INTRODUCTION

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The modified ortho-cleavage pathway plays a significant role in the aerobic bacterial degradation of chlorocatechols produced through converging pathways from various chlorinated aromatics (reviewed in references 12, 24, 47, 51, 55, and 56). Well-characterized examples include 3-chlorobenzoate (3-CB), 2,4-dichlorophenoxyacetic acid (2,4-D), and 1,2,4-trichlorobenzene, which are converted to the corresponding chlorocatechols by peripheral pathways and then catabolized to tricarboxylic acid cycle intermediates by the enzymes of the modified ortho-cleavage pathway encoded by the clcABDE genes from plasmid pAC27 of Pseudomonas putida (3, 20, 27) and the clc element (46) (or plasmid pB13 [4]) of Pseudomonas sp. strain B13, the tfdCDEF genes from plasmid pJP4 of Ralstonia eutropha (formerly Alcaligenes eutrophus) JMP134 (14, 45), and the tcbCDEF genes from plasmid pP51 of Pseudomonas sp. strain P51 (57, 59), respectively. Sequence homology and overall structural similarity among the modified ortho-cleavage pathway operons and the catechol ortho-cleavage pathway operon catBCA (1, 26) indicate an evolutionary relationship (51, 56). Each operon has a lysR-type regulatory gene (50) which is located upstream of and is divergently transcribed from the degradative gene clusters (9, 30, 48, 58). For the tfdCDEF operon of plasmid pJP4, however, tfdT, the lysR-type regulatory gene originally located upstream of the operon, is inactivated by an insertion sequence element, and its function has been taken over by distantly located tfdR (30, 61).

The regulatory mechanisms of the operons catRBCA and clcRABD have been studied both in vivo and in vitro (6-9, 26, 32-35, 41-44, 48; reviewed in reference 33). The regulators CatR and ClcR bind specifically to the catB and clcA promoter regions, respectively, and activate the expression of the degradative genes upon recognition of inducer. The inducers of the catBCA and clcABD operons have been identified as intermediates of their respective pathways, cis,cis-muconate (CCM) (43) and 2-chloro-cis,cis-muconate (2-CM) (35). In the 2,4-D degradation process, 2,4-dichloromuconate, the intermediate produced by TfdC, has been identified as an inducer of the tfdCDEF operon in vivo (18). DNA binding of the regulator TcbR to the tcbC promoter has been described (29), and initial in vivo characterization of the role of tcbR has been performed (30, 58). P. putida KT2442 harboring a plasmid containing the tcbRCD orfXEF genes was found to grow on 3-CB, while KT2442 containing the tcbCD orfXEF genes with an inactivated tcbR gene grew on 3-CB at a much lower rate (58). P. putida and R. eutropha strains harboring plasmids containing the tcbRCD orfXEF genes showed elevated (chloro)catechol 1,2-dioxygenase activity towards 3-chlorocatechol after cultivation on 3-CB (30, 58). This activity was not observed with cells of R. eutropha containing the tcbCD orfXEF genes with tcbR inactivated. These results indicated that tcbR was required for the efficient expression of tcbC, which encodes chlorocatechol dioxygenase (30, 58). Further analysis of this operon including the identification of inducer is yet to be done.

The cbnRAB orfXCD operon on plasmid pENH91, found in a 3-CB degradative bacterium R. eutropha NH9, is highly homologous to the tcbRCD orfXEF operon (95.6 to 100% identity at the amino acid level and identical 150-bp divergent promoter regions) and is responsible for the degradation of 3-chlorocatechol (38). In this paper, we report the transcriptional regulation of the cbnRAB orfXCD operon, which includes the identification of inducers and a change in the bending angle of the promoter region upon recognition of an inducer. These observations provide a view of a conserved transcriptional mechanism of regulation plus the independent evolution of inducer-recognizing specificity and DNA-binding property among the regulators of the ortho-cleavage pathway.

