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Jun 29, 2015 - and bifenthrin, and their metabolite 3-PBA as the sole carbon and energy sources for growth (5, 9). The esterase gene pytH, which is.
PbaR, an IclR Family Transcriptional Activator for the Regulation of the 3-Phenoxybenzoate 1=,2=-Dioxygenase Gene Cluster in Sphingobium wenxiniae JZ-1T Minggen Cheng, Kai Chen, Suhui Guo, Xing Huang, Jian He, Shunpeng Li, Jiandong Jiang Key Laboratory of Microbiological Engineering of Agricultural Environment, Ministry of Agriculture, College of Life Sciences, Nanjing Agricultural University, Nanjing, Jiangsu, China

The 3-phenoxybenzoate (3-PBA) 1=,2=-dioxygenase gene cluster (pbaA1A2B cluster), which is responsible for catalyzing 3-phenoxybenzoate to 3-hydroxybenzoate and catechol, is inducibly expressed in Sphingobium wenxiniae strain JZ-1T by its substrate 3-PBA. In this study, we identified a transcriptional activator of the pbaA1A2B cluster, PbaR, using a DNA affinity approach. PbaR is a 253-amino-acid protein with a molecular mass of 28,000 Da. PbaR belongs to the IclR family of transcriptional regulators and shows 99% identity to a putative transcriptional regulator that is located on the carbazole-degrading plasmid pCAR3 in Sphingomonas sp. strain KA1. Gene disruption and complementation showed that PbaR was essential for transcription of the pbaA1A2B cluster in response to 3-PBA in strain JZ-1T. However, PbaR does not regulate the reductase component gene pbaC. An electrophoretic mobility shift assay and DNase I footprinting showed that PbaR binds specifically to the 29-bp motif AATAG AAAGTCTGCCGTACGGCTATTTTT in the pbaA1A2B promoter area and that the palindromic sequence (GCCGTACGGC) within the motif is essential for PbaR binding. The binding site was located between the ⴚ10 box and the ribosome-binding site (downstream of the transcriptional start site), which is distinct from the location of the binding site in previously reported IclR family transcriptional regulators. This study reveals the regulatory mechanism for 3-PBA degradation in strain JZ-1T, and the identification of PbaR increases the variety of regulatory models in the IclR family of transcriptional regulators.

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he main metabolite in the degradation of insecticide pyrethroids in mammals (1), insects (2), fungi (3), and bacteria (4, 5) is 3-phenoxybenzoate (3-PBA), a diaryl ether compound. Because of the wide use of pyrethroids (6) and the stability of the diaryl ether compound itself (7), 3-PBA is typically detected as an important environmental contaminant. To date, two biodegradation systems of 3-PBA have been identified in bacteria (see Fig. S1 in the supplemental material): (i) the PobAB system in Pseudomonas pseudoalcaligenes POB310 (8), which is a type I Rieske nonheme iron aromatic-ring-hydroxylating oxygenase (RHO), and (ii) the type IV RHO PbaA1A2BC system in Sphingobium wenxiniae JZ-1T (9). Due to hydroxylation at different positions in the aromatic ring (9), 3-PBA is catabolized to protocatechuate and phenol in the first system and to 3-hydroxybenzoate and catechol in the second system. Although the molecular mechanism of 3-PBA degradation in bacteria has been well characterized (8, 9), the regulation of its degradation is still unknown. Strain JZ-1T can utilize pyrethroids, such as cypermethrin, cyhalothrin, deltamethrin, fenpropathrin, fenvalerate, permethrin, and bifenthrin, and their metabolite 3-PBA as the sole carbon and energy sources for growth (5, 9). The esterase gene pytH, which is responsible for the first step of hydrolyzing pyrethroids, is constitutively expressed (5), while the expression of the 3-PBA catabolic gene cluster pbaA1A2BC is induced by its substrate 3-PBA (9). PbaA1 and PbaA2 are the ␣ and ␤ subunits of the dioxygenase, respectively, PbaB is a ferredoxin component, and PbaC is a glutathione reductase (GR)-type reductase (9). The pbaA1, pbaA2, and pbaB genes are distributed together and form one gene cluster, while pbaC is not physically linked to the pbaA1A2B cluster (9). Although the transcription of pbaC is 3-PBA inducible, its transcriptional level is far lower than that of the pbaA1A2B cluster

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under the same conditions (9). Moreover, no candidate transcriptional regulator gene was found nearby the pbaA1A2B cluster. In the present study, by using a DNA affinity approach, an IclR family transcriptional regulator, PbaR, was identified as the transcriptional activator of the pbaA1A2B cluster in response to 3-PBA in strain JZ-1T. The regulatory mechanism, including the transcriptional start site (TSS), the binding site, and the core binding sequence, and the effect of the substrate on binding were investigated, and a regulatory model for PbaR on the pbaA1A2B cluster was also proposed. PbaR showed an unusual binding site that is distinct from those of previously reported members of the IclR family of transcriptional regulators. The identification of PbaR increases the variety of regulatory models within the IclR family of transcriptional regulators. MATERIALS AND METHODS Chemicals and media. Cypermethrin (⬎97% purity) and 3-PBA (98% purity) were purchased from J&K Scientific, Ltd. (Shanghai, China), were prepared as a 0.1 M stock solution in methanol, and were sterilized by

