Carbon Catabolite Repression of Organic Acids in Phosphate Solubilizing Klebsiella Species

Abstract

Carbon catabolite repression (CCR) governs uptake and metabolism of preferred and secondary carbon sources. CCR may have effect on carbon source dependent plant growth promoting (PGP) properties like phosphate (P) solubilization. Glucose is by far the best carbon source known for organic acid production linked P solubilization. But catabolite repression may prevent the preferential utilization of glucose or the organic acid production. PGP Bacteria with dual property of nitrogen (N2) fixation and P solubilization may solve dual purpose for plant providing it with fixed N2 and soluble P. We looked for the succinate mediated catabolite repression of the organic acid production linked P solubilization. Two strains of free living N2 fixing Klebsiella (SM6 and SM11) were isolated from rhizospheric soil that solubilized mineral phosphate (Ca-P and Rock P) by secretion of organic acid from glucose. HPLC analysis showed that oxalic acid was the major organic acid produced by both the strains when bacteria are grown in glucose. P solubilization was observed only when glucose was the sole carbon source and was completely inhibited in presence of succinate singly or along with glucose. Enzymes activities of glucose dehydrogenase (GDH; periplasmic glucose oxidation), isocitrate lyase (ICL) and glyoxylate oxidase (GO) responsible for oxalic acid production were estimated. The enzymes GDH, ICL and GO were repressed in succinate grown cells. While significant activity of ICL, the key enzyme for carbon flux through glyoxylate shunt and oxalic acid production during growth on glucose suggested that it could be inducible in nature, and its inhibition by succinate appeared to be similar to catabolite repression. Diauxic growth profile showed that succinate was not preferentially utilized over glucose. Expression studies carried out form glucose, succinate and glucose+succinate grown cells of both isolates showed that iclR, repressor of glyoxalate shunt, was expressed when growth media contained succinate and the repression was at transcriptional level. Oxalic acid production and expression of genes of glyoxylate shunt (aceBAK) was found only in glucose but not in succinate or glucose+succinate grown cells. iclR was inactivated out using allelic exchange system resulting in derepressed MPS phenotype by constitutive expression of glyoxylate shunt. Insertional inactivation of iclR resulted in increased activity of glyoxylate shunt enzymes even in succinate grown cells. An improved P solubilization upto ~53.8% and ~59% soluble phosphate release was attained in glucose+succinate grown SM6 iclR:kan and SM11 iclR:kan respectively compared to glucose grown cells which was otherwise absent or negligible in wild type cells grown in glucose+succinate. Both wildtype and iclR knockout strains showed similar IAA production. Inoculation of wheat seeds with wildtype SM6 and SM11 improved root length by ~1.2 fold and shoot length by ~1.2 fold. However, iclR knockout strains SM6 iclR:kan and SM11 iclR:kan increased root and shoot length by ~1.5 and ~1.4 folds respectively compared to uninoculated controls. The repressor knockout phosphate solubilizers may better serve the purpose of constitutive P solubilization even in field conditions where presence of other carbon sources (succinate) may repress preferential utilization of glucose and hence mineral phosphate solubilization phenotype of wildtype strains.