Identification of anthranilate and benzoate metabolic operons of Pseudomonas fluorescens and functional characterization of their promoter regions
© Retallack et al; licensee BioMed Central Ltd. 2006
Received: 17 November 2005
Accepted: 05 January 2006
Published: 05 January 2006
In an effort to identify alternate recombinant gene expression systems in Pseudomonas fluorescens, we identified genes encoding two native metabolic pathways that were inducible with inexpensive compounds: the anthranilate operon (antABC) and the benzoate operon (benABCD).
The antABC and benABCD operons were identified by homology to the Acinetobacter sp. anthranilate operon and Pseudomonas putida benzoate operon, and were confirmed to be regulated by anthranilate or benzoate, respectively. Fusions of the putative promoter regions to the E. coli lacZ gene were constructed to confirm inducible gene expression. Each operon was found to be controlled by an AraC family transcriptional activator, located immediately upstream of the first structural gene in each respective operon (antR or benR).
We have found the anthranilate and benzoate promoters to be useful for tightly controlling recombinant gene expression at both small (< 1 L) and large (20 L) fermentation scales.
Ideally, to facilitate control of gene expression for production of proteins in an organism like P. fluorescens, which has been developed as a robust recombinant protein expression system[1, 2], it is desirable to have a collection of expression cassettes. These cassettes would contain a variety of promoters that are tightly regulated, of differing strengths, induced under different growth conditions, and/or by different chemicals. These expression cassettes can then be linked to various genes of interest to achieve total control of those genes under typical fermentation conditions. High levels of gene expression are often obtained in the P. fluorescens system using the E. coli lac UV5 and tac promoters . Several bacterial promoters have been previously shown to be effective at driving transgene expression in pseudomonads including the bacteriophage λ PR and PL promoters [3, 4], which are regulated by a temperature sensitive repressor protein, and the Pseudomonas Pm, Pu, and Psal promoters [4, 5], which are regulated by alkyl- or halotoluenes (Pm and Pu) or salycilates (Psal) and the T7 early promoter , regulated by isopropyl-thiogalactopyranoside (IPTG).
Pseudomonads are capable of metabolizing a wide variety of aromatic hydrocarbons, including benzoate and anthranilate [7–11], which are inexpensive and non-toxic. Benzoate and anthranilate are converted to catechol by benzoate 1,2-dioxygenase together with 2-hydro-1,2dihydroxybenzoate dehydrogenase, and anthranilate 1,2-dioxygenase respectively. These enzymes are encoded by the benABCD and antABC operons, which have been identified in several organisms [9–16], and are often regulated by transcriptional activators belonging to the AraC/XylS family of transcriptional regulators. Transcriptional activators BenR and BenM have been described, which activate transcription of the benABCD operon [7, 17, 18]. Recently transcriptional activators of two different anthranilate operons have been described [10, 13].
We describe in this report the identification of the P. fluorescens strain MB214 benABCD and antABC operons, along with the genes coding for the transcriptional regulatory proteins BenR and AntR, respectively. The promoter regions of the benABCD and antABC operons have been defined, and regulation by BenR and AntR examined. A range of potential inducing compounds, in addition to benzoate and anthranilate, were tested including o-toluate, m-toluate, p-toluate and chlorinated anthranilates. Promoter elements are defined which are capable of driving heterologous gene expression at both small (1 L shake flask) and large (20 L fermentor) scales.
P. fluorescens MB214 can utilize anthranilate and benzoate as sole carbon source
Bacterial strains used in this study
Subsequent experiments conducted in M9 media with glucose, glycerol, citrate, or succinate as primary growth substrates indicated that benzoate and anthranilate catabolism in strain MB214 was inducible by addition of the individual aromatic acid substrates. The functional evidence for catabolic pathways inducible by benzoate and anthranilate, or their metabolic intermediates, led to a systematic isolation and evaluation of the corresponding regulatory switches for the development of gene expression systems.
