Transcriptional Modulation of Transport- and Metabolism-Associated Gene Clusters Leading to Utilization of Benzoate in Preference to Glucose in Pseudomonas putida CSV86

Microorganisms and culture conditions. Pseudomonas putida CSV86 was grown on 150 ml minimal salt medium (MSM) (composition per liter of distilled water: K₂HPO₄, 8 g; KH₂PO₄, 1 g; NH₄NO₃, 1 g; MgSO₄ · 7H₂O, 100 mg; MnSO₄ · H₂O, 1 mg; CuSO₄ · 5H₂O, 1 mg; FeSO₄ · 7H₂O, 5 mg; H₃BO₃, 1 mg; CaCl₂ · 2H₂O, 1 mg; NaMoO₄, 1 mg; pH 7.5; 34) in 500 ml capacity baffled Erlenmeyer flasks at 30°C on a rotary shaker (200 rpm) supplemented aseptically with the appropriate carbon source (%, wt/vol), such as aromatics (0.1%), glucose (0.25%), succinate (0.25%), or combinations like glucose (0.25%) plus benzoate (0.1%), succinate (0.25%) plus benzoate (0.1%), and succinate (0.25%) plus glucose (0.25%). All growth experiments were performed using a shake flask/batch method. Substrate concentrations selected and used in the present work are based on our previous studies where the diauxic growth profile with a distinct second lag phase (for example, on glucose plus benzoate and succinate plus glucose) was observed (9, 10). Escherichia coli strain BL21(DE3) was grown in LB medium (35) at 37°C on a rotary shaker (200 rpm).

Growth profile and [¹⁴C]substrate uptake by whole cells. P. putida CSV86 was grown on MSM supplemented with glucose (0.25%) plus benzoate (0.1%), benzoate (0.1%), or glucose (0.25%). Growth was monitored by spectrophotometrically measuring the optical density at 540 nm (Lambda 35; PerkinElmer, USA). The cells were harvested, washed twice, and resuspended in ice-cold sterile MSM so as to obtain an optical density at 540 nm (OD₅₄₀) of 0.2. Cell suspension (1 ml, prewarmed at 30°C for 10 min) was incubated with appropriate concentrations of either [¹⁴C]benzoate (2 μM, universally ring labeled, specific activity of 75 mCi · mmol⁻¹; ARC, St. Louis, MO, USA) or [¹⁴C]glucose (2 μM, universally labeled, specific activity of 140 mCi · mmol⁻¹; BRIT, India) at 30°C for 2 min in a water bath and rapidly filtered through premoistened hydrophobic polyvinylidene difluoride (PVDF) membrane (0.45 μm; Pall Corporation, USA). The filters were washed twice with sterile MSM (2 × 1 ml). Air-dried filters were dissolved in scintillation cocktail [2,5-diphenyloxazole (PPO; 0.4%) and 1,4-bis(5-phenyloxazol-2yl)benzene (POPOP; 0.025%) in toluene (scintillation grade)] and the radioactivity was counted using a liquid scintillation counter (Tri-Carb B2810TR; PerkinElmer, USA). The values were corrected for counts due to the reaction mixture without cells. To obtain the dry weight, the cell suspension (1 ml, OD₅₄₀ = 0.2) was centrifuged (20,000 × g for 20 min) and dried overnight at 37°C and its weight was recorded. The uptake (which includes primary substrate uptake as well as metabolism) is expressed as pmol substrate · min⁻¹ · mg⁻¹ dry cell weight.

Genome sequence retrieval and analysis.The draft genome sequence of Pseudomonas putida CSV86 (NZ_AMWJ00000000, 36) was screened for the presence of various aromatic compound transporters using the RAST server (37). Genomic sequences of several Pseudomonas species were accessed from the Pseudomonas Genome Database (38, http://www.pseudomonas.com ) or the National Centre for Biotechnology Information website (http://www.ncbi.nlm.nih.gov ). Analysis of the upstream region of the glc and ben loci for a putative bacterial promoter −10 and −35 region was done using the Softberry bacterial promoter prediction computer program BPROM (39; Softberry, Inc., Mount Kisco, NY, USA). The sequences with the highest score and an in frame start codon site were considered probable promoters.

