cloning, expression, and nucleotide sequence of the ... · the ohb dna region, and the...

APPLIED AND ENVIRONMENTAL MICROBIOLOGY,0099-2240/99/$04.0010

May, 1999, p. 2151–2162 Vol. 65, No. 5

Copyright © 1999, American Society for Microbiology. All Rights Reserved.

Cloning, Expression, and Nucleotide Sequence of thePseudomonas aeruginosa 142 ohb Genes Coding

for Oxygenolytic ortho Dehalogenationof Halobenzoates

TAMARA V. TSOI,1,2* ELENA G. PLOTNIKOVA,1† JAMES R. COLE,1,2

WILLIAM F. GUERIN,2 MICHAEL BAGDASARIAN,1,3 AND JAMES M. TIEDJE1,2,3

Center for Microbial Ecology,1 Department of Crop and Soil Sciences,2 and Department of Microbiology,3

Michigan State University, East Lansing, Michigan 48824

Received 25 September 1998/Accepted 18 February 1999

We have cloned and characterized novel oxygenolytic ortho-dehalogenation (ohb) genes from 2-chloroben-zoate (2-CBA)- and 2,4-dichlorobenzoate (2,4-dCBA)-degrading Pseudomonas aeruginosa 142. Among 3,700Escherichia coli recombinants, two clones, DH5aF*(pOD22) and DH5aF*(pOD33), converted 2-CBA to cate-chol and 2,4-dCBA and 2,5-dCBA to 4-chlorocatechol. A subclone of pOD33, plasmid pE43, containing the3,687-bp minimized ohb DNA region conferred to P. putida PB2440 the ability to grow on 2-CBA as a solecarbon source. Strain PB2440(pE43) also oxidized but did not grow on 2,4-dCBA, 2,5-dCBA, or 2,6-dCBA.Terminal oxidoreductase ISPOHB structural genes ohbA and ohbB, which encode polypeptides with molecularmasses of 20,253 Da (b-ISP) and 48,243 Da (a-ISP), respectively, were identified; these proteins are in accordwith the 22- and 48-kDa (as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) polypep-tides synthesized in E. coli and P. aeruginosa parental strain 142. The ortho-halobenzoate 1,2-dioxygenaseactivity was manifested in the absence of ferredoxin and reductase genes, suggesting that the ISPOHB utilizedelectron transfer components provided by the heterologous hosts. ISPOHB formed a new phylogenetic clusterthat includes aromatic oxygenases featuring atypical structural-functional organization and is distant from theother members of the family of primary aromatic oxygenases. A putative IclR-type regulatory gene (ohbR) waslocated upstream of the ohbAB genes. An open reading frame (ohbC) of unknown function that overlapslengthwise with ohbB but is transcribed in the opposite direction was found. The ohbC gene codes for a48,969-Da polypeptide, in accord with the 49-kDa protein detected in E. coli. The ohb genes are flanked by anIS1396-like sequence containing a putative gene for a 39,715-Da transposase A (tnpA) at positions 4731 to 5747and a putative gene for a 45,247-Da DNA topoisomerase I/III (top) at positions 346 to 1563. The ohb DNA regionis bordered by 14-bp imperfect inverted repeats at positions 56 to 69 and 5984 to 5997.

Chlorobenzoates (CBAs) constitute a favorable model forstudying the molecular mechanisms of degradation of haloge-nated aromatic compounds. The most-extensively studiedhalobenzoate degraders are those bacteria that possess a mod-ified chlorocatechol ortho-cleavage pathway. In these cases,halobenzoate is oxidized to the corresponding chlorocatechol,which is funneled into a modified ortho-cleavage route in whicha fortuitous removal of halogen occurs. The genes for thispathway have been isolated from a number of strains and usedto construct recombinant pathways for degradation of differenthalogenated aromatic xenobiotics (9, 41, 58, 59). The typicalproblem in the construction of a polychlorinated biphenyl-degrading microorganism by combining the modified ortho-cleavage and biphenyl oxidation pathways is the incompatibil-ity of the meta and ortho pathways. The simultaneousfunctioning of these pathways usually creates suicide products(57); e.g., the meta fission of 3-chlorocatechol produces anacylchloride, which irreversibly inactivates (phenyl)catechol

2,3-dioxygenases such as 2,3-dihydroxybiphenyl 2,3-dioxygen-ase (4, 9). Hence, alternative strategies, such as the use of CBAdehalogenases, which remove chlorine prior to the oxidation ofthe aromatic ring, would appear to be useful for avoiding thisincompatibility.

Oxygenolytic dehalogenation of 2-CBA was implicated in anumber of Pseudomonas strains (23, 24, 28, 61, 72, 82). Dihy-droxylation is frequently used by microbes as an initial step inthe aerobic attack of aromatic compounds. The multicompo-nent nonheme iron dioxygenase systems catalyzing the dihy-droxylation typically consist of two or three proteins that com-prise a short electron transfer chain, mobilizing electrons fromNADH, via flavin and 2Fe-2S redox centers, to the site ofdioxygen activation (12, 47). The three-component system typ-ically consists of an NADH: acceptor reductase componentcontaining flavin adenine dinucleotide, a chloroplast-type2Fe-2S ferredoxin, and a Rieske-type 2Fe-2S iron-sulfur pro-tein (ISP) that is the terminal oxygenase component. In two-component systems, reductase and ferredoxin components arecombined in the same protein. The two-component CBA 1,2-dioxygenase from 2-CBA-grown Burkholderia sp. strain 2CBS(29) is similar to the two-component plasmid-borne toluate1,2-dioxygenase from Pseudomonas putida mt-2 (41) and thetwo-component benzoate 1,2-dioxygenases from P. putida C-1(78) and Pseudomonas sp. strain B13 (35). This enzyme cata-lyzes the double hydroxylation of 2-halobenzoate with concom-

* Corresponding author. Mailing address: A540 Center for Micro-bial Ecology, Plant and Soil Sciences Building, Michigan State Uni-versity, East Lansing, MI 48824-1325. Phone: (517) 432-1536. Fax:(517) 353-2917. E-mail: [email protected].

† Present address: Institute of Ecology and Genetics of Microorgan-isms, Russian Academy of Sciences, Ural Branch, Perm 614081, Rus-sia.