	MATERIALS AND METHODS

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Bacteria, plasmids, media, chemicals, and enzyme assays. The strains and plasmids used in this study are listed in Table 1. Preparation of media was performed as described previously (35, 37). As a selective medium for the transconjugants of R. eutropha NH9D, basal salts medium for NH9 (37) was supplemented with 0.2% sodium citrate. 2-Chloromuconate was produced by the enzymatic conversion of 3-chlorocatechol with chlorocatechol 1,2-dioxygenase (ClcA) as described previously (34). Quantitative determination of -galactosidase activity in the reporter assay was performed by the method of Miller (35, 36). Each experiment was performed in triplicate.

DNA manipulations and construction of plasmids. Recombinant DNA techniques were performed according to standard procedures (49). Transformation of the plasmid pQF50 and its derivatives into P. putida PRS4020 was performed as described previously (35). Mobilization of the plasmid pCP13 and its derivatives into P. putida PRS4020 and R. eutropha NH9D was conducted based on the method of Franklin (19). Plasmids to test growth complementation were constructed as follows: a plasmid, pBLcbn2, containing the cbnAB orfXCD genes with the cbnA promoter region in pBluescript KS(), was constructed by using a 5.6-kb insert from pEKC1 which was cut out by using a unique EcoRI site 4 bp within the N terminus of cbnR (EcoRI*) in the cbnRAB orfXCD operon and an XhoI site located downstream of the cbnD gene (Table 1). A 1.1-kb EcoRI fragment containing cbnR truncated at the EcoRI* site was cut out from pBLcbn1 and inserted into the EcoRI site of pBLcbn2 to restore the original structure of the cbnRAB orfXCD operon, yielding pBLcbn3. From pBLcbn2 and pBLcbn3, the relevant fragments were excised and inserted into the cloning site of pCP13, yielding pCbn13ABCD and pCbn13RABCD, respectively. The broad-host-range vector pQF50 was used to produce the transcriptional fusion constructs (Table 1 and see Fig. 2).

Purification of protein. Induction and extraction of protein from Escherichia coli BL21(DE3)pLysS containing a construct of pT7-7 or pET16b and further purification of the protein with the histidine tag were conducted according to the His · Bind Resin Manual (36a). In order to partially purify CbnR and also to eliminate a contaminating protein of 27 kDa from crude lysate of cells with pT7cbnRHis, a heparin-agarose column was used as described previously (8). The fractions that showed specific binding to the cbnA promoter fragment by gel retardation assay were pooled and concentrated by Centricon 10 (Amicon, Mass.). To prepare the vector (pT7-7) control for pT7cbnR, the fractions corresponding to the ones containing activity with pT7cbnR were used. CbnRHis was purified further with His · Bind resin.

Gel retardation assay. DNA binding reactions were performed in 20-µl volumes consisting of 10 mM HEPES (pH 7.9), 10% glycerol, 100 mM KCl, 4 mM spermidine, 0.1 mM EDTA, 0.25 mM dithiothreitol, 1.5 µg of bovine serum albumin, 5 µg of heparin (Sigma, H-3125), approximately 0.1 ng of a DNA fragment and the tested protein. The DNA fragments were synthesized by PCR and labeled internally with [-³²P]dCTP, followed by a purification procedure (40). The binding reactions were initiated by the addition of CbnR and incubated for 20 min at room temperature. The binding reaction mixtures were then electrophoresed through 5% native polyacrylamide gel in 0.5× Tris-borate-EDTA buffer for 2 h at 130 V with circulation of cooling water in Bio-Rad Protean apparatus. The DNA probe containing the 252-bp cbnRA promoter region was made by PCR with primers CBNFT1 (5'-TTG GCT GCT GCA GCC ATG TTC CC-3') and CBNFT2 (5'-AAT GCG GAC GCA ACC TGC TTC ACT CG-3') and with pBLcbn1 as a template. A 336-bp fragment containing a part of the hydroxyquinol 1,2-dioxygenase gene from Burkholderia cepacia AC1100 was used as a nonspecific DNA probe. The fragment was synthesized by PCR with primers ORF4P1 (5'-GCC TGC AGC GGC CCC TTC CAT GTG-3') and ORF4P2 (5'-GCC TGC AGC CTC AAG CAT TTG ACC-3') and with pKS100 as a template (13).

