Soil General Microbiology Laboratory, National Institute of Agro-Environmental Sciences, 3-1-1 Kannondai, Tsukuba City, Ibaraki 305-8604, Japan1
Author for correspondence: Kiyotaka Miyashita. Tel: +81 298 38 8256. Fax: +81 298 38 8199. e-mail: kmiyas{at}s.affrc.go.jp
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ABSTRACT |
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Keywords: Burkholderia, chlorobenzoate dioxygenase, cbeABC, cbeR, catA
Abbreviations: 2CB, 2-chlorobenzoate; 3CB, 3-chlorobenzoate; 4CB, 4-chlorobenzoate; 3CC, 3-chlorocatechol; 4CC, 4-chlorocatechol; BSMG, basal synthetic medium + glucose; DA, Davis-adonitol medium; DHB, dihydrodihydroxybenzoate (3,5-cyclohexadiene-1,2-diol-1-carboxylic acid); PCBs, polychlorinated biphenyls
The GenBank accession number for the sequence reported in this paper is AB024746.
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INTRODUCTION |
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Bacterial degradation of chlorobenzoate via chlorocatechol is supposed to be one of the typical degradation pathways for chlorobenzoates. In this pathway, chlorobenzoates are converted to chlorocatechols by (chloro)benzoate dioxygenase and (chloro)benzoate-dihydrodiol dehydrogenase (Focht, 1996 ), and the chlorocatechols thus generated are transformed by the so-called modified ortho pathway enzymes (Harwood & Parales, 1996
; Reineke, 1998
; van der Meer et al., 1992
). The genes encoding these enzymes were apparently derived from the ortho pathway genes for catechol degradation (Daubaras & Chakrabarty, 1992
; Frantz & Chakrabarty, 1987
; Reineke, 1998
; van der Meer et al., 1992
). While the structure and expression of the modified ortho pathway genes have been extensively studied (McFall et al., 1998
; van der Meer et al., 1992
), those genes for (chloro)benzoate dioxygenase in bacteria that transform chlorobenzoates to chlorocatechols have not been sufficiently examined. Analysis of (chloro)benzoate dioxygenase genes is essential for the elucidation of the molecular mechanism of chlorobenzoate degradation.
Burkholderia sp. NK8 is a soil isolate that shows broad specificity for chlorobenzoate degradation, being capable of growth on 3CB and 4-chlorobenzoate (4CB). The current study was conducted to examine the genes for chlorobenzoate dioxygenase(s) responsible for the broad substrate specificity of NK8 for chlorobenzoates. The chlorobenzoate dioxygenase genes of NK8 were cloned and analysed using various genetic and enzymic methods.
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METHODS |
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Cloning of the benzoate dioxygenase genes.
Degenerate PCR primers were designed from highly homologous regions of benA of Acinetobacter sp. strain ADP1 and xylX of P. putida TOL plasmid pWW0 (Harayama et al., 1991 ). Using purified NK8 genomic DNA as template, PCRs were carried out with different degenerate primer combinations. The forward primer BAf1 [5'-GC(C/T)CA(C/T)GA(G/A)AGCCAGATTCCC-3'] with the reverse primer BAr2 [5'-GGTGGC(G/T)GC(G/A)TAGTTCCAGTG-3'] yielded an approximately 500 bp fragment, which was then cloned in the pCR2.1 TA cloning vector (Invitrogen) and sequenced with an ALFred DNA Sequencer (Pharmacia Biotech). The cloned PCR product was used to probe for the benzoate dioxygenase genes of Burkholderia sp. NK8. Purified NK8 genomic DNA was digested with various restriction endonucleases. Restriction fragments were separated on an agarose gel by electrophoresis and then blotted onto Hybond-N+ nylon membrane (Amersham). The Southern blot was probed with the 500 bp PCR product labelled using the DIG Nucleic Acid Detection Kit (Boehringer Mannheim). An approximately 5·3 kb EcoRI fragment was selected for cloning into pUC118 and pBluescript II KS(+). DNA fragments of about 55·5 kb recovered from the agarose gel were cloned and used to transform E. coli DH5
. Identification of positive clones was done by colony hybridization with the DIG-labelled PCR product. Putative clones were verified through direct colony PCR with the primers BAf1 and BAr2, followed by Southern blot analysis. The 868 bp EcoRIPstI segment at the left end of the cloned 5·3 kb EcoRI fragment in Fig. 1
was excised, labelled and used to probe NK8 genomic DNA for overlapping upstream fragments. Among the positive bands, the 8·1 kb HindIIIPstI fragment was selected for cloning into pBluescript II KS(+).