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itant release of halide, carbon dioxide, and the nonchlorinatedcatechol (29). The plasmid-borne genes cbdABC encoding thistwo-component enzyme complex were isolated and sequenced(33). The cbdABC sequences showed similarity to the Acineto-bacter calcoaceticus benABC genes, encoding benzoate 1,2-di-oxygenase (52% identity of the deduced amino acid sequenc-es), and to the P. putida mt-2 xylXYZ genes, encoding toluate1,2-dioxygenase (51% identity).

Pseudomonas aeruginosa 142, used in the present study, dif-fers from strain 2CBS in its ability to grow on both 2-CBA and2,4-dichlorobenzoate (2,4-dCBA), its ability to oxidate and de-halogenate all ortho-halogenated dCBAs and triCBAs (61, 62),and its possession of a three-component dioxygenase (62)rather than a two-component enzyme (29). The ortho-CBA1,2-dioxygenase activity requires molecular oxygen, NADH,and Fe(II) and results in conversion of 2-CBA to catechol anddCBAs to their respective chlorocatechols (Fig. 1). No dehy-drogenase activity was required for conversion of CBA to cat-echol, in accord with spontaneous resolution of the 2-halo-3,5-cyclohexadiene-1,2-diol-1-carboxylic acid intermediate tocatechol (29, 32, 33). The oxygenation (dehalogenation) reac-tion is followed by a separate catechol ortho-cleavage pathway(modified chlorocatechol ortho-cleavage pathway for dCBAsand tri-CBAs). The ortho-halobenzoate activity was resolvedinto three protein fractions, and a 13-kDa protein resemblinga Rieske-type 2Fe-2S ferredoxin was purified and character-ized (62).

Our major objective was to clone and characterize the genescontrolling dehalogenation of ortho-halobenzoates in P. aerugi-nosa 142. In this paper, we report on the isolation and expres-sion of the novel ohb genes in Escherichia coli, the constructionof a functioning recombinant pathway for growth on 2-CBA inP. putida, nucleotide sequence determination and analysis ofthe ohb DNA region, and the identification and phylogeneticplacement of the structural ohb genes.

MATERIALS AND METHODS

Bacterial strains and plasmids. P. aeruginosa 142 was provided by I. I. Staro-voitov (IBPhM, Russian Academy of Science, Puschino, Russia). This strain wasisolated from polychlorinated biphenyl-contaminated soil in Moscow Region,Russia, and readily grew on both 2-CBA and 2,4-dCBA (61). E. coli laboratorystrains used in this work were DH5aF9 (Bethesda Research Laboratories, Be-thesda, Md.), JM109 (79), and minicell-producing strain x925 (F1 minA minB thrleu thi) (69). Pseudomonas strains utilized were P. putida mt-2 (PB2440 r2m1)(3), P. putida KZ6R (Rifr) (83), and Pseudomonas sp. strain B13 (18). Plasmid

vector pSP329 (Tcr), a derivative of low-copy-number, broad-host-range (bhr)plasmid RK2 (IncP), contains an HaeII fragment from pUC18 carrying multiplecloning sites and the lacZ a-complementation gene block cloned into the plasmidpTJS75 (63). The plasmid pSP329 was a gift from Vladimir Ksenzenko (IBPhM).Other plasmids employed were E. coli vectors pUC19 (79) and BlueScript (Strat-agene, La Jolla, Calif.). Plasmid pRK2013, a Kmr Tra1 ColE1 derivative of RK2(17), was used as a helper in triparental matings.

Media and growth conditions. E. coli strains were maintained at 37°C onenriched Luria broth or Luria agar (48), minimal medium M9 (45), or twice-diluted C12-free medium K1 (83). Pseudomonas strains were routinely grown at30°C in medium K1. Growth substrates were added at the following concentra-tions: Glucose, 0.2% (wt/vol); benzoate and 4-hydroxybenzoate, 3.0 mM; CBAs,0.5 to 3 mM; catechol, 2 mM; and sodium acetate, 10 mM. Antibiotics wereadded as needed as follows (with concentrations in micrograms per milliliter):ampicillin, 30 to 300; tetracyclin, 15; kanamycin, 30; and rifampin, 50 to 200.Isopropyl-b-D-thiogalactoside (IPTG) and 5-bromo-4-chloro-3-indolyl-b-galac-topyranoside (X-Gal) were added when necessary to a final concentration of0.004% (wt/vol).

Isotopes, enzymes, and chemicals. L-[35S]methionine was purchased from Am-ersham Life Science, Inc. (Arlington Heights, Ill.), and Na2

35SO4 was obtainedfrom ICN Biochemicals, Inc. (Costa Mesa, Calif.). Enzymes and reagents werefrom New England Biolabs, Inc. (Beverly, Mass.), Boehringer GmbH (Mann-heim, Germany), Gibco BRL (Gaithersburg, Md.), Sigma Chemical Co. (St.Louis, Mo.), and Merck (Darmstadt, Germany).

Detection of dehalogenation activity. C12 was detected as described elsewhere(5, 74). I2 was measured by a modification of a previously described method (7).Modifications included the use of medium M9 or twice-diluted K1 for bacterialgrowth, 2.5 M citric acid buffer (obtained by mixing 66 ml of 2.5 M citric acid, 35ml of 2 M NH4OH, and 8 g of NH4H2PO4 for a final pH of 4), and 0.3 to 0.45%(wt/vol) Oxone solution in H2O. The assay allowed detection of iodide at con-centrations as low as 5 mM in K1 medium and 25 mM in M9 medium.

Conjugation. Plasmids pOD22 and pOD33 were transferred into Pseudomonasstrains KZ6R and B13 by triparental matings (17). Following a 6- to 8-h incu-bation at 30°C, the conjugational mix was washed off the filter (BA85; 0.45 mmpore size, Schleicher & Schuell, Dassel, Germany) (21) and transconjugants wereselected by plating on K1 agar containing benzoate and tetracycline (B13) orLuria agar containing tetracycline and rifampin (KZ6R). Fifty to 100 randomlychosen colonies were replica plated onto K1 agar containing the desired sub-strate.

Electrotransformations. DNA transformation of E. coli and P. putida PB2440cells was conducted by electroporation with an E. coli Gene Pulser (Bio-RadLaboratories, Hercules, Calif.). Competent cells were prepared from early-log-phase cultures, grown in Luria broth, according to the protocol of Dower et al.(19), which is based on washing the cells three times with cold deionized H2O(equal volume) and then concentrating them 200- to 400-fold in a 15% (wt/vol)glycerol solution. Cells were stored at 270°C in 50-ml aliquots.