S1 nuclease analysis. Bacterial RNA was isolated via the RNeasy total RNA isolation kit (Qiagen, Chatsworth, Calif.) from a 9-ml culture of P. putida PRS4020 containing the plasmid pNO50RAB' cultivated in Luria broth supplemented with 5 mM 3-CB. To prepare the DNA probe, primer CBNFT2 was end labeled with T4 kinase (Gibco BRL, Gaithersburg, Md.) and [-³²P]ATP followed by PCR synthesis of a 252-bp fragment containing the promoter region with a second primer, CBNFT1, and pBLcbn1 as a template. The PCR product was purified by QIA quick PCR purification kit (Qiagen). This double-stranded fragment was denatured by the addition of 1/10 volume of 2 N NaOH-2 mM EDTA (pH 8.0) and incubated at room temperature for 5 min. The denatured DNA was ethanol precipitated, washed with 70% ethanol, and resuspended in water. The labeled DNA fragment was hybridized with the RNA at 45°C overnight and incubated with S1 nuclease with the S1-Assay kit (Ambion, Austin, Tex.) according to the manufacturer's instructions. Sequencing reactions to juxtapose the protected DNA fragment resulting from the S1 reaction were performed with the SequiTherm cycle sequencing kit (Epicentre Technologies, Madison, Wis.) with CBNFT2 as the primer.

DNase I protection assay. DNase I footprinting experiments were conducted based on the method described previously (40), except that the binding reaction was performed in 20 µl of solution consisting of 100 mM Tris-HCl (pH 7.9), 1 mM EDTA, 4% glycerol, 100 mM KCl, 1 µg of bovine serum albumin, 1 µg of poly(dI-dC), approximately 10 ng of a DNA fragment plus the purified protein indicated. PCR was used to generate a 252-bp fragment spanning the cbnA promoter region using pBLcbn1 as a template. In each reaction, one of the primers, CBNFT1 or CBNFT2, was end labeled with T4 kinase (Gibco BRL) and [-³²P]ATP as described previously (40). To locate the footprint, sequencing reactions were performed as described above with either of the end-labeled primers, CBNFT1 or CBNFT2, and pBLcbn1 as the template.

In vitro transcription assays. In vitro transcription assays were performed as described previously (25, 35). The supercoiled template, pMPcbn1, and E. coli holo RNA polymerase (Epicentre Technologies) were used. For each reaction, 100 ng of purified CbnRHis and a 1 mM concentration of each of the chemicals tested as effector molecules were used.

DNA bending by circular permutation gel shift assay. Circular permutation gel shift assays were conducted by the same method as the gel retardation assay with the following modifications: the binding reaction mixtures contained 0.2 µg of purified CbnRHis, approximately 0.3 ng of labeled DNA fragments and 2 µg of heparin. In the inducer-containing samples, CCM was added at 1 mM in the binding reaction mixtures and at 0.5 mM in the running buffer. Electrophoresis was performed at 130 V for 5 h. Five DNA fragments of 257 bp containing the CbnR-binding sites at different positions from the ends to the middle were generated and labelled internally by PCR with [-³²P]dCTP by using the following primer pairs: MCBD1 (5'-GTT CGA TCC CGC GGT GGC TTC GCT CCA GAA GC-3') and MCBD2 (5'-GCG CCG GCC ATG CCG TCC AAT ACC-3'), MCBD3 (5'-TCC AGG GCT TGC ATC TGC CGC GTG ATGG-3') and MCBD4 (5'-TTG TCG GTT TGC CCG GTCC-3'), MCBD0 (5'-GGC GCT TGG CTG CTG CAG CCA TGT TCCC-3') and CBNFT2 (above), MCBD5 (5'-GAA ATA CTT GAG CTG CCGG-3') and MCBD6 (5'-TTC CGT CAC GCG TTG CTC CGT GAG GG-3'), and MCBD7 (5'-GTG CCT ATA TTA CGC AAA CC-3') and MCBD8 (5'-TTG GCC TCG GCC AGT TTC ATC ATG TAG CC-3'). The DNA fragments were purified with the QIA quick PCR purification kit (Qiagen). The bending angles were calculated as described previously (34).