Received 29 June 2015 Accepted 10 September 2015 Accepted manuscript posted online 18 September 2015 Citation Cheng M, Chen K, Guo S, Huang X, He J, Li S, Jiang J. 2015. PbaR, an IclR family transcriptional activator for the regulation of the 3-phenoxybenzoate 1=,2=dioxygenase gene cluster in Sphingobium wenxiniae JZ-1T. Appl Environ Microbiol 81:8084 –8092. doi:10.1128/AEM.02122-15. Editor: M. A. Elliot Address correspondence to Jiandong Jiang, [email protected]. Supplemental material for this article may be found at http://dx.doi.org/10.1128 /AEM.02122-15. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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TABLE 1 Strains and plasmids that were used in this study Strain or plasmid E. coli strains DH5␣

Source or reference(s)

Characteristic F⫺ ⌬(lacZYA-argF)U169 ␾80dlacZ⌬M15 recA1 endA1 thi-1 supE44 relA1 deoR hsdR17(rk⫺ mk⫹) phoA ␭⫺ relA1 ⫺ F ompT hsdS(rB⫺ mB⫺) gal dcm lacY1 (DE3)

TaKaRa

Sphingobium wenxiniae JZ-1T (⫽ DSM 21828T) JZ-MT JZ-MTC

3-PBA degrading strain, Smr pbaR-disrupted mutant from strain JZ-1T; Kmr Smr Mutant JZ-MT harboring pBpbaR; Gmr Smr Kmr

5, 9 This study This study

Plasmids pMD19-T pMpbapt pBBR1MCS-2 pBBR1MCS-5 pBpbaR pGEX-4T-1 pGEpbaR

TA clone vector, Apr pMD19-T containing pba cluster promoter, Apr Broad-host-range cloning vector, Kmr Broad-host-range cloning vector, Gmr pBBR1MCS-5 harboring pbaR, Kmr Expression vector, Apr pGEX-4T-1 harboring pbaR, Apr

TaKaRa This study 12 12 This study GE This study

BL21(DE3)

membrane filtration (pore size, 0.22 ␮m). The mineral salt medium (MSM) that was used in this study contained NaCl (1.0 g liter⫺1), NH4NO3 (1.0 g liter⫺1), K2HPO4 (1.5 g liter⫺1), KH2PO4 (0.5 g liter⫺1), and MgSO4·7H2O (0.2 g liter⫺1) and had a pH of 7.0. Luria-Bertani (LB) medium contained NaCl (5.0 g liter⫺1), yeast extract (5.0 g liter⫺1), and tryptone (10.0 g liter⫺1). Bacterial strains, oligonucleotides, plasmids, and culture conditions. The bacterial strains and plasmids that were used in this study are listed in Table 1, and oligonucleotide primers are listed in Table 2. Escherichia coli strains were used for recombinant DNA procedures and were grown at 37°C in LB medium or LB medium supplemented with antibiotics. Other bacterial strains were grown aerobically at 30°C in LB broth or LB agar. Expression of 3-PBA-degrading genes was induced in MSM supplemented with 0.5 mM 3-PBA. Ampicillin (Ap) and streptomycin (Sm) were used at concentrations of 100 ␮g ml⫺1. The growth of cells was evaluated by measuring the value of the optical density at 600 nm (OD600). Analysis of the transcriptional start sites of the pba cluster. The total RNA of strain JZ-1T was isolated using the MiniBest universal RNA extraction kit (TaKaRa, Dalian, China) according to the manufacturer’s instructions. The TSSs of the pbaA1A2B cluster and pbaC gene were detected by a primer extension assay as described by Lloyd et al. (10). Reverse transcription-PCR (RT-PCR) was performed using the 5=-6-fluorescein amidite (FAM)-labeled primers FAM-PE and FAM-PEC for the pbaA1A2B cluster and pbaC gene, respectively (Table 2). The RNA and primer mixture was annealed at 65°C for 10 min; deoxynucleoside triphosphates (dNTPs) and Moloney murine leukemia virus (MMLV) transcriptase were then added, and the mixture was incubated at 45°C for 45 min. The reaction was stopped by heating the mixture at 65°C for 10 min. The synthetic cDNA was purified by phenol-chloroform extraction and precipitated with ethanol; the DNA was then dissolved in 10 ␮l of double-distilled water (ddH2O). For analysis, 1 ␮l of the DNA solution was mixed with 8.5 ␮l of formamide and 0.5 ␮l of GeneScan-LIZ 500 size standards (Applied Biosystems), and the sample was analyzed on a 3730 DNA analyzer. The DNA sequencing reaction was prepared using the non-FAM-labeled primer PE or PEC (Table 2) as the sequencing primer. To test whether the pbaA1, pbaA2, and pbaB genes in the pba cluster were in one transcriptional unit, the sequence of pbaA1A2B was detected by PCR using cDNA as the template. Due to the difficulty in synthesizing large fragments by reverse transcription, the transcriptional unit test included PCR detection of 6 overlapping fragments in the pbaA1A2B cluster (Fig. 1C).

