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J. Biol. Chem., Vol. 281, Issue 13, 8450-8457, March 31, 2006
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From the Biotechnology Research Center, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
Received for publication, January 10, 2006 , and in revised form, January 31, 2006.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Carbazole is a dioxin-like recalcitrant compound with a dibenzopyrrole ring. The carbazole catabolic car gene cluster was first cloned from Pseudomonas resinovorans CA10 (3, 4) and then from several Gram-negative bacteria (5, 6). Via the upper carbazole catabolic pathway, carbazole is degraded to anthranilate and 2-hydroxypenta-2,4-dienoate by carbazole 1,9a-dioxygenase, meta-cleavage enzyme, and hydrolase (see Fig. 1A) (3, 4). Then, anthranilate 1,2-dioxygenase converts anthranilate to generate catechol, which is further degraded to tricarboxylic acid cycle intermediates via the 
-ketoadipate pathway (7). The 2-hydroxypenta-2,4-dienoate hydratase, 4-hydroxy-2-oxovalerate aldorase, and acetaldehyde dehydrogenase enzymes degrade 2-hydroxypenta-2,4-dienoate into pyruvate and acetyl-CoA (the meta pathway) (8).
In P. resinovorans CA10, the upper and meta pathway enzymes are encoded by the carCA10 gene cluster (carAaAaBaBbCAcAdDFECA10), where the carAa gene is duplicated (see Fig. 1B). Anthranilate 1,2-dioxygenase is encoded by the antABC operon. Previous study has shown that the Pant promoter drives the transcription of ant operon and an AraC/XylS family (9) activator AntR stimulates Pant in the presence of anthranilate (10). Both carCA10 and ant structural genes and the regulatory gene antR are carried on a 199,035-bp IncP-7 plasmid pCAR1, whose entire nucleotide sequence has been recently determined (11).
Nucleotide sequence analysis of other car gene clusters and their flanking regions has shown evolutionary trails of gene recruitment and rearrangement (6). Notably, the carJ3 gene cluster (carAaBaB-bCAcAdDJ3) that was isolated from Janthinobacterium sp. J3 shows 94.3% nucleotide sequence identity to the carCA10 gene cluster in the structural and intergenic DNA regions. High similarities in deduced amino acid sequence suggests no differences in functions of Car enzymes between CA10 and J3, which is proven in part by enzymatic and structural studies of CarAa and hydrolase proteins (12, 13). However, the flanking regions of carCA10 and carJ3 gene clusters are completely different (Fig. 1B). The first open reading frame (ORF)4 of the carCA10 gene cluster designated ORF9 is thought to be a fusion gene generated by the one-ended transposition of an insertion sequence (IS) ISPre1 plus the 5'-portion of antA into the ancestral ORFU13 of carJ3 gene cluster (6, 8), although it has not yet been known how anthranilate is metabolized and whether ant genes are present in Janthinobacterium. As the Pant promoter is located within the transposed region, the second copy of the Pant promoter also functions to drive transcription of the downstream carCA10 gene cluster (10). Instead, upstream of the carJ3 gene cluster, a putative regulatory gene carRJ3, which belongs to the GntR family (14) and shows relatively high similarities to FadR subfamily transcriptional regulators among the GntR family (15), is located in a divergent direction. Furthermore, the carFE genes do not exist downstream of the carJ3 gene cluster. From these observations, the carJ3 gene cluster is probably an ancestral form of the carCA10 gene cluster before the transposition of ISPre1, the recruitment of carFE, and the duplication of carAa.