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Determination of (chloro)benzoate dioxygenase activity.
NK8 cells were grown on succinate, benzoate or 3CB liquid medium to late exponential phase, harvested by centrifugation, washed three times with 20 mM potassium phosphate buffer (pH 7·5) and stored at -80 °C until used. The cells were thawed on ice, disrupted by sonication and ultracentrifuged at 164000 g for 40 min at 4 °C. The supernatants were evaluated for their ability to convert benzoate, 2CB, 3CB and 4CB, according to the method of Romanov & Hausinger (1994) except that the reaction mixture, with a total volume of 2 ml, contained 1 mM aromatic substrate and about 3040 mg protein in addition to 5 mM Na-MES (pH 6·5), 10 mM Fe(NH4)2(SO4)2, 100 mM NADH and 2 µM FAD. Aliquots of 500 µl, which were taken at the initiation of the reaction and at 15 or 30 min thereafter, were immediately added to 86 µl 7 M trichloroacetic acid in microtubes to precipitate the proteins. Samples were prepared for quantitative HPLC analysis according to Fetzner et al. (1989)
by adjusting the supernatant pH to about 6 with 5 M sodium hydroxide. After a second centrifugation, samples were diluted with 1 vol. HPLC solvent. (Chloro)benzoate dioxygenase activity of the supernatant was determined by measuring substrate consumption in the supernatant by HPLC (HP1100; Hewlett Packard) on an Eclipse XDB-C18 (Agilent Technologies) reversed phase column, using acetonitrile:10 mM H3PO4 (50:50, v/v) as the solvent at a flow rate of 1 ml min-1. Authentic benzoate, 2CB, 3CB, 4CB and catechol (all purchased from Wako Pure Chemicals), and 3-chlorocatechol (3CC) and 4-chlorocatechol (4CC) (both purchased from Tokyo-Kasei) standards were run to verify their respective retention times.
Construction of NK8 cbeA, cbeR and catA disruptant strains.
These disruptants were generated by omega () cassette interposon mutagenesis following the method of Schweizer (1992)
. In all gene disruptants, internal fragments of considerable length were excised, i.e. the 603 bp PstIHincII fragment in cbeA, the 341 bp StuIEcoRI fragment in catA and the 473 bp SphINruI fragment in cbeR, and replaced by the 1721 bp HindIII
gentamicin-resistance (Gmr) cassette (
aac) of pHP45aac, the 1773 bp SmaI
aac cassette of pHP45aac and the 2267 bp HindIII
hygromycin-resistance (Hmr) cassette (
hyg) of pHP45hyg (Blondelet-Rouault et al., 1997
), respectively. In all constructs, DNA fragments ranging from 1·5 to 2·2 kb flank the
cassette. The gene constructs and the MOB cassette of pMOB3 were sequentially cloned into pNOT322, which was used to transform E. coli strain S17-1
pir. Conjugation of the transformed S17-1
pir with NK8 cells was done according to Franklin (1985)
. Transconjugants were selected at 30 °C on DA agar plates containing gentamicin or hygromycin and were evaluated on DA agar plates with the appropriate antibiotic to separate double from single cross-overs. Allelic replacement of the wild-type genes by the
cassette-disrupted genes was verified by Southern hybridization analysis. Disruptants were evaluated for their ability to grow on benzoate, 3CB and 4CB containing the appropriate antibiotic. NK8 strains disrupted in their cbeA, catA and cbeR genes were named NDBA1, NCAD and NCRD, respectively.
Complementation of the cbeA disruptant.
The 12·5 kb HindIIIEcoRI fragment of NK8 that carries the cbecat gene cluster was cloned into the broad-host-range plasmid vector pJRD215 (Davison et al., 1987 ) to generate plasmid pBAC1, which was utilized to transform E. coli S17-1. The E. coli S17-1 transformant was conjugated with the NK8 cbeA disruptant NDBA1. Transconjugants of NDBA1 were evaluated for their ability to utilize benzoate, 3CB or 4CB as the sole carbon source.