Bacterial RNA isolation and cDNA synthesis.The total RNA was isolated from CSV86 cells (400 μl culture) grown on MSM in 500 ml baffled Erlenmeyer flasks at 30°C on a rotary shaker (200 rpm) containing glucose plus benzoate, glucose, naphthalene, benzoate, or succinate as a carbon source until either mid-log or late-log phase using the RNeasy Mini Kit (Qiagen, Germany). Contaminating traces of DNA from RNA preparations were removed by treating the sample with Ambion Turbo RNase-free DNase I (4 units; Thermo, USA) at 37°C for 60 min. The reverse transcription reactions were performed using the SuperScriptIII first strand cDNA synthesis kit (Thermo, USA). Each real-time (RT) reaction mix (10 μl) contained RNA (0.5 to 1 μg), gene specific primers (1 μM, Table 2), or random hexamers (2.5 ng · μl⁻¹ for RT-PCR) and deoxynucleoside triphosphates (dNTPs; 0.5 mM). The reaction mixture was incubated at 65°C for 5 min followed by incubation on ice for 1 min. Synthesis of cDNA (final vol 20 μl) was carried out by addition of 10 μl of cDNA synthesis mixture containing 10× RT buffer (2 μl), MgCl₂ (25 mM, 4 μl), dithiothreitol (DTT; 0.1 M, 2 μl), RNaseOUT (40 units, RNase inhibitor), and SuperScript III RT enzyme (200 units). The reaction mixture was incubated at 50°C for 2 h followed by termination at 85°C for 5 min. After first strand synthesis, the RNA template from the cDNA-RNA hybrid was removed by digestion with RNaseH (2 units) at 37°C for 30 min. cDNA synthesis mixture without reverse transcriptase and with RNase treated template was used as a negative control for subsequent PCR.

RT-PCR and qPCR.Primers used for RT-PCR (cotranscriptional analysis) and qPCR (relative expression analysis) are listed in Table 2. RT-PCR and qPCR amplicons obtained were electrophoresed on agarose gel, purified using GenElute (Sigma, USA), and sequenced (Xcelris, India). The organization of the glc and ben locus genes was examined by reverse transcribing the total transcripts into cDNA using random hexamer primers followed by performing endpoint PCR using specific primers (Table 2) yielding products with overlapping regions. Reaction mixture with RNase-treated and/or RNase-lacking reverse transcriptase preparations were used as templates in negative controls, while a separate PCR with CSV86 genomic DNA as the template was used as a positive control.

All qPCRs were performed using the Platinum SYBR Green qPCR SuperMix-UDG with ROX (Invitrogen, Thermo, USA) with the StepOnePlus Real-Time PCR system (Applied Biosystems, USA) in accordance with the manufacturer's protocols. Each reaction mixture contained cDNA (4 μl, diluted 1:100 in sterile Milli-Q water) and 500 nM of each primer (Table 2) per qPCR mixture (final vol 20 μl). The qPCR thermal cycling program was initial denaturation at 95°C for 10 min, followed by 40 cycles, each cycle consisting of denaturation at 95°C for 15 s and extension at 65°C for 1 min.

Transcription of rpoD, a housekeeping gene that encodes the σ⁷⁰ transcription factor, was used as an internal control (40). The expression levels of glucose (gbp) and benzoate transport (benE) genes in cells grown on MSM supplemented with glucose or benzoate, respectively, versus those of cells grown on MSM containing either aromatics or organic acids, was determined by the cycle value at which fluorescence of the genes of interest cross threshold cycle (C_T) levels, taking into account the correction for reaction efficiencies of each primer pair. Values from three independent experiments were used to calculate the means and standard errors.