Owing to high evolutionary relationship between the carCA10 and carJ3 gene clusters, we aimed to characterize regulatory elements of the carCA10 and carJ3 operons and to compare their effectiveness experimentally. In this study, the developmental process of car operon was clearly explained by the acquisition of the Pant promoter via the transposition of ISPre1, which resulted in the alteration of the regulatory mechanism. Furthermore, this study provides a unique property of the CarRJ3 repressor in binding to its operators.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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(Toyobo) was used for cloning experiments. E. coli BL21(DE3) (Novagen) was used for protein overproduction. The plasmids used in this study are listed in supplemental Table S1. P. resinovorans strains were routinely grown at 30 °C in Luria-Bertani (LB) medium (17) or MM1 (per liter: 2.2 g of Na2HPO4, 0.8 g of KH2PO4, 3.0 g of NH4NO3, 0.2 g of MgSO4 ·6H2O, 0.01 g of FeSO4 ·7H2O, 0.01 g of CaCl2 ·2H2O, and 0.05 g of yeast extract). Carbazole was dissolved in Me2SO at a concentration of 100 mg/ml followed by sterilization with a 0.2-µm pore-size membrane filter (8), and 1% of this solution was added to MM1. E. coli strains were grown at 37 °C in LB medium or 2x YT medium (per liter, 10 g of yeast extract, 16 g of bactotryptone, and 5 g of NaCl) (17). When appropriate, antibiotics were added at the following concentrations: ampicillin, 50 µg/ml; gentamicin, 15 µg/ml; and kanamycin, 50 µg/ml. For plate cultures, the above media were solidified with 1.6% agar (w/v). DNA Manipulation—Transformation of E. coli cells, plasmid preparation from E. coli cells, restriction enzyme digestion, and DNA ligation were carried out according to standard protocols (17). P. resinovorans strains were transformed by electroporation using a Gene Pulser II (Bio-Rad) (10). The primers used in this study are listed in supplemental Table S2.
Construction of Plasmids—A promoter search vector pME4510 (18) was digested at the HindIII/SfiI sites to replace the lacZ reporter gene with a 1.7-kbp HindIII-EcoRI fragment containing the luc+NF reporter gene from pSP-luc+NF (Promega), to yield pMEGluc. A series of transcriptional fusion plasmids, pMC375 through pMC1094, was constructed as follows: DNA fragments containing the region upstream of carAaCA10 were amplified from pBCA721 (8) with a series of forward primers, CARAA-F-BamHI-375 through CARAA-F-BamHI-1094, and a reverse primer CARAA-R-HindIII, which added appropriate restriction sites and a ribosome binding site for the luc+NF gene. The BamHI-HindIII fragment of the amplified DNA was ligated to the corresponding site of pMEGluc, to yield pMC375 through pMC1094.
pMC2065, containing the 2065-bp region just upstream of the carAaCA10 translation start point up to –200 relative to the transcription start point of ORF9, was constructed as described above using the primer pair ANTA-F-BamHI-253 (10) and CARAA-R-HindIII. pMJ1345R was constructed by ligation of the 2.6-kb BamHI-EcoRI fragment (carrying carRJ3) from pBJ3101 (6) along with the 572-bp EcoRI-HindIII fragment upstream of carAaJ3, which was amplified by PCR from pBJ3101 by the method described above, to the BamHI/HindIII-digested pMEluc. pMJ1345–1066 was constructed by cloning the 279-bp PstI-HindIII fragment upstream of ORFU13, which was amplified using CARAA-F-PstI-1345 and CARAA-R-HindIII-1066 from pBJ3101, to the corresponding site of pMEluc. pMJ1345–1066R was constructed by cloning of the same 279-bp PstI-HindIII fragment along with a 1.8-kb BamHI-PstI fragment carrying carRJ3 from pBJ3101 to the BamHI/HindIII-digested pMEGluc. Nucleotide sequences of the PCR-amplified fragments were confirmed to be identical with the reference sequences.
To overexpress CarRJ3 with a hexahistidine tag at the C terminus (C-ht-CarRJ3: CarRJ3-AlaAlaAlaLeuGluHis6) in E. coli BL21(DE3), pEJChtCarR was constructed as follows: carRJ3 was amplified from pBJ3101 (6) with CARRJ3-F-NdeI and CARRJ3-R-NotI primers, adding appropriate restriction sites. The nucleotide sequence of the PCR product was confirmed by sequencing. The 764-bp NdeI-NotI fragment from the PCR product was cloned into the corresponding site of pET-26b (Novagen), to yield pEJChtCarR.
To accumulate intermediates of the upper catabolic pathway, a series of plasmids, pBRJ3001, pBRJ3001
BbC, and pBRJ3001C, was constructed and used to transform P. resinovorans CA10dm1. The 1.7-kb PstI fragment within the carBa-carC region or the 56-bp AatI-PmaCI fragment within the carC gene was deleted by self-ligation from the 5.6-kb EcoRI fragment of carAaBaBbCAcAdJ3 on pBJ3001 (6), to yield pBJ3001BbC or pBJ3001C, respectively. Then, the EcoRI fragment of pBJ3001, pBJ3001BbC, or pBJ3001C was cloned into pBBR1MCS-2 (19) in the same direction as the tac promoter, to yield pBRJ3001, pBRJ3001BbC, or pBRJ3001C, respectively.