	RESULTS

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Complementation by CbnR of growth on 3-CB of bacteria harboring cbnABCD genes. The cbnABCD genes encode enzymes of the modified ortho-cleavage pathway which degrade 3-chlorocatechol converted from 3-CB (Fig. 1) (38). To examine if cbnR was required for growth of NH9 on 3-CB, a complementation test was conducted. Either pCbn13RABCD or pCbn13ABCD, which retained the cbnA promoter region, was mobilized into R. eutropha NH9D, a derivative of NH9 which is cured of the cbnRAB orfXCD genes, or P. putida PRS4020, a catR knockout strain. The growth rates of the resultant four strains on 3-CB were compared together with those of strains containing the vector control, pCP13. Strains NH9D and PRS4020 with pCbn13RABCD showed growth on the 3-CB plate, while the strains with pCbn13ABCD or pCP13 did not. This result indicated that cbnR was necessary for the growth of these bacteria on 3-CB.

View larger version (16K): [in this window] [in a new window]   FIG. 1.   Pathway for 3-CB degradation by R. eutropha NH9 and the genetic organization of the cbnRAB orfXCD operon. TCA, tricarboxylic acid.

Activation of the cbnA promoter by CbnR during growth in the presence of Ben or 3-CB. It was presumed that CbnR may activate the cbnA promoter based upon the regulatory systems of other ortho-cleavage operons (29, 33, 58). To analyze the function of cbnR for the expression of the degradative genes in vivo, we used P. putida PRS4020 (catR knockout strain) as a host for reporter plasmids (Fig. 2). The cells were grown in basal synthetic medium (BSM) (1) supplemented with 10 mM glucose, 10 mM glucose, and 5 mM benzoate (Ben), or 10 mM glucose and 5 mM 3-CB for 18 h at 30°C. When the cells containing pNO50RAB' were grown on glucose with either Ben or 3-CB, the transcription from the cbnA promoter was activated 17- and 6.8-fold, respectively, compared to that in the cells grown on glucose alone (Table 2). The activation in the presence of Ben or 3-CB was not seen for the cells with pNO50AB', which did not contain cbnR. These results indicated that cbnR was a positive regulator of the transcription from the cbnA promoter.

View larger version (13K): [in this window] [in a new window]   FIG. 2.   Diagram of the inserts of the constructs used for the cbnA promoter activity assay.

Purification of CbnRHis protein. Specific binding of CbnR to the cbnA promoter was demonstrated by gel retardation assay using the crude protein from E. coli BL21(DE3)pLysS containing pT7cbnR. In order to simplify protein purification, a plasmid expressing cbnR with six His codons on its C terminus, pT7cbnRHis, was constructed. The crude soluble protein from E. coli BL21(DE3)pLysS containing pT7cbnRHis showed binding activity to the cbnA promoter, although a considerable portion of the protein was secluded in inclusion bodies. When the crude protein was directly purified with the nickel affinity column, two major bands appeared in the sodium dodecyl sulfate-polyacrylamide gel electrophoresis profile. One of the bands apparently corresponded to the calculated molecular mass of CbnRHis (32.9 kDa), and the other was a polypeptide of about 27 kDa. This 27-kDa protein was also produced from the cells of the vector control. To remove this contaminating protein, a heparin-agarose column was employed followed by the nickel affinity column, and the 32.9-kDa protein was recovered at more than 90% purity (Fig. 3, lane 3). The amino acid sequence of the N terminus of this protein was found to be identical to that of the deduced sequence of CbnRHis. The function of CbnRHis was evaluated with respect to partially purified wild-type CbnR in vivo and in vitro in the following sections.

