Bacterial strain, culture conditions and reagents
Isolate P. aeruginosa P4 [MCC No. 2365] used as the model strain in all the experiments, was kindly gifted by Prof. G. Naresh Kumar, Department of Biochemistry, The M S University of Baroda, India. The strain was grown and maintained on Pseudomonas agar (Hi-Media Laboratories, Mumbai, India) at 30°C, as required. The culture media, dextrose, Tris base and other routine analytical-grade salts and reagents were procured from SD Fine Chemicals Ltd., Hi-media Laboratories and Merck Life Science Pvt. Ltd. Specific molecular biology reagents like RNase, Taq DNA polymerase with its buffer and dNTPs, lysozyme, diethyl pyrocarbonate (DEPC), RNAse Zap solution, Trizol etc. were procured from Sigma Aldrich.
Physiological experiments
A single colony of P4 was aseptically inoculated in sterile 3 ml of Luria broth and bacterial growth was facilitated by overnight incubation in a rotary shaker maintained at 30 ˚C and 150 rpm (Scigenics Biotech Pvt Ltd). The freshly grown culture was harvested by centrifugation (8000 rpm for 5 minutes at room temperature), washed twice with sterile normal saline and finally resuspended in 1 ml of the same under aseptic condition. This cell suspension was used as inoculum for further experiments.
An aliquot of fresh inoculum was used to inoculate 100 ml of Tris-buffered minimal medium supplemented with 100 mM glucose and micronutrient cocktail (Buch et al. 2008) to give an initial cell density of OD600nm 0.01–0.03. Tris-minimal medium supplemented with 10 mM Pi was used as a P-sufficient condition while the same with 0.066 mM Pi was used to represent P-limited condition. Batch culture studies were performed by shaking 250 ml conical flasks containing 100 ml of the inoculated media on a rotary shaker (30 ˚C, 150 rpm). Two-milliliter culture samples were withdrawn aseptically at regular intervals to measure bacterial growth as a function of OD600nm and media pH. The observations were continued until the pH of the medium decreased to less than 5, under both P-sufficient and P-deficient conditions. At the point of termination, cell-free culture supernatants were obtained by centrifuging the harvested cultures (10,000 rpm, 5 minutes, room temperature) and stored at -20 ˚C until further analysis of metabolites.
The glucose concentration in the growth medium was monitored using the GOD-POD kit (ARKRAY Healthcare Pvt. Ltd., India). The difference between the glucose concentrations measured in the initial and terminal samples were used to calculate the total glucose depleted during the bacterial growth; while the difference between the total glucose depleted and gluconic acid produced was used to measure the amount of glucose consumed. Biomass yield was calculated as dry cell mass produced per unit of glucose consumed. The dry cell mass was determined by centrifuging a fixed volume of the terminal bacterial culture, discarding the supernatant, washing the cells using distilled water and weighing the cell pellet dried at 60 ᵒC for 3 days. Growth rate (µ) and specific glucose depletion rate (QGlc) were calculated using log phase culture growth recorded as OD600nm; for which the cell number was derived using the proportionality constant of 1 OD 600nm = 1.5 109 cells ml− 1 (Buch et al., 2008).
The effect of artificial root exudates (ARE) on growth and secondary metabolite production by P4 was monitored using a similar experimental setup, where 100 mM glucose in the Tris-buffered minimal medium composition described above was replaced using a synthetic root exudate cocktail. The final concentrations of ARE components in the medium were adjusted as follows: 18.4 mM glucose, 18.4 mM fructose, 9.2 mM xylose, 9.2 mM sodium citrate, 18.4 mM sodium gluconate, 13.8 mM sodium succinate, 9.2 mM alanine, 9.2 serine, 9.2 glycine. The choice and the level of the carbon source ingredients in ARE was based on their known occurrence in peanut root exudates and the other ARE compositions used for similar study (Baudoin et al., 2003; Dutta et al., 2013)
Metabolite analysis
Cell-free culture supernatants derived as above, from cultures at the point of experimental termination were subjected to various analytical techniques to estimate the levels of selected metabolites. A portion of the stored culture supernatants was filtered through 0.2 µm nylon membranes (MDI Advanced Microdevices, India) before subjecting to HPLC analysis (equipment from Waters India Pvt. Ltd.).
Gluconic acid levels were quantified using an RP-18 column operated at room temperature using a mobile phase of 20 mM NaH2PO4 at a flow rate of 1.0 ml min− 1. The column eluates were monitored using a UV detector at 210 nm and gluconic acid was detected and quantified by comparing the retention time and peak areas obtained for pure gluconic acid standard (10 mM) subjected to similar analytical conditions.