For plasmid transformation, 1 to 10 ng of supercoiled DNA in deionized H2Owas used. Ligated DNA was precipitated and redissolved in deionized H2O.From 10 to 50 ng of ligated DNA was used for E. coli cells, and 50 to 500 ng wasused for transformation of Pseudomonas strains. Transformants were selected onLuria agar containing the appropriate antibiotic.

DNA isolation and manipulation. Chromosomal DNA was isolated by themethod of Marmur (46). Routine screening of strains for the presence of plasmidDNA, preparation of low-copy-number plasmid DNA, digestion of DNA withrestriction endonucleases and exonuclease Bal31, treatment with alkaline phos-

FIG. 1. Molecular mechanism and involvement of the ohbAB genes in oxygenolytic ortho dehalogenation of halobenzoates. The three-component 1,2-dioxygenaseis responsible for oxygenation followed by fortuitous ortho dehalogenation of halobenzoates in P. aeruginosa 142 (62). The ohbAB genes isolated in the present workencode two subunits of the terminal oxygenase ISPOHB of the ortho-halobenzoate 1,2-dioxygenase and presumably require reductase and ferredoxin components of aheterologous host. The hypothetical dihydrodiol intermediate 2-chloro-(chloro)cyclohexadiene-1,2-diol-1-carboxylic acid is presumed to spontaneously lose carbondioxide and halogenide (29, 62). FAD, flavin adenine dinucleotide; FMN, flavin mononucleotide.

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phatase and the Klenow fragment of DNA polymerase, DNA ligation, and DNAelectrophoresis in agarose gels were performed according to standard proce-dures (45). Recovery of DNA from agarose gels was performed according to themethod of Dretzen et al. (20) or by using a DNA Cleanup kit (Promega,Madison, Wis.). Plasmid DNA for sequencing was purified by using a PromegaWizard 373 kit.

Construction of the gene library. Chromosomal DNA from strain 142 waspartially digested with restriction endonuclease Sau3A. After separation in a0.8% agarose gel, the fraction of fragments in the size range of 5 to 20 kb wasrecovered. This DNA was ligated to pSP329 DNA which had been linearized anddephosphorylated at its BamHI site. Following transformation of DH5aF9 cells,Tcr white colonies were selected on Luria agar containing tetracycline, X-Gal,and IPTG. The recombinant colonies were replica plated onto M9 mediumcontaining glucose as the growth substrate and in the presence of 2-CBA and2,4-dCBA. After 2 days of incubation, colonies were analyzed for production ofcatechol by the p-toluidine test of Parke (54).

High-performance liquid chromatography analysis. Benzoates and their prod-ucts were analyzed by isocratic reverse-phase chromatography on a 250- by 4-mmC18 column (Hibar RT E; Merck). The eluent, a mixture of 0.1% H3PO4 andacetonitrile (at ratio of either 80/20 or 66/33, depending on the expected prod-ucts), was applied at a flow rate of 1.5 ml/min. Compounds were detected bymeasuring UV absorbance at 230 nm. Products were identified by comparison ofretention times with those of authentic standards.

Minicell assay. In the minicell assay, single colonies of fresh transformants ofstrain x925 were assayed as previously described (69, 74).

Induction study. Induction of degradative enzymes in strain 142 was assessedin labeling experiments using Na2

35SO4. Cells were serially transferred threetimes in twofold-diluted nutrient broth (Difco, Detroit, Mich.), harvested in earlystationary phase, and washed in phosphate-buffered saline (PBS) (8.5 g of NaCl,0.6 g of Na2HPO4 and 0.3 g of KH2PO4 per liter of H2O; pH 7.0). Washed cellswere resuspended in PBS, and after incubation at room temperature for 6 h(starvation), the cell suspension was amended with the substrate/inducer (20mM) and isotope (0.4 mCi). Control cells were also suspended in PBS, but noinducer or isotope was added. After 68 h of incubation at room temperature, thecells were harvested, washed in PBS, and resuspended in deionized H2O. Lysedsamples containing 15 mg of total protein (44) were then separated by sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (40). After being stained, thegels were exposed to Kodak X-OMAT AR film (Eastman Kodak, Rochester,N.Y.) for 5 days.

DNA sequencing and sequence analysis. Determination of the nucleotidesequence of the entire DNA insert from plasmid pOD33 was done by usingdeletional variants and internal primers synthesized at the Michigan State Uni-versity Macromolecular Synthesis Facility. Each DNA strand was completelysequenced at least four times. Automated fluorescent sequencing was done at theMichigan State University DNA Sequencing Facility. Primers were designed byusing the LASERGENE software package (DNASTAR Inc., Madison, Wis.).Primary sequence editing was performed with Sequencer V.3, (Gene Code Cor-poration, Madison, Wis.) and DNASTAR. The BLAST program (1), withBEAUTY postprocessing (77), was used for similarity searches of the nonredun-dant NCBI sequence database (National Center for Biotechnology Information,National Institutes of Health, Bethesda, Md.). An initial multiple alignment wasdesigned by using the CLUSTAL W Program, version 1.7 (73), offered by BCMSearch Launcher (Human Genome Center, Baylor College of Medicine, Hous-ton, Tex.). This was used as a starting alignment to create a hidden Markovmodel-based alignment with the SAM-T98 program (38). Unrooted Fitch-Mar-golash dendrograms were derived from the alignment by using the SEQBOOT,PROTDIST, FITCH and CONSENSE programs of the PHYLIP package (27).

Nucleotide sequence accession number. The nucleotide sequence of the ohbDNA region has been deposited with GenBank (accession no. AF121970).

RESULTS

Isolation and expression of the ohb genes in E. coli cells.Among 3,700 recombinant clones of the library of total DNAfrom P. aeruginosa 142, two positive colonies were identified bythe p-toluidine method. Plasmids from these clones, desig-nated as pOD22 and pOD33, contained overlapping DNAinserts of about 8 and 6 kb in size, respectively (Fig. 2). StrainpOD22 was characterized by 90% segregational instability,whereas plasmid pOD33 was stable and therefore was theplasmid primarily used in further studies. StrainsDH5aF9(pOD22) and DH5aF9(pOD33) stoichiometricallyconverted 2-CBA into catechol and 2,4-dCBA and 2,5-dCBAinto 4-chlorocatechol, when grown in M9 medium in the pres-ence of different CBAs (Table 1), and released iodide into themedium when grown in the presence of 2-iodobenzoate (2-IBA). Release of chloride into K1 medium also was detected.We concluded that these recombinant plasmids contained the

ortho-halobenzoate 1,2-dioxygenase genes (ohb) of strain 142and that these genes were expressed in E. coli.

