JOURNAL OF BACTERIOLOGY, Oct. 2002, p. 5714–5722 0021-9193/02/$04.00⫹0 DOI: 10.1128/JB.184.20.5714–5722.2002 Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Vol. 184, No. 20
Differential Expression of Two Catechol 1,2-Dioxygenases in Burkholderia sp. Strain TH2 Katsuhisa Suzuki,1* Atsushi Ichimura,2 Naoto Ogawa,1 Akira Hasebe,1 and Kiyotaka Miyashita1† National Institute for Agro-Environmental Sciences, 3-1-3 Kan-nondai, Tsukuba, Ibaraki 305-8604,1 and Department of Bioengineering, Nagaoka University of Technology, Nagaoka 940-2188,2 Japan Received 22 April 2002/Accepted 13 July 2002
Burkholderia sp. strain TH2, a 2-chlorobenzoate (2CB)-degrading bacterium, metabolizes benzoate (BA) and 2CB via catechol. Two different gene clusters for the catechol ortho-cleavage pathway (cat1 and cat2) were cloned from TH2 and analyzed. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis showed that while both catechol dioxygenases (CatA1 and CatA2) were produced in BA-grown cells, CatA1 was undetectable when strain TH2 was grown on 2CB or cis,cis-muconate (CCM), an intermediate of catechol degradation. However, production of CatA1 during growth on 2CB or CCM was observed when cat2 genes were disrupted. The difference in the production of CatA1 and CatA2 was apparently due to a difference in inducer recognition by the regulators of the gene clusters. The inducer of CatA1 was found to be BA, not 2CB, by using a 2-halobenzoate dioxygenase gene (cbd) disruptant, which is incapable of transforming (chloro)benzoate. It was also found that CCM or its metabolite acts as an inducer for CatA2. When cat2 genes were disrupted, the growth rate in 2CB culture was reduced while that in BA culture was not. These results suggest that although cat2 genes are not indispensable for growth of TH2 on 2CB, they are advantageous. that strain TH2 has two catechol 1,2-dioxygenases. What is more interesting is that while either protein was induced during growth on BA, a protein with a molecular mass of 32 kDa was not observed when TH2 cells were grown on 2CB (44), indicating that the genes encoding the two catechol 1,2-dioxygenases in strain TH2 are expressed differentially. Several catechol-degrading bacteria possess two sets of catechol 1,2-dioxygenases, CatA1 and CatA2 (1, 19, 23). CatA1 and CatA2 of Frateuria sp. strain ANA-18 (23) are very similar to those of Acinetobacter lwoffii K24 (19). The catechol dioxygenases of both strains have been purified and analyzed (19, 23). While some differences in enzyme properties between CatA1 and CatA2 have been found, the significance of possessing two Cat enzyme systems in these bacteria is not clear. In this study, we analyzed the structure of the two sets of genes for catechol degradation of Burkholderia sp. strain TH2. The significance of possessing two sets of cat genes is discussed in terms of the difference in inducer recognition.
Chlorobenzoates (CBs) are key intermediates in the degradative pathways of polychlorinated biphenyls (PCBs). Therefore, the degradation of CBs has been extensively studied in conjunction with that of PCBs. CBs degraded by aerobic bacteria are often converted to nonaromatic chloro-cyclohexadiene-1,2-diol-1-carboxylic acid (DHB) by (chloro)benzoate dioxygenase and then to chlorocatechols by DHB dehydrogenase (11). The chlorocatechols yielded are then catabolized via a modified ortho-cleavage pathway (21, 29, 32) or a metacleavage pathway (18, 33). In 2-chlorobenzoate (2CB)-degrading bacteria, such as Burkholderia sp. strain TH2 (44) and Pseudomonas sp. strain 2CBS (9), degradation of 2CB is initiated by 2-halobenzoate dioxygenase (CbdABC) (13), giving rise to 2-chloro-3,5-cyclohexadiene-1,2-diol-1-carboxylic acid (2-Cl-DHB), which is presumed to spontaneously lose carbon dioxide and halogenide and generate catechol (10). Accordingly, DHB dehydrogenase is not necessary for 2CB degradation in these bacteria. Catechol degradation in these 2CBdegrading bacteria, however, has not been studied extensively at the genetic level. Burkholderia sp. strain TH2 has 2-halobenzoate dioxygenase genes (cbdABC) nearly identical to those of Pseudomonas sp. strain 2CBS (13, 44). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of proteins induced when strain TH2 was grown on 2CB or benzoate (BA) showed that the N-terminal sequences of the induced proteins with molecular masses of 34 and 32 kDa were similar to those of catechol 1,2-dioxygenases of other bacteria (44), suggesting
MATERIALS AND METHODS Bacterial strains, plasmids, and culture conditions. The bacterial strains and plasmids used in this study are listed in Table 1. Burkholderia sp. strains TH2 and NDBA1 were cultivated at pH 7.0 and 30°C with vigorous shaking in basal salt (BS) medium (28) containing 5 mM BA, 5 mM 2CB, or 0.1% (wt/vol) glucose as the sole source of carbon. Escherichia coli cells were grown in a Luria-Bertani medium (37) at 37°C with vigorous shaking. When required, ampicillin (50 g/ml), kanamycin (50 g/ml), or gentamicin (20 g/ml) was added to the growth medium. DNA manipulations. Small- and large-scale preparations of plasmid DNAs from E. coli were obtained using the alkaline lysis method (37) or using a QIAprep Miniprep kit (Qiagen, Hilden, Germany). Genomic DNAs were prepared using a Puregene DNA isolation kit (Gentra Systems, Inc., Minneapolis, Minn.). The other techniques used for DNA manipulation were standard procedures (37). Induction. TH2, TD2, TCD1, TCD2, and TBD1 strains were grown overnight at 30°C in BS medium (28) (2 ml) containing 0.1% glucose (wt/vol). The cells were harvested by centrifugation at the end of the exponential phase and suspended in fresh BS medium (100 ml) containing 5 mM BA, 5 mM 2CB, 5 mM