Mean values from three independent experiments where reaction were performed in duplicate were used to calculate individual differences in gene expression according to a mathematical model proposed by Livak and Schmitten (41). Briefly, ΔC_T was calculated by the equation ΔC_T = C_{T(target gene)} − C_T(rpoD). The ΔC_T of samples from cells grown on compound A were compared with those from cells grown on B by using the equation ΔΔC_T = ΔC_T(A) − ΔC_T(B). For benzoate transport genes A is benzoate- and B is glucose- or succinate-grown condition. For glucose transport genes A is glucose- and B is benzoate- or succinate- or naphthalene-grown condition. The expression profile of a representative of the benzoate transport gene, benE, and glucose transport gene, gbp, was monitored every 2 h during the growth of CSV86 in the presence of glucose plus benzoate (with benzoate-grown cells as the inoculum). The expression of these genes at the time of inoculation, i.e., at 0 h, was considered basal expression.

Enzyme assays.Cells grown under appropriate conditions were harvested by centrifugation, washed, resuspended (1 g in 5 ml) in Tris-Cl buffer (50 mM, pH 7.5), and disrupted by sonication (4 cycles, each cycle consisting of 15 pulses [11 W] of 1 s each with an interval of 5 min between cycles). The cell lysate was centrifuged at 30,000 × g for 30 min at 4°C to obtain a clear supernatant, referred as cell extract (CFE). Catechol 1,2-dioxygenase (C12DO) activity was monitored as described previously (42). Briefly, the reaction mixture (1 ml) contained Tris-Cl buffer (50 mM, pH 7.5), catechol (100 μM), and an appropriate amount of CFE. The activity was calculated by monitoring the formation of cis,cis-muconate (ε_{260 nm} = 16,000 M⁻¹ · cm⁻¹) spectrophotometrically and specific activity was reported as nmol · min⁻¹ · mg⁻¹ protein. Glucose 6-phosphate dehydrogenase (Zwf) activity was monitored as described previously (43). Briefly, the reaction mixture (1 ml) contained Tris-Cl buffer (50 mM, pH 7.5), glucose 6-phosphate (4 mM), NADP (200 μM), and an appropriate amount of CFE. The activity was calculated by monitoring the formation of NADPH (ε_{340 nm} = 6,220 M⁻¹ · cm⁻¹) spectrophotometrically and specific activity was reported as nmol · min⁻¹ · mg⁻¹ protein. Protein estimation was performed by the Bradford method using bovine serum albumin (BSA) as the standard (44).

Expression and purification of recombinant GBP and ATPase.The constructs of gbp (45) or glcK (primers, Table 2) in pET28a(+) were transformed into E. coli BL21(DE3). A single transformed colony was inoculated in 5 ml LB containing kanamycin (40 μg · ml⁻¹) at 37°C on a rotary shaker (200 rpm). Cultures (OD₅₄₀ = 0.8 to 1.0) were induced with isopropyl-β-d-thiogalactopyranoside (IPTG; 100 μM) for 4 h at 37°C for GBP and 12 h at 16°C for ATPase. Under these conditions GBP and ATPase gave maximum expression of protein. Cells were harvested and resuspended in binding buffer (Tris-Cl, 10 mM, pH 7.5; MgCl₂, 1 mM) for GBP and lysis buffer (Tris-Cl, 25 mM, pH 8.0; NaCl, 150 mM, and glycerol, 20%) for ATPase. Cell extracts were prepared by sonication as described earlier.

The soluble fraction of cell extract was loaded onto Ni-nitrilotriacetic acid column (10 mg of CFE protein per ml of column volume) preequilibrated with binding or lysis buffer (5 times column volume). The unbound proteins were removed by washing the column with binding or lysis buffer (5 column volumes). The bound proteins were eluted using a linear gradient of imidazole (0 to 250 mM) in binding or lysis buffer (10 column volumes). The eluted fractions were analyzed for protein OD at 280 nm and activity (glucose binding or ATPase) by SDS-PAGE (12%; 46). Active fractions were pooled and dialyzed against binding and lysis buffer, respectively, for 4 h at 4°C and used for further studies.