Characterization of the anthranilate and benzoate metabolic operons
Anthranilate operon homologues. Percent amino acid identity/similarity shown below. AntA: P. aeruginosa (UNI_TREMBL:Q9I0X0); P. putida (UNI_TREMBL:Q93SR3),, and P. resinovorans (GenPept: NP_758565). AntB: P. aeruginosa (UNI_TREMBL:Q9I0SR8); P. putida (UNI_TREMBL:Q90W9); P. resinovorans (GenPept: NP_758547). AntC: P. aeruginosa (UNI_TREMBL:Q90W8); P. putida (UNI_TREMBL:Q93SR4), P. resinovorans (GenPept: NP_758546). Anthranilate dioxygenase transcriptional activator AntR: P. resinovorans (GenPept: NP_758551).
P. aeruginosa homologue (%identity/similarity)
P. putida homologue (identity/similarity)
P. resinovorans homologue (%identity/similarity)
P. fluorescens AntA
P. fluorescens AntB
P. fluorescens AntC
P. fluorescens AntR
Benzoate operon homologues. Percent amino acid identity/similarity shown below. BenR: XylS of P. aeruginosa (UNI_TREMBL:Q9I0W3) and P. putida (UNI_SPROT:XYS3_PSEPU). BenA: P. aeruginosa toluate 1,2 dioxygenase alpha subunit (UNI_TREMBL:Q9I0W4); P. putida BenA (UNI_TREMBL:Q88I40). BenB: P. aeruginosa toluate 1, 2-dioxygenase small subunit (UNI_TREMBL:Q9I0W5); P. putida BenB (UNI_TREMBL:Q88I39). BenC: P. aeruginosa toluate 1,2 dioxygenase subunit (UNI_TREMBL:Q9WWV0); P. putida BenC (UNI_TREMBL:Q88I38). BenD: P. aeruginosa cis 1,2-dihydroxycyclohexa-3,4-diene carboxylate dehydrogenase (UNI_TREMBL:Q9I0W7); P. putida BenD (UNI_TREMBL:Q88I37).
P. aeruginosa homologue (% identity/similarity)
P. putida homologue (% identity/similarity)
P. fluorescens BenA
P. fluorescens BenB
P. fluorescens BenC
P. fluorescens BenD
P. fluorescens BenR
Isolation of the Pant and Pben promoters
Overexpression of transcriptional activator increases Pant activity
In an attempt to improve the activity of the anthranilate inducible promoter, the promoter fragment was amplified to include the putative transcriptional activator ORF directly upstream of antA. This PCR fragment was cloned upstream of the lacZ reporter gene of pDOW1017. MB101 was transformed with the resulting plasmid, pDOW1035, and activity upon induction with anthranilate was assessed. The addition of the transcriptional activator in multicopy demonstrated faster and greater induction compared to Pant713 (Figure 3B), which does not contain the entire transcriptional activator ORF. The activity of the culture harbouring pDOW1035 peaks early, then declines between 6 and 24 hours. This is likely due to the rapid decline in anthranilate concentration, compared to the culture carrying pDOW1029 (data not shown). Similar experiments were performed with a fragment containing benR and the Pben509 fragment, however no difference in promoter activity were observed (data not shown).
Inactivation of the benABCD operon transcriptional activator
Alternative inducers of Pant and Pben promoters
Mapping transcriptional start sites of Pben and Pant promoters
Total RNA was isolated from an anthranilate induced culture of MB101 carrying pDOW1029 (Pant713). Primer extension analysis was performed using the lacZPE2 primer. An extension product was observed corresponding to the adenosine nucleotide 31 bases upstream of the 3' end of the Pant713 clone (Figure 7B). This start site is consistent with the spacing of the putative -10 and -35 promoter sequences shown in italics and underlined in Figure 7D.
Analysis of Pben and Pant activity at the 20 L fermentation scale
The antR-Pant construct pDOW1035 was also tested at the 20 L scale. MB101 carrying pDOW1035 was grown under standard fermentation condition, with glucose as a carbon source. Following induction with either 5 mM or 10 mM anthranilate, promoter activity was observed (Figure 8B). The culture induced with 10 mM anthranilate showed a higher level of activity than that induced with 5 mM anthranilate, as expected. Activity was not detected until very late in the fermentation unlike Pben278, which showed an increase in activity between 6 and 24 hours (Figure 8A).
