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Use of multifunctional plant growth promoting rhizobacteria (PGPR) for managing plant growth and health could not only facilitate higher positive effects on plants but also enable their predominant rhizospheric prevalence. While multi-functional PGPR are common, those harbouring both direct and indirect traits of growth promotion are relatively fewer. The present work aimed at isolating and characterizing the otherwise unusual multipotential PGPR with P-solubilizing ability in combination with broad-spectrum biocontrol abilities from diverse soils and analysing their relative prevalence. Primary screening yielded 50 isolates with varying P-solubilizing potential; of which only 8 showed In vitro antibiosis of E. coli. Selected 14 isolates with varying degree of P-solubilizing and antibacterial potential were evaluated for siderophore, HCN and indole-3-acetic acid (IAA) production. While all selected isolates produced HCN, 13 of them produced IAA and 10 showed siderophore production, at varying levels. Biochemical characterization of these isolates indicated that siderophore production was maximum with fluorescent Pseudomonas isolates while isolates of Enterobacteriaceae family were best IAA producers. However, molecular characterization of isolates capable of efficient P-solubilization along with strong ability to exhibit all the three biocontrol traits, identified them as Pseudomonas spp., typically P. aeruginosa. Overall, these results indicate that categorically P. aeruginosa species are likely to predominate as rhizobacteria with coexistence of discrete abilities to solubilize P as well as produce IAA, siderophore and HCN. The study also implies relatively higher metabolic versatility of P. aeruginosa species as compared to other members of fluorescent Pseudomonas family; thus, accounting for their rhizospheric abundance.

Citric acid secretion by fluorescent pseudomonads has a distinct significance in microbial phosphate solubilization. The role of citrate synthase in citric acid biosynthesis and glucose catabolism in pseudomonads was investigated by overexpressing the Escherichia coli citrate synthase (gltA) gene in Pseudomonas fluorescens ATCC 13525. The resultant~2-fold increase in citrate synthase activity in the gltA-overexpressing strain Pf(pAB7) enhanced the intracellular and extracellular citric acid yields during the stationary phase, by about 2-and 26-fold, respectively, as compared to the control, without affecting the growth rate, glucose depletion rate or biomass yield. Decreased glucose consumption was paralleled by increased gluconic acid production due to an increase in glucose dehydrogenase activity. While the extracellular acetic acid yield increased in Pf(pAB7), pyruvic acid secretion decreased, correlating with an increase in pyruvate carboxylase activity and suggesting an increased demand for the anabolic precursor oxaloacetate. Activities of two other key enzymes, glucose-6-phosphate dehydrogenase and isocitrate dehydrogenase, remained unaltered, and the contribution of phosphoenolpyruvate carboxylase and isocitrate lyase to glucose catabolism was negligible. Strain Pf(pAB7) demonstrated an enhanced phosphate-solubilizing ability compared to the control. Co-expression of the Synechococcus elongatus PCC 6301 phosphoenolpyruvate carboxylase and E. coli gltA genes in P. fluorescens ATCC 13525, so as to supplement oxaloacetate for citrate biosynthesis, neither significantly affected citrate biosynthesis nor caused any change in the other physiological and biochemical parameters measured, despite approximately 1.3-and 5-fold increases in citrate synthase and phosphoenolpyruvate carboxylase activities, respectively. Thus, our results demonstrate that citrate synthase is rate-limiting in enhancing citrate biosynthesis in P. fluorescens ATCC 13525. Significantly low extracellular citrate levels as compared to the intracellular levels in Pf(pAB7) suggested a probable limitation of efficient citrate transport.

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Genetic engineering of fluorescent pseudomonads for various industrially, agriculturally and environmentally important bioprocesses often involves the use of suitable plasmids. Plasmid-mediated alterations in host physiology and metabolism are poorly understood for this group of organisms. Thus, we investigated the metabolic perturbations in Pseudomonas fluorescens 13525 due to the independent and combined presence of broad-host-range plasmids, pBBR1MCS-2 (copy number 30) and pUCPM18 derived pAB4 and pAB8 (copy number 14-16). Presence of pAB4 and pAB8 not only significantly increased the growth rate and glucose utilization of P. fluorescens 13525, but also increased glucose dehydrogenase activity and gluconic acid production indicating enhanced direct oxidative pathway for glucose catabolism. Additionally, increased secretion of pyruvic, acetic, and citric acids caused faster media acidification in presence of pAB4 and pAB8. Simultaneous presence of pAB4/pAB8 in Pf (pAB48) and pAB4/pBBR1MCS-2 in Pf (pAB4BBR1MCS-2) reduced their respective copy numbers to nearly half. Pf (pAB48) demonstrated further increase in direct oxidation pathway without altering growth and glucose depletion rates, as compared with single transformants. Conversely, pBBR1MCS-2 plasmid did not greatly alter P. fluorescens 13525 metabolism when present independently but masked the effects imposed by pAB4 when present in its combination. In conclusion, P. fluorescens 13525 redesigns its metabolism in response to the presence of plasmids irrespective of their nature, by enhancing anaplerosis with a simultaneous reduction in catabolism as indicated by increased pyruvate carboxylase and decreased citrate synthase activities, respectively. Such information will be helpful for vector designing during genetic engineering of fluorescent pseudomonads.

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Citation: Disha Patel and Aditi Buch, Aerobic L-tartrate Utilization by Bacillus Isolates, J Pure Appl Microbiol., 2019; 13(4):2045-2054. https://doi.org/10.22207/JPAM.13.4.16 Abstract Microbial utilization of uncommon C 4 dicarboxylate L-tartrate is largely anaerobic, with aerobic L-tartrate utilization known for few bacterial species including Rhodopseudomonas sphaeroides and Pseudomonas putida. Aerobic L-tartrate-utilizing microbes could be industrially relevant owing to the efficient nature of the bioprocess and catalytic versatility of tartrate dehydrogenase (TDH) responsible for aerobic catabolism of L-tartrate. Present work involves isolation and characterization of Bacillus strains capable of aerobic L-tartrate utilization and its correlation with occurrence of TDH activity. Two out of 37 isolates, IC1-G and IC1-Y were identified as Bacillus megaterium spp. showing efficient aerobic growth, utilizing ~3.7 and 2.8 mM L-tartrate respectively at the end of 48 h. Several organic acids possibly including oxalic, succinic and citric acids were secreted as by-products of L-tartrate metabolism. Utilization of L-tartrate directly correlated with induction of TDH activity by ~3.2 and 5.2 folds in IC1-G and IC1-Y respectively, when grown in presence of L-tartrate as compared to when grown on citrate. Overall, this study contributes Bacillus as only the third genus capable of aerobic, TDH mediated L-tartrate utilization. These Bacillus isolates thus offer potential targets to develop an industrially relevant bioprocess and biocatalyst.