* Corresponding author. Mailing address: National Institute for Agro-Environmental Sciences, 3-1-3 Kan-nondai, Tsukuba, Ibaraki 305-8604, Japan. Phone: 81 298 38 8309. Fax: 81 298 38 8199. E-mail:
[email protected]. † Present address: National Institute for Agrobiological Sicences, 2-1-2 Kan-nondai, Tsukuba, Ibaraki 305-8602, Japan. 5714
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TABLE 1. Bacterial strains and plasmids used in this study Strain or plasmid
Strains Burkholderia sp. strain Burkholderia sp. strain Burkholderia sp. strain Burkholderia sp. strain Burkholderia sp. strain Burkholderia sp. strain E. coli DH5␣ E. coli S17-1 E. coli BL21(DE3) Plasmids pBluescript II KS(⫹) pET14b pET16b pJRD215 pMOB3 pNOT322 pHP45⍀aac pBP144 pBB311 pJPBC pPCA2 pCAD pCAU pCASL3 p14CA1 p14CA2 pCA218 pCA218⍀ pNCA218⍀ pMNCA218⍀ pCB18 pCB18⍀ pNCB18⍀ pMNCB18⍀ pDB90 pDBSC pDBSC⍀ pNDBSC⍀ pMNDBSC⍀ p16TLXYZ
Genotype or description
TH2 NDBA1 TD2 TCD1 TCD2 TBD1
Reference or source
BA⫹ 2CB⫹ 3CB⫺ 4CB⫺ BA⫺ 2CB⫺ 3CB⫺ 4CB⫺ cbd disruptant, BA⫺ 2CB⫺ 3CB⫺ 4CB⫺ cat1 disruptant of TH2 cat2 disruptant of TH2 benD disruptant of TH2 supE44 ⌬lacU169(80lacZ⌬M15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1 recA thi pro hsdR⫺M⫹ pR4::2- Tc::Mu-Km::Tn7(Tpr/Smr) F⫺ ompT hsdSB(rB⫺ mB⫺) gal dcm (DE3)
44 12 44 This study This study This study 37 43 Novagen
Apr, cloning vector Apr, Expression vector Apr, Expression vector Kmr Smr, broad-host-range vector with multiple-cloning sites Kmr Cmr, mob⫹ sacB⫹ Apr, cloning vector Gmr 8-kbp PstI fragment, hybridized with the benA probe, cloned into PstI site of pBluescript II KS(⫹) 7.2-kbp BamHI fragment, hybridized with the benA probe, cloned into BamHI site of pBluescript II KS(⫹) 8-kbp KpnI-XbaI fragment of pBP144 cloned into KpnI-XbaI site of pJRD215 5.2-kbp PstI fragment, hybridized with the catA2 probe, cloned into PstI site of pBluescript II KS(⫹) 3.7-kbp SalI fragment, hybridized with the SalI-PstI fragment probe from pPCA2, cloned into SalI site of pBluescript II KS(⫹) 8-kbp NotI fragment, hybridized with the NotI-PstI fragment probe from pPCA2, cloned into NotI site of pBluescript II KS(⫹) 2.3-kbp SalI-NotI fragment of pCAU cloned into the SalI site of pBluescript II KS(⫹) 2.5-kbp XhoI fragment of pBB311 cloned into the XhoI site of pET14b 3.3-kbp NcoI-XhoI fragment of pPCA2 cloned into the NcoI-XhoI site of pET14b 5.2-kbp PstI fragment of pPCA2 cloned into the PstI site of pUC18 pCA218 derivative; Apr Gmr pNOT322 derivative containing the XbaI-SmaI insert of pCA218⍀; Apr Gmr pNCA218⍀ derivatives; mob⫹ sacB⫹ Apr Gmr 7.2-kbp BamHI fragment of pBB311 cloned into the BamHI site of pUC18 pCB18 derivative; Apr Gmr pNOT322 derivative containing the XbaI-SmaI insert of pCB18⍀; Apr Gmr pNCB18⍀ derivatives; mob⫹ sacB⫹ Apr Gmr 9-kbp BamHI fragment, encoding benD, cloned into pBluescript II KS(⫹) pDB90 derivative: Apr pDBSC derivative: Apr Gmr pDBSC⍀ derivatives: Apr Gmr pNDBSC⍀ derivatives: mob⫹ sacB⫹ Apr Gmr XbaI-BamHI fragment encoding xylXYZ cloned into the XbaI-BamHI site of pET16b
37 Novagen Novagen 6 40 40 2 This study This study This study This study This study
3-chlorobenzoate (3CB), 5 mM 4-chlorobenzoate (4CB), 5 mM cis,cis-muconate (CCM), or 0.1% (wt/vol) glucose. The cells were grown until late exponential phase. The time required depended on the strain and the inducer used and was at least 24 h. Conjugation and electroporation. Conjugation was performed using a method described previously (12). Electroporation was done with a Gene Pulser (BioRad, Hercules, Calif.) as specified by the manufacturer. Enzyme assay in Burkholderia sp. strain TH2. Cells of strain TH2 induced with BA or 2CB were suspended in PGE buffer (34), and cell extracts were prepared by sonication. Protein concentrations were determined using the method described by Bradford (3). Catechol-1,2-dioxygenase activity was assayed spectrophotometrically by monitoring the increase in CCM concentration as indicated by the absorbance at 260 nm (A260) (7). The activity is expressed as the rate of conversion to CCM per minute per milligram of protein. The assay for catechol 2,3-dioxygenase was done by the method of Nozaki (27). Cloning of the ben-cat1 gene cluster. Using the forward primer BAf1 and the reverse primer BAr2 (12), designed from the highly homologous regions of benA of Acinetobacter strain ADP1 and xylX of Pseudomonas putida TOL plasmid pWW0, part of the benA gene was amplified by PCR using KOD polymerase (Toyobo) and strain TH2 total DNA as a template. The PCR product was used as a probe for the benA gene of strain TH2. The chromosomal DNA of strain TH2 was completely digested with BamHI or PstI. Restriction fragments were separated on a 0.7% (wt/vol) agarose gel by electrophoresis and then blotted onto a Hybond-N⫹ nylon membrane (Amersham Pharmacia Biotech, Little
This study This This This This This This This This This This This This This This This This This
study study study study study study study study study study study study study study study study study