Benzoate induces Pben509
The work presented here describes the anthranilate and benzoate catabolic operons of P. fluorescens strain MB101. The anthranilate dioxygenase large and small subunits (AntA and AntB) and reductase (AntC) are most closely related to the anthranilate dioxygenase and reductase recently described in P. resinovorans. Although the transcriptional activator, AntR, is also closely related to the transcriptional activator of P. resinovorans, the chromosomal arrangement of P. fluorescens antR gene in relation to the antABC is different than that of P. resinovorans . The P. fluorescens antR gene is divergently transcribed from the antABC operon, similar to the arrangement of the anthranilate dioxygenase operon of B. cepacia . The P. fluorescens AntR represents further evidence of an AraC/XylS type transcriptional activator regulating anthranilate dioxygenase expression. Transcriptional activators for anthranilate operons have only recently been described [10, 13]. Studies of antABC promoter activation presented here indicate that AntR may be the limiting factor in expression from the Pant promoter. Addition of antR in multicopy results in significant improvement of Pant activity (Figure 3) indicating that the presence of presumably higher levels of inducer alone was not sufficient to promote high levels of gene expression from the Pant promoter. It is possible that either AntR is produced at low levels, or that the protein binds weakly to the promoter region. In either case, increasing the overall concentration of the protein would result in improved promoter activity. The rapid metabolism of anthranilate in the pDOW1035 harbouring strain supports the observation that overexpression of antR results in improved Pant activity, since increased activity of the chromosomally located Pant promoter would result in increased expression of the anthranilate dioxygenase, and subsequently, increased anthranilate metabolism. Although AntR shows high homology to other AraC/XylS transcriptional activators, we were unable to detect a consensus XylS type DNA binding site (TGCA-N6-GGNTA)  upstream of the Pant core promoter. Further investigation of AntR expression and elucidation of its binding site in the Pant promoter may help to improve promoter activity. The Pben promoter fragment, however, does appear to have a XylS type DNA binding site as shown in italics in Figure 2B. The sequences TGCG-N6-GGATA and TGCA-N6-GGATA, separated by 5 bp, are located immediately upstream of the putative -35 signal. Further investigation is required to determine whether these sequences are important for Pben activation. Unlike the antR-Pant system, increasing the copy number of benR did not improve Pben activity (data not shown).
Anthranilate and benzoate have been shown to be inducers of the Pant and Pben promoters, respectively, as expected. Not unexpectedly, several other compounds were found to induce the promoters. m-toluate, which has been shown to induce other benzoate operons, including those of Rhodococcus sp. strain 19070  and P. putida mt-2 , was found to induce the Pben promoter 13-fold over background. o-toluate, which has not been previously reported to induce either benzoate or anthranilate operon promoters, was found to induce the Pant promoter 19-fold. However, o-toluate induction of the Pben promoter, along with p-toluate induction, was only 2.5 to 3-fold over background. The anthranilate promoter was also inducible with 6-chloro-anthranilate. The fact that 6-chloro-anthranilate was not metabolized significantly by P. fluorescens strain MB101 indicates that 6-chloro-anthranilate acts as a gratuitous inducer, and that anthranilate itself, and not necessarily a metabolite of anthranilate, may act as an inducer. Benzoate was shown to be the inducer of the Pben promoter by deleting the genes coding for the benzoate dioxygenase large and small subunits. As shown in Figure 9, the Pben promoter remains active in the absence of benzoate metabolism.
For Pben and Pant to be used as part of an expression system, it is important to identify inducing compounds that are not metabolized by the host organism. We have shown that the Pben and antR-Pant promoters can drive heterologous gene expression at the 20 L fermentation scale. Although the Pben promoter appears to be repressed under the glucose feed conditions used at the 20 L fermentations scale, the antR-Pant promoter construct was found to be active. However, a change in carbon source from glucose to glycerol alleviated the observed Pben repression at the 20 L scale. Additional fermentation optimization may result in improved promoter activity and heterologous gene expression. Further strain development, such as chromosomal deletion of the anthranilate and benzoate dioxygenases should also improve expression during the course of induction and simplify the induction process, in that the inducer will not need to be added more than once during the course of induction to maintain expression levels.