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TaKaRa

Identification of a promoter DNA-binding protein by DNA affinity purification. To identify the probable transcription factor that binds to the promoter DNA of the pbaA1A2B cluster, a DNA affinity purification approach was conducted (11). Cells of strain JZ-1T were grown in 100 ml of LB medium containing 0.5 mM 3-PBA and were harvested by centrifugation (12,000 ⫻ g for 3 min) at 4°C. After being washed with TN buffer (50 mM Tris-HCl [pH 7.4] and 50 mM NaCl) twice, the precipitated cells were resuspended in 15 ml of binding buffer (50 mM Tris-HCl [pH 7.4], 1 mM EDTA, 1 mM dithiothreitol [DTT], 100 mM NaCl, 0.05% [vol/vol] Triton X-100, and 10% [vol/vol] glycerol) and were then disrupted by sonication at 4°C. The suspension was centrifuged at 12,000 ⫻ g for 30 min at 4°C and concentrated to 1 ml using an Amicon ultrafiltration device. The pba promoter area sequence (⫺359 to ⫹137 bp relative to the translation start codon) was amplified using the biotin-modified primer pair bio-pbapt-F/pbapt-R (Table 2). The DNA was then purified using the E.Z.N.A. gel extraction kit (Omega, USA). Approximately 200 pmol of the promoter DNA was immobilized on streptavidin magnetic beads (NEB, England) according to the manufacturer’s protocol. The beads were then incubated with 1 ml of crude protein extracts (obtained as described above) at 25°C for 1 h with slow shaking. Unbound proteins were removed by washing the beads with binding buffer 4 times. Finally, the specifically bound proteins were eluted with 400 ␮l of low-NaCl-concentration elution buffer A (50 mM Tris-HCl [pH 7.4] and 0.5 M NaCl) and high-NaCl-concentration elution buffer B (50 mM Tris-HCl [pH 7.4] and 1.0 M NaCl). The eluted proteins were separated by 12% polyacrylamide sodium dodecyl sulfate (SDS)-gel electrophoresis, and 4 bound proteins with molecular masses of ⬍44.3 kDa were digested with trypsin and identified using matrix-assisted laser desorption ionization–time of flight/ time of flight (MALDI-TOF/TOF) mass spectrometry. The polypeptide peaks were compared with those of computed peptide sequences from the draft genome sequence of strain JZ-1T. Genetic disruption and complementation. Two DNA fragments, corresponding to 350-bp flanks upstream and downstream of the pbaR gene, were amplified using the primer pairs MTF1-F/MTF1-R and MTF3F/MTF3-R, respectively. They were linked to a kanamycin resistance gene that was amplified from plasmid pBBR1-MCS2 (12) with primer pair MTF2-F/MTF2-R by overlap extension PCR. This DNA fragment for homologous recombination was electroporated into strain JZ-1T cells. A positive genetic disruption mutant, JZ-MT, was achieved by serial culture on a kanamycin-containing medium until the pbaR gene could no longer

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TABLE 2 Oligonucleotides that were used in this study Function and oligonucleotide

Sequence (5= ¡ 3=)a

Primer extension FAM-PE FAM-PEC PE PEC

FAM-ATTTGCCATGTCCATCTCCTATA FAM-TCATTGCGACCGATCCTTCGAACCC ATTTGCCATGTCCATCTCCTATA TCATTGCGACCGATCCTTCGAACCC

Transcriptional unit analysis pba-F1 pba-R1 pba-F2 pba-R2 pba-F3 pba-R3 pba-F4 pba-R4 pba-F5 pba-R5 pba-F6 pba-R6

AACGGTATAGTCAAAGCCAGCATAT CGCATCCCAGTTGCCAAAAATCA CGGACGGCTACCACAACAAGCTC AACACGGTCATCACCAGCGAAC AGAGATGCGAGCCCGGTTAGGCGAT AAGCGCGGGCTCCTTCTTCGGCGTA CGAAAATGTCGCCCTCTGTCACCGA TCGGCAGGAACCGTTTGTCGAAATC GCCATGCCAGTTCGGACAACCC GCCTCGATCCCCGGAACACC CGGACGGACGTGAATTTACGGTCAA CTAGTGTTGCGTCTCAGGGATGGTG

DNA affinity and EMSA bio-pbapt-F pbapt-F pbapt-R pbapt-d-R pbapt-d-F pbapt-m-R pbapt-m-F pbapt-m2-R pbapt-m2-F pcpt-F pcpt-R ck-F ck-R

