INTRODUCTION

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The chlorinated benzoic acids (CBA) are a common class of pollutants that occur in the environment as a result of intentional introduction (e.g., in the form of herbicides) or incomplete bacterial metabolism of some accidentally released chemicals (e.g., polychlorinated biphenyls) (46). Bacteria possess a remarkable assortment of metabolic pathways for biodegradation of CBA, and the innate ability of bacteria to degrade CBA has been exploited for bioremediation of contaminated sites (47). Several aerobic degradation pathways have been characterized at the biochemical and genetic levels. The most intensively studied pathway is encoded by the clc genes of Pseudomonas putida that specify intradiol ring fission of 3-chlorocatechol, a metabolite generated by nonspecific activity of benzoate or toluate dioxygenases with 3-chlorobenzoate (3CBA) (19). In contrast, the cba-encoded pathway involves a dioxygenase and a dehydrogenase that convert 3CBA, 4CBA, or 3,4-dichlorobenzoate to the vicinal diol intermediates protocatechuate (PCA) and 5-Cl-PCA (Fig. 1A) (40, 41). Other CBA degradation operons include the cbd-encoded pathway of Burkholderia cepacia (22) and the ohb-encoded pathway of Pseudomonas aeruginosa (60), both of which specify dioxygenase-mediated conversion of 2CBA to catechol; the fcb pathway of Arthrobacter globiformis for conversion of 4CBA to 4-hydroxybenzoate by a coenzyme A ligase and a hydrolase (61); and an Alcaligenes sp. pathway that converts 3CBA to 3-hydroxybenzoate (31, 32). Proven or putative regulatory factors for these pathways are encoded by genes closely linked to the catabolic operons. These factors include ClcR, a LysR-like regulator for clc (9); CbdR, an AraC-like regulator for cbd (22); and OhbR, an IclR-like regulator for ohb (60).

View larger version (31K): [in this window] [in a new window]   FIG. 1.   (A) Physical and restriction map of 3CBA catabolism transposon Tn5271 found on plasmid pBRC60 of C. testosteroni BR60. The solid arrows indicate various genes, including cbaABC, which encode the dioxygenase (CbaAB) and dehydrogenase (CbaC) that convert 3CBA to PCA or 5-Cl-PCA; cbaR, which codes for a regulatory protein (this study); the transposase tnpA gene of the two copies of IS1071; and an ORF that codes for a truncated aryl coenzyme A ligase-like product (ORF 8). Flanking tnpA are the left and right inverted repeats of IS1071 (open triangles). (B) Restriction map of EcoRI fragment 11 (E11) from pBRC60 and schematic diagrams of lacZ constructs made to study expression of cbaA and cbaR. Note that lacZ (grey boxes) is not drawn to scale.

The cbaABC operon is located in composite transposon Tn5271 (Fig. 1A) found on the self-transmissible IncP plasmid pBRC60 (38, 67). Despite the potential for being mobilized to members of various subclasses of the class Proteobacteria (14, 20, 21, 47, 65), the natural host range of this plasmid includes primarily members of the  subclass of the Proteobacteria, particularly strains that degrade PCA, the product of CbaABC-mediated catabolism of CBA, by an extradiol pathway (39). From an ecological point of view, little is known about factors that limit the horizontal spread of this mobile pathway. We therefore sought to better understand regulation of cbaABC expression, not only because horizontal transfer in contaminated environments brings this operon into a variety of genetic backgrounds and we are curious about whether the capacity to effectively regulate cbaABC plays a role in determining the host range of these genes, but also because cbaABC encodes an additional pathway by which bacteria metabolize CBA and thus has potential applications in bioremediation of contaminated sites. We therefore characterized induction of cbaABC in the original host, and in this report we show that a cis-encoded MarR-like regulator, CbaR, plays a role in modulating expression of cbaABC. We also present evidence that a chromosomally encoded protein may be involved in regulating this operon.