It is important to identify and develop several recombinant expression systems to allow for flexibility and reliability in protein production. The availability of more than one inducible expression system will allow for differential expression of proteins during production. Differential expression of chaperones, foldases or disulfide bond isomerases from these alternate expression systems may aid in overall yield of active target protein. We have shown that the benzoate and anthranilate promoters examined in this work can be used to drive recombinant protein expression in P. fluorescens at both small (shake flask) and large (20 L) scales. Further development of these promoter systems will aid in expression of complex proteins in P. fluorescens.
Bacterial strains and growth conditions
Plasmids Used in This Study
New England Biolabs
400 bp benR fragment
3' benR + 5' benC
20 L fermentation
P. fluorescens fermentations were conducted in standard, aerated 20 L research fermentors with a mineral salts medium derived from Riesenberg, et al. . Cultures were grown at 32°C and pH 6.5–7.0 was maintained through the addition of aqueous ammonia. Agitation and sparged airflow rates were gradually increased during the growth phase of the fermentation to control dissolved oxygen at a positive level (15% of saturation) but were fixed at maximum levels when these were reached. The cultures were operated as either a glucose or glycerol fed-batch or as a glycerol batch (glycerol concentration <200 g/L). The fermentation process was divided into an initial cell growth phase (typically 24–30 hours) and a gene expression (induction) phase in which inducer was added (anthranilate or sodium benzoate solution delivered as a bolus or fed) to initiate recombinant gene expression.
General cloning methods
Primers Used in This Study
Construction of benzoate promoter fusions
The Pben509 promoter fragment was amplified using the ben40PL and ben40PU primers with MB214 genomic DNA as template. The resulting fragment was digested with Pac I and BamH I and cloned into pDOW1017 to produce the Pben509::lacZ fusion pDOW1019. The Pben278 promoter fragment was amplified using primers benL278 and ben40PU, using pDOW1026 as template. PCR product was purified on a Microcon YM-100 column (Millipore, Billerica, MA). The PCR product was digested with Pac I and BamH I and cloned into the same sites of pNEB193 to produce pDOW1022. The BamH I-Pac I fragment of pDOW1022 containing the Pben278 fragment was then cloned into pDOW1017 to produce pDOW1028, a Pben278::lacZ fusion.
Construction of anthranilate promoter fusions
The Pant713 Promoter was amplified using primers antPU and antPL and MB214 genomic DNA as the template. The resulting fragment was cloned into pDOW1017 to produce the Pant713::lacZ fusion pDOW1029. The Pant311 promoter was amplified using primers ant311 and antPL+SD using MB214 genomic DNA as the template. The PCR product was purified with a Microcon YM-100 column (Millipore) and the volume brought up to 50 μl with 5 mM Tris-Cl, pH8.0 (~100 ng/μl). The PCR product was cloned into the pGEM-T Easy vector (Promega) and sequenced using M13F and M13R primers. The pGEM clone was digested with BamH I and Pac I then cloned into the same sites of pDOW1033 creating a Pant311::phoA fusion, pDOW1055.
Construction of promoter fusions with transcriptional activators
The putative benzoate operon transcriptional activator gene that lies upstream of benA was amplified using the primers benact5 and benact3, using MB214 genomic DNA as template. An ~1.2 kb fragment was isolated using the Prep-a-Gene kit (Biorad, Hercules, CA), digested with Pme I and Hind III and cloned into the same sites to pNEB193 (New England Biolabs) to produce pDOW1021. The Pme I – Hind III fragment of pDOW1021 was then cloned into the same sites of pDOW1019, upstream of the Pben509::lacZ fusion.