Biotin-CCCGGTTTTGGTCCCATTGGT CCCGGTTTTGGTCCCATTGGT AAGAGCCATGAACGGGAGAAT TACAAAAATAAGACTTTCTATTATATTATCGACAA TAGAAAGTCTTATTTTTGTATAGGAGATGGACATG TACCCCGGTTTTAGACTTTCTATTATATTATCGAC CTAAAACCGGGGTATTTTTGTATAGGAGATGGACA CAAGCCGTACGGCAACCAACCAACCATATTATCGACAAATCTGCCGAATT GGTTGGTTGCCGTACGGCTTGGGGGGTATAGGAGATGGACATGGCAAATC GCCTTCTTGGCTTCGAACTGGC GATCAGAACGTCATAATAGCTC CCTGGTCAGAAAATCCGCCAAG ATATCCTCCCTTCGTCCCGTGA

Gene expression pbaR-F2 pbaR-R2

AGCTGGAATTCGGTACAGTTGATAAGGCAT (EcoRI) TATGACTCGAGTCAGTTGTCTGACATCAGGCCAAGC (XhoI)

Genetic disruption MTF1-F MTF1-R MTF2-F MTF2-R MTF3-F MTF3-R MTT-F MTT-R pbaRC-F pbaRC-R

ACATCCCATCCCAAATCGGACGCC TGCTTTCTCTTCTGATACGGCACCAAACGACTTCA TGCCGTATCAGAAGAGAAAGCAGGTAGCTTGCAG GCGCCTCCTGTCAGAAGAACTCGTCAAGAAGGC GAGTTCTTCTGACAGGAGGCGCAGGAGCTCGTTC TCGCCGTTGCAGTGTTCCAATCCG ATGATAATTCGCCGAGCCCCGATGC CCAGTCATAGCCGAATAGCCTCTCC TTGACTCGAGACATCCCATCCCAAATCGGACGCC (XhoI) GATTACTAGTGCAAAGCGGTTGACGGAGCCAA (SpeI)

DNase I footprinting pbapt-F3 pbapt-R3 FAM-M13F(⫺47) M13R(⫺48)

AGTTGCACGCTCAATGGCGAAACCA ATTTGCCATGTCCATCTCCTATACA FAM-CCCAGTCACGACGTTGTAAAACG AGCGGATAACAATTTCACACAGGA

a

Restriction sites are in boldface, and nucleotide sequences that are different from the template are underlined.

be detected by PCR. At every generation, the pbaA1A2B genes were verified by PCR. The functional pbaR gene was amplified with the primer pair pbaRC-F/pbaRC-R and inserted into the broad-host-range plasmid pBBR1-MCS5 (12) to generate pBpbaR. The plasmid pBpbaR was elec-

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troporated into the ⌬pbaR mutant JZ-MT to obtain the pbaR-complemented strain JZ-MTC. RT-qPCR. Strain JZ-1T, the corresponding ⌬pbaR mutant strain JZMT, and the pbaR-complemented strain JZ-MTC were cultured in an LB

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FIG 1 (A) TSS of the pbaA1A2B cluster as determined by primer extension assay. By comparing the location of the reverse-transcribed PCR fragment with that of the standard sequence, the TSS was determined to be A. (B) TSS of pbaC as determined by primer extension assay. C was determined to be the TSS. (C) Schematic diagram of the transcriptional regulation of PbaR. Three DNA fragments that are located at different positions in the genome are shown. A 5-kb fragment conserved with plasmid pCAR3 is displayed on the left, the pbaA1A2B gene cluster is in the middle, and the pbaC gene is on the right. The ellipse represents the protein PbaR, which activates the transcription of the pbaA1A2B cluster in the presence of 3-PBA (displayed as a solid line). PbaR has no regulatory effects on pbaC (displayed as a dashed line). The scale bar represents 500 bp. The amplification fragments for transcriptional unit evaluation are shown under the pbaA1A2B cluster as lines. (D) PCR amplification of the pba cluster using total RNA and cDNA as the templates. The amplified products were detected by electrophoresis. Lanes 1, 3, 5, 7, 9, and 11 show samples using RNA as the template (negative controls), and lanes 2, 4, 6, 8, 10, and 12 show samples using cDNA as the template. Lanes M, molecular size markers. (E) DNA elements in the pbaA1A2B cluster promoter. The ⫺35 and ⫺10 boxes are shown in boxes, and the TSS is shown by an arrowhead. The PbaR-binding site and the ribosome-binding site (RBS) are indicated by a line above the sequence. (F) DNA elements in the pbaC promoter.

