ABSTRACT
The effective elimination of xenobiotic pollutants from the environment can be achieved by efficient degradation by microorganisms even in the presence of sugars or organic acids. Soil isolate Pseudomonas putida CSV86 displays a unique ability to utilize aromatic compounds prior to glucose. The draft genome and transcription analyses revealed that glucose uptake and benzoate transport and metabolism genes are clustered at the glc and ben loci, respectively, as two distinct operons. When grown on glucose plus benzoate, CSV86 displayed significantly higher expression of the ben locus in the first log phase and of the glc locus in the second log phase. Kinetics of substrate uptake and metabolism matched the transcription profiles. The inability of succinate to suppress benzoate transport and metabolism resulted in coutilization of succinate and benzoate. When challenged with succinate or benzoate, glucose-grown cells showed rapid reduction in glc locus transcription, glucose transport, and metabolic activity, with succinate being more effective at the functional level. Benzoate and succinate failed to interact with or inhibit the activities of glucose transport components or metabolic enzymes. The data suggest that succinate and benzoate suppress glucose transport and metabolism at the transcription level, enabling P. putida CSV86 to preferentially metabolize benzoate. This strain thus has the potential to be an ideal host to engineer diverse metabolic pathways for efficient bioremediation.
IMPORTANCE Pseudomonas strains play an important role in carbon cycling in the environment and display a hierarchy in carbon utilization: organic acids first, followed by glucose, and aromatic substrates last. This limits their exploitation for bioremediation. This study demonstrates the substrate-dependent modulation of ben and glc operons in Pseudomonas putida CSV86, wherein benzoate suppresses glucose transport and metabolism at the transcription level, leading to preferential utilization of benzoate over glucose. Interestingly, succinate and benzoate are cometabolized. These properties are unique to this strain compared to other pseudomonads and open up avenues to unravel novel regulatory processes. Strain CSV86 can serve as an ideal host to engineer and facilitate efficient removal of recalcitrant pollutants even in the presence of simpler carbon sources.
INTRODUCTION
Pseudomonas spp. are ubiquitous and metabolically versatile bacteria present in a wide range of ecological niches like soil, water, and in association with other living organisms. They have the ability to adapt to different environmental conditions and assimilate an array of compounds (1). This is reflected in their large genome size (∼5 to 6 Mb), great genome plasticity, unique gene organization, and ability to transmit traits through horizontal gene transfer (2). These properties make Pseudomonas a promising candidate organism for bioremediation. For effective remediation, the candidate organism(s) should possess the ability to mineralize xenobiotics efficiently through diverse metabolic routes even in the presence of mixture of carbon sources. However, a bottleneck is the presence of simpler carbon source like sugars and organic acids, leading to a shift in the metabolic preference of organisms and thus reducing the efficiency of the bioremediation process. Carbon catabolite repression (CCR), the regulatory process present in bacteria which inhibits the utilization of nonpreferred (complex) carbon sources in the presence of preferred (simple) ones, is not very well understood in Pseudomonas, compared to Enterobacteria and Firmicutes (3, 4, 5). In Pseudomonas, organic acids are the most preferred and aromatic compounds are the least preferred carbon sources, while glucose occupies a position between the two (5). Attempts are being made to engineer microbes for coutilization or preferential utilization of various carbon sources (6, 7, 8).
Pseudomonas putida CSV86 (here referred to as CSV86), is a soil bacterium that displays a novel property of utilization of aromatics like naphthalene, benzyl alcohol, benzoate, phenylacetic acid, and phenylpropanoids prior to glucose (9, 10). Draft genome sequence analysis and validation corroborate the capability of CSV86 to metabolize various aromatic compounds (11). Glucose transport in Pseudomonas is an energy-driven process mediated by low- and high-affinity transport pathways (12). In P. putida KT2440, glucose is metabolized using a combination of Entner-Doudoroff, Embden-Meyerhof-Parnas, and pentose phosphate pathways. This leads to overproduction of NADPH, which helps the strain combat different environmental stresses, including the presence of aromatics or xenobiotic pollutants. Most of the glucose consumed via low-affinity pathways is metabolized to gluconate and 2-ketogluconate (13). In CSV86, glucose is transported solely by an active, high-affinity glucose ABC-transport system and metabolized via intracellular phosphorylation pathway (14). Although cells showed the ability to convert glucose into gluconate, they failed to respire as well as to utilize gluconate or 2-ketogluconate as the source of carbon and energy, suggesting that the low-affinity transport pathway is absent in CSV86 (15). Compared to other pseudomonads, CSV86 behaves differently. Therefore, it is interesting to study the modulation/regulation of metabolic pathways when cells are grown on a double carbon source like glucose plus benzoate.
