Sequential uptake of aldoses over fructose and enhanced phosphate solubilization in Rhizobium sp. RM.

Rhizobium sp. RM solubilized tri-calcium phosphate (TCP: 324-463 µg ml-1) and rock phosphate (RP: 36-46.58 µg ml-1) in the presence of common rhizospheric sugars-glucose, arabinose, xylose and their combinations. Fructose, though did not support RP solubilization individually, surprisingly solubilized significantly higher phosphate when combined with aldoses. The highest TCP (644 µg ml-1) and RP (75 µg ml-1) solubilization was achieved in fructose + glucose combination. Presence of gluconate, malate and oxalate in culture supernatant indicated functioning of periplasmic glucose oxidation, the non-phosphorylative arabinose dehydrogenase pathway and the tricarboxylate (TCA) cycle, respectively. Aldoses, when present together, were co-utilized (monoauxic growth) however, when added with fructose, prevented the uptake of fructose yielding a typical diauxic growth. This presented an unusual sequential utilization of aldoses over a ketose (fructose) in strain RM. The prevention of fructose uptake by aldoses was investigated through real-time expression of key genes coding fructose transport proteins and initial enzymes of sugar metabolism. Fructose was actively transported via fructose-specific ABC transporters as suggested by upregulation of frcB and frcC only in fructose and fructose growth phases of fructose + aldose combinations. The probable route of initial fructose metabolism involved either fructokinase and/or xylose isomerase, as confirmed by enzyme activities. The upregulation of hfq and hprK genes only in aldose phase of fructose + aldose combinations suggested their possible involvement in governing the preferential utilization. The novel aspects of this study are enhanced organic acid mediated P solubilization in fructose + aldose combinations and a rare hierarchy of aldoses over fructose which is possibly regulated at the level of fructose transport and fructokinase. KEY POINTS: • Sugars when provided in different dual combinations, supported enhanced P solubilization from complex phosphate sources like TCP and RP in Rhizobium sp. RM. • Transcriptional status of genes in cells of RM when grown in different individual sugars and their combinations suggested that fructose might be a less preferred carbon source and hence was utilized after aldoses with the possible regulation by Hfq and HPrK. • First study to present a unique phenomenon of sequential utilization of aldoses (glucose, arabinose and xylose) over fructose in a concentration-independent manner in Rhizobium sp. RM. and to present the effect of dual combinations of sugars on organic acid mediated P solubilization trait of rhizobia.

Glucose and arabinose dependent mineral phosphate solubilization and its succinate-mediated catabolite repression in Rhizobium sp. RM and RS.

Rhizobium sp. RM and RS are Vigna radiata root nodule isolates with the ability to solubilize tricalcium phosphate and rock phosphate under 50 mM Tris-Cl buffering conditions. Rhizobium sp. RM and RS were unique as they could produce two different organic acids, gluconic acid and oxalic acid using glucose and arabinose, respectively, which are two of the most prominent sugars present in the rhizospheric soil. However, P solubilization in these isolates was repressed in the presence of succinate resembling the phenomenon of catabolite repression. RM and RS produced 24 mM and 20 mM gluconic acid in presence of glucose which was repressed to 10 mM and 8 mM, respectively, in glucose + succinate conditions. Similarly, RM and RS produced 28 mM and 23 mM oxalic acid in arabinose containing media which was repressed to 9 mM and 8 mM, respectively, in the presence of arabinose + succinate. Results of enzyme activities indicated 66% repression of quinoprotein glucose dehydrogenase in glucose + succinate compared to glucose grown cells and 84% repression of glyoxylate oxidase in arabinose + succinate compared to arabinose grown cells. This is perhaps the first report on mechanism of P solubilization in rhizobia through utilization of two different sugars, glucose and arabinose and its repression by succinate. Succinate-mediated catabolite repression of arabinose is the unique aspect of this study.

Succinate irrepressible periplasmic glucose dehydrogenase of Rhizobium sp. Td3 and SN1 contributes to its phosphate solubilization ability.

Td3 and SN1 are phosphate-solubilizing nodule rhizobia of Cajanus cajan and Sesbania rostrata, respectively. They solubilized 423 µg/mL and 428 µg/mL phosphate from tricalcium phosphate through the secretion of 19.2 mM and 29.6 mM gluconic acid, respectively, when grown in 100 mM glucose. However, 90% and 80% reduction in phosphate solubilization coupled to the production of 40 mM (Td3) and 28.2 mM (SN1) gluconic acid was observed when the two isolates were grown in 50 mM succinate + 50 mM glucose. Our results illustrate 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 6.3 to 4.8 in SN1. Hence, repression had no genetic basis and is purely due to the biochemical interplay of protons and other cations.

Genome sequence and comparative genomics of Rhizobium sp. Td3, a novel plant growth promoting phosphate solubilizing Cajanus cajan symbiont.

Rhizobium sp. Td3 is a Sesbania plant growth promoting, Cajanus cajan nodulating rhizobia. Studying its whole genome was important as it is a potent phosphate solubilizer with constitutive gluconic acid production ability through operation of the periplasmic glucose oxidation pathway even under conditions of catabolite repression. This is in contrast to the other explored 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. Genome comprises of a single chromosome of size 5,606,547 bp (5.6 Mb) with no symbiotic plasmid. Rhizobium leguminosarum bv. viciae USDA2370 was the closest whole genome known. 109 genes responsible for diverse plant growth promoting activities like P solubilization, synthesis of acetoin, nitric oxide, indole-3 acetic acid, exopolysaccharide, siderophore and trehalose have been identified. Flagellar proteins, genes encoding antibiotic and metal resistance, enzymes required for combating oxidative stress as well as attachment and colonization in the plant rhizosphere are also present. Availability of genome sequence of such a versatile plant growth promoting agent will help in exploiting all the phyto-beneficial traits of Td3 for its use as a biofertilizer.

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.