Induction—Cells of P. resinovorans CA10 or its derivatives grown overnight in LB medium were washed twice with CNF buffer (per liter: 2.2 g of Na2HPO4, 0.8 g of KH2PO4) and resuspended in the same volume of CNF buffer. MM1 supplemented with 0.1 mg/ml of ammonium succinate as a carbon and nitrogen source and with or without 0.02 mg/ml carbazole as an inducer was inoculated with one-tenth volume of cell suspension and incubated at 30 °C with reciprocal shaking (300 strokes/min).
Reverse Transcription-PCR—After a 2-h induction, total RNA was prepared from CA10 cells using NucleoSpin RNAII (Macherey-Nagel). For semi-quantitative reverse transcription-PCR, the first-strand cDNA was synthesized from 2 µg of total RNA with the specific primer CARE-12R by SuperScript III reverse transcriptase (Invitrogen) at 55 °C for 60 min and diluted with 9 volumes of H2O. PCR was performed in a 25-µl mixture containing 1 µl of the diluted cDNA solution and 0.4 µM of the primer set with cycles of denaturation at 95 °C for 30 s, annealing at 60 °C for 30 s, and extension at 72 °C for 2 min. The optimal number of cycles was selected for each amplified fragment. The following primer sets were used for PCR (supplemental Table S2): ORF9-carAa (1167 bp), ORF9–13F and CARAA-11R; carAa-carBa (1231 bp), CARAA-3F and CARBA-15R; carBa-carAc (2051 bp), CARBA-4F and CARAC-7R; carAc-carD (2121 bp), CARAC-2F and CARD-8R; carD-ORF33 (1100 bp), CARD-3F and ORF33–10R; ORF33-carF (1509 bp), ORF33–8F and CARF-3R; and carF-carE (1710 bp), CARF-1F and CARE-12R. As an internal reference, a 1061-bp DNA was amplified with the primer pair 16S-2F and 16S-13R from the cDNA synthesized from 16S ribosomal rRNA with 16S-13R. As a negative control, the reverse transcription reaction mixture without SuperScript III reverse transcriptase was used for PCR. The PCR products were analyzed by 1.0% agarose gel electrophoresis.
Luciferase Reporter Assay—After induction for the appropriate time, cells of P. resinovorans CA10 or CA10dm1 containing the reporter plasmid (supplemental Table S1) were harvested by centrifugation (2400 x g, 5 min, 4 °C) and were broken by ultrasonic treatment in sonication buffer (25 mM Tris-HCl (pH 8.0), 2 mM EDTA, and 10% (v/v) glycerol). The supernatant was collected by centrifugation (21,600 x g, 30 min, 4 °C). The relative light units (RLUs) of 1 µg of total protein was measured using a Centro LB960 (Berthold Technologies) according to the method described previously (10). Each sample was assayed independently at least three times. The induction rate was defined as the ratio of luciferase activity (RLU/µg) of cells after induction in the presence of carbazole to that in its absence.
Primer Extension—Total RNA was prepared from cells of P. resinovorans CA10 or those containing pMJ1345R induced for 2 h, using NucleoSpin RNA II. 2 pmol of the IRD800-labeled specific primer (ORF9 –13R, ORFU13–2, and CARR-1) was annealed to 10 µg of total RNA after denaturation at 65 °C for 5 min and was extended using SuperScript III reverse transcriptase (Invitrogen) at 55 °C for 30 min. The extended product was purified by phenol/chloroform extraction and ethanol precipitation and was then dissolved in 2 µl of H2O and 1 µl of IR2 stop solution (Li-Cor). The solution was denatured at 95 °C for 2 min and subjected to electrophoresis using a Li-Cor model 4200L-2 auto-DNA sequencer (Li-Cor) together with the sequence ladder obtained using the same primer.