Chalfont, England). The PCR product was labeled with the DIG labeling kit as described by the supplier (Boehringer GmbH, Mannheim, Germany). Hybridization was carried out using the standard method (37) with the digoxigeninlabeled PCR product. Immunological detection was performed with the digoxigenin luminescence detection kit (Boehringer). DNA fragments of 6.5 to 7.5 kbp from the BamHI digestion and DNA fragments of 7.5 to 8.5 kbp from the PstI digestion were recovered from a 0.7% (wt/vol) agarose gel and inserted into the BamHI and PstI sites of pBluescript II KS(⫹), respectively. E. coli DH5␣ was transformed using the ligation mixture and screened for the existence of the benA-cat gene cluster by colony hybridization with the benA probe of strain TH2. Cloning of cat2 genes. To clone the gene for catechol 1,2-dioxygenase (catA2), on the basis of the N-terminal amino acid sequence of the protein induced by 2CB only (44), the mixed forward primer 5⬘-ATGGA(T/C)AT(T/C)AAGACC ATCGA(T/C)-3⬘ was synthesized. The mixed reverse primer 5⬘-GATCTGGGT GGT(G/C)AGCTTGCG-3⬘ was synthesized on the basis of the C-terminal amino acid sequence of CatA2 of A. lwoffii K24 (19). The PCR product was used as a probe for the catA2 gene of strain TH2. The chromosomal DNA of TH2 was completely digested with PstI. DNA fragments of 4.7 to 5.7 kbp were recovered from a 0.7% (wt/vol) agarose gel and inserted into the PstI site of pBluescript II KS(⫹). E. coli DH5␣ was transformed using the ligation mixture and screened for the existence of cat2 genes by colony hybridization with the catA2 probe. DNA sequencing and sequence analysis. DNA sequencing of both strands of pBB311, pBP144, pPCA2, pCASL3, and pCAD was performed using a Thermo Sequenase cycle-sequencing kit (Amersham Pharmacia Biotech) and a Li-Cor
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model 4200L(S)-2 automated DNA sequencer. Sequence analysis and homology searches were done using Genetyx-Mac (v10.1) software (Software Development, Tokyo, Japan). Expression of catA1 and catA2 in E. coli. The catA1 and catA2 genes were expressed using the T7 RNA polymerase-promoter system (45). Plasmids p14CA1 and p14CA2 were each introduced into E. coli BL21(DE3) bearing the T7 RNA polymerase gene for expression of the target proteins. Cells bearing each plasmid were grown at 30°C overnight in 2⫻ YT medium containing ampicillin. The culture was diluted 100-fold into a prewarmed fresh 2⫻ YT medium containing ampicillin and grown at 30°C. When the A600 of the culture reached 0.8 to 1.0, 1.0 mM isopropyl--D-thiogalactopyranoside (IPTG) was added to induce gene expression. After 4 h of culturing with IPTG, cells were harvested by centrifugation. Construction of cat1 and cat2 disruptants of strain TH2. The cat1 and cat2 gene clusters were each disrupted by ⍀ interposon mutagenesis by using the method described by Schweizer (40). Plasmid pCB18 (Table 1) was digested with EcoT22I and ApaI to remove the region of the cat1 operon, blunted with T4 DNA polymerase (Takara Shuzo Co., Kyoto, Japan), then ligated to a SmaI ⍀Gmr cassette of pHP45⍀aac (2). Plasmid pCA218 (Table 1) was digested with NotI and NaeI to remove the region of the catBAC genes, blunted with T4 DNA polymerase, and then ligated to a SmaI ⍀Gmr cassette of pHP45⍀aac (2). The resultant pCB18⍀ and pCA218⍀ plasmids were digested with XbaI and SmaI, respectively, and then blunted with T4 DNA polymerase. These blunted XbaISmaI fragments were ligated to the EcoRV site of pNOT322, resulting in pNCB18⍀ and pNCA218⍀ plasmids, respectively. The MOB cassette of pMOB3 was subsequently cloned into pNCB18⍀ and pNCA218⍀. The resultant pMNCB18⍀ and pMNCA218⍀ plasmids were each introduced into E. coli S17-1 and then sequentially transferred into strain TH2 by conjugation. Transconjugants were selected at 30°C on a BS plate (28) containing 0.2% (wt/vol) ribitol and gentamicin and evaluated in the presence of 5% sucrose on a BS plate containing ribitol and gentamicin to separate double from single crossovers. Allelic replacement of the wild-type cat1 and cat2 by the ⍀ cassette-disrupted cat1 and cat2 was verified using Southern blot analysis. The disruptants of cat1 and cat2 operons of strain TH2 were named TCD1 and TCD2, respectively. Induction of CatA1 and CatA2. The induction of expression of catA1 and catA2 was examined using strains TH2, TCD1, TCD2, and TD2, which is a 2-halobenzoate-1,2-dioxygenase gene disruptant (44). TH2, TCD1, and TCD2 cells were grown on BS medium (28) containing BA, 2CB, or CCM (5 mM) as the sole source of carbon and energy and harvested at the end of the exponential phase by centrifugation. TD2 cells, which cannot convert (chloro)benzoate to (Cl)-DHB, were grown on BS medium (28) amended with 0.1% (wt/vol) glucose and 5 mM (chloro)benzoate. Cell extracts were prepared by sonication using PGE buffer (34), and the protein concentrations were determined using the method described by Bradford (3). The production of CatA1 and CatA2 was examined using SDS-PAGE and Western blot analysis. Immunoblots. Using synthetic peptides designed on the basis of a common amino acid sequence (WHSTPDGKYSGFHD) from TfdC of Burkholderia sp. strain NK8 (accession no. AB050198) (21) and CbnA of Ralstonia eutropha NH9 (accession no. AB019032) (29), which is also conserved in TetC of Pseudomonas chlororaphis RW71 (31), antiserum was prepared by Sawady Technology Co. Proteins (10 g) from TH2, TCD1, TCD2, and TD2 cells grown on various substrates were separated by SDS-PAGE (12% polyacrylamide) and transferred onto a membrane (Clear blot membrane-p; ATTO Co., Tokyo, Japan) by electroblotting. Immunoreactive proteins were detected on Western blots by enzyme immunoassay using an ECL Western blot analysis system (Amersham Pharmacia Biotech). Measurement of growth rate. The growth of strains TH2, TCD1, and TCD2 was determined in the presence of BA or 2CB as follows. Overnight cultures (0.5 ml) grown on BS medium (28) containing 0.1% (wt/vol) glucose were harvested by centrifugation. The harvested cells were suspended in 1 ml of sterilized water and inoculated in 50 ml of fresh BS medium (28) containing 5 mM BA or 5 mM 2CB as the sole source of carbon and energy in 200-ml flasks and incubated at 30°C with shaking. At 1-h intervals, samples (0.5 ml) were taken and the A600 was measured. benD disruption. The benD gene was disrupted by deleting most of the gene and using ⍀ interposon mutagenesis by the method of Schweizer (40). To construct a plasmid for the benD gene disruption, a 9-kbp BamHI fragment containing the benD gene was cloned by colony hybridization using a probe from a 2.8-kbp BamHI-PstI fragment of pBP144. The resultant pDB90 plasmid was digested with SacI, and the 8-kbp SacI fragment was ligated by intramolecular ligation. The resultant pDBSC plasmid was digested with SphI to remove the benD region, blunted with T4 DNA polymerase (Takara Shuzo Co.) and ligated with a SmaI ⍀Gmr cassette of pHP45⍀aac (2). The resultant plasmid, pDBSC⍀,