In the present study, we demonstrate that in CSV86, glucose and benzoate transport genes are clustered at the glc and ben loci, respectively. Results suggest that genes from these loci are transcribed as two independent polycistronic messages and are regulated at the transcription level in a substrate-dependent manner, leading to preferential utilization of aromatics in P. putida CSV86.
RESULTS
Independent cotranscription of glc and ben locus genes.In CSV86, the glucose transport genes are arranged in a single cluster, 5′-gbp-glcF-glcG-glcK-oprB-3′, at the glc locus (Fig. 1A, Table 1). These genes are annotated as follows: GBP, periplasmic glucose binding protein; GlcF and GlcG, inner membrane glucose ABC transporter proteins; GlcK, a cytoplasmic ATPase component; and OprB, an outer membrane porin. BLAST analysis revealed that they are arranged as a single cluster in Pseudomonas (Fig. 1A). The benzoate transport genes were found to be arranged as 5′-benK-catA-benE-benF-3′ at the ben locus in CSV86 (Fig. 1B and Table 1). These genes are annotated as BenF, an outer membrane porin belonging to the OprD family, and two inner membrane secondary transport proteins, BenE (benzoate:H+ symporter) and BenK (aromatic acid:H+ symporter [AAHS] of the major facilitator superfamily [MFS]). The catA gene which encodes catechol 1,2-dioxygenase (C12DO), a key enzyme involved in benzoate metabolism, is also a part of the ben locus, flanked by benK and benE genes. The gene arrangement observed in CSV86 is similar to that reported in P. putida KT2440 but different compared to other Pseudomonas spp., where additional copies of transport (AAHS) and catechol degradation (catB, catC) genes are present in the vicinity (Fig. 1B).
Gene organization, transcription, and carbon-source-dependent relative gene expression of glc and ben loci in Pseudomonas putida CSV86. Organization of genes involved in glucose transport (A) and benzoate transport and metabolism (B) from different Pseudomonas spp. The annotation of genes from CSV86 is given in Table 1. The putative σ70-dependent promoter sequences from glc and ben loci are underlined. Numbers inside the arrows indicate the gene length (bp) while those in between the arrows indicate the intergenic distance. Numbers with negative signs indicate overlapping genes. Panels C and D represent the overlapping RT-PCR strategy and product analysis to determine the cotranscription of genes at glc and ben loci, respectively. The observed products (in kb) are for the glc locus [gbp and glcF (0.69) (i), glcF and glcG (1.2) (ii), glcG and glcK (0.85) (iii), glcK and oprB (0.7) (iv), gbp-glcF-glcG (1.67) (I), glcF-glcG-glcK (1.86) (II), and glcG-glcK-oprB (2.13) (III)] and for the ben locus [benK and catA (0.95) (1), catA and benE (1.5) (2), and benE and benF (1.4) (3)]. PCRs were performed using cDNA as the template (t, test), without reverse transcriptase (n, negative control) and using genomic DNA as the template (p, positive control). Relative gene expression level (fold change) of glucose transport genes gbp, oprB, and glcG from glucose-grown cells compared to naphthalene (black)-, benzoate (blue)-, and succinate (green)-grown cells (E) and of benzoate transport genes benE and benK from benzoate-grown cells compared to glucose (red)- and succinate (green)-grown cells (F). Transcription of rpoD was used as an internal control to normalize gene expression. Experiments were performed independently 3 times with reactions in duplicates.