Overproduction and Purification of C-ht-CarRJ3—E. coli BL21(DE3) harboring pEJChtCarR was grown overnight in 2x YT medium, and then 5 ml of the same medium was inoculated with 100 µl of overnight culture. After incubation at 30 °C with reciprocal shaking at 300 strokes/min for 3 h, isopropyl--D-thiogalactoside was added to the culture to a final concentration of 0.2 µM, and gene expression was induced at 30 °C for 3 h. After induction, the cells were collected by centrifugation (3,300 x g, 5 min, 4 °C) and broken by ultrasonic treatment in sonication buffer. The supernatant was then collected by centrifugation (21,600 x g, 30 min, 4 °C) and subjected to metal chelation chromatography using the QuickPick IMAC metal affinity kit (Bio-Nobile) according to the manufacturer's instructions. The purified protein sample was analyzed by SDS-PAGE.
DNase I Footprinting—DNase I footprinting was performed as described by Endoh et al. (20). A 276-bp DNA region spanning from –193 to +83 relative to the transcription start point of ORFU13 was amplified by PCR with the primer pairs 5'-biotin-labeled ORFU13–1 (Invitrogen) and 5'-IRD800-labeled CARR-1 (Aloka) or 5'-IRD800-labeled ORFU13–1 and 5'-biotin-labeled CARR-1. Approximately 100 fmol of the labeled PCR products was purified using a MicroSpin S-400 HR column (Amersham Biosciences) to remove excess primers and then were bonded to 60 µl of Streptavidin MagneSphere Paramagnetic Particles (Promega). The magnetic particles were washed with 60 µl of BW buffer (10 mM Tris-HCl (pH 7.5), 1 mM EDTA, and 2 M NaCl) and suspended in 100 µl of RM buffer (25 mM HEPES-NaOH (pH 7.8), 50 mM KCl, 5 mM MgCl2, 0.05 mM EDTA, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, and 5% glycerol). After the addition of 0.4, 0.8, 1.6, or 3.2 µg of the purified C-ht-CarRJ3, the mixture was incubated for 5 min at room temperature and then for 45 s at room temperature with 3 units of DNase I (Takara Bio). An equal volume of stop solution (4 M NaCl and 0.1 M EDTA) was immediately added to the reaction mixture to inactivate the DNase. The magnetic particles in the reaction mixture were collected, washed with equal volumes of BW buffer and TE buffer (10 mM Tris-HCl (pH 8.0) and 1 mM EDTA), and then suspended in 2 µl of H2O and 1 µl of IR2 stop solution (Li-Cor). After incubation of the magnetic particle solution at 95 °C for 2 min, an aliquot of the supernatant was subjected to electrophoresis on a Li-Cor model 4200L-2 auto-DNA sequencer (Li-Cor) together with the sequence ladder obtained using the same 5'-IRD800-labeled primer.
Electrophoretic Mobility Shift Assay—Electrophoretic mobility shift assay (EMSA) was performed using a DIG Gel Shift Kit 2nd generation (Roche Applied Science) according to the manufacturer's instructions as described by Endoh et al. (20). A 102-bp probe from –79 to +23 relative to the transcription start point of ORFU13 was obtained by PCR from pBJ3101 with the primer sets F1 and R1, followed by 3'-end labeling of the PCR products with digoxigenin-11-ddUTP. Four 21-bp DNA fragments, Pa (–55 to –35), Pb (–42 to –22), Pc (–24 to –4), and Pd (–11 to +10) were obtained by annealing equimolar concentrations of complementary synthetic oligonucleotides, F-Pa and R-Pa, F-Pb and R-Pb, F-Pc and R-Pc, and F-Pd and R-Pd, respectively (supplemental Table S2). The DNA-protein binding reaction was performed with 10 µl of binding buffer (20 mM HEPES, pH 7.6, 1 mM EDTA, 10 mM (NH4)2SO4, 1 mM dithiothreitol, 0.2% (w/v) Tween 20, and 30 mM KCl) containing 8 fmol of the digoxigenin-11-ddUTP-labeled probe and 0.1 µg of nonspecific competitor poly[d(A-T)]. When appropriate, 0.8, 8, or 80 pmol of unlabeled specific competitor was added. An increasing amount (from 50 to 400 ng) of the purified C-ht-CarRJ3 was mixed to samples and incubated for 15 min at room temperature, before the addition of 2.5 µl of loading buffer (60% of 0.25x TBE buffer (22.25 mM Tris, 22.25 mM boric acid, and 5 mM EDTA, pH 8.0), 40% glycerol, and 0.2% bromphenol blue (v/v)). The sample was fractionated on a 6% native polyacrylamide gel in 0.25x TBE buffer. After electrophoresis, the labeled DNAs were electroblotted onto Hybond-N+ (Amersham Biosciences) nylon membranes and detected using the CSPD detection system (Roche Applied Science) with LAS-1000 plus (Fujifilm).