Physical mapping of the ohb DNA region. Attempts to re-clone the DNA inserts from plasmids pOD22 and pOD33 intomulticopy E. coli vector plasmids pUC19, pGEM, and Blue-Script failed due to the instability of the resulting constructs inboth the DH5aF9 and JM109 strains. Further cloning experi-ments to locate and isolate the functional ohb region weredone with vector pSP329. Some nonfunctional deletion sub-clones were produced with vectors BlueScript SK(1) andKS(1). The overlapping region of the DNA inserts in plasmidspOD22 and pOD33 (Fig. 2, positions 1.1 to 6 kb on the map ofpOD33) was approximately 4.9 kb in size and presumably con-tained all of the information necessary to control halobenzoateoxidation in E. coli cells. This was confirmed by subcloningrestriction fragments of the DNA insert from plasmid pOD33.The 5.3-kb EcoRI-HindIII and 4.75-kb KpnI-HindIII frag-ments conferred on the host the ability to oxidize 2-CBA, asdetermined with p-toluidine (Fig. 2, plasmids p33E10 andp33K21, respectively), whereas the 3.35-kb SalI fragment (Fig.2, plasmid p33G1) proved to be nonfunctional. ExonucleaseBal31-derived deletions allowed the determination of the lo-cation of the functionally active ohb gene region within the3.7-kb DNA fragment (Fig. 2, plasmid pE43).

Expression of the ohb genes in Pseudomonas cells. The re-combinant plasmids pOD22 and pOD33 were introduced intoPseudomonas sp. strains PB2440, KZ6R, and B13 by eitherconjugation or transformation. Each of these strains is capableof degrading catechol via the ortho-cleavage pathway. StrainB13 additionally harbors the modified ortho pathway for oxi-dation of chlorocatechol (18). Recombinants were plated ontosolidified K1 medium containing 2-CBA or 2,4-dCBA as thesole carbon source and incubated for up to 12 weeks with nospecific growth being observed. However, cells ofPB2440(pOD33) and KZ6R(pOD33) released small amountsof iodide (10 to 20 mM) when grown in the presence of 2-IBA(200 mM) in K1 mineral medium containing glucose or acetate;this suggested that the expression of the ohb genes was insuf-ficient to allow growth on the halobenzoate.

Plasmid pE43 showed improved expression of the ohb genesin E. coli. While no C12 was found in overnight cultures ofDH5aF9(pOD33) in the presence of 2-CBA (0.5 mM), 20 mMC12 was measured in DH5aF9(pE43), with the concentrationreaching 95 mM by 24 h of incubation, compared to 70 mM forpOD33. PB2440(pE43) transformants selected on Luria agarcontaining tetracycline were replica plated on K1-tetracyclineagar containing 2-CBA (2.5 mM) as the carbon source. After afew weeks of incubation, all replicants formed colonies thatreproducibly grew on 2-CBA in 1 to 2 days in subsequenttransfers. Repeated transformation showed that a shorter ini-tial incubation period was needed, along with less 2-CBA (1.25mM). The clones contained plasmid DNA of the same struc-ture as that isolated from E. coli.

Batches of PB2440(pE43) inoculated from stationary-phase2-CBA cultures grew on 2-CBA at concentrations of up to 2mM (Fig. 3). Notably, growth on 1 and 1.5 mM concentrationsof the substrate was completed in 30 and 48 h, respectively.However, an extended lag period was characteristic for growthon 2 mM 2-CBA. Doubling times in the growth phase with allthree concentrations of the substrate were comparable, sug-gesting that higher concentrations of 2-CBA may be toxic tocells in the initial phase of growth.

No growth was detected on dCBAs. The p-toluidine test,however, indicated the production of catechols from dCBAs, inagreement with the inability of PB2440 to grow on chlorocat-echol. A resting-cell assay (Fig. 4) showed that complete con-

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sumption of 2-CBA was observed in about 1.5 h, with nodetectable catechol product, in agreement with this strain’sability to grow on 2-CBA. The initial rates of oxidation of2,4-dCBA, 2,5-dCBA, and 2,6-dCBA were similar to that ob-served for 2-CBA; however, the transformation did not con-tinue to completion, probably due to toxicity of the oxidationproduct(s). Unlike in E. coli, chlorocatechol was not detect-able, suggesting nonproductive transformation of chlorocat-echol by the recipient cells.

The ohb-encoded proteins. As shown in Fig. 5, minicellscontaining plasmid pOD33 (lane 2), p33K21 (lane 3), pOD22(lane 4), or pE43 (lane 5) synthesized three new polypeptideswith apparent molecular masses of 48, 47, and 22 kDa. Nospecific bands in addition to those featured for the vectorpSP329 (lane 1) were found for the deletion variants (data notshown) except for pE33-1 (lane 6). In the last case, both bands(48 and 47 kDa) were replaced by a band of 24 kDa, possiblya remnant of one of the former polypeptides. These polypep-tides could be products of overlapping genes, or they couldhave resulted from partial proteolysis. Sequencing results (seebelow) confirmed the former suggestion.

The synthesis of cellular proteins in strain 142 was assessedupon amendment of cells in phosphate-buffered saline withbenzoate, 2-CBA, 2,4-dCBA and 2,5-dCBA, catechol, or4-chlorocatechol. Controls were cells provided with succinatebut no substrate. No protein synthesis occurred in the absenceof substrate/inducer (Fig. 6, lane 1) or with 2,5-dCBA (lane 3),the latter in accordance with the inability of the strain to growon 2,5-dCBA as a sole source of carbon and indicating that2,5-dCBA was not being recognized as an inducer. Comparisonof the polypeptide patterns produced upon addition of 2-CBA(lane 4) or 2,4-dCBA (lane 2) with those characteristic forother substrates/inducers (lanes 5 to 8) showed that only twoadditional bands, with apparent molecular masses of 48 and 22kDa, could be attributed to chlorobenzoate induction. Thesedata were in accord with the results of the E. coli assay and thenucleotide sequence determination and analysis.