medium with appropriate antibiotics to an OD600 of 0.6. The cells were harvested and washed twice with MSM. Expression of 3-PBA degradation genes in washed cells was induced in MSM (the final cell density corresponded to an OD600 of 2.0) at 30°C for 3 h in the presence of 0.5 mM 3-PBA. MSM with 0.5 mM glucose was used as the control. Total RNA was extracted as described above; genomic DNA (gDNA) was digested with gDNA Eraser (TaKaRa, China) at 42°C for 2 min. Reverse transcription was then conducted with 1 ␮g of gDNA-removed RNA using random primers. The cDNA was synthesized by incubation at 37°C for 15 min with PrimeScript reverse transcriptase (RTase; TaKaRa), and the reaction was stopped by heating the mixture at 85°C for 5 s. Every sample was diluted 5-fold to serve as a template in quantitative PCR (qPCR). The qPCR was performed in an Applied Biosystems 7300 real-time PCR system (Applied Biosystems, USA) using the SYBR Premix Ex Taq RT-PCR kit (Tli RNaseH Plus; TaKaRa, China) as described in the manufacturer’s instructions. The transcription of the 16S rRNA gene was set as an internal standard, and relative expression was quantified according to the 2⫺⌬⌬CT threshold cycle (CT) method (13). Purification of PbaR and electrophoretic mobility shift assay (EMSA). The pbaR gene was amplified with the primer pair pbaR-F2/ pbaR-R2 and was then linked to the EcoRI and XhoI sites of pGEX-4T-1 to generate the expression plasmid pGEpbaR. E. coli BL21(DE3) cells harboring pGEpbaR were cultured in LB medium supplemented with 100 mg liter⫺1 of ampicillin at 37°C to an OD600 of 0.6, and 0.3 mM isopropyl-␤D-thiogalactopyranoside (IPTG) was then added to induce the expression of glutathione S-transferase (GST)-labeled PbaR. After 12 h of incubation

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at 16°C, the cells were harvested from 200-ml cultures by centrifugation, resuspended in phosphate-buffered saline (PBS) (Na2HPO4, 1.44 g liter⫺1; KH2PO4, 0.24 g liter⫺1; NaCl, 8.0 g liter⫺1; and KCl, 0.2 g liter⫺1; pH 7.4), and disrupted by sonication at 4°C. The suspension was centrifuged at 12,000 ⫻ g for 30 min at 4°C. The supernatant was then mixed with 5 ml of GST-Sefinose resin (Sangon Biotech) for 30 min at 4°C. After two washes with PBS, a final concentration of 2 U ml⫺1 thrombin (Sigma) was added to cleave the GST-tagged PbaR from the resin at 4°C for 12 h. The thrombin was then removed using Benzamidine Sepharose (GE). The resulting protein was concentrated using an Amicon ultrafiltration device. Finally, PbaR was stored in 30% glycerol with 1 mM DTT at ⫺70°C. Protein concentrations were quantified using the Bradford method with bovine serum albumin (BSA) as a standard, and the purity of PbaR was assayed using 15% SDS-PAGE. For EMSA, a nonradioactive strategy was implemented according to the method described by De la Cruz et al. (14). The wild-type 469-bp promoter DNA probe of the pbaA1A2B cluster was amplified using pbaptF/pbapt-R; a 430-bp promoter sequence of pbaC was amplified using the primer set pcpt-F/pcpt-R. Nucleotides in the mutated promoter DNA were introduced by primers (listed in Table 2), and sequences were amplified using overlapping extension PCR. The fragment with the deleted 29-bp motif sequence (AATAGAAAGTCTGCCGTACGGCTATTTTT) was amplified by overlap extension PCR using primers pbapt-F, pbaptd-R, pbapt-d-F, and pbapt-R. Approximately 20 ng of a promoter probe was mixed with an increasing concentration of purified PbaR in a binding buffer (100 mM Tris-HCl [pH 8.0], 50 mM KCl, 5% [vol/vol] glycerol,

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250 ␮g ml⫺1 BSA, and 1 mM DTT). A nonspecific DNA sequence (a sequence in pbaA2) was used as the negative control. The effects of 3-PBA on the binding of PbaR to the promoter probes were evaluated by adding 0.5 mM 3-PBA to the reaction system. The mixture was incubated at 25°C for 20 min and was then separated by 6% (vol/vol) native polyacrylamide gel electrophoresis in 0.5⫻ Tris-glycine-EDTA. The DNA or DNA-protein complexes were visualized by ethidium bromide staining. DNase I footprinting. DNase I footprinting assays were performed as described by Wang et al. (15). The promoter region of pbaA1A2B was PCR amplified with the primer pair pbapt-F3/pbapt-R3 and inserted into the plasmid pMD19-T to generate pMpbapt. To prepare fluorescent FAMlabeled probes, DNA was amplified with Pfu DNA polymerase (TaKaRa, Dalian, China) from the plasmid pMpbapt using the primers FAMM13F(-47) and M13R(-48). The FAM-labeled probes were purified with the E.Z.N.A. gel extraction kit (Omega, USA) and were quantified with a NanoDrop 2000c (Thermo Scientific, USA). For each assay, 400 ng of probe was incubated with 120 nM PbaR in a total volume of 40 ␮l. After incubation for 30 min at 25°C, 10-␮l volumes containing approximately 0.015 U of DNase I (Promega) and 100 nmol freshly prepared CaCl2 were added and further incubated for 1 min at 25°C. The reaction was stopped by adding 140 ␮l of DNase I stop solution (200 mM unbuffered sodium acetate, 30 mM EDTA, and 0.15% SDS). Samples were first extracted with phenol-chloroform and then were precipitated with ethanol, and the pellets were dissolved in 30 ␮l of Milli-Q water. The preparation of the DNA ladder, electrophoresis, and data analysis were performed as described by Wang et al. (15), except that the GeneScan-LIZ 500 size standard (Applied Biosystems) was used. Nucleotide sequence accession number. The sequence of the 5-kb DNA fragment has been deposited in the GenBank database under the accession number KT222890.