Expression of NK8 cbeABCD in E. coli.
The NK8 cbeABCD gene cluster was amplified by PCR with the 77-mer forward primer 5 '-CCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACCATGTCCGCCATCACCGACAAAGCCAGTCAGCTCG-3' and the 29-mer reverse primer 5'-CAGGATCCATAGCGAATCTTCTCGTACAC-3'. The amplified fragment was digested with XbaI/BamHI and cloned into the XbaI/BamHI sites of pET14b to generate plasmid p14BEP. E. coli strain HMS174(DE3) was transformed with p14BEP carrying the correct fragment, as verified by sequencing. Transformants were cultured overnight at 30 °C in 2xYT medium (Sambrook et al., 1989 ) containing ampicillin. The cultures were diluted 50-fold with pre-warmed fresh 2xYT-ampicillin medium and again grown at 30 °C to an OD600 of 0·40·6, at which point IPTG was added to a final concentration of 0·5 mM to induce expression of the cbeABCD genes. After 3 h of culture with the inducers, cells were harvested, washed three times with 20 mM potassium phosphate buffer (pH 7·5) and stored at -80 °C until used for the preparation of crude cell-free extracts for enzyme assay by HPLC.
Determination of the products of CbeABCD.
Products generated from (chloro)benzoates by CbeABCD were determined using whole cells of E. coli HMS(DE3)/p14BEP. Cells freshly harvested from 150 ml culture grown as described above were washed with 1 vol. 20 mM potassium phosphate buffer (pH 7·5) and resuspended in 45 ml of the buffer. To aliquots of 10 ml, substrates were added to a final concentration of 2 mM and the reaction mixtures were incubated at 30 °C in a shaking water bath. Samples (1 ml) taken at selected time points were immediately centrifuged at 20000 g for 10 min at 4 °C. Aliquots (500 µl) of the supernatant were mixed with an equal volume of HPLC solvent, centrifuged and subjected to HPLC analysis at A203.
Transcriptional fusion studies.
Various DNA fragments from the NK8 cbecat region were ligated immediately upstream of the promoterless lacZ gene of the reporter plasmid pQF50 (Farinha & Kropinski, 1990 ) to generate several lacZ transcriptional fusion plasmids as shown in Fig. 3
. The plasmid pFJ50cbeRcatAcbeA' contains the complete cbeR and catA genes and the truncated cbeA gene fused to lacZ; pFJ50cbeRcatA::
hygcbeA' differs from pFJ50cbeRcatAcbeA' in having its catA gene disrupted by the
hyg cassette; pFJ50cbeR'catAcbeA' is similar to pFJ50cbeRcatAcbeA' except that cbeR is incomplete; in pFJ50cbeR'catA' and pFJ50cbeRcatA', the truncated catA is fused to lacZ. These lacZ transcriptional fusion plasmids were introduced by electroporation into PRS4020, the catR knockout mutant of P. putida (Parales & Harwood, 1993
). Transformed PRS4020 cells were assayed for ß-galactosidase activity. Induction of the lacZ gene in PRS4020 transformants was performed by pre-culturing the cells overnight at 30 °C on LB medium containing gentamicin and carbenicillin. One hundred microlitres of the pre-culture was used to inoculate 10 ml basal synthetic medium (Aldrich et al., 1987
) containing 10 mM glucose (BSMG), or BSMG supplemented with 5 mM benzoate, 3CB or 4CB, or with 0·1 or 0·05 mM catechol, or 5 mM cis,cis-muconate (Celgene), then grown for 17 h at 30 °C. ß-Galactosidase activity was assayed according to the method of Miller (1972)
. All assays were done in triplicate. ß-Galactosidase activity was expressed in Miller units [nmol nitrophenol generated min-1 (mg protein)-1].