Determination and analysis of the nucleotide sequence ofthe ohb DNA region. The nucleotide sequence of the 6,052-bpDNA insert of plasmid pOD33 contains six open readingframes (ORFs) in both orientations (Fig. 2 and 7). The ORFdesignated ohbA has two possible translation start sites, at

FIG. 2. Physical map and organization of the ohb DNA region. Physical maps of the 6.052-kb DNA insert of plasmid pOD33 and its deletion variants are presented.The overlapping region of the DNA insert of plasmid pOD22 is shown at the top. Plasmids pK33A16, pK33H7, and pK33A20 were constructed with the BlueScriptvector; for the other variants, vector pSP329 was used. Phenotypes are indicated to the right of the plasmid names. The DNA insert of plasmid pOD33 has beensequenced by using the deletion variants and internal primers. The locations and orientations of ORF1 (top), ORF2 (ohbR), ORF3 (ohbA), ORF4 (ohbB), ORF5(ohbC), and ORF6 (tnpA) are shown in the lower part of the figure, along with the calculated molecular masses of the corresponding polypeptides. Restriction sitesare shown, with their locations indicated in parentheses.



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positions 2636 and 2783. However, the first ATG codon is notpreceded by a putative Shine-Dalgarno site, whereas an ex-tended 59-AAGAGGAGGGAGAG-39 sequence is located up-stream of the ATG codon, at position 2783. The deducedmolecular masses for the ohbA, ohbB, and ohbC gene productswere in accord with those of polypeptides synthesized in E. coli(Fig. 5) and in parental strain 142 (Fig. 6). The ohbB and ohbCgenes are located within nearly the same DNA locus but aretranscribed in opposite directions, in agreement with data forsubclone pE33-1 (Fig. 5).

The deduced sequence of OhbB showed similarity to largesubunits of terminal oxygenases (ISPs) from multicomponentprimary dioxygenases. It features the conservative clusterCys64-His66-Cys84-His87, in agreement with the consensus se-quence Cys-X-His-X15-17-Cys-X2-His for Rieske-type iron-sul-fur clusters (12, 47), and conserved residues of asparagine(Asn171), aspartate (Asp182), and glutamate (Glu177) in addi-tion to two histidine residues (His185 and His190) that aresupposedly involved in ligation of nonheme iron centers ofdioxygenases (12) (Fig. 7). OhbB had recognizable overall lev-els of identity of 46, 39, 37, 36, and 34% (based on multiplealignment), respectively, to the putative BphA1c and BphA1dproteins from catabolic plasmid pNL1 of Sphingomonas aro-maticivorans F199 (GenBank accession no. AF079317), NagGof the salicylate-5-hydroxylase from Pseudomonas sp. strain U2(31), the putative OhbB protein from P. aeruginosa JB2 (Gen-Bank accession no. AF087482), and the putative a-ISP ORF2protein from Burkholderia sp. strain DNT (70). A large numberof a-ISPs from aromatic oxygenases had recognizable, albeitweak, similarity, attributed mostly to a conservative N-terminaldomain. These findings, along with functional characterizationof recombinants, identify OhbB as the a-subunit of the ISPOHBfrom the ortho-halobenzoate 1,2-dioxygenase. Multiple-align-ment analysis included the 10 best-matching (BLAST search)sequences plus arbitrarily chosen AntB from Acinetobacter sp.strain ADP1 (11) and CbdC from Burkholderia sp. strain 2CBS(33). This analysis showed that OhbB and the five best-match-

FIG. 3. Growth of the recombinant P. putida PB2440(pE43) on 2-CBA. Batch cultures were grown aerobically in mineral medium K1 with 2-CBA as the sole sourceof carbon at concentrations of 1, 1.5, and 2 mM. Aliquots were taken at certain time points to measure optical densities (OD) at 600 nm (solid lines) and 2-CBAconcentrations (broken lines).

TABLE 1. Conversion of CBAs by recombinant E. coliDH5aF9(pOD33) and DH5aF9(pOD22)

Strainand substrate

Time,h

Concn ofremaining

substrate, mM

Product concn, mM

4-Chlorocatechol Catechol

DH5aF9(pOD33)2-CBA 0 98 0 0

20 93 0 1230 64 0 4645 52 0 52

2-CBA plusIPTGa

0 101 0 0

20 89 0 12.530 65 0 4245 57 0 47.5

2,4-dCBA 0 98 0 020 86 8.5 030 68 34 045 58 38 0

2,5-dCBA 0 88 0 020 75 10.4 030 54 36.5 0

Benzoate 0 85 0 020 82 0 030 71 0 045 63 0 0

DH5aF9(pOD22)2-CBA 0 144 0 0

45 94 0 432,4-dCBA 0 94 0 0

45 77 22 02,5-dCBA 0 164 0 0

45 132 22 0

a IPTG was used to determine whether expression of the ohb genes is en-hanced by Plac; however, the levels of expression did not differ from those of cellsgrown in the absence of IPTG.



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ing sequences appear to form a cluster to the exclusion of theother sequences analyzed (Fig. 8A).

OhbA showed recognizable lengthwise similarity to 14 se-quences of small, b-ISP subunits. Similar to OhbB, the fivesequences best matching OhbA were, in order of descendingdegree of identity, BphA2c and BphA2d from strain F199,OhbC from strain JB2, NagH from strain U2, and ORFX fromstrain DNT, with the level of identity ranging from 45 to 27%.As shown in Fig. 8B, the OhbA clustered with these b-ISPsubunits, in good agreement with the phylogenetic placementof OhbB. Presumably, OhbA is the b-ISP of the ortho-halo-benzoate 1,2-dioxygenase. (Omitted from the trees are NahG[formerly NahAc2] and NahH [formerly NahAd2] from Co-mamonas testosteroni GZ42, whose sequences are not availablefrom databases but were reported to be nearly identical tothose of NagG and NagH from strain U2 [31, 84], ORFX fromstrain DNT [70] [which is nearly identical to NagH, with onlytwo amino acid replacements], and the truncated version ofORF2 that was found in the same position in the 2-nitrotolu-ene operon in Pseudomonas sp. strain JS2 [53].)