J. BACTERIOL. was digested with EcoRV. The EcoRV fragment was ligated to the EcoRV site of pNOT322, resulting in pNDBSC⍀. The MOB cassette of pMOB3 was subsequently cloned into pNDBSC⍀. The resultant plasmid, pMNDBSC⍀, was introduced into E. coli S17-1 and then sequentially transferred into Burkholderia sp. strain TH2 by conjugation. The strain of the double crossovers was separated as described above. Allelic replacement of the wild-type benD by the ⍀ cassettedisrupted benD was verified by Southern blot analysis. The disruptant of the benD gene of TH2 was named TBD1. HPLC. A high-pressure liquid chromatography (HPLC) assay was performed on an Eclipse XDB-C18 column in an HP 2000 model (Hewlett Packard) using an acetonitrile-water solvent system containing 10 mM H 3PO 4 (50:50, vol/vol) at a flow rate of 1.0 ml/min. Cloning of xylXYZ genes on the TOL plasmid in E. coli. The xylXYZ genes on the TOL plasmid (15) were amplified by PCR with the 77-mer forward primer 5⬘-GCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACCA TGACCATGACAATGCACCTGGGGCTCGACTATA-3⬘ and the 30-mer reverse primer, 5⬘-CAGGATCCCTAGGCGCTGGCGGCGAACTTC-3⬘. The amplified fragment was digested with XbaI-BamHI and cloned into the XbaIBamHI site of pET16b to generate p16TLXYZ. The relevance of the inserted fragment of the plasmid was verified by sequencing. E. coli BL21(DE3) was transformed with p16TLXYZ. The transformant was grown overnight in 1 ml of Luria-Bertani medium (37) with ampicillin. The cells were harvested and suspended in 100 ml of M9 medium (37) containing glucose (10 mM). IPTG was added to the culture to produce a final concentration of 0.5 mM to induce expression from the T7 promoter of the pET vector and grown overnight. BA was subsequently added to the overnight culture. Samples of the culture medium (1 ml) were collected at various time points. Metabolites in the samples were detected by HPLC. Measurement of DHB dehydrogenase activity. Strain TH2, TCD1, and TBD1 cells were grown on BS medium (28) containing 5 mM of 2CB as the sole source of carbon and harvested at the end of the exponential phase by centrifugation. The harvested cells were subsequently suspended in 15 ml of 50 mM phosphate buffer (pH 7.4) amended with 2 mM BA. The cells were incubated at 30°C with vigorous shaking. At various time points, 1.0-ml samples were collected. The DHB dehydrogenase activity of the samples was assayed by HPLC monitoring of the quantity of DHB remaining in the reaction mixture. Chemicals. CCM was purchased from Celgene Co., Warren, N.J. BA, 2CB, 3CB, and 4CB were obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Nucleotide sequence accession number. The nucleotide sequences of the bencat1 gene cluster and the cat2 genes reported in this paper have been deposited in the DDBJ, EMBL, and GenBank nucleotide sequence databases under accession no. AB035483 and AB035325, respectively.
RESULTS Catechol 1,2-dioxygenase activity of Burkholderia sp. strain TH2. Burkholderia sp. strain TH2 and Pseudomonas sp. strain 2CBS, 2CB-degrading bacteria, have nearly identical 2-halobenzoate dioxygenase genes (cbdABC) (44). In strain 2CBS, the catechol yielded is degraded mainly via the meta-cleavage pathway (9). Accordingly, we tried to detect catechol 2,3-dioxygenase activity in strain TH2 by using the method described by Nozaki (27). However, catechol 2,3-dioxygenase activity was not detected in cell extracts from either BA- or 2CB-grown cells (data not shown). On the other hand, high activity for catechol 1,2-dioxygenase was detected in BA- and 2CB-grown cells of strain TH2 (820 and 650 nmol min⫺1 mg⫺1, respectively). Therefore, these results indicated that TH2 utilizes catechol from BA and 2CB via the ortho-cleavage pathway. Cloning and sequencing of ben-cat1 genes. We obtained one of the cat gene clusters of strain TH2 by cloning genes for benzoate dioxygenase. Southern hybridization showed that the benA probe hybridized with a ca. 8-kbp band from PstI digestion and a ca. 7-kbp band from BamHI digestion, which were cloned into pBluescript II KS(⫹), generating pBP144 and pBB311, respectively (Table 1). The contiguous nucleotide sequence of the two insert fragments from pBP144 and pBB311
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FIG. 1. Schematic representation of the ben-cat1 and cat2 genes. The open arrows indicate locations and directions of transcription of ORFs. IS1599 is indicated by a gray box. The orientation of the open arrow in IS1599 indicates the direction of transcription of the ORF within it. The thick solid line above the gene map indicates the DNA fragment from plasmids pBB311, pBP144, pCASL3, pPCA2, and pCAD that were sequenced in both strands. The open box indicates the omega cassette used for gene disruption. Abbreviations for restriction endonucleases: A, ApaI; B, BamHI; ET22, EcoT22I; EV, EcoRV; H, HindIII; N, NotI; Na, NaeI; Nc, NcoI; P, PstI; S, SalI, Sc, SacI; Sm, SmaI; Sp, SphI; Xb, XbaI; X, XhoI. aa, amino acids.