The anthranilate operon transcriptional activator that lies upstream of antA was amplified using the 3'antactiv primer and antPL+SD primer using MB214 genomic DNA as template. An ~1.3 kb fragment was purified using the Prep-a-Gene kit (Biorad), digested with Hind III and BamH I and cloned into the same sites to pNEB193 to produce pDOW1020. The BamH I -Hind III fragment of pDOW1020 was then cloned into the same site of pDOW1017 to produce pDOW1035.
Strains of interest were grown overnight (30°C shaking at 250 rpm) in 1× M9 supplemented with 1% (w/v) glucose, 1 mM MgSO4 and trace elements. Strains were then subcultured and induced with indicated concentrations of anthranilate or benzoate. β-galactosidase activity of samples was analyzed in a 96-well format. For each sample well, buffer was prepared as follows: 152 μl Z buffer (0.06 M Na2HPO4-7H2O, 0.04 M NaH2PO4-H2O, 0.01 M KCl, 0.001 M MgSO4-7H20) was mixed with 8 μl 1 M β-mercaptoethanol. Buffer was prepared in bulk quantities: to each 900 μl of this mix were added 1 drop 0.1%SDS and 2 drops CHCl3, and vortexed to mix. In each well, 144 μl of the above reaction mix and 16 ul of cells were combined. The microtiter plate was sealed and vortexed for 10 seconds, then equilibrated at room temperature for 5 minutes before addition of 50 μl 4 mg/ml 2-nitrophenyl β-D-galactopyranoside (ONPG). When a significant yellow color developed, 90 μl stop solution (1 M Na2CO3) was added and time was recorded. Reactions were read at A420 and A550, soon after stopping the reaction; cell density was read at A600. Miller Units were calculated as follows: 1000 * ((A420 - (1.75*A550))/(time (in minutes) * 0.1 * A600)). An average of triplicate wells is reported for each sample.
Analysis of anthranilate and benzoate concentrations
HPLC analyses of culture supernatant was carried out on a Hewlett-Packard Series 1100 HPLC using an isocratic method capable of separating anthranilate, benzoate, catechol, 6-chloroanthranilate and o-toluate. The separation column was a ZORBAX Eclipse XDB-C8 (4.6 × 150 mm, 5 μm; Agilent P/N 993967-906) equipped with a Supelguard Discovery C18 guard column (4 × 20 mm; Supelco P/N 505129). The mobile phase contained 25% acetonitrile in 25 mM sodium dihydrogen phosphate (NaH2PO4), pH 2.5, and the flow rate was 1.0 mL/min. The method used a standard sample injection volume of 2.5 μL and compounds were detected by monitoring absorbance at 254 nm. Under these conditions, compound retention times were as follows: catechol (2.20 min), 6-chloroanthranilate (3.01 min), anthranilate (3.46 min), benzoate (4.98 min), and o-toluate (6.53 min). Area responses were linear (R2 0.99) for calibration standards ranging from 0.5 to 5.0 mM.
An overnight culture of MB101 carrying the appropriate plasmid was grown in 1× M9 supplemented with 1% glucose (w/v), 1 mM MgSO4 and trace elements. The culture was induced with 5 mM benzoate or anthranilate as appropriate for 8 or 24 hours. Cells were pelleted and total RNA isolated using RNeasy maxi kit (Qiagen). The RNA was resuspended to a final volume of 200 μl and treated with 10 units DNAseI (RNase free) (Ambion) according to manufacturer's protocol. Following DNAseI treatment, the RNA was purified using an RNeasy midi or mini column (Qiagen). RNA concentration was determined using Ribogreen (Molecular Probes).
Primer Labeling:1 μl 10 μM primer (either lacZPE or lacZPE2), 1 μl 10× T4 kinase buffer, 5 μl 32P γ ATP (50 uCi, Amersham Biosciences, Pistcataway, NJ), 1 μl T4 kinase, 2 μl ddH2O, was incubated at 37°C for 30–60 minutes. 5 μl of the reaction was reserved to use for sequencing ladder. 20 μl TE was added to the remaining 5 μl and purified using a G25 sephadex column (Amersham-Pharmacia) to remove unincorporated nucleotides, yielding a final concentration of 0.2 μM labelled primer.