Substrate interaction studies: interaction with rGBP.Interaction of rGBP with glucose, benzoate, or succinate was studied by surface plasmon resonance (Biacore S200; GE Healthcare Life Sciences, UK) as described previously (47). rGBP was dialyzed against HEPES buffer (HEPES, 10 mM, pH 7.4; NaCl 150 mM; Biacore Surfactant P20, 0.05%). rGBP (100 μg · ml⁻¹) in HEPES buffer was immobilized on carboxymethyl dextran (CM5) gold chip surface by a nonspecific amine coupling method. The CM5 chip was activated using N-ethyl-N-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC)/N-hydroxysuccinimide (NHS). For immobilization, rGBP in sodium acetate (10 mM, pH 4.0) was injected with a flow rate of 10 μl · min⁻¹ for 720 s. A pulse of ethanolamine (1 M, pH 8.0) was given to remove nonspecifically bound protein and quench the reaction. Different concentrations of glucose (0.05, 0.1, 0.2, 0.4, 0.8, 1.6, 3.2, 6.25, 12.5, 25, 50, and 100 μM), benzoate, or succinate (0.4, 0.8, 1.6, 3.2, 6.25, 12.5, 25, 50, 100, and 1,000 μM) were used for the binding studies at a flow rate of 30 μl · min⁻¹, with contact and dissociation times of 90 and 240 s, respectively. Injections were performed on both protein and blank surfaces. The data were corrected with appropriate blank (HEPES buffer alone) and analyzed by using a steady-state one-site-binding saturation model.

Substrate interaction studies: ATPase activity.The activity of purified recombinant ABC transporter component ATPase was carried out by monitoring the release of inorganic phosphate using the malachite-green-based spectrophotometric assay (48) with minor modifications. The assay was performed as follows. rATPase (1 μg) was incubated at 30°C in a mixture (20 μl) containing Tris-Cl (20 mM, pH 8.0), NaCl (100 mM), and MgCl₂ (5 mM). The reaction was started by the addition of ATP (final concentration 1 mM), followed by incubation for 20 min. Reactions were terminated by the addition of EDTA (50 mM, 2 μl). The inorganic phosphate was determined by adding sterile Milli-Q water (0.8 ml) followed by malachite green color reagent (0.2 ml), prepared as described previously (48). The absorbance was measured at 630 nm after 30 min incubation at 30°C. The inorganic phosphate concentration was calculated using the standard graph obtained using KH₂PO₄. All data were corrected for autohydrolysis of ATP (reaction mixture without purified protein), which was found to be insignificant. The effect of succinate or benzoate on the ATPase activity was monitored by carrying out an assay in the presence of 2 and 10 mM succinate or benzoate. The percent activity was calculated by considering the ATPase activity without succinate or benzoate as 100%.

Substrate utilization studies by HPLC.To determine the residual concentration of benzoate and succinate from the spent medium of CSV86, cultures were grown on benzoate, succinate, or succinate plus benzoate (succinate-adapted cells were used as the inoculum to check if cells displayed any preference for succinate over benzoate) and harvested at specific times of growth. The culture supernatants were acidified with H₂SO₄ (final concentration 5 mM) and filtered through a 0.45-μm nylon syringe filter (Axiva, India). Samples were analyzed by HPLC (Jasco, Japan) using an Aminex HPX-87H column (300 by 7.8 mm; Bio-Rad, USA) and a Jasco MD-2010 Plus UV detector (Jasco, Japan) at 204 nm. The solvent system was H₂SO₄ (5 mM) in Milli-Q water with a flow rate of 0.6 ml · min⁻¹ and an operating temperature of 65°C. The authentic benzoate and succinate were prepared in MSM, acidified, and analyzed as HPLC standards.

Statistical analysis.For all experiments, the means and standard errors were calculated using values obtained from triplicates of at least three independent experiments, unless otherwise specified.