RESULTS

Determination of the TSS of the pba cluster. Three promoters were predicted by the Berkeley Drosophila Genome Project (BDGP) Neural Network Promoter Prediction online program (http://www.fruitfly.org/seq_tools/promoter.html) with a score of ⬎0.6 in the region 300 bp upstream of the pbaA1 gene. To determine where transcription starts, a primer extension experiment was performed. The TSS A was the 59th base upstream of the translational start codon (Fig. 1A and E). The ⫺10 box TTCGGC and the ⫺35 box GCAGAATA were predicted according to the TSS (Fig. 1E). The TSS of pbaC was determined to be a C 32 bp upstream of the initiation codon, and the ⫺10 box AGCCGTCC and the ⫺35 box TTGAAA of the pbaC promoter were also predicted (Fig. 1B and F). All 6 of the overlapping fragments (F1 to F6) within the pbaA1A2B cluster were amplified when cDNA was used as the template (Fig. 1C), indicating that the pbaA1, pbaA2, and pbaB genes were in one operon and transcribed in a single unit. PbaR specifically binds to pbaA1A2B promoter DNA. According to the results from the primer extension experiment, a 496-bp promoter area sequence for the pba cluster (⫺300 to ⫹196 bp related to its TSS) was amplified using a biotin-labeled primer and was immobilized on streptavidin-coated magnetic beads. DNAbinding proteins were analyzed by SDS-PAGE (Fig. 2). According to previously reported studies, molecular masses of most transcriptional regulators are approximately or less than 35 kDa. For examples, BenR is 36.4 kDa (16), PnpR is 35 kDa (17), NicR2 is 28 kDa (15), and PcaR is 32 kDa (18). Therefore, proteins in the bands with molecular masses of ⬍44.3 kDa were further analyzed (Fig. 2). The four protein bands were excised from the gel and digested with trypsin; the peptides were then sequenced using MALDI-TOF/TOF mass spectrometry. The peptides were compared with the proteins in the database

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FIG 2 SDS-PAGE analysis of pba promoter-binding proteins that were enriched by a DNA affinity approach. Lane 1 shows proteins that were eluted by 0.5 M NaCl, lane 2 shows proteins that were eluted by 1.0 M NaCl, and lane 3 shows a protein standard marker with the molecular masses (in kilodaltons) shown on the right. Four bound bands, marked with a, b, c, and d on the left, were digested with trypsin and were identified using MALDI-TOF/TOF mass spectrometry.

that was translated from the draft genome sequence of strain JZ-1T, and each identified protein is listed in Table 3. The proteins in bands a, c, and d were deduced to be ribosomal protein, ribosomal protein, and protein S930S ribosomal, respectively, none of which showed any relationship with transcription. However, the protein in band b was deduced to be a transcriptional regulator that corresponded to a 762-bp gene with GTG as the initiation codon. This protein shows 99% identity with an unidentified putative IclR family transcriptional regulator (open reading frame 229 [ORF229]) from the carbazole-degrading plasmid pCAR3 (GenBank accession number AB270530) in Sphingomonas sp. strain KA1. The protein that we identified was designated PbaR (3-PBA dioxygenase gene regulator) because of its role in the transcriptional regulation of the pbaA1A2B cluster. PbaR contains 253 amino acids and has a calculated pI of 8.8 and a molecular mass of 27.7 kDa. The N-terminal amino acids 3 to 52 form a helix-turn-helix (HTH_XRE superfamily) domain (see Fig. S2 in the supplemental material), which is a characteristic of the IclR family of transcriptional regulators (19). PbaR was overexpressed in E. coli BL21(DE3) from the expression vector pGEX-4T-1 and was detected by SDS-PAGE (see Fig. S3 in the supplemental material). Purified PbaR was used in an TABLE 3 Bound proteins that were identified by TOF mass spectrometry Bound band

Protein and function

MMa (kDa)

a b c d

rplB, large-subunit ribosomal protein L2 IclR family transcriptional regulator Translation initiation factor 3 Protein S930S, ribosomal

30.38 27.67 19.68 19.09

a

MM, molecular mass.