	MATERIALS AND METHODS

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Bacterial strains, plasmids, and growth conditions. Strains and plasmids used in this study are listed in Table 1. Escherichia coli was routinely grown at 37°C in Luria-Bertani medium (1% [wt/vol] tryptone, 0.5% [wt/vol] yeast extract, 0.5% [wt/vol] NaCl) containing ampicillin (250 mg liter¹), kanamycin (40 mg liter¹), or chloramphenicol (50 mg liter¹), as required. Comamonas testosteroni strains (formerly Alcaligenes sp. strains [see below]) were grown as described below in minimal medium A (64) containing 10 mM succinate, aromatic substrate at a concentration of 4 mM, and chloramphenicol (50 to 100 mg liter¹), as required. All chemicals and antibiotics were purchased from Sigma-Aldrich (Oakville, Ontario, Canada). When necessary, growth media were solidified by adding agar to a final concentration of 1.6% (wt/vol).

DNA sequencing and sequence analysis. To add to the phenotypic characteristics previously determined for 3CBA-degrading strain BR60(pBRC60) (67), both strands of the first 523 bases of the 16S rRNA gene were sequenced by MIDI Labs (Newark, Del.) by using primers that anneal beginning at positions 5 and 531 in the 16S rRNA gene of E. coli. The sequences of both strands of Tn5271 DNA between IS1071L and cbaA (Fig. 1A) were determined by the chain-terminating dideoxy method with an ABI Prism automated sequencer (Biotechnology Research Institute, University of Ottawa, Ottawa, Ontario, Canada) using appropriate primers and pBRE11 (Table 1) as the template. This DNA was analyzed for similarities to entries in the GenBank nonredundant database by using the BLAST (5) network service of the National Center for Biotechnology Information, Bethesda, Md. (http://www.ncbi.nlm.nih.gov). A divergently transcribed open reading frame (ORF) upstream of cbaA was identified and designated cbaR. Because the putative product of this ORF showed similarity to MarR and various other transcriptional regulators that are known to respond to aromatic compounds or control aromatic catabolism genes (see below), this region was taken into consideration when we designed the various lacZ expression constructs described below. DNA between cbaC and IS1071R (Fig. 1A) had been sequenced previously and was not taken into consideration as it contains ORFs whose products show similarity to proteins involved in uptake of aromatic compounds (unpublished results) and a truncated aryl coenzyme A ligase (14).

Determination of cbaA transcriptional start site and reverse transcription (RT)-PCR assay. The transcriptional start of cbaA was determined by primer extension analysis as described elsewhere (53). Briefly, C. testosteroni BR60(pBRC60) was grown to the mid-log phase in minimal medium A containing 3CBA, which maximally induced 3CBA degradation activity (see below). Total RNA was isolated, and primer CBAAR (5'-CATGAGGCCGCCCATCG-3'; complementary to the N-terminal coding region of CbaA) was annealed to it and extended with Moloney murine leukemia virus reverse transcriptase (New England Biolabs). During extension, [-³²P]dCTP (Amersham Canada, Oakville, Ontario, Canada) was included in the reaction mixture to label the product. The extension product and the product of a sequencing reaction conducted with the same primer (using pMP14.10 as the template) were resolved by electrophoresis on a denaturing 8% polyacrylamide sequencing gel, and DNA was detected by autoradiography; both of these analyses were performed by using standard methods (55).