Putative gene ohbR, upstream of ohbA, was identified as amember of the IclR family of transcriptional regulators. OhbRshowed overall levels of identity of 31.6, 30.6, and 26%, re-spectively, to GylR, the glycerol operon repressor from Strep-tomyces coelicolor (68); KdgR, the pectin degradation repres-sor from E. coli (37, 50); and IclR, the repressor of aceBAK, theglyoxylate bypass operon from E. coli (71). The OhbR se-quence features an N-terminal conservative a-helix–turn–a-helix 20-amino-acid stretch (Fig. 7) that strongly resembles thepreviously identified DNA-binding domain for the IclR family

proteins and a larger group of DNA-binding proteins (49, 50,60, 68). Although the dehalogenation activity in E. coli wasunregulated, 2-CBA and 2,4-dCBA induced the synthesis of48- and 22-kDa polypeptides in strain 142 cells. Whether ohbRis responsible for this induction remains unknown. OhbR alsoshowed overall levels of identity of 30, 27, 24, and 22%, re-spectively, to the recently released sequences of the proteinencoded by putative gene ORF007 from catabolic plasmidpNL1 in strain F199 (GenBank accession no. AF079317),SC5A7 from Streptomyces coelicolor A3(2) (EMBL accessionno. AL031107), and CatR (EMBL accession no. X99622) andPcaR (26) from Rhodococcus opacus 1CP.

OhbC features an ATP-binding cassette (ABC) transporterfamily signature pattern (accession no. PS00211), i.e.,Val143SerGlnGlyGluLeuArgValIsoGlyValLeuSerLeuAla157(Fig. 7). BLAST search results found no matches with greaterthan 15% identity. However, the superfamily of ABC trans-porter proteins, involved in transfer of solutes across the cellmembrane, is known to be highly diverse (64).

A product of the putative top gene upstream of the ohbgenes was similar to a number of DNA topoisomerases III andI that are involved in the resolution of DNA replication inter-mediates during either vegetative replication or conjugativeDNA transfer and are frequently found on transmissible plas-mids (6, 42). The four best-matching sequences (in order ofdecreasing degree of identity) were human TopIII (34), TopIfrom Methanococcus jannaschii (10), TopI from Mycoplasmagenitalium (30), and TraE from plasmid RP4 (GenBank acces-sion no. L10329), with overall levels of identity ranging from 33to 25%.

The sequence at positions 4668 to 6052, starting 27 bp down-stream of the ohbB termination codon, is 99% identical to thesequence of insertion element IS1396 from Serratia marcescens

FIG. 4. Oxidation of 2-CBA, 2,4-dCBA, 2,5-dCBA, and 2,6-dCBA by restingcells of the recombinant strain P. putida PB2440(pE43). 2-CBA grown cells wereharvested and then resuspended in fresh K1 medium, which was amended withdifferent CBAs. The reaction mixtures were incubated at room temperature withaeration. Triplicate samples were taken at certain time points to measure theconcentrations of CBA and (chloro)catechol.

FIG. 5. Synthesis of plasmid-encoded proteins in E. coli minicells. Shown isan autoradiogram of [35S]methionine-labeled proteins synthesized in strain x925minicells containing plasmids. Lanes: 1, pSP329; 2, pOD33; 3, p33K21; 4,pOD22; 5, pE43; 6, pE33-1. Samples were prepared according to the method ofPlatt (56), and 20-ml aliquots of each were loaded onto SDS–12.5% polyacryl-amide gels. Molecular mass standards (SDS-7; Sigma) were as follows: bovineserum albumin (66.2 kDa), hen egg white ovalbumin (45 kDa), glyceraldehyde-3-phosphate dehydrogenase (36 kDa), carbonic anhydrase (29 kDa), trypsinogen(24 kDa), trypsin inhibitor (20.1 kDa), and a-lactalbumin (14.2 kDa).



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R plasmid R471a (39), except that sequence corresponding tothe left end of IS1396 is missing (Fig. 7). (Both plasmidsselected from the original library [pOD33 and pOD22] havethe same physical structure in this region [Fig. 2], indicatingthat it is unlikely that the missing stretch resulted from a DNArearrangement during cloning.) The IS-associated putativetransposases (tnpA genes) from strain 142 and S. marcescenswere nearly identical (96%) and also exhibited 46% identity toTnpA from P. putida ML2 (GenBank accession no. U25434).Lesser degrees of similarity were found to a number of bacte-rial transposases from a family of sparsely dispersed IS ele-ments (8, 39). The 35-bp stretch at positions 5963 to 5997 ofthe ohb DNA region (Fig. 7) is identical to the 35-bp imperfectinverted repeat (IR) bordering IS1396 on the right (39). Aleft-end IR matching that of IS1396 is missing from the ohbDNA; however, the 14-bp stretch CCTTCATCCGTCGC atpositions 56 to 69 (Fig. 7) forms an imperfect IR with a 14-bpstretch, GCGGCAGGTGAAGG, bordering the IS1396-likesequence at positions 5984 to 5997.

DISCUSSION

Cloning and characterization of the ohb genes confirmed anoxygenolytic mechanism of ortho dehalogenation of chloroben-zoates in strain 142 that was implied previously (62, 65). How-ever, our study showed that the ortho-halobenzoate 1,2-dioxy-genase activity in recombinant E. coli and Pseudomonas strainsis manifested by the ohbAB-encoded terminal oxygenaseISPOHB alone, implicating utilization of electron transfer com-ponents provided by the recipient cell. Given the prolonged

incubation required for initial growth of the PB2440(pE43)transformants on 2-CBA, it is possible that an unspecified hostmutation, perhaps leading to constitutive or higher-level produc-tion of electron transfer components, complements ISPOHB.The apparent inability of CBAs to induce synthesis of electrontransfer components in strain 142 indirectly supports this in-ference. On the other hand, 2-CBA toxicity also appears toaffect growth and rates of degradation in both recombinantPB2440(pE43) (present study) and parental strain 142 (61).

The optimum concentration of 2-CBA for growth wasaround 2.5 to 3.0 mM for parental strain 142 (61), compared to1.5 mM for recombinant PB2440(pE43); this could arguably bedue to cognate ferredoxin or reductase components in theparent. However, in later experiments we have expressed theohb genes in biphenyl-degrading C. testosteroni VP44, and theresulting recombinant, VP44(pE43), readily mineralized 10mM each 2CBA and 2-chlorobiphenyl (36).

The interchangeability of electron transfer components be-tween evolutionarily related primary dioxygenases was de-scribed previously (12, 31, 81). Utilization of a heterologoushost’s ferredoxin oxidoreductase by aromatic oxygenases wasalso reported earlier (67, 80, 81). The expression of Pseudo-monas mendocina KR1 toluene-4-monooxygenase activity in E.coli and other Pseudomonas strains did not require the reduc-tase component, TmoF, although the latter enhanced this ac-tivity by at least twofold (81). Similarly, Pseudomonas sp. strainU2 naphthalene dioxygenase ISPNAG genes nagAc and nagAdalong with ferredoxin gene nagAb allowed the oxidation ofindole to indigo in E. coli, while reductase gene nagAa wasapparently not required (31).