corresponds to 11,171 bp; it contains 11 open reading frames (ORFs) in either orientation (designated ORFI-1 to ORFI-5 and ORFII-1 to ORFII-6) (Fig. 1). Based on the results of homology search of the database using the deduced amino acid sequences of the ORFs ORFI-1 ORFI-3, ORFI-4, and ORFI-5 are apparently the genes for the large subunit (benA) and the small subunit (benB) of the terminal oxygenase, the reductase component (benC) of the benzoate dioxygenase, and the DHB dehydrogenase (benD), respectively (8, 12, 14, 20, 25, 26). The deduced amino acid sequences of ORFII-1, ORFII-3, and ORFII-4 are similar to catA, catB, and catC of the catechol degradation gene cluster of the proteobacteria, respectively (Table 2), indicating that ORFII-1, ORFII-3, and ORFII-4 are the genes for catechol 1,2-dioxygenase (19, 23), CCM-lactonizing enzyme (41, 42), and muconolactone isomerase (41, 42), respectively. They are designated catA1, catB1, and catC1 (Fig. 1), since additional cat genes were found later. The N-terminal amino acid sequence of CatA1 was identical to that of the 34-kDa protein (IP2) found in the SDS-PAGE profile of TH2 cells grown in BA (44). ORFII-2 encodes a protein with an amino acid sequence very similar to the LysR-type transcriptional regulator involved in catechol and benzoate catabolism (12, 17, 36). ORFII-2 is designated catR1 because it is transcribed divergently from the catA gene and possibly regulates transcription of the catA promoter. The overall structure of the ben-cat gene cluster of strain TH2 is very similar to the (chloro)benzoate dioxygenase (cbe)
and catechol dioxygenase (cat) gene cluster of Burkholderia sp. strain NK8 (12). The identity of the predicted amino acid sequence in the corresponding genes between strains TH2 and NK8 was ca. 85 to 91%. BenA of strain TH2 (352 amino acids), however, is smaller than CbeA of NK8 (452 amino acids) (12), because the C-terminal region of the benA gene of strain TH2 TABLE 2. Identity between strain TH2 Cat proteins and those of other bacteriaa % identity to: TH2 Cat protein
TH2
ANA-18
Cat2
NK8 Cat
Cat1
Cat2
Cat1
Cat2
ADP1 Cat
PRS2000 Cat
100 51.8 51.8 100 100 59.2 59.2 100 100 56.5 56.5 100 100 54.9 54.9 100
86.2 49.2 91.2 58.6 85.9 54.3 88.5 54.9
81.4 50.7 56.5 60.4 71.7 60.0 46.6 46.1
56.4 80.1 57.7 79.0 55.4 69.8
81.7 50.7 57.1 60.4 70.7 58.9 46.3 46.1
51.5 79.7 57.7 77.3 55.4 68.8 56.0 80.8
55.3 50.3 57.2 60.8 68.5 56.8 43.0 40.3
45.8 51.9 63.0 57.4 56.5 70.8 49.1 48.2
Cat1
CatA1 CatA2 CatB1 CatB2 CatC1 CatC2 CatR1 CatR2
K24
a Gene products, bacterial strains, and database accession numbers are as follows: CatA, CatB, CatC, and CatR of Burkholderia sp. strain NK8 (12) (AB024746); CatA1, CatB1, CatC1, and CatR1 of A. lwoffii K24 (19) (U77658); CatA2, CatB2, and CatC2 of A. lwoffii K24 (19) (U77659); CatA1, CatB1, CatC1, and CatR1 of Frateuria sp. strain ANA-18 (23) (AB009343); CatA2, CatB2, CatC2, and CatR2 of Frateuria sp. strain ANA-18 (23) (AB009373); CatA, CatB, CatC, and CatM of Acinetobacter sp. strain ADP1 (24, 35, 41) (M76991); CatA, CatB, CatC, and CatR of P. putida PRS2000 (17) (U12557).
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is truncated by the insertion of an IS element. The IS element, designated IS1599, is 1,349 bp long, including the 13-bp inverted repeats (IR) at both ends. External to the IRs, a 4-bp (CTAA) direct duplication was found. ORFI-2 in IS1599 encodes a polypeptide of 367 amino acids; its sequence is similar to the transposase of Ralstonia metallidurans (54% identity) (39). ORFII-5 encodes a polypeptide of 103 amino acids, which shows similarity to the N-terminal portion of CbeE of strain NK8 (12). ORFII-5, however, is much shorter than CbeE of strain NK8 (401 amino acids). The function of cbeE, a homolog of the benE gene of Acinetobacter sp. strain ADP1, is unknown. ORFII-6 comprises 513 bp nucleotides, corresponding to 170 amino acids, with no homolog in the database. Sequences corresponding to ORFII-6 were not found in the gene cluster of strain NK8 (12). Degradative function of ben genes. Truncation of the 3⬘ region of benA by the insertion of IS1599 raised the question whether BenA is dysfunctional. Accordingly, pJPBC containing the benABCD genes was introduced into a derivative of strain NK8 (strain NDBA1) in which the gene for the large subunit of benzoate dioxygenase (cbeA) was deleted by interposon mutagenesis (BA) (12). Strain NDBA1 harboring pJPBC did not grow in BS medium containing BA as the sole source of carbon (data not shown), demonstrating that the ben gene cluster of strain TH2 cannot confer on strain NDBA1 the ability to degrade BA. This result suggests that the benzoate dioxygenase system of strain TH2 is dysfunctional. Cloning and sequencing of cat2 genes. The N-terminal amino acid sequence of IP3 (44), a 32-kDa protein whose production was induced on BA or 2CB, is different from that of CatA1 of strain TH2 but is similar to that of CatA2 of A. lwoffii K24 and Frateuria sp. strain ANA-18 (19, 23). This suggests that strain TH2 has an additional catechol 1,2-dioxygenase (CatA2). To clone the catA2 gene, the forward primer was designed from the N-terminal sequence of IP3, together with the reverse primer based on the sequence of CatA2 of A. lwoffii K24 (19), to amplify a portion of the gene for the IP3 protein. A ca. 5.2-kbp PstI fragment of TH2 genomic DNA, which was hybridized with the DIG-labeled PCR product, was cloned into pBluescript II KS(⫹), yielding pPCA2. The overlapping NotI fragment (8 kbp) and SalI fragment (3.7 kbp) were subsequently cloned into pBluescript II KS(⫹), generating pCAU and pCAD plasmids, respectively (Fig. 1). The combined nucleotide sequence of the inserts of pPCA2, pCASL3 containing a 2.3-kbp SalI-NotI fragment from pCAU, and pCAD is 8,820 bp, which contains eight ORFs in either orientation (designated ORFIII-1 to ORFIII-8). From the results of database searches, ORFIII-1, ORFIII-2, ORFIII-3, and ORFIII-5 were designated catB2, catA2, catC2, and catR2, respectively. As shown in Table 2, the CatA2, CatB2, CatC2, and CatR2 sequences of strain TH2 are similar to those of A. lwoffii K24 (19) and Frateuria sp. strain ANA-18 (23). ORFIII-4 encodes a protein of 393 amino acids, which showed the highest similarity to flavohemoprotein of Ralstonia eutropha (43% identity) (5). ORFIII-6, encodes a protein of 532 amino acids which is a member of the NtrC/NifA family of regulators (50% identity to Xy1R) (22). ORFIII-7 and ORFIII-8 were found upstream and downstream, respectively, of the cat2 genes. Neither ORF, however, showed any similarity to other proteins in the database.
J. BACTERIOL. TABLE 3. Activity of catechol 1,2-dioxygenases with different substrates
Substrate
Catechol 3-Chlorocatechol 4-Chlorocatechol 4-Methylcatechol
Relative activitya (%)
Sp act b (nmol min⫺1 mg of crude protein⫺1)
CatA1
CatA2
CatA1
CatA2
100 4.4 18.1 39.5
100 ⬍0.1 2.5 13.1
94.8
3,920
a Expressed as a percentage of the specific activity with catechol which is set at 100%. b Catechol 1,2-dioxygenase activity was assayed spectrophotometrically at 260 nm with ring cleavage product inferred to be CCMs.