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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12-kb carCA10 gene cluster constitutes an operon, which is transcribed from the Pant promoter. Transcriptional Pattern of the carCA10 Operon—The transcription start point of the Pant promoter is located at 1865 nucleotides (nt) upstream of the carAaCA10 translation start point. Reporter plasmid pMC2065 carries a transcriptional fusion of the 2065-bp fragment just upstream of carAaCA10 (Fig. 1B), which contains the 200-bp region sufficient for the stimulation of Pant in the presence of anthranilate (10). Luciferase expression of pMC2065 was increased with time in the presence of carbazole in CA10 cells, but low level expression was also detected in the absence of carbazole (Fig. 3A). This result is consistent with the previous work, which showed by Northern hybridization that the transcription of the carAa gene is constitutive but is largely induced when CA10 grows on carbazole or anthranilate (10). On the other hand, luciferase expression of pMC1094, which carries a transcriptional fusion of a 1094-bp fragment truncating the putative transposed region within ORF9 from pMC2065 (Fig. 1B), was constitutive at the same level as the basal expression by pMC2065 (Fig. 3B). These results suggest that another promoter for the constitutive transcription of the carCA10 operon is located within the 1094-bp region upstream of carAaCA10, whereas the Pant promoter is required for the inducible transcription of carAaCA10.
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70-dependent promoters (21). This result was confirmed by a luciferase reporter assay using a deletion series of the 1094-bp region upstream of carAaCA10 in P. resinovorans CA10. The expression levels of pMC446 and pMC426 were the same as that of pMC1094, but truncation of the –35 box (pMC416 and pMC406) or the whole promoter region (pMC375) greatly reduced the promoter activity (Fig. 4C). Therefore, the promoter required for the constitutive expression of the carCA10 operon was designated PcarAa. Transcriptional Pattern of the carJ3 Operon—To analyze the transcriptional pattern of the carJ3 operon, we constructed a reporter plasmid pMJ1345R that carries a transcriptional fusion of the 3146-bp region just upstream of the carAaJ3 translation start point containing the putative regulatory gene carRJ3 (Fig. 1B). To compare the transcriptional pattern of pMJ1345R with that of pMC2065, luciferase expression of pMJ1345R was monitored periodically in the heterologous genetic background of P. resinovorans CA10. Luciferase expression of pMJ1345R was increased in the presence of carbazole but was decreased after 4 h of induction (Fig. 3C). This result suggests that carAaJ3 is transcribed from a promoter regulated differently from the Pant promoter. In addition, low level expression in the absence of inducer was observed, suggesting that a PcarAa-like promoter sequence, which is conserved upstream of carAaJ3 (Fig. 4B), is also involved in the constitutive transcription of the carJ3 operon.
To identify a transcription start point of carAaJ3 mRNA that is induced in the presence of carbazole, primer extension was performed using the ORFU13–2 primer. From total RNA of CA10 harboring pMJ1345R induced in the presence of carbazole, we detected two extension products whose positions corresponded to adenine and thymine bases located 71 and 66 nt upstream of the ORFU13 translation start point, respectively (Fig. 5A). Putative –35 and –10 sequences (5'-TTGACA-N17-TAATCT-3') precede the 71-nt upstream adenine at a 5-nt distance (Fig. 5B). This promoter was designated the Pu13 promoter.
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Involvement of CarRJ3 in the Regulation of the Pu13 Promoter— pMJ1345–1066R, which contains carRJ3 gene and the Pu13 promoter region (Fig. 1B), showed a significant induction of luciferase expression (193 RLU/ng in the presence of carbazole and 6.2 RLU/ng in the absence) with an induction rate of 31.0. On the other hand, pMJ1345–1066, which truncates carRJ3 but only contains the Pu13 promoter region from –201 to +69 relative to the transcription start point of ORFU13 (Fig. 1B), showed constitutive expression with an induction rate of 1.5 at significantly high levels (3740 RLU/ng the presence of carbazole and 2250 RLU/ng in the absence). This result suggests that CarRJ3 is a repressor of the Pu13 promoter.