The ohbAB-encoded ISPOHB of the ortho-halobenzoate 1,2-dioxygenase forms a cluster with several other terminal oxyge-nases that are distant from the rest of the members of thefamily of primary oxygenases (Fig. 8). In this cluster, theISPOHB from strain 142 and the putative ISPBPH from strainF199 are more deeply branching and are outside a tight inter-nal cluster formed by ISPOHB from strain JB2 (GenBank ac-cession no. AF087482), NagG from strain U2 (31), the ORF2protein from strain DNT (70), NahG from strain GZ42 (84),and the truncated ORF2 protein from strain JS42 (53). Onlythe ISPOHB from strain 142 (present study) and the ISPSALfrom strain U2 were assigned functions experimentally. Char-acteristically, these structurally related ISPOHBs and theISPSAL were capable of promiscuous use of an energy supplysystem provided by the host bacterium. The ISPSAL genesnagG and nagH alone enabled E. coli to convert salicylate togentisate, albeit with a low level of activity. Addition of ferre-doxin gene nagAb increased this activity, while reductase genenagAa again was not required for salicylate oxidation in E. coli(31). Structural evidence indicated that a loose association withelectron transfer components might be characteristic of thiscluster. Indeed, the ohbAB from strain 142 and bphA1dA2dfrom strain F199 (GenBank accession no. AF079317) are notassociated with any electron component genes, while anotherdeeply branching ISPBPH (bphA2cA1c) from the same strain,F199, is preceded by a Rieske 2Fe-2S-type ferredoxin gene,bphA3, in what could be viewed as an intermediate stage ingene assembly. Evolutionary relationships between tightly re-lated genes from strains DNT, U2, and GZ42 were previouslydiscussed (31). In all three strains was found the same dualoxygenase operon arrangement, reductase-ISPX-ferredoxin-ISPY, in which two ISPs share the same set of electron transfercomponents. Fuenmayor et al. (31) concluded that the ISPSALgenes nagG and nagH (ORF2 and ORFX in DNT; nahG andnahH in GZ42) were acquired as an insert in a preexistingnaphthalene (nitrotoluene) degradation pathway. However,

FIG. 6. Induction of protein synthesis in P. aeruginosa 142 by (chloro)ben-zoates. Shown is an autoradiogram of 35S-labeled proteins synthesized in strain142 upon induction by potential substrates/inducers. Lanes: 1, no substrate; 2,2,4-dCBA; 3, 2,5-dCBA; 4, 2-CBA; 5, benzoate; 6, 4-chlorocatechol; 7, catechol;8, succinate. Each sample loaded onto the SDS–10% polyacrylamide gel con-tained 37.5 mg of total protein. Molecular mass standards (Bio-Rad) were asfollows: rabbit muscle phosphorylase B (96 kDa), bovine serum albumin (66.2kDa), hen egg white ovalbumin (45 kDa), bovine carbonic anhydrase (31 kDa),and soybean trypsin inhibitor (21.5 kDa).



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FIG. 7. Nucleotide sequence of the 6,052-bp ohb DNA region of P. aeruginosa 142. Shown are the six ORFs along with their deduced amino acid sequences.Single-letter symbols for amino acids are aligned with the second nucleotide of each codon. Conserved amino acid residues in sequences of OhbA, OhbB, OhbC, andOhbR are shown in boldface (for an explanation, see the text). Possible Shine-Dalgarno sites and a potential transcription terminator for the ohbB are underlined. Alsoshown are locations of some restriction sites (indicated above the sequence) and the IS1396-like sequence and bordering imperfect IRs (,, below sequence; ., abovesequence).



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functional evidence found by Fuenmayor et al. (31) and struc-tural evidence presented in the form of the recently releasedsequence of an ohb operon from strain JB2 (GenBank acces-sion no. AF087482) suggest otherwise. In this JB2 ohb operon,reductase ohbA, ohbBC (ISPOHB), and ferredoxin ohbD genesare in the same gene order and are highly similar to iso-functional genes from the nag, nah, and dnt operons. It appearsthat these dual oxygenase-containing operons might have beenassembled from preexisting reductase-ISPX (nahGH, nagGH,or ORF2-ORFX)-ferredoxin and independently evolved ISPY(nahAcAd, nagAcA, or dntAcAd) DNA regions. The unaccom-panied ORFG1 protein from strain RW1 (2) and the productof the bphA1e-bphA2e genes (associated with a reductase gene

[bphA4] from strain F199) (GenBank accession no. AF079317)showed pairwise levels of similarity to the OhbB from strain142 in the range of 30%, intermediate between the similarityvalues seen in the OhbB cluster and the levels of similaritybetween OhbB and the BphA LB400(25)-BphA1 B4 (Gen-Bank accession no. U95054)-TcbAa P51 (75) cluster. Interest-ingly, the three ISPBPHs from strain F199, similar to the ISPO-

HBs, are among six sets of putative aromatic oxygenase ISPgenes dispersed within a 184-kb sequence of catabolic plasmidpNL1; however, only two ferredoxin genes (bphA3) and twoferredoxin oxidoreductase genes (bphA4) were annotated forthe entire sequence. Overall, our sequence analysis suggestedcoevolution of the a-ISP and b-ISP genes, whereas electron

FIG. 7—Continued.



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transfer components might not be obligatory for these oxyge-nases and could have been independently acquired via hori-zontal gene transfer.

Although our analysis was limited to a few sequences bestmatching that of ohbAB, in a larger picture, recent findings byother investigators also point to independent evolution of in-dividual components of primary oxygenases. In B. cepaciaDBO1, the terminal oxygenase gene (ophA2) was found to lietogether with the dihydrodiol dehydrogenase gene (ophB)while reductase gene ophA1 was found 7 kb away (13). GenesdxnA1 and dxnA2, encoding dioxin dioxygenase ISPDXN, werefound on a DNA fragment that did not code for electrontransport components in Sphingomonas sp. strain RW1, whilethe cognate ferredoxin (fdx1) and reductase (redA2) geneswere isolated from two other, separate DNA regions (2). Sim-ilar to S. aromaticivorans F199, several other putative ISPgenes were found dispersed on the RW1 genome (2). Thoseincluded ORFG1, which was found to be similar to OhbB (Fig.8A); the pair ORFG5 (a-ISP) and ORFG6 (b-ISP), whichdemonstrated recognizable similarity to OhbB and OhbA (Fig.8B); and ORFG4 (a putative a-ISP), which alone controlledconversion of indole to indigo in E. coli (2).