Specific activity of recombinant catechol 1,2-dioxygenase with different substrates. The low sequence similarity between CatA1 and CatA2 (52% identity [Table 2]) suggests differences in their enzymatic properties. Therefore, each catA gene was expressed with an E. coli expression system to examine the substrate specificities of the two catechol 1,2-dioxygenases of strain TH2. Crude cell lysates were used as enzyme solutions. Since the productivity of CatA2 was much higher than that of CatA1, as observed by SDS-PAGE, the results are shown as percentages relative to the activity against catechol (Table 3). Both enzymes showed higher activity against catechol than against the substituted catechols, and CatA1 had a higher activity against some substituted catechols than did CatA2. Disruption of each cat gene cluster. SDS-PAGE analysis showed that while CatA1 and CatA2 were produced in BAgrown cells of strain TH2, CatA1 was not produced in 2CBgrown cells (44). To examine the roles of the cat1 and cat2 genes in the catabolism of BA and 2CB, we disrupted each cat operon by deletion, thus generating strains TCD1 and TCD2. Each disruptant was grown on a BS agar plate with BA or 2CB as the sole source of carbon. The growth phenotypes of TCD1 and TCD2 were BA ⫺ 2CB ⫹ and BA ⫹ 2CB ⫹, respectively. To detect CatA1 in a cell extract precisely, Western blotting was done using the antiserum against the oligopeptide designed from the common amino acid sequences of TfdC and CbnA. The antiserum was expected to be reactive with the catechol dioxygenases. An immunoreactive band appeared at 34 kDa but not 32 kDa in TH2 cells grown on BA. The 34-kDa band disappeared in cells grown on 2CB (Fig. 2B). These results indicate that the antiserum reacts with CatA1 but not with CatA2. Use of the antiserum clearly showed that when grown on 2CB, CatA1 was not produced in the TH2 cells but in the TCD2 (cat2 disruptant) cells (Fig. 2B). It is thus shown that in 2CB degradation, CatA1 and CatA2 function as catechol 1,2-dioxygenase in strains TCD2 and TH2, respectively. However, the growth rate of a 2CB culture of strain TCD2 was lower than that of strains TH2 and TCD1, suggesting that cat2 is more effective than cat1 in allowing TH2 to metabolize 2CB (Table 4). On the other hand, strain TCD2 grew as fast as strain TH2 in a BA culture, suggesting that CatA1 is as effective as CatA2 in metabolism of BA. Induction of cat expression. The differential expression of cat1 and cat2 implies a difference of the inducers between the two gene clusters. The cbe-cat gene cluster in strain NK8, which is very similar to the ben-cat gene cluster in strain TH2,
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FIG. 2. (A) SDS-PAGE analysis of strain TH2, TCD1, and TCD2 proteins induced by different substrates. (B) CatA1 protein detected by Western blot analysis with a polyclonal rabbit antiserum raised against synthetic peptides (see Materials and Methods). The approximate molecular masses of marker proteins (kilodaltons) are indicated on the left. CatA positions are indicated by the arrows.
is regulated by CbeR, with BA, 3CB, and 4CB themselves as actual inducers (12). In contrast, 2CB is not the effector of CbeR. Since the CatR1 of TH2 exhibits 88.5% identity to the CbeR of NK8 (Table 2), induction of the cat1 genes by BA, 3CB, and 4CB was anticipated. Accordingly, we examined the induction of CatA1 production using strain TD2, a cbd disruptant that is incapable of growing on BA and CBs. The strain was grown on a BS glucose medium supplemented with BA or CBs. As shown in Fig. 3, CatA1 was produced in the presence of BA, 3CB, and 4CB but not in the presence of 2CB. It is thus obvious that BA, 3CB, and 4CB are actual inducers of CatA1 production. Induction of CatA1 or CatA2 production during growth on CCM, a well-known inducer of the cat operon of P. putida (30), was examined by growing strains TH2, TCD1, and TCD2 in the presence of 5 mM CCM. Western blot analysis showed that CatA1 was produced in strain TCD2 but not in strain TH2. In contrast, CatA2 was produced by strains TH2 and TCD1 during growth on CCM, as shown by SDS-PAGE analysis (Fig. 4). It was thus obvious that although CCM or its metabolite acted as an inducer of CatA2 production irrespective of the presence of the cat1 gene cluster, it induced CatA1 production only when the cat2 gene cluster was deleted. The productivity of CatA1 induced by CCM in strain TCD2, however, was much lower than that induced by BA in strain TH2 (Fig. 2 and 4). To detect the small amount of CatA1 in strain TCD2, a longer exposure was necessary, resulting in the spurious bands in the right-hand lane (Fig. 4). DHB dehydrogenase activity. It was unexpected that strain TCD1, a cat2 disruptant, could not grow on BA since the isofunctional cat2 genes were present in this strain. In addition to the 2-halobenzoate dioxygenase system (CbdABC), which is involved in the conversion of BA to DHB (10), DHB dehydrogenase is necessary for the conversion of BA to catechol (25). The enzyme is not required for the conversion of 2CB. A possible reason for the failure of strain TCD1 to grow on BA
was that benD, a gene for DHB dehydrogenase, is involved in the conversion of DHB to catechol in strain TH2, but becomes dysfunctional following disruption of the cat1 gene cluster (in strain TCD1). To clarify the function of the benD gene, the gene in strain TH2 was deleted. As a result, the benD disruptant, strain TBD1, was not capable of growing on BA as a sole source of carbon (data not shown). To test whether DHB dehydrogenase activity is absent in strains TCD1 and TBD1, HPLC was used to monitor metabolite transformations by 2CB-grown cells. Since DHB is not commercially available, the DHB peak must be identified. DHB is produced from BA by XylXYZ (25), and so metabolites of E. coli BL21(DE3)p16TLXYZ grown in an M9 medium containing BA and glucose (10 mM) were analyzed to examine the retention time of DHB by HPLC. As shown in Fig. 5, the peak of BA (peak I) disappeared after 5 h. In contrast, peak II increased over time, indicating that this peak represents DHB. The quantities of DHB in reaction mixtures of strains TH2, TCD1, and TBD1 were compared. In the TH2 mixture, the area of the DHB peak remained low for 4 h (Fig. 6), while in the TCD1 and TBD1 mixtures, it increased over time, indicat-
TABLE 4. Growth rate of strains TH2, TCD1, and TCD2 in BA and 2CB culturesa Generation time (min) with induced C sourceb:
Strain
TH2 TCD1 TCD2
BA
2CB
359 ⫾ 20 NGc 373 ⫾ 7
176 ⫾ 20 183 ⫾ 12 253 ⫾ 20
a Strains TH2, TCD1, and TCD2 were grown in BS medium (28) containing 5 mM BA or 5 mM 2CB. b Mean ⫾ standard deviation. c NG, no growth.