View larger version (86K): [in this window] [in a new window]   FIG. 3.   Sodium dodecyl sulfate-polyacrylamide gel electrophoresis profile of purification of CbnRHis. Lanes: 1, total protein from induced cells of BL21(DE3)pLysS containing pT7cbnRHis (100 µg); 2, protein purified by heparin-agarose column (50 µg); 3, CbnRHis purified by heparin-agarose column and nickel column (5 µg); M, protein molecular mass markers. Sizes are shown in kilodaltons.

Confirmation of the function of CbnRHis. Partially purified CbnR and purified CbnRHis were subjected to gel retardation assays with a 252-bp fragment containing the cbnA promoter region. The two proteins showed the same retardation mobility of the cbnA promoter fragment (Fig. 4, lanes 3 to 5 and 6 to 8). To test if CbnRHis had the same function as CbnR in vivo, growth complementation tests and reporter analysis were conducted. In growth complementation, strains NH9D and PRS4020 harboring pCbn13RHABCD grew on 3-CB, as did strains with pCbn13RABCD. In reporter analysis, PRS4020 containing pNO50RHAB' exhibited activation of transcription in the presence of either Ben or 3-CB to the same levels as PRS4020 containing pNO50RAB' did (data not shown). These results indicated that CbnRHis had the same function as CbnR in these in vitro and in vivo experiments.

View larger version (61K): [in this window] [in a new window]   FIG. 4.   Gel retardation assay demonstrating specific binding of CbnR and CbnRHis to the cbnA promoter region. A 252-bp fragment containing the cbnA promoter region was used as probe in lanes 1 to 8. A 336-bp fragment containing part of the hydroxyquinol 1,2-dioxygenase gene of B. cepacia AC1100 was used in lanes 9 to 12. Lanes: 1 and 9, no protein; 2 and 10, partially purified protein from BL21(DE3)pLysS/pT7-7 at 0.5 µg; 3, 4, 5, and 11, partially purified CbnR at 0.05, 0.1, 0.5, and 0.5 µg, respectively; 6, 7, 8, and 12, CbnRHis at 0.05, 0.1, 0.5, and 0.5 µg, respectively.

Features of the cbnA promoter region bound by CbnR. The transcriptional start site of the cbnA promoter was determined by S1 nuclease analysis; the A on the template strand is marked by an arrow in Fig. 5 and was located 47 bp upstream from the translational start codon of the cbnA gene. The precise site of CbnR binding on the cbnA promoter was determined by the DNase I protection assay (Fig. 6). In the absence of inducer, approximately 60 bp from 76 to 19 (relative to the +1 of transcription) were protected from DNase I cleavage by CbnR. Both partially purified CbnR and CbnRHis exhibited the same protected patterns on the sense and antisense strands. These footprints were very similar to those of TcbR bound to the tcbC promoter (29). The addition of CCM or 2-CM up to 1 mM in the binding reaction mixture did not change the footprinting pattern of CbnR to the cbnA promoter. Besides the inverted repeat containing T-N₁₁-A located in the recognition binding site (RBS) (50), there was a second inverted repeat (Fig. 6c) whose location is similar to those described recently by the study of BlaA, a LysR type regulator for -lactamase genes of Streptomyces cacaoi (31).

View larger version (97K): [in this window] [in a new window]   FIG. 5.   S1 nuclease assay to determine the transcriptional start site of cbnA. Sequencing reactions of the bottom strand of the cbnA promoter region are shown as A, T, G, and C. The products of S1 nuclease assay are shown in lanes 1 and 2 (marked by arrow). In lanes 1 and 2, 20 and 10 µg of in vivo-derived RNA were used, respectively.