Gene annotation for glc and ben loci of Pseudomonas putida CSV86
Sequence analysis of glc and ben loci led to identification of putative σ70 (sigma 70)-dependent promoters upstream of each operon, suggesting that they are independent transcription units (Fig. 1A and B). The PCR amplification yielding products of overlapping regions of mRNAs from the cells grown on glucose or benzoate by reverse transcriptase PCR (RT-PCR) (Fig. 1C and D) suggests that five genes of the glc locus and four genes of the ben locus were transcribed as two independent polycistronic transcripts. Further, the substrate-dependent relative gene expression of these loci was measured using quantitative real-time PCR (qPCR). The transcript levels of the glc locus genes gbp, glcG, and oprB were found to be significantly higher (35- to 405-fold) in the glucose-grown cells compared to naphthalene-, benzoate-, or succinate-grown cells (Fig. 1E). The benzoate-grown cells showed 15- to 24-fold higher transcript levels for the ben locus (benE and benK) compared to glucose- or succinate-grown cells (Fig. 1F). These results indicate that genes at the glc or ben loci are cotranscribed independently and are induced by glucose or benzoate, respectively.
Substrate-dependent transcriptional modulation of ben and glc loci.The first step in carbon utilization is the transport of substrate inside the cell. Time-dependent [14C]substrate uptake studies from cells grown on glucose plus benzoate exhibited significantly higher benzoate uptake in the first log phase than in the second log phase (Fig. 2). Glucose uptake increased progressively from the second lag phase, attaining a maximum in the second mid-log phase. The observed growth rates (h−1) were 0.68 and 0.07 in the first and second log phases of the diauxic growth pattern compared to 0.54 or 0.23 for cells grown on benzoate or glucose alone, respectively. The relative transcript level of benE (representative of ben locus) was observed to be 3.7-fold higher in the first log phase, which decreased significantly to −6-fold during the second lag phase (Fig. 2). The relative transcript level of gbp (representative of glc locus) was observed to be low (range, −13 to −5) in the first log phase and increased significantly (4-fold) during the later phases of growth (Fig. 2). The transcription analysis and biochemical data suggested that glucose was unable to suppress benzoate transport and metabolism. On the contrary, benzoate suppressed the utilization of glucose.
Correlation between gene expression and substrate transport activity during the growth of Pseudomonas putida CSV86 on glucose plus benzoate. Benzoate-grown culture was used as an inoculum. Filled circles represent the diauxic growth profile on glucose (0.25% wt/vol) plus benzoate (0.1% wt/vol). Thin bars represent the relative expressions of benE (yellow, representative of the ben locus) and gbp (orange, representative of the glc locus) during growth. The expression of gbp and benE at the time of inoculation (i.e., at 0 h) was considered basal expression; values were normalized to rpoD expression at each time point. Thick bars represent the transport of [14C]benzoate (cyan) and [14C]glucose (blue) by whole cells. Maximum uptake observed for [14C]benzoate (81 ± 15 pmol · min−1 · mg−1 cells) and [14C]glucose (775 ± 7 pmol · min−1 · mg−1 cells) was considered 100% (control). The specific growth rate (h−1) observed in the first log (benzoate utilization) and second log (glucose utilization) phases were 0.68 and 0.07 on glucose plus benzoate compared to 0.24 or 0.54 for cells grown on glucose or benzoate alone, respectively. The experiment was done independently three times in triplicate and the graph represents data with standard error bars.
Succinate does not repress benzoate but does suppress glucose transport and metabolism in CSV86.On succinate plus benzoate, irrespective of succinate- or benzoate-grown inoculum, CSV86 showed a single log phase with significantly high benzoate uptake and C12DO activity (Fig. 3A). High-pressure liquid chromatography (HPLC) analysis revealed the utilization of benzoate (by 9 h) and succinate (by 11 h) in the mid- and late-log phases, respectively (Fig. 3B). These results suggested that succinate was unable to suppress benzoate transport as well as metabolism and that both were cometabolized. These observations are interesting and contrary to earlier reports, where organic acids are known to suppress the degradation of aromatics in other Pseudomonas spp. (16, 17, 18, 19). On succinate plus glucose, CSV86 showed a diauxic growth profile with significantly reduced glucose transport (95 pmol · min−1 · mg−1 cells) in the first log phase, which increased to 1,200 pmol · min−1 · mg−1 cells in the second log phase, indicating utilization of succinate in the first log phase and of glucose in the second log phase.