Regulation of 3CBA degradation activity. Control of 3CBA catabolism in C. testosteroni BR60(pBRC60) was studied by first determining which growth substrates other than 3CBA were capable of inducing 3CBA degradation activity. As discussed above, CbaABC-mediated metabolism of 3CBA generates PCA and 5-Cl-PCA, which are then degraded by an extradiol (meta) ring fission pathway (38). Because benzoate, 3-hydroxybenzoate, 4-hydroxybenzoate, and PCA also induce this pathway (41, 62), we tested whether 3CBA degradation activity was induced by growth on these four aromatic substrates. Control cells were grown on succinate, a nonaromatic tricarboxylic acid cycle intermediate that presumably does not induce activity. O₂ uptake assays were conducted as described previously (41). Briefly, cultures were grown to the mid-log phase on one of the substrates mentioned above, harvested, and washed, and the respiration rates of cells with the various test compounds were determined polarographically. For each growth substrate, two or three independent trials were performed, and O₂ consumption was measured with replicate samples of the culture from each trial. Spontaneous deletion of Tn5271 occurs at a high frequency (66), and we therefore assessed the loss of the 3CBA degradation genes for cultures grown on carbon sources other than 3CBA by diluting and plating samples on minimal medium A agar containing 3CBA (Tn5271-proficient cells) or succinate (total count). During the incubation period used in these experiments, there was no noticeable loss of Tn5271.

Construction and insertion of cbaA and cbaR expression constructs and measurement of LacZ activity. Additional studies were conducted to investigate the effect of CbaR on cbaA expression. Two translational fusion constructs were used: cbaRA'-'lacZ, which included the gene cbaR, and cbaA'-'lacZ1, which lacked it (Fig. 1B). Expression of cbaR was studied by using three translational fusion constructs which contained different amounts of DNA upstream of the cbaR start codon (Fig. 1B). Finally, a control expression construct (promoterless lacZ) was also designed. Table 1 provides information on the structures and derivations of the relevant plasmids used in these studies. The nucleotide sequences of the junctions between cbaA' and 'lacZ in pBRCW34L and between cbaR' and 'lacZ in pBRCW27L were determined to confirm that both genes were in frame with lacZ. The various expression constructs were then cloned into suicide delivery vector pUTCm or pUTCmMCS.3 (Table 1) so that lacZ was transcribed in the direction opposite the direction of transcription of the chloramphenicol resistance gene, and they were mobilized from E. coli CC118pir and inserted into the chromosome of C. testosteroni BR6020 (Table 1) by triparental mating (41). BR6020 is a pBRC60/Tn5271-deficient derivative of BR60 and thus is not able to grow on 3CBA. This strain allowed expression to be measured in a genetic background lacking plasmid/Tn5271-encoded functions. Transconjugants were recovered by plating cells on minimal medium A agar containing 4-hydroxybenzoate, chloramphenicol, and 5-bromo-4-chloro-3-indolyl--D-galactopyranoside (X-Gal). Because insertion of the expression constructs into the chromosome was random, we tested whether the location of transposition affected LacZ levels by isolating four independent transconjugants for each construct and measuring the LacZ activity of succinate-grown cells, as described below. Levels could vary by as much as twofold; therefore, the transconjugant that had the lowest LacZ activity was used as the sole representative in subsequent studies in which expression with that construct was measured. Transposition of the expression constructs into the chromosome as single copies was confirmed by Southern blotting. A Tn5271-proficient background, which restored the ability to grow on 3CBA, was created for the representative strains harboring an expression construct by transferring pBRC60 from C. testosteroni BR6024(pBRC60) and recovering transconjugants on minimal medium A agar containing 3CBA, chloramphenicol, and X-Gal (41). The LacZ levels in cells harboring the expression constructs were measured as described elsewhere (55). Cultures were grown on succinate (a noninducing substrate) and 3CBA or benzoate (inducing substrates). The averages ± standard errors based on three independent trials are reported below for each carbon source, and the statistical significance of observed effects was determined by a paired t test (P = 0.05).