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RESULTS |
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At a concentration of 5 mM substrate in batch cultures, NK8 cells pre-cultured in 3CB grew fastest in 3CB medium, with a doubling time of approximately 4 h. Growth with 4CB and benzoate were slower, with doubling times of about 5·5 and 11 h, respectively. Interestingly, the lag phase with 4CB was rather long, reaching about 45 h in batch cultures inoculated with 3CB-grown cells. Corresponding values for benzoate and 3CB are 20 and 10 h, respectively. The reason for the long lag phase with 4CB is not clear. Even in batch cultures inoculated with 4CB-grown cells, the lag phase in 4CB was consistently longer than those in 3CB and benzoate media. Nevertheless, once the exponential phase commenced, NK8 cells grew rapidly, and as in 3CB medium, the transition to stationary phase in 4CB was clearly defined.
Degradation of chlorobenzoates
NK8 cells grown on succinate, benzoate or 3CB were disrupted by sonication and the supernatants were subjected to enzyme assay. The lysate of NK8 cells grown on succinate did not transform benzoate, 2CB, 3CB or 4CB (Table 2), indicating the absence of constitutive expression of the chlorobenzoate dioxygenase genes. Lysates of benzoate- or 3CB-grown cells, on the other hand, showed considerable conversion of the aromatic substrates. The relative activities for the chlorobenzoates are not remarkably different between benzoate- and 3CB-grown NK8 cells. It is noteworthy that the activity of NK8 cell lysate against 2CB is comparable to that against 3CB. These data suggest that NK8 has a (chloro)benzoate dioxygenase that possesses a broad substrate specificity.
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Nucleotide sequence analysis of this 12·5 kb DNA region revealed the presence of 9 ORFs. ORFs 59 are transcribed in the same direction (from left to right in Fig. 1), while ORFs 14 are transcribed divergently. After comparisons with related sequences in the database, ORFs 69 were designated cbeA, cbeB, cbeC and cbeD, respectively (Fig. 1
). CbeA showed the highest amino acid sequence identity to CbdA (64%) (Table 3
), the large subunit of the terminal oxygenase of the 2-halobenzoate 1,2-dioxygenase of B. cepacia 2CBS (Haak et al., 1995
); CbeB to XylY (60%), the small subunit of the terminal oxygenase of toluate 1,2-dioxygenase of P. putida TOL plasmid pWW0 (Harayama et al., 1991
); and CbeC to XylZ (54%), the reductase component of toluate 1,2-dioxygenase. Probably, CbeABC comprise an aromatic ring hydroxylase that belongs to group IB of Baties classification (Batie et al., 1992
) as indicated by its closeness to the above-mentioned group IB hydroxylases. cbeD encodes a protein with an amino acid sequence that resembles those of the cis-diol dehydrogenase encoded by xylL on the TOL plasmid pWW0 (57% identity) and benD in Acinetobacter sp. ADP1 (56% identity).
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The deduced amino acid sequences of ORFs 2 and 3 are similar to those of catC (63% identity) and catB (57% identity), respectively, of ADP1. Therefore, they were designated catC and catB, respectively. ORF1 encodes a protein with a deduced amino acid sequence that is 55% identical to that of benE of ADP1, the function of which is unknown (Collier et al., 1998 ). The gene was named cbeE.
cbeA disruption and complementation
To ascertain the function of the cbeABCD gene cluster in chlorobenzoate catabolism, cbeA was disrupted by interposon mutagenesis (Fig. 1
). The disruption was confirmed by Southern hybridization. The disruptant strain NDBA1 failed to grow on 3CB, 4CB or benzoate, the substrates degraded by the wild-type strain NK8. This observation indicates that cbeA is involved in the catabolism of benzoate and monochlorobenzoates. The disruptant strain NDBA1 was complemented by the 12·5 kb HindIIIEcoRI fragment containing the cbecat gene cluster harboured by pBAC1. The complemented disruptant (NDBA1/pBAC1) grew on benzoate, 3CB and 4CB (data not shown), indicating that the cbecat DNA region restored in the disruptant the ability to catabolize benzoate and monochlorobenzoates.
Expression of cbeABCD in E. coli
E. coli HMS174(DE3) transformed with p14BEP, a derivative of expression vector pET14b which carries the cbeABCD genes, was cultured in the presence of IPTG to induce the expression of the genes. The cells were harvested, washed and disrupted by sonication. Cell-free extracts transformed 2CB, 3CB, 4CB and benzoate. Activity was greatest for benzoate followed by 4CB, 3CB and 2CB (Table 2). While the value for 2CB is relatively low compared to those of NK8 cell lysates, this result, together with that of the disruption of cbeA, confirms that the cbeABCD genes are involved in the oxidation of 2CB, 3CB, 4CB and benzoate in NK8.