Metabolic analysis of Pseudomonas putida CSV86 on succinate plus benzoate. Panels A and B represent the growth profile, whole-cell benzoate uptake rate (%), and C12DO activity of CSV86 cells grown on succinate (0.25% wt/vol) plus benzoate (0.1% wt/vol) using succinate (all green symbols, A)- or benzoate (all blue symbols, B)-grown inocula. The circles (green or blue) represent growth profile on succinate plus benzoate, squares (green or blue) represent whole-cell [14C]benzoate uptake, and bars (green or blue) represent C12DO activity. The specific growth rate (h−1) observed was 0.51 on succinate plus benzoate compared to 0.79 or 0.54 on succinate or benzoate alone, respectively. Panels C and D represent the concentrations of benzoate and succinate determined by HPLC from the spent media of culture grown on various carbon sources. Solid lines with circles represent growth profiles on succinate (green, succinate inoculum), benzoate (blue, benzoate inoculum), or succinate plus benzoate (pink, succinate inoculum), respectively. Dashed lines with squares represent the succinate concentration in the spent media of succinate (green)- or succinate plus benzoate (pink)-grown conditions, respectively. Dotted lines with inverted triangles represent the benzoate concentration in the spent media of benzoate (blue)- or succinate plus benzoate (pink)-grown conditions, respectively. The experiment was done independently a minimum of three times in triplicate, and the graph represents data with standard error bars.
Succinate and benzoate suppress glucose transport at the transcriptional level.When glucose-grown cells were spiked with benzoate or succinate, transcription analysis showed the repression of gbp within 20 min (Fig. 4A). The repression was stronger for benzoate- (−12-fold) compared to succinate-spiked cells (−4.6-fold). Beyond 20 min, the expression level for gbp was found to be similar (range, −4.6- to −2-fold) in succinate- or benzoate-spiked cells. During this period the expression of benE in benzoate-spiked glucose-grown cells increased within 20 min to 3.4-fold and peaked at 60 min to 12.6-fold, suggesting rapid induction of benE (Fig. 4A). The reduction in the glucose uptake was observed within an hour of succinate and 2 h of benzoate spiking of glucose-grown cells (Fig. 4B). It was also observed that benzoate-spiked glucose-grown cells showed a steady increase in benzoate uptake until 4 h, which increased significantly by 6 h. Further, at the metabolic level in the cell extracts the activity of Zwf (glucose 6-phosphate dehydrogenase, an enzyme involved in glucose metabolism) reduced significantly from 341 to 114 nmol · min−1 · mg−1 protein within 2 h in succinate-spiked cells compared to benzoate-spiked glucose-grown cells (288 nmol · min−1 · mg−1 protein) (Fig. 4C). At the 4 h time point the Zwf activity from succinate- or benzoate-spiked cells was comparable. The activity of C12DO (a ring-cleaving enzyme involved in benzoate metabolism) was increased from 9.6 to 372 nmol · min−1 · mg−1 protein by 2 h and attained maximum (457 nmol · min−1 · mg−1 protein) by 4 h (Fig. 4C).
Relative expression and functional analysis of benzoate, glucose transport genes, and metabolic enzymes from Pseudomonas putida CSV86. Cells were grown on glucose as the carbon source until 16 h and spiked with succinate (0.25% wt/vol), benzoate (0.1% wt/vol), or glucose (0.25% wt/vol) and subjected to analysis. (A) Relative expression (fold change) of gbp (representative of glucose transport) from glucose-grown cells spiked with glucose (red bars), benzoate (blue bars), or succinate (green bars) and benE (yellow bars, representative of benzoate transport) from glucose-grown cells spiked with benzoate. The expression of gbp and benE genes at the time of spiking (16 h glucose-grown cells) was considered basal expression. Expression was normalized to internal control, rpoD. (B) Percent whole-cell [14C]glucose uptake by glucose-grown cells spiked with glucose (red diamond), benzoate (blue triangle), or succinate (green square). The [14C]benzoate uptake of glucose-grown cells spiked with benzoate is depicted in yellow circles. The percent uptake was calculated considering glucose uptake at 0 min as 100% (930 pmol · min−1 · mg−1 cells) and benzoate uptake at 360 min as 100% (63 pmol · min−1 · mg−1 cells). (C) Specific activity of Zwf from glucose-grown cells spiked with glucose (red hatched bars), benzoate (blue hatched bars), or succinate (green hatched bars); and C12DO from glucose-grown cells spiked with glucose (dark green hatched bars) or benzoate (yellow hatched bars). The experiment was done independently a minimum of three times in triplicate, and the graph represents data with standard error bars.