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J. BACTERIOL.
FIG. 3. (A) SDS-PAGE analysis of strain TD2 proteins induced by different substrates. (B) CatA1 protein detected by Western blot analysis with a polyclonal rabbit antiserum raised against synthetic peptides (see Materials and Methods). Approximate molecular masses of marker proteins (kilodaltons) are indicated on the left. The CatA1 position is indicated by the arrows.
ing the absence of DHB dehydrogenase in strains TCD1 and TBD1. DISCUSSION Burkholderia sp. strain TH2 has 2-halobenzoate dioxygenase genes (cbdABC) nearly identical to those of Pseudomonas sp. strain 2CBS (13, 44), which is involved in the utilization of BA and 2CB as the sole source of carbon (44). In Pseudomonas sp. strain 2CBS, BA and 2CB are converted to DHB and 2Cl-DHB, respectively, by 2-halobenzoate-1,2-dioxygenase (CbdABC) (9, 10). Strain 2CBS, however, could not utilize BA as a sole source of carbon and energy. This is presumably due to the lack of a gene for DHB dehydrogenase in strain 2CBS, which is necessary for the metabolism of BA but not for 2CB. The cbd gene cluster is apparently specialized to 2-halobenzoate metabolism since this cluster is devoid of a gene for DHB dehydrogenase. Strain TH2, in contrast, can utilize BA as a sole source of carbon. The presence and absence of DHB dehydrogenase activity in strain TH2 and in the benD disruptant strain TBD1, respectively, indicate that benD functions as the gene for DHB dehydrogenase and is involved in BA degradation in strain TH2. Moreover, the lack of DHB dehydrogenase activity in the cat1 disruptant, TCD1, was similar to that of TBD1 (Fig. 6). In strain TCD1, the cat1 gene cluster including the regulatory gene (catR1) and the divergent promoter region of catA and catR was replaced by omega interposon mutagenesis (Fig. 1). A possible explanation for the absence of DHB dehydrogenase activity in strain TCD1 might be that the upstream region including catR1 is required for benD transcription. The differential production of CatA1 and CatA2 suggests that the expression of the cat1 and cat2 genes is controlled by the different regulators. It is likely that the expression of the cat1 and cat2 genes is regulated by CatR1 and CatR2, respectively. CatR1 and CatR2 are members of the LysR-type transcriptional regulator family (16, 38). Most LysR family members require an inducer molecule, which presumably interacts
with the protein and causes transcriptional activation. In the well-studied LysR-type transcriptional regulator in the cat gene cluster, CCM is the effector (30). In contrast, a chromosomal fusion reporter assay showed that the CbeR of NK8, a homologue of CatR1 of strain TH2, activated the catA promoter in the presence of BA, 3CB, or 4CB (12). However, 2CB was not the effector of CbeR (12). Similar results were obtained with the production of CatA1 of strain TH2 by using TD2 as a 2-halobenzoate dioxygenase (cbd) disruptant, which is incapable of transforming BA and CBs (Fig. 3). BA, 3CB,
FIG. 4. (A) SDS-PAGE analysis of strain TH2, TCD1, and TCD2 proteins induced during growth on CCM. (B) CatA1 protein detected by Western blot analysis with a polyclonal rabbit antiserum raised against synthetic peptides (see Materials and Methods). Approximate molecular masses of marker proteins (kilodaltons) are indicated on the left. CatA positions are indicated by the arrows.
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FIG. 5. Oxidation products of BA resulting from IPTG-induced expression of the xylXYZ genes on the TOL plasmid in E. coli BL21 (DE3). Data were obtained by HPLC analysis at A230 of BA subjected to conversion by E. coli BL21(DE3)p16TLXYZ grown on M9 medium containing 2 mM BA and 10 mM glucose. Data shown are for samples taken at the indicated times.
and 4CB, but not 2CB, induced the production of CatA1. The lack of production of CatA1 during the growth of strain TH2 on 2CB might be due to the inability of CatR1 to respond to 2CB to initiate transcription. CatA2, in contrast, was induced in TH2 cells during growth on 2CB as well as on CCM or BA (Fig. 2 and 4). When TD2, the cbd disruptant, was grown on BS medium with glucose and 2CB, catechol 1,2-dioxygenase activity was not detected in the cell extracts (data not shown), indicating that 2CB is not the actual inducer of CatA2. Although the possibility that CatA2 is induced by BA itself cannot be excluded, CCM or an intermediate of CCM degradation is probably the actual inducer of cat2 gene expression during growth on BA, as well as on 2CB. Production of CatA1 during growth on 2CB or CCM was observed in strain TCD2, a cat2 disruptant, but not in the wild type (Fig. 4). The presence of functional cat2 genes might have suppressed the expression of the cat1 genes during growth on CCM. One possible explanation is that a high level of intracellular CCM or intermediate of CCM degradation is required for the induction of CatA1. During growth of strain TH2 on 2CB, the substrate is converted to catechol by 2-halobenzoate dioxygenase (CbdABC), which is induced by 2CB (44). The catechol generated is partially converted by the background
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level of CatABC to the intermediates of catechol degradation, which, in turn, activates cat2 gene expression. In this case, the activity of Cat2 enzymes is apparently high enough to keep the intracellular level of the intermediates of catechol degradation under the threshold for stimulating cat1 gene expression. In strain TCD2, catechol generated from 2CB is converted into the intermediates of catechol degradation by the background levels of Cat1 enzymes. When the activity of CatB1 or CatC1 produced in the cell is low relative to that of the upstream enzyme, CCM or muconolactone, respectively, accumulates in the cell. The difference in enzyme activity of CatA1B1C1 might cause the higher intracellular levels of an intermediate of catechol degradation, which, in turn, initiates expression of the cat1 operon. Examination of the accumulation of CCM in culture media showed that the amount of CCM in strain TCD2 culture was about three times that in TH2 culture (data not shown), indicating that CatB1 activity is lower than CatA1 activity. If the concentration in the culture medium reflects the intracellular level, CCM might be the actual inducer of CatA1. The possible significance of the intracellular level of CCM in the catechol degradation has also been reported for Acinetobacter sp. strain ADP1 (4). When CCM was added exogenously, production of CatA1 was observed in strain TCD2 but not in strain TH2 (Fig. 4). In strain TH2, exogenous CCM might be taken into the cell and degraded very rapidly, so that high internal levels of this compound are not achieved. Although CatA2 is not indispensable for the growth of strain TH2 on 2CB, the presence of cat2 genes is beneficial. As shown in Table 4, the doubling time of BA-grown TCD2 cells was nearly equal to that for strain TH2. That is, the cat2 operon is not required to utilize BA efficiently. On the other hand, the doubling time of 2CB-grown cells of strain TCD2 was longer than those of strains TH2 and TCD1. Although CCM acts as an inducer of Cat1 production in strain TCD2, the productivity of the enzyme is very low compared with that induced by BA in strain TH2 (Fig. 3 to 5). The slow growth of strain TCD2 might be explained by the low effectiveness of CCM as an inducer of CatA1. Collectively, these results indicate the importance of catechol metabolism in 2CB degradation.