Sequencing ladder: The Promega fmol kit, along with 1 μM labelled primer was used to generate a sequencing ladder. Plasmid template used corresponds with that contained in the strain from which RNA was isolated for the extension reaction.
Primer Extension reaction: 10–20 μg of total RNA was mixed with 0.2 pmol primer to yield a final volume of 12 μl, and incubated at 70C for 10 min. To this was added 4 μl 5× Superscript II buffer, 2 μl 1 M DTT, 1 μl 10 mM dNTPs, 1 μl Superscript II or Thermoscript Reverse transcriptase (Invitrogen) and incubated 42°C (Superscript) or 55°C (Thermoscript) for 1 hour. 4 μl of sequencing stop solution was added to the reaction. All reactions were heated at 70°C for 2 minutes immediately before being loaded onto a 6% Long Ranger (Biowhittaker, Rockland, ME)/8 M urea/1.2× TBE gel next to the sequencing ladder. The gel was run in 0.6× TBE buffer, then dried, exposed to phophor screen, and imaged on the Typhoon phophorimager (Molecular Dynamics).
Inactivation of benzoate transcriptional activator
A DNA fragment containing a portion of the open reading frame (ORF) upstream of the benA gene was amplified by PCR using MB214 genomic DNA as template with the benactKO-for and benactKO-rev primers. JM109 was transformed with the resulting product cloned into the pCR2.1 TOPO vector (Invitrogen). Transformants were screened for insert by colony PCR using M13F and M13R primers, and the positive clones were further confirmed by DNA sequencing. The resulting plasmid, pDOW1131, was used to insertionally inactivate the corresponding ORF. P. fluorescens strain MB101 was transformed with pDOW1131, selecting on LB agar supplemented with tetracycline. Primers benactKO-for and either M13F or M13R were used to confirm insertion of the plasmid into the desired ORF by colony PCR. The resulting strain was named DC284.
Construction of P. fluorescens ΔbenAB ΔproC ΔpyrF
Following the method previously described for construction of gene deletions in P. fluorescens , the plasmid pDOW1139 was constructed to facilitate deletion of the benAB genes as follows. The 3' portion of the benR gene and the 5' portion of the benC gene were amplified using MB214 genomic DNA as template. The benR region was amplified using primers H3_5'benAKOclean and benKOmega. The benC region was amplified using primers H3_3'benBKOclean and invbenKOmega. The benR and benC fragments were fused using primers H3_5'benAKOclean and H3_3'benBKOclean with both fragments as template. The expected 1.1 kb fragment was gel-purified using Qiaex II (Qiagen) and cloned into Srf I digested pDOW1261-2. P. fluorescens MB101 derivative DC164  was transformed with the resulting plasmid, pDOW1139. Transformants were selected by plating on LB-proline-uracil with tetracycline for selection. Since the plasmid could not replicate in P. fluorescens, tetracycline resistant colonies isolated following transformation resulted from the plasmid integration into the chromosome. The site of plasmid integration was analyzed by PCR. To obtain strains that have lost the integrated plasmid by recombination between the homologous regions, single colonies of the transformants were inoculated into liquid LB supplemented with 250 μg/mL each proline and uracil (LB-proline-uracil), grown overnight, and then plated onto LB-proline-uracil and 500 μg/mL 5'-fluoroorotic acid (FOA) to counter select for the loss of the plasmid . Isolates having the phenotype expected (i.e., tetracycline sensitive, uracil auxotrophic, and FOA resistant) were selected. DNA from the resulting strain (DC253) was analyzed by PCR to confirm removal of the benAB region using primers 5'benA_seq, seq_3'benB, M13R21, 1261-8378F and 1261-103R.
The authors would like to thank John D. Stankowski, Angel Salazar, Miracle Foy and Joe Illeman for excellent technical assistance. We also thank Lawrence Chew, Carrie Schneider, Tom Ramseier and Mani Subramanian for helpful discussions. This work was supported by the Dow Chemical Company.
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