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FIG 3 Electrophoretic mobility shift assays. (A) PbaR binds to the pbaA1A2B cluster promoter. Each lane contains 20 ng of DNA probe. The first 5 lanes show samples incubated without the substrate 3-PBA, and the next 5 lanes show samples incubated with 0.5 mM 3-PBA. The concentrations of PbaR, increasing from left to right, are shown above the lanes. The control DNA was a 163-bp fragment that was amplified from the pbaA2 gene. (B) Electrophoretic mobility shift assays of PbaR binding to the pbaC promoter. The DNA probe that was used in the first 2 lanes was the pbaA1A2B promoter, which was used as the positive control, and the next 8 lanes contain pbaC promoter DNA. The 3rd to 6th lanes were incubated without 3-PBA, and the 7th to 10th lanes were incubated with 0.5 mM 3-PBA. (C) EMSA of PbaR binding to the promoter DNA with the 29-bp motif deleted. The first three lanes are wild-type pbaA1A2B promoter DNA, which was used as the control, and the next three lanes are 29-bp motif-deleted DNA probes. The sample in each lane was incubated with 0.5 mM 3-PBA. (D) Electrophoretic mobility shift assays of PbaR binding to the mutant pbaA1A2B cluster promoter DNA. The nucleotide se-

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EMSA to test its binding capacity for pbaA1A2B promoter DNA. When 40 nM PbaR was added, a DNA-protein complex was formed (Fig. 3A). No DNA-protein complex was found for the nonspecific control DNA (sequence in pbaA2) (Fig. 3A). These results indicate that PbaR can specifically bind to the pbaA1A2B promoter DNA. Furthermore, the substrate 3-PBA had no effect on the binding (Fig. 3A), which is in agreement with previous reports that IclR-type positive regulators can bind to their target DNA regions in the absence of effectors (19, 20). To test the ability of PbaR to bind to the pbaC promoter, EMSA was conducted similarly to the way it was conducted for the pbaA1A2B promoter. The EMSA results showed that PbaR did not bind to the pbaC promoter (Fig. 3B). PbaR is a transcriptional activator of the pbaA1A2B cluster. Cells of the wild-type strain JZ-1T, the pbaR knockout mutant JZ-MT, and the pbaR-complemented strain JZ-MTC were incubated in MSM supplemented with 0.5 mM 3-PBA or cypermethrin as the sole carbon resource, and the growth (OD600) was evaluated every 8 h. As shown in Fig. 4, ⌬pbaR mutant strain JZ-MT grew much slower than JZ-1T, while the growth curve of the pbaR-complemented strain was similar to that of the wild-type strain. The results showed that pbaR was essential for the growth of strain JZ-1T on 3-PBA and cypermethrin. Cells of strains JZ-1T, JZ-MT, and JZ-MTC were also incubated with or without 3-PBA, the total RNA of these cells was extracted, and reverse transcription-PCR was performed. The transcriptional levels of the pbaA1, pbaA2, pbaB, and pbaC genes were evaluated. As shown in Fig. 4C, the transcriptional levels of pbaA1, pbaA2, and pbaB in the wild-type strain JZ-1T, when supplemented with 3-PBA, were 96-, 114-, and 100-fold higher, respectively, than their levels in the absence of 3-PBA. In the mutant strain JZ-MT, the transcriptional levels of pbaA1, pbaA2, and pbaB were virtually unchanged by the presence or absence of 3-PBA. In the pbaR-complemented strain JZ-MTC, the transcriptional levels of pbaA1, pbaA2, and pbaB were similar to those of the wild-type strain JZ-1T. However, no significant differences in transcriptional levels were observed for the reductase gene pbaC among the strains JZ-1T, JZ-MT, and JZ-MTC under the same conditions. These results indicate that PbaR is a transcriptional activator of the pbaA1A2B cluster but that it has no regulatory effect on the pbaC gene. Binding site of PbaR. DNase I footprinting was performed to identify the PbaR-binding site in the promoter region of the pbaA1A2B cluster. It was found that PbaR protected the 29-bp motif AATAGAAAGTCTGCCGTACGGCTATTTTT, which is located from bp ⫹16 to ⫹44 relative to the TSS in the pbaA1A2B promoter (Fig. 5). This binding region was located between the ⫺10 box and the ribosome-binding site (Fig. 1E). If the 29-bp motif was deleted, EMSA showed that the incomplete DNA probe could not bind to PbaR (Fig. 3C). Within the 29-bp motif, there is a palindromic se-

quence 5=-GCCGTACGGC-3= in the PbaR-binding site was mutated to 5=-C CCCGGTTTT-3=. The first 3 lanes show wild-type pbaA1A2B promoter DNA, which was used as the positive control, and the next 3 lanes contain mutant promoter DNA. The sample in each lane was incubated with 0.5 mM 3-PBA. (E) EMSA of PbaR binding to promoter DNA with the 29-bp motif mutated. The sequence besides the palindrome was mutated to GGTTGGTTGGTT-pa lindrome-TTGGGGG. The first lanes are mutated promoter DNA, and the next three lanes are wild-type promoter DNA. The sample in each lane was incubated with 0.5 mM 3-PBA.

