobia are symbiotic, diazotrophic bacteria which utilizes C4 acids and other key intermediates of TCA cycle. C4 acids like succinate acts as the preferred carbon source repressing the utilization of other sugars, by a mechanism known as succinate mediated catabolite repression (SMCR). Organic acid production plays a paramount role in mineralization of P present in soil. In soil conditions where multiple carbon sources are present, presence of C4 acids may lead to hierarchical carbon source utilization by repressing sugar utilization. This repression of sugar utilization in soil may compromise with the beneficial rhizobial plant growth promotion traits. Hence, the mechanism of SMCR governing sequential carbon source utilization may also govern the PGP traits of rhizobia. Soil and plant root exudates possess various range of carbon sources where some organisms prefer C4 acids over other sugar sources and fail to produce acids responsible for MPS phenotype. In the P solubilizing rhizobia used in this study, solubilization of P is due to the secretion of gluconic acid by the enzyme glucose dehydrogenase (GDH). Therefore, our main aim was to generate and characterize mutants with relieved SMCR or enhanced P solubilization which can serve as a better biofertilizer for promoting plant growth. After several rounds of transformation, seven mutants with unique transposition sites showing altered P solubilization were generated and characterized for change in growth, pH, P release, and GDH activity under normal and repressing conditions. We observed that few of our transformants gave slight pink zone when spot inoculated on TRP GS compared to wild type Td3. TΔ3 and TΔ6 showed maximum GDH activity in glucose containing Pikovskaya’s broth followed by wild type. But under repressing conditions, wild type showed highest activity followed by TΔ6 and then TΔ3. pH of media decreased with an increase in P release indicating that mutation might have altered acid production and hence P release.