View larger version (101K): [in this window] [in a new window]   FIG. 6.   DNase I footprint of CbnRHis and CbnR on the cbnA promoter region. (a and b) Top strand (a) and bottom strand (b) of the cbnA promoter region. Lanes: 1, no protein; 2, CbnRHis (0.5 µg); 3, partially purified CbnR (1 µg); 4, partially purified protein from BL21(DE3)pLysS/pT7-7 (1 µg). (c) Schematic diagram of the cbnA promoter region protected from DNase I digestion by CbnRHis. The protected nucleotides are shown by the brackets. The vertical arrows indicate sites of hypersensitivity to DNase I digestion. The thick horizontal arrows show the inverted repeats containing T-N11-A, regarded as a motif of LysR regulatory systems (50). The horizontal dotted arrows above the bottom strand indicate the second imperfect inverted repeat similar to those described recently (31). The horizontal solid lines under the top strand and above the bottom strand indicate the 35 and 10 regions of the cbnA promoter and the divergently transcribed cbnR promoter. The regions from nucleotide 76 to 49 and from 44 to 19 are suggested to be the RBS and the ABS, respectively (33). The numbering is relative to the transcriptional start site of cbnA.

Identification of the inducers by in vitro transcription assay. The results of the LacZ assays suggested that CCM and 2-CM act as the inducers of the cbnA promoter. Considerable activity, however, was also shown when the cells containing a truncated CbnA gene (pNO50RA') were grown in glucose with Ben (Table 2). The possibility that either Ben or catechol may also serve as an inducer could not be excluded. To examine the effect of these compounds on the transcriptional activation by CbnR, in vitro transcription assays were performed with purified CbnRHis and potential inducer compounds. Figure 7 shows that cbnA transcripts were produced only when either CCM or 2-CM was added to the reaction mixture (lanes 12 and 14). No transcript was produced with other compounds, including Ben (lane 4) or catechol (lane 8). Therefore Ben or catechol does not serve as an inducer for the cbnA promoter. The transcriptional start sites of the products with CCM or 2-CM were determined by S1 nuclease assay and were confirmed to be the same as that derived by in vivo assay (data not shown).

View larger version (91K): [in this window] [in a new window]   FIG. 7.   In vitro transcription assay demonstrating the requirement of both CbnR and inducer. In lanes 1 to 14, 0.1 µg of CbnRHis was used in the lanes with even numbers, while it was not added to the lanes with odd numbers. Lanes: 1 and 2, no chemicals; 3 and 4, Ben; 5 and 6, 3-CB; 7 and 8, catechol; 9 and 10, 3-chlorocatechol; 11 and 12, CCM; 13 and 14, 2-CM; 15, marker (a transcript of 428 bases derived from plasmid template pJET41) (16, 35) is shown as a faint band near the arrow marked cbnA. All of the chemicals were used at 1 mM concentrations. RNA-1 is the transcript from the ColE1 ori of the supercoiled plasmid pMP7.

Bending of the cbnA promoter region. The presence of hypersensitive sites in the center of the footprint region of the cbnA promoter suggested that changes in DNA conformation might occur upon binding of CbnRHis. To examine this potential change with or without inducer, circular permutation gel shift assays were performed. Lanes 1 to 5 of Fig. 8 indicate that binding of CbnRHis caused bending of the promoter fragment; the bending angle was estimated to be 78°. When CbnRHis was bound to the promoter in the presence of CCM, relaxation of the bending angle to 54° was observed (lanes 6 to 10). The bending angles of three independent experiments varied by no more than 7.7%. Partially purified CbnR gave the same bending angles as CbnRHis (data not shown).

View larger version (76K): [in this window] [in a new window]   FIG. 8.   Circular permutation gel shift assay demonstrating CbnRHis bending of the cbnA promoter region in the absence (lanes 1 to 5) and presence (lanes 6 to 10) of 1 mM CCM. The ca. 60-bp region that showed a footprint by CbnR was distributed in different positions from the ends (lanes 1 and 6, left ends; lanes 5 and 10, right ends) to the center (lanes 3 and 8) of 257-bp fragments. The calculated bending angles were 78° and 54° in the absence and presence of CCM, respectively.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

Transcriptional activation of the cbnA promoter by cbnR has been characterized both in vivo and in vitro. In growth complementation studies, cbnR was demonstrated to be necessary for growth on 3-CB for R. eutropha NH9D (3-CB derivative of NH9) and P. putida PRS4020 (catR knockout strain), when they harbor the cbnABCD degradative genes. CbnR has also been demonstrated to be a positive regulator by transcriptional reporter and in vitro transcription assays.