Succinate and benzoate might interact directly with periplasmic GBP (glucose-binding protein) and/or cytoplasmic GlcK (ATPase) components of the ABC transporter and may thereby inhibit or reduce glucose transport. The purified rGBP failed to show any interaction with succinate or benzoate (0.4 to 1,000 μM), and the affinity toward glucose as measured by surface plasmon resonance (SPR) remained unaffected (Fig. 5). In the presence of succinate or benzoate (1 and 5 μM), rGBP showed 100% [14C]glucose binding activity. The purified rGlcK (ATPase) showed 85 to 95% activity (100% activity = 91.2 ± 10.2 nmol · min−1 · mg−1 protein) in the presence of succinate or benzoate (10 mM). Similarly, these substrates might interact with enzymes involved in glucose and/or benzoate metabolism. The activities of Zwf and C12DO remained unaffected in the presence of succinate and benzoate. These results suggest that succinate and benzoate do not interact directly with the glucose transport components (rGBP, rGlcK) or metabolizing enzymes (Zwf, C12DO).
Interaction of rGBP with solutes (glucose, benzoate, or succinate) by surface plasmon resonance. The binding of rGBP to glucose (A), benzoate followed by glucose (B), and succinate followed by glucose (C), with respective glucose saturation profiles. Bars in the panel represent blank (BLK, red), solute-free HEPES buffer (0, blue), glucose (G, green), benzoate (B, orange), and succinate (S, magenta). The Kd (dissociation constant) values given in the inset are determined by plotting the substrate saturation plot (0.045 to 100 μM). Note the x axis for the saturation plots for panels A to C. The experiment was done independently two times in duplicate. The trends observed in both experiments were similar. The best profile is depicted here.
DISCUSSION
Pseudomonas spp. display a hierarchical carbon utilization pattern, viz. organic acids > glucose > aromatic substrates. Although Pseudomonas strains are reported to be versatile xenobiotic degraders, regulatory features like catabolite repression of their metabolic pathways restrict their potential for efficient bioremediation. Pseudomonas putida CSV86 showed unique behavior in utilization of aromatics in preference to glucose. Based on the annotation and biochemical and transcriptional studies, the glucose ABC transporter in CSV86 is comprised of GlcF, GlcG, GlcK, and glucose-specific GBP. Analysis of the positional relationship of orthologous genes present at the glc locus revealed that glucose transport genes were arranged as a single cluster in Pseudomonas genomes. Similar analysis for orthologues among bacterial genomes revealed that the majority of orthologous transporter genes are present in conserved strings in the same gene order and are mostly arranged as operons (20, 21). These observations suggest that there is a positive selection of clustering of related genes forming an operon in the genome organization or under a common regulatory mechanism. The identification of a putative promoter in the upstream region of the glc locus and its transcription as a polycistronic mRNA indicate that all five genes are cotranscribed as a single transcription unit. In CSV86, the ben locus harbors benzoate transporter and metabolism genes that are also cotranscribed as a single polycistronic mRNA. Both glc and ben operons showed respective carbon-source-dependent inducible transcription.
In pseudomonads glucose represses the metabolism of aromatic compounds like toluene, methylphenol, phenylacetic acid, styrene, and phenanthrene (5, 7, 18, 22, 23, 24, 25). Interestingly, in CSV86, glucose failed to repress benzoate transport and metabolism at a transcriptional as well as at a biochemical level. This result was supported by the observed significantly higher C12DO activity (249 nmol · min−1 · mg−1 protein) in the first log phase, which decreased to 20 nmol · min−1 · mg−1 protein during the second log phase with a concomitant increase in the Zwf activity from 8 (first log phase) to 28 nmol · min−1 · mg−1 protein (second log phase) when cells were grown on glucose plus benzoate (10). Zwf activity in the presence of benzoate remained uninduced by glucose (10). Zwf activity from aromatic- or succinate-grown cells was found to be comparable and 4- to 5-fold lower than that from glucose-grown cells (9, 10). This is in contrast to the observed Zwf activity from P. putida KT2440, wherein benzoate- or xylene-grown cells showed 3- to 4-fold higher activity than citrate-grown cells (26). The enzyme induction pattern in CSV86 correlates well with the suppression of transcription of glc locus in the presence of benzoate observed in the present study. Transcriptional and biochemical analyses correlate well with the diauxic growth profile indicating regulation of glucose transport at the transcriptional level, leading to the preferential utilization of benzoate.