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FIG 4 Growth of wild-type strain JZ-1T (WT), the pbaR knockout mutant (MT), and the pbaR-complemented strain (MTC) on 3-PBA (A) and cypermethrin (B). (C) Transcriptional expression analysis of pbaA1, pbaA2, pbaB, and pbaC in the JZ-1T (WT), the pbaR knockout mutant (MT), and the pbaR-complemented strain (MTC) in the presence of 0.5 mM glucose (G) or 0.5 mM 3-PBA (S). The transcriptional level of the 16S rRNA gene was used as an internal standard, and the data in each column were calculated by the 2⫺⌬⌬CT threshold cycle (CT) method using 3 replicates.

quence, 5=-GCCGTACGGC-3= (Fig. 5). To determine whether the palindromic sequence is essential for PbaR binding, it was mutated to 5=-CCCCGGTTTT-3=. EMSA showed that the mutated DNA probe displayed no binding to PbaR (Fig. 3D). Additionally, to determine the role of other 19-bp sequences in binding, the 29-bp motif was mutated to GGTTGGTTGGTTGCCGTACGGCTTGGGGG (mu-

tated sequences underlined), and no stable binding complex was formed when this mutated DNA probe was used (Fig. 3E). These results indicate that the palindromic sequence plays an important role in PbaR sequence recognition and that the motif sequence besides the palindrome sequence is important for the stability of the DNA-protein complex.

FIG 5 DNase I footprinting analysis of the PbaR-binding site in the pba promoter. A total of 400 ng of 6-carboxyfluorescein-labeled DNA probe was incubated with 0 nM PbaR (red line) or 120 nM PbaR (blue line) in the presence of 0.5 mM 3-PBA. The PbaR-protected region is shown in a box, and the protected sequence is shown at the bottom. The palindromic sequence in the protected region is underlined.

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Transcriptional Activator for 3-PBA Catabolism

DISCUSSION

ACKNOWLEDGMENTS

PbaR, an IclR family transcriptional activator that regulates the transcription of the pbaA1A2B cluster, was identified in this study. The pbaR gene was not located together with the pbaA1A2B cluster and the pbaC gene (Fig. 1C). PbaR shares 99% identity with a putative transcriptional regulator from the plasmid pCAR3 in Sphingomonas sp. KA1 (21). Moreover, a 5-kb fragment containing the pbaR gene was highly conserved with the corresponding fragment on pCAR3 (99.9% similarity). Plasmid pCAR3, which is deficient in conjugative transfer, contains a complete set of genes that are involved in carbazole mineralization (22). PbaR was found to share similarity with IclR-type transcriptional regulators (see Fig. S2 in the supplemental material). Interestingly, IclR-type transcriptional regulators, including PbaR, that are involved in the catabolism of xenobiotics are described as activators (19, 23); examples include PobR in Acinetobacter calcoaceticus (24, 25), PcaR in Pseudomonas putida (18), and NdpR in Rhodococcus opacus (26). In addition, the binding of PbaR to pbaA1A2B promoter DNA was effector independent (Fig. 3A). This phenomenon is very common among IclR-type transcriptional regulators (23), for which the effector is not essential for the regulator to bind DNA but is essential for the interaction with RNA polymerase (27). The binding sites of most IclR family transcriptional regulators, including MhpR, GenR, and PcaR, are located upstream of their TSSs (19). For example, the binding sites of PobR and PcaU are located at bp ⫺55 to ⫺89 and bp ⫺48 to ⫺93 relative to their TSSs (24, 28), respectively. However, in the case of PbaR, the binding site was located between the ⫺10 box and the ribosome-binding site (bp ⫹16 to ⫹44 relative to its TSS). This sort of downstream binding region typically represents a repressor-binding site (19, 23). On the other hand, the binding sites of IclR family transcriptional regulators seem to be diverse and no conserved sequences have been found. MhpR, the 3-(3-hydroxyphenyl)propionic acid degradation regulator, binds to a 17-bp palindrome sequence, GGTGCACCTGGTGCACA (29); GenR, the 3-hydroxybenzoate and gentisate catabolism regulator, binds to the palindrome ATTCC-N7(5)-GGAAT (30); PcaR binds to a threerepeat 10-bp DNA sequence, TTTGTTCGAT (18, 27). With regard to PbaR, it specifically binds to the 29-bp motif AATAGAA AGTCTGCCGTACGGCTATTTTT, and the palindromic sequence (GCCGTACGGC) is essential for PbaR binding. Therefore, there is no uniform model for the binding pattern of IclRtype transcriptional regulators (19, 23), and PbaR increases the diversity of IclR-type transcriptional regulators with respect to the binding model. In the PbaA1A2BC dioxygenase system, pbaA1A2B is in a transcriptional unit while pbaC is in another transcriptional unit. Although the expression of the pbaC gene is induced by 3-PBA, the expression of the pbaC gene was much lower than that of pbaA1A2B at the transcriptional level (Fig. 4). Furthermore, there is no evidence demonstrating that PbaR regulates the transcription of the pbaC gene (Fig. 3 and 4). These results indicate that another regulator might exist for the transcription of the pbaC gene in response to 3-PBA. However, this suggestion requires further investigation. Because the reductase component of a dioxygenase system is typically not specific (21, 31–33), we speculate that the lower expression of the pbaC gene might be compensated for by other isoenzymes, making catabolic progress successful.

This work was supported by grants from the Chinese National Science Foundation for Excellent Young Scholars (31222003), the Outstanding Youth Foundation of the Jiangsu Province (BK20130029), the Program for New Century Excellent Talents in University (NCET-12-0892), and the Fundamental Research Funds for the Central Universities (KYZ201422).

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