To facilitate the purification of CarRJ3, a C-terminal histidine-tagged CarRJ3 (C-ht-CarRJ3) was overproduced and purified from a crude extract of E. coli transformant in a single step by metal ion affinity chromatography. The His-tagged CarRJ3 was purified to apparent homogeneity (supplemental Fig. S1). The molecular mass of the purified protein was 30 kDa, which was consistent with the deduced molecular mass of C-ht-CarRJ3 (29.3 kDa).
To demonstrate the direct repression of the Pu13 promoter by CarRJ3 protein, DNase I footprinting was performed using the 276-bp region from –193 to +83 that contains Pu13 and PcarR promoter regions. The 65-bp region, from –57 to +8 on the sense strand or from +13 to –52 on the antisense strand, was protected from DNase I digestion, although intensities of bands corresponded to –11 and –28 on the antisense strand were not changed (Fig. 6). No further spreading of the protection region was observed even when the concentration of CarRJ3 was increased (data not shown). Within the protection region, we found two putative operators similar to the consensus sequence of FadR subfamily members (15), OI (5'-TTGTAGAACAA-3') from –50 to –40 and OII (5'-TGGTAGAACAA-3') from –6 to +5 (Fig. 5B).
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C or pBRJ3001BbC, together with the reporter plasmid pMJ1345–1066R. The induction rate in CA10dm1 harboring pBRJ3001BbC was 1.3 and that of the negative control pBBR1MCS-2 was 1.2 (Table 1). This result shows that neither carbazole nor 2'-aminobiphenyl-2,3-diol stimulates the promoter activity of Pu13. When pBRJ3001C was used, luciferase expression was increased 89-fold. As the disruption of carC results in the accumulation of 2-hydroxy-6-oxo-6-(2'-aminophenyl)hexa-2,4-dienoate (HOADA), this result shows that HOADA is the true inducer of Pu13 under the control of CarRJ3. The induction rate in CA10dm1 harboring pBRJ3001 was significantly lower than that in CA10dm1 harboring pBRJ3001C (Table 1).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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The GntR family of regulators, which contains a conserved N-terminal, helix-turn-helix DNA-binding motif, is distributed among diverse bacterial groups and regulates many biological processes (15). Almost all of the GntR family members that control the degradation of aromatic compounds are repressors, although transcriptional regulation by repressors is rare for catabolic pathways (22). FadR is the only member of the GntR family whose structure has been solved in complex with its target DNA, 5'-TGGTCCGACCA-3' (23) or 5'-TGGTACGACCA-3' (24). FadR-DNA contact points are found on the central C-G base pair (assigned as 0), the two Gs at –3 and –4, and the A at +5 of the target DNA. The Arg-45 and Arg-35 of FadR specifically contact the Gs at –3 and –4, respectively, whereas Thr-44, Thr-46, and Thr-47 are involved in the interaction with the central C-G base pair. The alignment of the N-terminal, DNA-binding domains of FadR, CarRJ3, and GntR (Fig. 8A) shows that Arg-45 is conserved, whereas Arg-35 and the three threonines are not, in accordance with an alignment of the GntR family based on the structure of FadR (15). The variety of residues aligned to Arg-35 of FadR implies the difference of their recognizing bases at –4 (Fig. 8B).
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60-bp range of DNA. Given that OI exists just downstream of the –10 element of Pu13 and that OII locates between the –35 elements of Pu13 and PcarR, it is deduced that CarRJ3 preferentially represses Pu13 rather than PcarR. An analysis of the relative expression of these two promoters would further clarify the autoregulation mechanism of CarRJ3.