Both CCM and 2-CM have been identified as inducer molecules. In reporter analysis, the PRS4020 cells containing a plasmid with a truncated cbnA gene, pNO50RA', exhibited some elevated transcription in the presence of Ben. Therefore, it remained possible that the cbnA promoter could be induced by Ben or catechol. However, neither Ben nor catechol induces transcriptional activation by purified CbnRHis in vitro. The reason for the in vivo activation of the pNO50RA' construct is unclear. It is possible that the activity observed was induced by CCM supplied by the host-encoded catechol dioxygenase (CatA). Although catR, the activator of the host catA gene (26), is disrupted in PRS4020, there may be some low-level constitutive expression of catA which results in the conversion of Ben to CCM, allowing the activation of the cbnA promoter. It is also possible that there are additional CbnR binding sites in the cbnA gene which add to the complexity of the cbnA promoter regulation. Both repressor and activator internal binding sites have been observed with other LysR family members, including CatR (6, 10). Although the activity observed with construct pNO50RA' in the presence of Ben cannot be explained, the in vitro transcription assays clearly demonstrated that CCM and 2-CM function as inducers for cbnA promoter expression.

It is puzzling that CbnR recognizes CCM as well as 2-CM as an inducing molecule. The enzymes of the cbn operon can catabolize catechol to a certain extent. Chlorocatechol 1,2-dioxygenase (CbnA) can utilize catechol as a substrate (15, 47), and chloromuconate cycloisomerase (CbnB) has maintained some activity against nonchlorinated CCM (47, 52). However, 3-oxoadipate enol-lactone, the product from CCM by CbnB, is presumed not to be catabolized further by the enzymes encoded by cbnCD (51). CatR and ClcR activate their regulated promoters significantly only in the presence of the intermediates of their respective pathways: CCM for CatR (43) and 2-CM for ClcR (35). The clcABDE operon is expressed when cells are grown in Ben because CatR binds to and activates the clcA promoter (34, 35, 42). However, CatR cannot bind to or activate the cbnABCD promoter (unpublished observations). It is possible that in addition to utilizing chlorocatechols as carbon substrates, the chlorocatechol dioxygenase enzymes act as important detoxifying agents. Catechol could be toxic to pseudomonads as chlorocatechols are (21). Because CatR cannot activate the expression of CbnA to detoxify catechol as it would ClcA, CbnA expression by CbnR upon recognition of CCM could be beneficial for the cell to decompose catechol rapidly.

The clcABDE operon was isolated from 3-CB-degrading bacteria (5) and thus functions to degrade 3-chlorocatechol in the original isolate. The high homology between the cbnAB orfXCD and the tcbCD orfXEF genes (38) suggests that the cbnAB orfXCD genes may have evolved to degrade more highly chlorinated catechols compared to the clcABDE genes. The recognition of more highly substituted catechols or corresponding muconates by the regulators of the ortho-cleavage pathway is mostly unknown. It has been demonstrated that 2,4-dichoromuconate acts as an inducer of tfdCDEF expression (18). However, the range of inducing molecules for these operons has not been explored. Since the products from the tcbCD orfXEF operon degrade dichlorocatechols and 3,4,6-trichlorocatechol, TcbR (CbnR) probably recognizes corresponding chloromuconates as inducers. Nevertheless, CbnR also has the ability to recognize CCM as an inducer, which is characteristic of CatR.