Cloning and overexpression of cbaR Affinity-purified CbaR for gel shift studies and DNase I protection assays (described below) was obtained as follows. The gene coding for CbaR was PCR amplified by using forward primer CBAR3958 (5'-cgggatccCTTGCAAGAGATCCTCGA-3') and reverse primer CBAR3440 (5'-ccgagctcCTACTCCTGAGGAGATTC-3') (lowercase letters indicates nucleotides that are not native to cbaR, and the underlined nucleotides in the two sequences are restriction sites for BamHI and SacI, respectively). CBAR3958 is complementary to the portion of cbaR coding for the N terminus but lacks the native start codon, while CBAR3440 is complementary to the C-terminal coding portion of cbaR up to and including the native stop codon. The amplification product was initially cloned into pCRScript SK(+) digested with SmaI (resulting in pMP132.4), and the insert was sequenced to confirm that PCR amplification had not introduced mutations. cbaR was then placed downstream from and in frame with DNA coding for the N-terminal affinity tag MRGSH₆GS by digesting pMP132.4 with BamHI and SacI and cloning the ~550-bp fragment that was generated into pQE30 digested with the same enzymes, which resulted in pQE30cbaR. CbaR was then overexpressed and affinity purified with an Ni-nitriloacetate column as recommended by the manufacturer (Qiagen, Mississauga, Ontario, Canada), dialyzed twice for ~18 h against glycerol storage buffer (300 mM NaCl, 50 mM NaH₂PO₄, 50% [vol/vol] glycerol; pH 8.0) by using a Pierce Slyde-A-Lyzer cassette (10-kDa molecular mass cutoff; MJS BioLynx, Brockville, Ontario, Canada), and stored at 20°C. The protein concentration was determined by using the Bradford reagent (Bio-Rad Laboratories, Mississauga, Ontario, Canada) with bovine serum albumin as the standard. Based on the results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the purified extract contained traces of contaminating proteins or degradation products (see Results). 3A). Therefore, as a control to confirm that CbaR caused the effects observed in gel shift assays (see below), an extract was prepared as described above from cells harboring the expression construct with no insert.

Gel shift assays. Binding of CbaR to DNA containing the cbaA or cbaR transcriptional start site was characterized by gel shift assays using three DNA templates, shown schematically in Fig. 3B. In the initial assays we tested interactions with two templates, templates 1 and 2. Template 1 putatively contained the transcriptional start site of cbaR, which was roughly mapped in expression studies (see below), and spanned nucleotides (nt) 4098 (BglII site) to 4446 (NotI site). Template 2 contained the transcriptional start site of cbaA (determined by primer extension analysis [see below]), as well as sufficient upstream DNA so that cbaA expression was normal (as determined in expression studies with construct cbaA'-'lacZ1 [see below]). The latter sequence spanned nt 4446 (NotI site) to 4705 within cbaA (ScaI site). Templates 1 and 2 were obtained by digesting pMP14.10 with NotI and BamHI and gel purifying the ~400- and ~260-bp fragments, respectively. For more detailed gel shift studies we used a 186-bp fragment (molecular weight, 114,000) that was similar to template 2 but slightly shorter at both ends (Fig. 3B). The sequence of this third template, template 3, is shown in Fig. 2B, and it was generated by PCR with primers PCBA4480B (5'-CTCGGGTAAACACCTAGA-3') and CBAA4665 (5'-TCGCACCAAATCTTCGT-3'). The amplification product was agarose gel purified and quantified by UV spectroscopy. All templates were end labeled with digoxigenin-11-ddUTP by using terminal transferase as recommended in the instructions for a DIG Gel Shift kit (Roche Molecular Biochemicals, Laval, Québec, Canada). Templates were separated from unincorporated label by spin column chromatography (Qiagen) and treated as described below with respect to binding.