Only six residues were conserved among the small subunitsof primary dioxygenases (Fig. 7), reflecting the highly diversenature of b-ISP subunits, which might be involved in deter-mining substrate specificity, perhaps along with the large a-ISPsubunit (12). Whereas small subunits of the ISPs from strainsU2 and DNT showed (by BLAST search) recognizable simi-larity only to the members of the identified cluster, OhbAshowed recognizable lengthwise similarity to 14 b-ISPs for avariety of substrates, including mono- and polyaromatic com-pounds (Fig. 8B), possibly suggesting a broader substrate rangefor ISPOHB. The ohb genes isolated in this study dehaloge-nated a variety of halobenzoates, and 2-CBA-grown cells ofstrain 142 oxidized 4- and 5-chloroanthranilate (62) as well as2-trifluoromethylbenzoate and anthranilate (65). Comparative

studies of the substrate ranges of the terminal oxygenases fromthe new cluster perhaps will be helpful in gaining a furtherunderstanding of the evolution of new degradative activities.The previous hypothesis that ortho-halobenzoate 1,2-dioxyge-nases and anthranilate 1,2-dioxygenases from different organ-isms should be closely related, if not the same (65), was notconfirmed. Sequence analysis of the ISPOHBs from strains 142and JB2 and of the ISPCBD from strain 2CBS (33) (Fig. 8A) didnot reveal a specific relationship between these iso-functionalgenes. The large evolutionary distance separating the ohb andcbd genes suggests separate origins of the dehalogenation ac-tivities.

The strain 142 ohb genes are embedded in a transposon-likecontext, implying the likely involvement of horizontal genetransfer in the evolution of the ortho-halobenzoate 1,2-dioxy-genase activity (Fig. 2 and 7). Insertion of the IS1396-likesequence adjacent to a potential transcription terminator (56)of ohbB is consistent with the acquisition of structural genes byIS elements via targeting their terminator or promoter regions(15, 16, 39, 52). Testing for transmissibility of the ohb DNAregion was hindered by the failure to sustain the DNA insert ofpOD33 on E. coli-specific vectors. However, the transposablenature of the dehalogenation activity in strain JB2 was previ-ously demonstrated (55). IS1396 is a member of an insertionelement family that is broadly but sparsely dispersed amonggenomes of gram-positive bacteria, cyanobacteria, and broad-host-range plasmids from gram-negative bacteria (14, 16, 39,76). This, and the similarity of the putative Top protein to theplasmid RP4 topoisomerase (TraE) (42), could imply a plas-mid origin for the ohb DNA region; however, plasmids havenot been reported in strain 142 (62), and finding only two ohbclones among 3,700 recombinants was consistent with theirbeing chromosome-borne genes. Comparison of nucleotide se-quences from halobenzoate-degrading strains 142 and JB2 re-vealed that the IS1396-like sequence from strain 142 at posi-tions 4673 to 5533 (Fig. 7) was 94% similar to the sequence at

FIG. 8. Unrooted Fitch-Margolash dendrograms of large a-ISP subunits of aromatic oxygenases and OhbB (A) and of small b-ISP subunits and OhbA (B). Thehorizontal axes are scaled in terms of expected numbers of amino acid substitutions per site. The numbers on branches refer to the percent confidence estimated bybootstrap analysis (100 replications). The proteins are labeled by trivial abbreviations. (A) In descending order of overall sequence similarity, the proteins were theputative BphA1c and BphA1d from S. aromaticivorans F199 (GenBank accession no. AF079317), NagG of the salicylate-5-hydroxylase from Pseudomonas sp. strainU2 (31), putative OhbB from P. aeruginosa JB2 (GenBank accession no. AF087482), the hypothetical ORF2 of the aromatic dioxygenase from Burkholderia sp. strainDNT (70), the putative BphA1e from strain F199 (GenBank accession no. AF079317), the hypothetical ORFG1 protein of the aromatic dioxygenase fromSphingomonas sp. strain RW1 (2), the putative BphA1 from Pseudomonas sp. strain B4 (GenBank accession no. U95054), BphA of the biphenyl dioxygenase fromBurkholderia sp. strain LB400 (25), TcbAa of the chlorobenzene dioxygenase from Pseudomonas sp. strain P51 (75), AntA of the anthranilate dioxygenase fromAcinetobacter sp. strain ADP1 (11), and CbdC of the 2-halobenzoate 1,2-dioxygenase from B. cepacia 2CBS (33). (B) In descending order of overall sequence similarity,the proteins were the putative BphA2c and BphA2d from strain F199 (GenBank accession no. AF079317), the putative OhbC from strain JB2 (GenBank accessionno. AF087482), NagH from strain U2 (31), the putative BphA2e from strain F199 (GenBank accession no. AF079317), the hypothetical ORFG6 from strain RW1 (2),CmtAc of the p-cumate dioxygenase from P. putida F1 (22), CarAb of the carbazole dioxygenase from Sphingomonas sp. strain CB3 (66), BenB of the benzoate1,2-dioxygenase from strain ADP1 (formerly A. calcoaceticus) (51), AntB from strain ADP1 (11), the putative BphA2 from Rhodococcus erythropolis TA421 (DDBJaccession no. D88021), an unknown MtmX from Streptomyces argillaceus (43), and DxnA2 of the dioxin dioxygenase from strain RW1 (2).



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positions 8302 to 9161 upstream of the putative ohbABCDgenes of strain JB2 (GenBank accession no. AF087482). In thelatter, the remnant of IS1396-like sequence is separated fromohbB (an analog of the ohbB gene in strain 142) by an IS21-likesequence containing the tnpAB genes, by a LysR-type putativeohbR, and by the reductase ohbA gene. Although the dehalo-genation genes from strain 142 and JB2 are clearly divergent(Fig. 8), it appears that the same IS1396-like insertion elementmight have been involved in assembly of the ortho-chloroben-zoate pathway in both strains.

ACKNOWLEDGMENTS

This work was supported by the Great Lakes and Mid-Atlantic EPAHazardous Substance Research Center and by the Strategic Environ-mental Research and Development Program (SERDP), with addi-tional contributions being provided by the National Science Founda-tion Center for Microbial Ecology (DEB 9120006).

We are thankful to Gerben Zylstra, Bob Hausinger, Vladimir Ro-manov, and Sergey Selifonov for useful discussion of the results.

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