Effect of succinate on phosphate solubilization in nitrogen fixing bacteria harbouring chick pea and their effect on plant growth.

Diverse nitrogen fixing bacteria harbouring chick pea rhizosphere and root nodules were tested for multiple plant growth promoting traits like tricalcium phosphate (TCP) and rock phosphate (RP) solubilization, production of ammonia, indole 3-acetic acid, chitinase, phytase and alkaline phosphatase. Isolates belonged to diverse genus like Enterobacter, Acinetobacter, Erwinia, Pseudomonas, Rhizobium, Sinorhizobium, Ensifer, Klebsiella, etc. Most isolates solubilized TCP and RP along with the lowering of media pH, indicating acidification to be the chief mechanism behind this solubilization. However, lowering of media pH and P release decreased by 32-100% when media was supplemented with succinate, a major component of plant root exudates indicating succinate mediated repression of P solubilization. Maximum TCP and RP solubilization with P release of 850µg/mL and 2088µg/mL was obtained with lowering of media pH up to 2.8 and 3.3 for isolate E43 and PSB1 respectively. This pH drop changed to 4.4 and 4.8 with 80% and 87% decrease in P solubilization in the presence of succinate. Maximum 246µg/mL indole 3-acetic acid production in Lh3, 44.8U/mL chitinase activity in MB3, 11.3U/mL phytase activity in I91 and 9.4U/mL alkaline phosphatase activity in SM1 were also obtained. Most isolates showed multiple PGP traits which resulted in significant plant growth promotion of chick pea plants. Present study shows repression of P solubilization by succinate for various bacterial groups which might be one of the reasons why phosphate solubilizing bacteria which perform well in vitro often fail in vivo. Studying this repression mechanism might be critical in understanding the in vivo efficacy.

Organic acid mediated repression of sugar utilization in rhizobia.

Rhizobia are a class of symbiotic diazotrophic bacteria which utilize C4 acids in preference to sugars and the sugar utilization is repressed as long as C4 acids are present. This can be manifested as a diauxie when rhizobia are grown in the presence of a sugar and a C4 acid together. Succinate, a C4 acid is known to repress utilization of sugars, sugar alcohols, hydrocarbons, etc by a mechanism termed as Succinate Mediated Catabolite Repression (SMCR). Mechanism of catabolite repression determines the hierarchy of carbon source utilization in bacteria. Though the mechanism of catabolite repression has been well studied in model organisms like E. coli, B. subtilis and Pseudomonas sp., mechanism of SMCR in rhizobia has not been well elucidated. C4 acid uptake is important for effective symbioses while mutation in the sugar transport and utilization genes does not affect symbioses. Deletion of hpr and sma0113 resulted in the partial relief of SMCR of utilization of galactosides like lactose, raffinose and maltose in the presence of succinate. However, no such regulators governing SMCR of glucoside utilization have been identified till date. Though rhizobia can utilize multitude of sugars, high affinity transporters for many sugars are yet to be identified. Identifying high affinity sugar transporters and studying the mechanism of catabolite repression in rhizobia is important to understand the level of regulation of SMCR and the key regulators involved in SMCR.

Derepression of Mineral Phosphate Solubilization Phenotype by Insertional Inactivation of iclR in Klebsiella pneumoniae.

The mode of succinate mediated repression of mineral phosphate solubilization and the role of repressor in suppressing phosphate solubilization phenotype of two free-living nitrogen fixing Klebsiella pneumoniae strains was studied. Organic acid mediated mineral phosphate solubilization phenotype of oxalic acid producing Klebsiella pneumoniae SM6 and SM11 were transcriptionally repressed by IclR in presence of succinate as carbon source. Oxalic acid production and expression of genes of the glyoxylate shunt (aceBAK) was found only in glucose but not in succinate- and glucose+succinate-grown cells. IclR, repressor of aceBAK operon, was inactivated using an allelic exchange system resulting in derepressed mineral phosphate solubilization phenotype through constitutive expression of the glyoxylate shunt. Insertional inactivation of iclR resulted in increased activity of the glyoxylate shunt enzymes even in succinate-grown cells. An augmented phosphate solubilization up to 54 and 59% soluble phosphate release was attained in glucose+succinate-grown SM6Δ and SM11Δ strains respectively, compared to glucose-grown cells, whereas phosphate solubilization was absent or negligible in wildtype cells grown in glucose+succinate. Both wildtype and iclR deletion strains showed similar indole-3-acetic acid production. Wheat seeds inoculated with wildtype SM6 and SM11 improved both root and shoot length by 1.2 fold. However, iclR deletion SM6Δ and SM11Δ strains increased root and shoot length by 1.5 and 1.4 folds, respectively, compared to uninoculated controls. The repressor inactivated phosphate solubilizers better served the purpose of constitutive phosphate solubilization in pot experiments, where presence of other carbon sources (e.g., succinate) might repress mineral phosphate solubilization phenotype of wildtype strains.

Repression of oxalic acid-mediated mineral phosphate solubilization in rhizospheric isolates of Klebsiella pneumoniae by succinate.

Two strains of Klebsiella (SM6 and SM11) were isolated from rhizospheric soil that solubilized mineral phosphate by secretion of oxalic acid from glucose. Activities of enzymes for periplasmic glucose oxidation (glucose dehydrogenase) and glyoxylate shunt (isocitrate lyase and glyoxylate oxidase) responsible for oxalic acid production were estimated. In presence of succinate, phosphate solubilization was completely inhibited, and the enzymes glucose dehydrogenase and glyoxylate oxidase were repressed. Significant activity of isocitrate lyase, 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.