The transposition of ISPre1 along with the Pant promoter from the ant operon into the carCA10 operon alters the pathway-specific regulator from a GntR-type repressor CarRJ3 to an AraC/XylS-type activator AntR. The alteration of the pathway-specific regulator then directs the catabolic operon of a xenobiotic compound to be induced by anthranilate, which is a naturally occurring compound formed through tryptophan degradation (25). Therefore, anthranilate triggers the induction of both the carCA10 and ant operons, which accelerate carbazole catabolism via both the meta pathway and -ketoadipate pathway. As ammonium is only released through the dioxygenation of anthranilate by anthranilate 1,2-dioxygenase followed by spontaneous deamination and decarboxylation, the completely simultaneous transcriptional activation of the carCA10 and ant operons suggests the effective utilization of carbazole as a nitrogen source in the CA10-type regulatory circuit. In contrast to their simultaneous activation, the expression of the carJ3 operon decreased earlier than that of the carCA10 operon even in the presence of carbazole (Fig. 3, A and C), and the derepression of Pu13 was insufficient compared with the carRJ3-deficient background. These results may be attributable to the instability of HOADA, which is hydrolyzed by hydrolase (26) and is degraded spontaneously with a half-life of 6.1 min (27).
Retrospective studies have indicated that the construction of a catabolic operon and the acquisition of regulatory elements seem to be relatively independent events (28, 29) and that mobile genetic elements play a major role in the in situ spread and de novo construction of catabolic operons in bacteria, allowing bacterial communities to rapidly adapt to new xenobiotics (30). This study showed that the transposition of ISPre1 replaced the regulated promoter of the car operon, which is a relatively recent event in the process of car operon construction. As another example, it has been reported that ISPre1 is also located on the naphthalene catabolic IncP-9 plasmid pDTG1 from Pseudomonas putida NCIB9816–4 and separates a regulatory gene and a salicylate hydrolase gene (nahRG) from the sal operon (nahTHINLOMKJY), which encodes catechol meta-cleavage enzymes (31). Another naphthalene catabolic IncP-7 plasmid, pND6 –1 from Pseudomonas sp. ND6, also has these genes in an identical arrangement but lacks ISPre1 (32). The former study suggests that ISPre1 integrated after the acquisition of the nahRGTHINLOMKJY genes and that it interrupts the nahG transcript to prevent the expression of downstream genes through polar effects. In fact, NCIB9816–4 degrades naphthalene via the catechol ortho pathway catalyzed by induced chromosome-encoded enzymes rather than the meta pathway catalyzed by plasmid-encoded enzymes that are constitutively expressed at low levels (33). These observations suggest that ISPre1 is involved in the genetic rearrangement and resultant transcriptional alteration of several catabolic operons for xenobiotic compounds in Pseudomonas.
The growing repertoire of available bacterial genome sequences has proven that the evolution of the regulatory circuit is inextricably linked to the organization of the genome to meet the control needs of each different organism in a changing environmental niche (34). Thus, it is hypothesized that a host strain harboring a catabolic plasmid alters the regulatory pattern of a plasmid-borne catabolic operon in response to its genetic background, transcriptional network, and metabolic capacity. As the present study aimed at a comparative analysis of regulatory elements between the carCA10 and carJ3 operons, both were analyzed in Pseudomonas of the same genetic background. The possibility remains that the regulatory circuit of each car operon is equally effective in its domestic genetic background, but no information about the downstream catabolic pathways in Janthinobacterium is available. It will be necessary to compare the expression of car operons and the resultant carbazole-degrading activities in Pseudomonas and Janthinobacterium backgrounds. Furthermore, the monitoring of the regulatory circuits of pCAR1 in various organisms will provide new insights into the expression range of catabolic operons on mobile genetic elements.
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FOOTNOTES |
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The on-line version of this article (available at http://www.jbc.org) contains Fig. S1 and Tables S1 and S2.
1 Supported by Research fellowships of the Japan Society for the Promotion of Science for Young Scientists.
2 Present address: Dept. of Industrial Chemistry, Shibaura Institute of Technology, 3-9-14 Shibaura, Minato-ku, Tokyo 108-8548, Japan.
3 To whom correspondence should be addressed. Tel.: 81-3-5841-3064; Fax: 81-3-5841-8030; E-mail: anojiri{at}mail.ecc.u-tokyo.ac.jp.
4 The abbreviations used are: ORF, open reading frame; IS, insertion sequence; RLU, relative light unit; EMSA, electrophoretic mobility shift assay; nt, nucleotide(s); HOADA, 2-hydroxy-6-oxo-6-(2'-aminophenyl)hexa-2,4-dienoate.
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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