Total siderophores were quantitated using Chromo-azurol S solution assay of P4 culture supernatants and expressed as EDTA equivalents (Leclère et al. 2009). Siderophore pyochelin was detected and quantified by HPLC using an isocratic gradient comprising of 70 % Solvent A (H2O, 0.1 % trifluoroacetic acid) and 30 % Solvent B (95 % acetonitrile, 0.1 % trifluoroacetic acid) and a flow rate of 1 ml/min at 254 nm (Youard et al. 2007). Pyochelin standard was prepared using layer chromatography (TLC) of the supernatant of 48 h old P4 culture grown on King’s B medium. A 50 µl aliquot of the culture supernatant was spotted on pre-coated Silica Gel G sheets (0.2 mm; Xtra SILG/UV254, Macherey Nagel GmbH & Co. KG), air-dried and chromatographed with the solvent system, chloroform:acetic acid:ethanol (50:5:2.5 vol/vol) (Farmer and Thomas 2004). The appearance of characteristic yellow fluorescence upon visualization under UV light at Rf ~0.35 cm; turning typical red-brown when sprayed with 0.1 M FeCl3 in 0.1 M HCl indicated the presence of pyochelin. The silica portion at around the appropriate spot was scraped and used to extract pyochelin in 100 µl of methanol, for further use as the reference standard in HPLC. The area under the appropriate peak, as compared with the standard, was used as a measure to quantify pyochelin. An untargeted metabolite detected in HPLC chromatograms was further identified using LC-MS and HR-LC-MS, respectively obtained as an outsourced service from SAIF, IIT, Bombay and Sophisticated Instrumentation Centre for Applied Research and Testing (SICART), Vallabh Vidyanagar, Gujarat, India. LC-MS was performed using LCQ Fleet and TSQ Quantum Access with Surveyor Plus HPLC System, Thermo Scientific, USA while HR-LC-MS was performed using 1290 Infinity UHPLC System, Agilent Technologies, USA by employing the aforementioned chromatography conditions with minor modifications. Pyoverdine production was determined by measuring characteristic fluorescence at 400 ± 10/460 ± 10 nm excitation/emission of 0.1 ml culture supernatants and expressed as relative fluorescence units (RFU) (Dumas et al. 2013). Pyocyanin levels were measured spectrophotometrically at 690 nm using the culture supernatants extracted with chloroform (1:2 v/v) and were calculated using the extinction coefficient of ε = 4,130 M− 1 cm− 1 at pH 7 (Dietrich et al. 2006). IAA levels were determined spectrophotometrically at 535 nm using Salkowski reagent (Ahmad et al. 2008), against a standard curve generated using pure IAA. Each metabolite level was normalized to the amount of glucose consumed, to calculate their production yields.
Effect of nature and concentration of carbon source on production of secondary metabolites was determined in a time course analysis. Supernatants derived from culture aliquots harvested at regular time intervals during growth on 100 mM Tris-buffered medium with (i) 100 mM glucose and (ii) ARE, as sole carbon source. were used to measure the production of total siderophores, pyocyanin and IAA.
RT-qPCR based expression analysis of selected genes
P4 culture was allowed to grow under shaking conditions on P-sufficient and P-limited media and the cells were harvested across two growth stages (i) the experimental endpoint and (ii) mid-log phases. The cells were immediately used for total RNA isolation using Qieasy RNA isolation kit as per manufacturer’s recommendations with minor variations. RNA yield and purity were determined as A260/A280 and A260/A230 ratios using QIAxpert nano-spectrophotometer. Subsequently, 1 µg of RNA was reverse transcribed to yield cDNA using oligo(dT)18 primers at 42°C using Maxima H Minus reverse transcriptase (ThermoFisher Scientific) under following conditions: 25 ºC for 10 minutes, 42 ºC for 50 minutes and 85 ºC for 5 minutes. The cDNA synthesized was stored at − 80°C until further use. Subsequently, the real-time PCRs were conducted in Qiagen Rotor-Gene Q3000 5Plex HRM platform using Kapa SYBR Fast Universal qPCR kit as per manufacturer’s instructions. The thermal cycle program used was as follows: initial denaturation at 95 ºC for 10 min followed by 35 cycles of denaturation at 95 ºC for 10 s, primer annealing 60 ºC for 1 min and extension at 72 ºC for 1 min, with a final extension at 72°C for 10 min. Specific gene fragments were amplified using primer sets either designed using Primer3 tool or adopted from published literature (Huang et al. 2009; Lopez-Medina et al. 2015; Supplementary material-I]. Specific amplification was verified using melt curve analyses. For relative quantification, the expression of each target gene was normalized to rpoD used as an internal control. Relative fold change in expression of the target gene under P-limitation as compared to P-sufficiency was determined using 2−ΔΔCt method (Livak and Schmittgen 2001). Before subjected to real-time PCR, the specificity of the selected primer pairs was validated by using them for standard PCR amplification using P4 genomic DNA and confirming the presence of the desired amplicon as a single band with predicted size on ethidium bromide-stained 2 % agarose gel (Supplementary material-I).
Statistical analysis
Experimental data was derived from substantial number of replicate observations and expressed as standard statistical measures as indicated in the table footnote and the figure legends. For comparing the parameters across the two experimental conditions the statistical significance of differences was determined by two-tailed student's t test computed using Microsoft Excel (Microsoft office version 10).