Artificial citrate operon confers mineral phosphate solubilization ability to diverse fluorescent pseudomonads

Abstract

Citric acid is a strong acid with good cation chelating ability and can be very efficient in solubilizing mineral phosphates. Only a few phosphate solubilizing bacteria and fungi are known to secrete citric acids. In this work, we incorporated artificial citrate operon containing NADH insensitive citrate synthase (gltA1) and citrate transporter (citC) genes into the genome of six-plant growth promoting P. fluorescens strains viz., PfO-1, Pf5, CHAO1, P109, ATCC13525 and Fp315 using MiniTn7 transposon gene delivery system. Comprehensive biochemical characterization of the genomic integrants and their comparison with plasmid transformants of the same operon in M9 minimal medium reveals the highest amount of ∼7.6±0.41 mM citric and 29.95±2.8 mM gluconic acid secretion along with ∼43.2±3.24 mM intracellular citrate without affecting the growth of these P. fluorescens strains. All genomic integrants showed enhanced citric and gluconic acid secretion on Tris-Cl rock phosphate (TRP) buffered medium, which was sufficient to release 200-1000 µM Pi in TRP medium. This study demonstrates that MPS ability could be achieved in natural fluorescent pseudomonads by incorporation of artificial citrate operon not only as plasmid but also by genomic integration.

Figure 1. Citrate synthase activity of P. fluorescens PfO-1 transformants.

The activity was estimated with transformants containing pAB8 (vector control, denoted as Km), pAB7 (denoted as AB7), pK167A, pR163L and pY145F along with the untransformed native strain (WT). Cultures were grown on M9 minimal medium with 100 mM glucose to mid log or stationary phase. Activity is represented in the units of nmole/min/mg total protein. The values are depicted as Mean ± S.E.M of 4 independent observations. # Comparison of parameters with WT, § with respect to vector control AB8, ¶ comparison of parameter between Y145F and AB7/R163L/K167A. ###, §§§, ¶¶¶: P<0.001; ##, §§, ¶¶: P<0.01; #, §, ¶: P<0.05.

Figure 2. Organic acid profile and enzyme activities of P. fluorescens PfO-1 transformants.

Activities of key enzymes (A) using stationary phase cultures grown on M9 medium with 100 mM glucose were represented in the units of nmoles/min/mg total protein. Intracellular and extracellular citric acid levels (B) are represented in grey bars and black bars respectively. Gluconic-pyruvic-acetic acid levels (C) and citric acid levels were estimated from stationary phase cultures grown on same media. Results are expressed as Mean ±S.E.M of 4 independent observations. WT is native untransformed strain, Gm indicates vector control containing pUCPM18Gm, Km indicates vector control containing pAB8 and YC indicates tansformant with artificial citrate operon pYC; KG indicates vector control with both the pUCPM18 and pAB8 plasmids cotransfromed; YF indicates transformant with pY145F; YFCitC and YFCitM indicate cotransformants carrying plasmid pY145F along with pCitC and pCitM, respectively. * comparison of parameters with wild type control; $ comparison of parameters with vector control pAB8, # comparison between parameters of AB7 and YF.***,$ $ $,###: P<0.001; **,$ $,##: P<0.01; *,$,#: P<0.05.

Figure 3. Construction of P. fluorescens genomic integrants carrying artificial citrate operon.

Natural fluorescence and antibiotic resistance of genetically modified P. fluorescens strains (A); P. fluorescens pYC plasmid transformants (a), P. fluorescens genomic integrants (b) and P. fluorescens wild type (c) grown on Pseudomonas agar plates containing ampicillin and gentamycin. Schematic representation of the genomic region of Pseudomonas genome showing the integrated mini transposon and gel picture showing confirmation of integrants by PCR amplification of genomic DNA isolated from P. fluorescens wild type and genomic integrants using Tn7-Gm (510 nt from the start site of Gm^r gene) and cs reverse primer (PCR product∼2800 bp) (B).

Figure 4. Effect of P. fluorescens yc operon genomic integrants on citric acid accumulation, citric and gluconic acid secretion.

Intracellular citric acid levels (A), extracellular citric (B) and gluconic (C) acid levels (mM) estimated from the stationary phase cultures of wild type (WT), plasmid transformants containing yc operon (pYC), yc operon genomic integrants (Int) of different P. fluorescens strain PfO-1, Pf5, CHAO1, ATCC13525 (13525), P109 and Fp315 grown in TRP minimal medium for 96–120 h. Results are expressed as mean ± S.E.M of 4–8 independent observations, † comparison of parameters with WT; ‡ comparison of parameters between plasmid transformants (pYC) and genomic integrants (Int). †††, ‡‡‡: P<0.001; ††,‡‡: P<0.01; †,‡: P<0.05.

Figure 5. Phosphate solubilisation activities of P. fluorescens yc operon genomic integrants and plasmid transformants.

Zone of clearance and PSI (Phosphate solubilisation index) (A and B) in Pikovskaya's agar medium during 96–120 h of growth. Zone of colouration (C) and Pi release (D) of transgenic P. fluorescens strains in TRP broth and TRP agar medium during 96–120 h of growth. WT: wild type strain; pYC: P. fluorescens with pYC plasmid; Int: P. fluorescens yc operon genomic integrant. Results are expressed as Mean ±S.E.M of 4 independent observations. * Comparison of parameters with wild type control; ‡ comparison between parameters of plasmid transformants (pYC) and genomic integrants (int).***, ‡‡‡: P<0.001; **,‡‡: P<0.01; *,‡: P<0.05.