DNase I protection assays. The specific areas bound by CbaR were determined by DNase I protection assays. The two templates ([³²P]PCBA and [³²P]CBAA) were identical to the 186-bp gel shift template described above, except that one of the primers used during amplification had been previously radioactively end labeled using [-³²P]ATP (Amersham Canada Ltd.) and T4 polynucleotide kinase so that only the noncoding strand ([³²P]PCBA) or the coding strand ([³²P]CBAA) was phosphorylated. Following amplification, PCR products were separated from unincorporated label by spin column chromatography (Qiagen). ³²P-labeled DNA templates (~6 ng) were bound to various amounts of CbaR with and without 4 mM 3CBA or 4 mM 3-carboxybenzoate (see below) as described above for the gel shift assays; the only differences were that the binding buffer contained 5 mM MgCl₂ and 2.5 mM CaCl₂ and the reaction volume was 12 µl. After binding, 2.5 × 10³ U of DNase I (Sigma-Aldrich) was added, and template DNA was digested at 22°C as follows. For [³²P]PCBA, 4-µl samples were removed from the reaction mixture at 30, 60, and 120 s and combined into one tube containing 1.5 µl of stop solution (500 mM EDTA); for [³²P]CBAA, 1.5 µl of stop solution was added after 60 s of digestion. Three microliters of a glycerol loading buffer was then added, and samples were placed in a boiling water bath for 3 min. A 3-µl aliquot was applied to a denaturing sequencing gel (8% [wt/vol] polyacrylamide, 8 M urea), and sequencing ladders (generated with the appropriate primer using pMP14.10 as the template) were included in adjacent lanes. Fragments were resolved electrophoretically and visualized by autoradiography.

Nucleotide sequence accession numbers. The nucleotide sequences reported in this paper have been deposited in the GenBank database under accession numbers U18133 (for CbaR) and AF345907 (for 16S ribosomal DNA).

	RESULTS

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Classification of strain BR60(pBRC60). The identity of 3CBA-degrading strain BR60(pBRC60), originally described as an Alcaligenes sp. strain (67), was not firmly established, and we therefore sequenced a 523-nt portion of the 16S rRNA gene. This portion of the 16S rRNA gene was identical to the corresponding portion of the 16S rRNA gene of the C. testosteroni type strain, and because BR60(pBRC60) has several genetic and metabolic similarities to other C. testosteroni strains (see below), Alcaligenes sp. strain BR60 is reclassified as C. testosteroni BR60.

Identification of cbaR and determination of cbaA transcriptional start. BLAST analysis of DNA upstream of Tn5271 revealed the presence of a divergently oriented ORF beginning 667 bp upstream of cbaA (Fig. 1). This ORF was designated cbaR and codes for a 19.4-kDa protein that exhibits identity to various members of the MarR family of transcriptional regulators (Table 2). Because many proteins belonging to this family respond to specific aromatic compounds or are involved in regulating genes for metabolism of aromatic compounds, we focused on the effect of CbaR on expression of cbaA (see below).

View larger version (72K): [in this window] [in a new window]   FIG. 2.   (A) Transcriptional initiation site of cbaA, as determined by primer extension analysis of total RNA isolated from 3CBA-grown C. testosteroni BR60(pBRC60). The extension product was loaded onto a sequencing gel next to four sequencing reaction mixtures (lanes A, C, T, and G) from reactions conducted with the same primer. The primer extension lane was digitally manipulated to enhance image quality. (B) RT-PCR assay to compare levels of cbaA mRNA in succinate-grown (uninduced) and 3CBA-grown (induced) BR60(pBRC60). The RT step was performed with the primer used for primer extension analysis (see Materials and Methods). For reference purposes, the PCR signal obtained with whole cells is also shown. The lengths of DNA size standards are indicated on the right.

Induction of 3CBA degradation activity and expression of cbaA In order to determine which growth substrates induced 3CBA degradation activity, we initially investigated control of 3CBA catabolism in C. testosteroni BR60(pBRC60) by performing O₂ uptake assays with whole cells, and the results are shown in Table 3. As expected, maximal 3CBA degradation activity was induced by 3CBA. Benzoate, which is not a substrate of this pathway and is not channeled via the PCA extradiol pathway (50), gratuitously induced 3CBA degradation activity (~29% of the maximum activity). Similarly, benzoate degradation activity was gratuitously induced by 3CBA (~22% of the maximum activity observed following growth on benzoate). Succinate, a nonaromatic carbon source, did not induce 3CBA activity, nor did 3-hydroxybenzoate, 4-hydroxybenzoate, and PCA, three aromatic carbon sources that are channeled through the PCA extradiol ring fission pathway, suggesting that the cba pathway, at least with respect to 3CBA, responds to the parent substrate but not to downstream metabolites that are common to these degradative pathways (i.e., PCA or the ring fission products). We observed good reproducibility between trials for all cultures except those grown on benzoate. For unknown reasons, cells exhibited variable lag phases (18 to 36 h) in the three trials before the onset of logarithmic growth. This apparently affected O₂ uptake rates with benzoate but not with the other carbon sources (data not shown). The results presented here are the results for the trial in which the log phase was achieved within 18 h, the time necessary for the other growth substrates.