FIG. 6. Amount of DHB remaining in reaction mixtures of strains TH2, TCD1, and TBD1 provided with BA. DHB was monitored by HPLC analysis. The amount of DHB was determined by measuring the area under the DHB peak.
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Since cat genes highly homologous to the cat2 gene cluster of strain TH2 were found in phylogenetically distant strains, A. lwoffii K24 (19) and Frateuria sp. strain ANA-18 (23), strain TH2 might have acquired the cat2 gene cluster by gene transfer, making it efficient in the degradation of 2CB. ACKNOWLEDGMENTS This work was supported by a grant from the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN). REFERENCES 1. Aoki, K., T. Konohana, R. Shinke, and H. Nishihara. 1984. Two catechol 1,2-dioxygenases from an aniline-assimilating bacterium, Frateuria species ANA18. Agric. Biol. Chem. 48:2097–2104. 2. Blondelet-Rouault, M.-H., J. Weiser, A. Lebrihi, P. Branny, and J.-L. Pernodet. 1997. Antibiotic resistance gene cassettes derived from the ⍀ interposon for use in E. coli and Streptomyces. Gene 190:315–317. 3. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248–254. 4. Cosper, N. J., L. S. Collier, T. J. Clark, R. A. Scott, and E. L. Neidle. 2000. Mutations in catB, the gene encoding muconate cycloisomerase, activate transcription of the distal ben genes and contribute to a complex regulatory circuit in Acinetobacter sp. strain ADP1. J. Bacteriol. 182:7044–7052. 5. Cramm, R., R. A. Siddiqui, and B. Friedrich. 1994. Primary sequence and evidence for a physiological function of the flavohemoprotein of Alcaligenes eutrophus. J. Biol. Chem. 269:7349–7354. 6. Davison, J., M. Heusterspreute, N. Chevalier, V. Ha-Thi, and F. Brunel. 1987. Vectors with restriction site banks. V. pJRD215, a wide-host-range cosmid vector with multiple cloning sites. Gene 51:275–280. 7. Dorn, E. and H.-J. Knackmuss. 1978. Chemical structure and biodegradability of halogenated aromatic compounds. Substituent effects on 1,2-dioxygenation of catechol. Biochem. J. 174:85–94. 8. Eby, D. M., Z. M. Beharry, E. D. Coulter, D. M. Kurtz, Jr., and E. L. Neidle. 2001. Characterization and evolution of anthranilate 1,2-dioxygenase from Acinetobacter sp. strain ADP1. J. Bacteriol. 183:109–118. 9. Fetzner, S., R. Mu ¨ller, and F. Lingens. 1989. Degradation of 2-chlorobenzoate by Pseudomonas cepacia 2CBS. Biol. Chem. Hoppe-Seyler 370:1173–1182. 10. Fetzner, S., R. Mu ¨ller, and F. Lingens. 1992. Purification and some properties of 2-halobenzoate 1,2-dioxygenase, a two-component enzyme system from Pseudomonas cepacia 2CBS. J. Bacteriol. 174:279–290. 11. Focht, D. D. 1996. Biodegradation of chlorobenzoates, p. 71–80. In T. Nakazawa, K. Furukawa, D. Haas, and S. Silver (ed.), Molecular biology of Pseudomonas. American Society for Microbiology, Washington, D.C. 12. Francisco, P. B., Jr., N. Ogawa, K. Suzuki, and K. Miyashita. 2001. The chlorobenzoate dioxygenase genes of Burkholderia sp. NK8 involved in the catabolism of chlorobenzoates. Microbiology 147:121–133. 13. Haak, B., S. Fetzner, and F. Lingens. 1995. Cloning, nucleotide sequence, and expression of the plasmid-encoded genes for the two-component 2-halobenzoate 1,2-dioxygenase from Pseudomonas cepacia 2CBS. J. Bacteriol. 177:667–675. 14. Haddad, S., D. M. Eby and E. L. Neidle. 2001. Cloning and expression of the benzoate dioxygenase genes from Rhodococcus sp. strain 19070. Appl. Environ. Microbiol. 67:2507–2514. 15. Harayama, S., M. Rekik, A. Bairoch, E. L. Neidle, and L. N. Ornston. 1991. Potential DNA slippage structures acquired during evolutionary divergence of Acinetobacter calcoaceticus chromosomal benABC and Pseudomonas putida TOL pWW0 plasmid xylXYZ, genes encoding benzoate dioxygenases. J. Bacteriol. 173:7540–7548. 16. Henikoff, S., J. C. Wallace, and J. P. Brown. 1990. Finding protein similarities with nucleotide sequence databases. Methods Enzymol. 183:111–132. 17. Houghton, J. E., T. M. Brown, A. J. Appel, E. J. Hughes, and L. N. Ornston. 1995. Discontinuities in the evolution of Pseudomonas putida cat genes. J. Bacteriol. 177:401–412. 18. Kaschabek, S. R., T. Kasberg, D. Muller, A. E. Mars, D. B. Janssen, and W. Reineke. 1998. Degradation of chloroaromatics: purification and characterization of a novel type of chlorocatechol 2,3-dioxygenase of Pseudomonas putida GJ31. J. Bacteriol. 180:296–302. 19. Kim, S. I., S.-H. Leem, J.-S. Choi, Y. H. Chung, S. Kim, Y.-M. Park, Y. K. Park, Y. N. Lee, and K.-S. Ha. 1997. Cloning and characterization of two catA genes in Acinetobacter lwoffii K24. J. Bacteriol. 179:5226–5231. 20. Kitagawa, W., K. Miyauchi, E. Masai, and M. Fukuda. 2001. Cloning and characterization of benzoate catabolic genes in the gram-positive polychlorinated biphenyl degrader Rhodococcus sp. strain RHA1. J. Bacteriol. 183: 6598–6606. 21. Liu, S., N. Ogawa, and K. Miyashita. 2001. The chlorocatechol degradative genes, tfdT-CDEF, of Burkholderia sp. strain NK8 are involved in chlorobenzoate degradation and induced by chlorobenzoates and chlorocatechols. Gene 268:207–214.
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