Biochemical Basis of Repression of Mineral Phosphate Solubilization (MPS) Phenotype in Rhizobial Species

Abstract

The present study focuses on investigating the mechanism of phosphate solubilization in nitrogen fixing rhizobia, as such strains may provide dual advantage to the plants by providing fixed N and free P. Td3 and SN1 are phosphate solubilizing nodule rhizobia of Cajanus cajan and Sesbania rostrata respectively. They solubilized 423μg/mL and 428μg/mL phosphate from tricalcium phosphate through secretion of 19.2mM and 29.6mM gluconic acid respectively, when grown in 100mM glucose. However, 90% and 80% reduction in phosphate solubilization coupled to production of 40mM (Td3) and 28.2mM (SN1) gluconic acid was observed when the two isolates were grown in 100mM glucose+50mM succinate (repression medium). Our results illustrated the role of succinate irrepressible glucose dehydrogenase (gcd) in phosphate solubilization and the role of succinate: proton symport in succinate mediated repression of phosphate solubilization in these rhizobia. Calcium ion supplementation reduced the 88% and 72% repression in P solubilization to 18% and 9% in Td3 and SN1 respectively coupled to a reduction in media pH from 6.8 to 4.9 in Td3 and 4.8 in SN1. Hence, succinate mediated repression of P solubilization had no genetic basis and was purely due to the biochemical interplay of protons and other cations, which was in contrast to other reported rhizobial strains, in this regard. Studying the genome of Td3 was important as it had succinate irrepressible glucose dehydrogenase and could not solubilize phosphate in glucose+succinate conditions, in spite of gluconic acid production which was enough to achieve mineral phosphate solubilization. This is in contrast to the other explored rhizobial phosphate solubilizers. Rhizobial isolates sequenced so far are known to lack components of the direct glucose oxidation pathway and cannot produce gluconic acid on its own. Here, we present the genome sequence of Rhizobium sp. Td3 that comprises of a single chromosome of size 5,598,346 bp (5.6 Mb) with no symbiotic plasmid. Rhizobium leguminosarum bv. viciae USDA2370 was the closest genome known. Genome annotations and mining identified approximately 544 genes probably responsible for diverse phytobeneficial activities including mineral phosphate solubilization, organic phosphate mineralization, siderophore production and transport, nodulation, N2 fixation, acetoin production, nitric oxide synthesis, auxin biosynthesis, flagellar proteins, exopolysaccharide biosynthesis, metal resistance, stress response, multidrug resistance, etc. Availability of genome sequence of such a versatile plant growth promoting bio-agent will help in deeper understanding of a unique genome and the phytobeneficial traits it bears for better exploitation of the organism for field applications. RNA-seq analysis of Td3 grown in glucose and succinate revealed that SMCR exhibited by Td3 is only partial as the genes that are supposedly reported to be repressed by succinate in other rhizobia were not repressed in Td3. This may be attributed to the prevalence of an altered SMCR in Td3 or because the regulation of SMCR was not the transcriptional level. Also, expression of the DctM dicarboxylate transporters which function by co-transporting protons with succinate validates the model proposed in this study for the repression in phosphate solubilization by succinate. The detailed investigations of these mechanisms would instigate better understanding of the rhizobial behaviour in soil. Such studies would help to gain further insights into extending the possible practical applications of the strain as a bio fertilizing agent.