View larger version (54K): [in this window] [in a new window]   FIG. 3.   Levels of cbaA expression in C. testosteroni BR6020 harboring different lacZ constructs. Expression was measured with a growth substrate that did not induce 3CBA degradation activity (succinate) or did induce this activity (benzoate or 3CBA) in Tn5271-deficient cells (pBRC60) and Tn5271-proficient cells (+pBRC60). The data are averages based on three independent trials; the error bars indicate standard errors. 3Cba, 3-chlorobenzoate.

Expression of cbaR and gel shift and DNase I footprinting assays with affinity-purified CbaR. In order to better understand the gene coding for CbaR, we investigated expression of cbaR with various LacZ constructs, not only to determine whether the levels of cbaR expression were affected by growth conditions and genetic backgrounds that affected cbaA expression but also to roughly map the transcription starting point. LacZ activity in BR6020 harboring cbaR'-'lacZ1 and cbaR'-'lacZ2 was very low (~20 Miller Units), but compared to the control (see above) the activity was significant. These levels of expression were observed in Tn5271-deficient cells grown on succinate or benzoate and in Tn5271-proficient cells grown on succinate or 3CBA, implying that Tn5271-encoded functions, particularly CbaR, did not have an effect on cbaR expression. With BR6020 harboring cbaR'-'lacZ3 in both genetic backgrounds and grown under the same conditions, the activity was negligible (~1 Miller unit). This implied that cbaR transcription began before nt 4098 (i.e., the BglII site defining the end of construct cbaR'-'lacZ3) but after nt 4446 (i.e., the NotI site defining the end of construct cbaR'-'lacZ2, in which expression was observed) (Fig. 1B).

View larger version (32K): [in this window] [in a new window]   FIG. 4.   (A) Analysis of affinity-purified CbaR by SDS-PAGE. Ten micrograms of protein was loaded onto the gel. The positions of protein size standards (in kilodaltons) are indicated on the left. (B) Schematic diagram of the three DNA templates used in gel shift assays. Template 1 putatively contains the transcriptional start site of cbaR, while templates 2 and 3 harbor the transcriptional start site and promoter of cbaA (PcbaA). (C to F) Unless indicated otherwise, each binding reaction mixture contained 0.4 µg of CbaR, 4 ng of DNA, and an aromatic compound at a concentration of 4 mM. The positions of unbound template (Free DNA), CbaR-PcbaA DNA complex 1 (C1), and CbaR-PcbaA DNA complex 2 (C2) are indicated. Template 3 was used for all assays except those whose results are shown in panel C, which tested the effect of CbaR on the mobility of templates 1 and 2. For template 2, the effect of including 3CBA in the binding reaction mixture was also tested. (D) Assay to determine the minimum concentration at which 3CBA interfered with CbaR binding to PcbaA DNA. The concentration of 3CBA was varied, while the amounts of CbaR and DNA were kept constant. (E) Stoichiometry of CbaR-PcbaA DNA complex formation. The amount of CbaR per reaction mixture was varied, while the amount of DNA was kept constant. For reference purposes, the approximate molar ratios of CbaR to DNA are also shown. (F) Effects of benzoate and various hydroxylated benzoates on CbaR binding to PcbaA DNA. 3Cba, 3-chlorobenzoate.