The specific binding of CbnRHis and CbnR to the cbnA promoter has been shown by gel retardation and DNase I protection assays. CbnRHis had the same function as CbnR in growth complementation, the reporter assay, and the bending assay of the cbnA promoter with and without inducer. Therefore, functional equality of CbnRHis with CbnR has been verified in vivo and in vitro. A similar observation about a His tag on the C terminus has been reported for NhaR, a LysR-type regulator of Na⁺/H⁺ antiporter gene of E. coli (2), suggesting the unaltered function of a C-terminally His-tagged LysR-type protein. On the other hand, the crude soluble protein from E. coli BL21(DE3)pLysS containing pEHiscbnR did not show binding activity to the cbnA promoter (data not shown). Although the N-terminal part of LysR family proteins functions as the DNA binding domain, addition of amino acids on the N terminus does not necessarily abolish binding activity. The addition of a polypeptide to the N terminus of SpvR, a LysR-type regulator of virulence genes of Salmonella dublin, showed little effect on the specific binding to the regulated promoters and activation of the promoters (22). It is not known whether the protein produced from the construct pEHiscbnR does not have the binding activity to the cbnA promoter or is exclusively retained in the inclusion bodies.

Under the established conditions for gel retardation assays (40, 58), crude soluble protein from BL21(DE3)pLysS containing either pT7cbnR or pT7cbnRHis always formed aggregates with the cbnA promoter fragment in the well of the electrophoresis gel. The addition of heparin in the binding reaction dissolved the aggregate, and the protein-DNA complex was able to migrate in the gel. It was presumed that CbnR formed these aggregates because of its highly basic nature (calculated pI = 10.3 by Genetyx Software [Software Development Co., Tokyo, Japan]). Heparin has been used successfully in the study of the formation of splicing complexes on mRNAs, where it was presumed to quench the nonspecific binding of components in the nuclear extract with the highly negatively charged RNA (28). In our study, it is possible that the negatively charged heparin was able to cancel the electrostatic force among the complexes of CbnR(His)-DNA, but not the specific binding between CbnR(His) and the DNA fragment.

The length and the location of the footprinting pattern of CbnR to the cbnA promoter, with hypersensitive sites localized in the center, resemble those of CatR to the catB promoter (in the presence of the inducer CCM) (43) and of ClcR to the clcA promoter (8, 35). Thus, it is suggested that the region from nucleotide 76 to 49 is the RBS and the region from 44 to 19 is the activation binding site (ABS) (33), as shown in Fig. 6c. The RBS contains the conserved T-N₁₁-A motif critical for binding by the LysR-type regulators (33, 50). Further binding of the regulator to ABS helps RNA polymerase bind to the promoter region, and thus to activate transcription (33). Although both CbnR and CatR recognize CCM as an inducer, the extended footprinting pattern of CbnR in the absence of the inducer, encompassing both the RBS and ABS of the cbnA promoter, resembles that of ClcR to the clcA promoter (8, 35). This is in contrast to the restricted footprinting pattern of CatR to the RBS of the catB promoter in the absence of CCM (43). The DNA binding domain of the LysR-type regulators is believed to be located in the N terminus, and the regions responsible for inducer recognition are believed to be located in the middle of the protein (50). The difference in extent of the footprinting pattern without CCM between cbn and cat suggests that the evolution of the DNA binding domain and the inducer recognition region(s) could be discrete.

The ABS binding pattern of CbnR without inducer seemed similar to that of ClcR. However, the addition of an inducer, CCM or 2-CM, to the binding reaction mixture did not cause a change in the footprinting pattern of CbnR to the cbnA promoter, while addition of 2-CM caused a shift in the footprinting pattern of ClcR to the ABS of the clcA promoter region (35). It remains to be elucidated if the apparent lack of change in the footprinting pattern of CbnR is an intrinsic characteristic of the CbnR-cbnA promoter system or is due to experimental conditions.

The relaxation of the bending angles of cbnA promoter region bound by CbnR(His) upon the addition of inducer was similar to those shown by CatR (41) and ClcR (34). The footprinting patterns and changes in DNA bending upon addition of inducers of the cat, clc, and cbn promoters suggest that the outline of the transcriptional mechanism of these regulatory systems is conserved. Our present study further indicates independent evolution of the regulatory genes in terms of their DNA binding and recognition of inducer molecules.