Organic acids like acetate, fumarate, pyruvate, and succinate are known to repress metabolism of benzyl alcohol, methylphenol, and chlorocatechol in pseudomonads (16, 17, 18, 19). However, in CSV86, succinate failed to repress benzoate transport and metabolism, leading to cometabolism of succinate and benzoate. Intriguingly, benzoate consumption was faster than that of succinate, as evident from HPLC analysis. When grown on succinate plus glucose, CSV86 cells showed a diauxic profile with glucose transport and metabolism in the second phase, similar to what has been reported for other Pseudomonas spp. (27). The important concern raised by this experimental data is whether the observed repression of the glc locus at the transcription level is due to benzoate per se or to one of its degradation metabolites, like succinate. To answer this, we studied the glc locus transcript levels and glucose transport as well as metabolism at the biochemical level. The glc locus genes were repressed when glucose-grown cells were spiked with benzoate or succinate. Besides transcription regulation, the observed reduction in biochemical activity could probably be due to direct interaction of aromatic or organic acids with proteins involved in glucose transport and metabolism. The inability of succinate or benzoate to interact with or inhibit the glucose transport components (GBP and/or GlcK) or the activity of the metabolic enzyme (Zwf) suggested that repression of the glucose transport system is more likely to be at transcriptional level. These results are interesting and not reported so far from any of the aromatic-compound-degrading bacterial strains.
In Pseudomonas, the key regulators proposed to be involved in catabolite repression are likely to be Crc, Hfq, and HexR proteins and small RNAs like CrcZ and CrcY (7, 28, 29, 30, 31, 32, 33). The draft genome analysis of CSV86 reveals the presence of genes encoding putative Crc, Hfq, and HexR proteins and small RNAs like CrcZ and CrcY. However, the unique carbon utilization hierarchy displayed by CSV86 compared to other Pseudomonas strains suggests the probable existence of a different regulatory mechanism(s). It will be interesting to study the interplay among these components so as to understand more about these regulatory features in CSV86. The properties of preferential utilization of aromatics over glucose and cometabolism with succinate by P. putida CSV86 have potential applications in the field of bioremediation. Further, the strain can serve as an ideal host to engineer degradation pathways for diverse aromatic pollutants, as it can metabolize them efficiently, even in the presence of glucose or organic acids.
MATERIALS AND METHODS
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.
List of primers used in this study
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 (CT) 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, ΔCT was calculated by the equation ΔCT = CT(target gene) − CT(rpoD). The ΔCT of samples from cells grown on compound A were compared with those from cells grown on B by using the equation ΔΔCT = ΔCT(A) − ΔCT(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 (ε260 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 (ε340 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.
ACKNOWLEDGMENTS
We thank Veenita Shah for her help with the SPR studies. We thank Santosh Noronha, Chemical Engineering Department, Indian Institute of Technology, Bombay, for use of an HPLC facility.
A.C. and A.M. thank the Department of Biotechnology (DBT) and the Council of Scientific and Industrial Research (CSIR), Government of India, respectively, for research fellowships. S.K.A. thanks the Department of Science and Technology, India, for the award of the Sir J. C. Bose National Fellowship (no. SERB/F/2569/2013-14, awarded 29 July 2013) and the Department of Atomic Energy, India, for the award of the Raja Ramanna Fellowship (no. DAE/10/1[20]/2014/RRF-R&D-II/3208, awarded 11 March 2015). P.P. thanks the Department of Science and Technology, Government of India, for the research grant.
FOOTNOTES
- Received 8 June 2017.
- Accepted 16 July 2017.
- Accepted manuscript posted online 21 July 2017.
- Copyright © 2017 American Society for Microbiology.