View larger version (91K): [in this window] [in a new window]   FIG. 5.   (A) DNase I protection assay to determine specific regions of PcbaA DNA protected by CbaR. Template DNA was end labeled with 32PO4 on the noncoding strand (32P-PCBA) or the coding strand (32P-CBAA). Various amounts of CbaR were added, and the effect of 4 mM 3CBA (3Cba) or 4 mM 3-carboxybenzoate (3Crba) on CbaR protection of two regions (sites I and II) was determined. A sequencing reaction conducted with the appropriate primers was included during electrophoretic resolution of DNase I-digested samples. (B) Sequences of both strands of the template DNA used for the assay, indicating the positions of sites I and II relative to the transcriptional start site of cbaA (position +1); the putative 10 hexamer; and the start codon of cbaA (ATG). Inverted repeats found in site I and modified versions of these repeats in site II are indicated by arrows, and bases present in both sites are indicated by boldface type.

	DISCUSSION

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Classification of strain BR60(pBRC60). We reclassified Alcaligenes sp. strain BR60(pBRC60) as C. testosteroni BR60(pBRC60) based on several metabolic and genetic similarities between this bacterium and various other strains of the -proteobacterium C. testosteroni (59, 62). The metabolic similarities include an inability to grow on various carbohydrates (67); induction of the PCA meta ring fission enzymes by growth on 3-hydroxybenzoate, 4-hydroxybenzoate, benzoate, or PCA (41); and a lower growth rate with benzoate than with hydroxylated benzoates (62; Providenti, unpublished data). The genetic similarities are the presence of the same partial 16S ribosomal DNA sequence (see above) and highly homologous genes for PCA meta ring fission pathway enzymes (J. Mampel, M. A. Providenti, R. C. Wyndham, and A. M. Cook, unpublished data).

Control of 3CBA degradation activity. We initially hypothesized that cbaABC was negatively regulated by CbaR, which was consistent with the predominant role of the MarR family of proteins (Table 2) (see below), but studies with the cbaA expression constructs implied that the major role of CbaR may be to modulate gratuitous induction by compounds like benzoate (see above). Gel shift experiments showing that benzoate only weakly disrupted CbaR binding to P_cbaA supported this model (Fig. 4F). Furthermore, previous work that showed that the cbaR region is not required for cbaABC-mediated growth on 3CBA (41) suggested that CbaR is not essential for cbaA expression. Because studies with Tn5271-deficient cells harboring construct cbaA'-'lacZ1 showed that growth on benzoate (Fig. 3) or growth on succinate in the presence of 3CBA (data not shown) induced cbaA expression, we propose that there is a chromosomally encoded regulator that, along with CbaR, controls cbaABC. 3CBA and benzoate gratuitously activate each other's pathways and exhibit similar induction spectra with respect to 3-hydroxybenzoate, 4-hydroxybenzoate, and PCA (Table 3), and we therefore hypothesize that there is a shared regulator for the two pathways. The cbaABC regulatory system may thus have similarities to CatR activation of both the chromosomally encoded catBCA operon for catechol metabolism and the plasmid-encoded pheBA operon for phenol metabolism (26, 44). Alternatively, we may have observed a cross-activation phenomenon, like induction of the lower pathway of the plasmid-encoded xyl genes in response to benzoate despite the absence of XylS, the normal activator (10, 24, 28), or CatR- and ClcR-mediated cross-activation of the clc- and cat-encoded catabolic pathways (37, 45).

CbaR: expression of the gene and functional characterization. The gene that encodes CbaR appears to be expressed at a very low level (see above), and neither benzoate nor 3CBA, two growth substrates that increased cbaA expression, altered the level of cbaR expression. In addition, CbaR itself does not appear to exert control over expression of its own gene. This conclusion is suggested by the observation that the levels of cbaR expression were the same regardless of the presence of Tn5271, which provided a trans-encoded CbaR (see above), and the observation that CbaR did not bind to DNA spanning the region putatively containing the cbaR transcription start site (see above) (Fig. 4C). Furthermore, no sequences similar to the two binding sites of CbaR were detected when the complete region upstream of cbaA was analyzed.