Analysis of HPLC peaks emerging during enzyme reaction by whole cells of E. coli cbeABCD+ showed that benzoate generates catechol while 4CB produces 4CC. Oxidation of 3CB gives rise to 4CC and 3CC as the major and minor intermediate products of oxidation, respectively (Fig. 2), while 2CB yields not only 3CC but also catechol (apparently in equal amounts), suggesting that CbeABC lacks absolute regiospecificity.
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Co-expression of the cbeA and catA genes
In strain NK8, catA and cbeA are separated from each other by only 115 bp, and both lie upstream of and are transcribed divergently from cbeR (Fig. 1). Neither the -10 and -35 bacterial promoter-like sequences nor the consensus motif of LysR-type regulator recognition site exist in the catAcbeA intergenic region. Thus, it is likely that catA and cbeA are co-transcribed, a possibility that is further indicated by the inability of the catA disruptant NCAD to grow on (chloro)benzoates. To determine whether cbeA transcription is initiated from the catAcbeA intergenic region, the
hyg cassette was inserted into catA of the lacZ transcriptional fusion plasmid pFJ50cbeRcatAcbeA' to generate pFJ50cbeRcatA::
hygcbeA' (Fig. 3
), which was then introduced into PRS4020. There was no induction of ß-galactosidase activity even in the presence of inducers (Fig. 4
), indicating that cbeA is exclusively co-transcribed with catA.
Inducers of cbeA expression in NK8
A cbeA::lacZ transcriptional fusion construct was introduced into the NK8 wild-type genome by allelic replacement to generate strain NBALZ. HPLC analysis confirmed that the cbeA disruption had blocked the conversion of 3CB, 4CB and benzoate by NBALZ. Addition of 3CB, 4CB or benzoate to the BSMG medium increased ß-galactosidase activity by more than 70-fold compared to those grown without these aromatics (Fig. 5). The addition of 2CB, on the other hand, did not increase the activity. cis,cis-Muconate induced ß-galactosidase activity to a level comparable to those induced by benzoate, 3CB or 4CB. Catechol, added at a concentration low enough to allow NK8 growth, also induced ß-galactosidase activity to a level almost half of those induced by benzoate, 3CB and 4CB. On the other hand, 3CC and 4CC did not induce ß-galactosidase activity. These results are consistent with those obtained with PRS4020 harbouring the transcriptional fusion plasmid pQF50 derivatives (Fig. 4
), and thus confirm 3CB, 4CB, benzoate and cis,cis-muconate as inducers of cbeA expression.
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DISCUSSION |
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Similar or even greater ability to considerably transform all the monochlorinated benzoate isomers was previously reported for the toluate 1,2-dioxygenase of B. cepacia WR401, an o-toluate degrader (Reineke, 1998 ). However, notable differences between this enzyme and NK8 (chloro)benzoate dioxygenase are evident. Relative to benzoate, NK8 CbeABC activities against the three isomeric monochlorobenzoates ranged from 28 to 61% (Table 2
), while WR401 has activities estimated from Reineke (1998)
of approximately 90, 110 and 50% for 2CB, 3CB and 4CB, respectively. Moreover, unlike WR401, which is a natural toluate degrader, NK8 could not grow on o-, m- or p-toluate medium. The clustering in NK8 of the cbe genes with the catechol degradation genes further justifies the classification of NK8 CbeABC as a (chloro)benzoate dioxygenase.
The CbeABC has a relaxed regiospecificity
Strain NK8 (chloro)benzoate dioxygenase appears to lack absolute regioselectivity as revealed by the species of intermediates it generates from the asymmetrical substrates 2CB and 3CB (Fig. 2). 2CB binds with NK8 CbeABC either as 2CB or 6CB, apparently with equal affinity. The subsequent 1-2 dioxygenation of 2CB gives rise to the unstable intermediate 2-chloro-3,5-cyclohexadiene-1,2-diol-1-carboxylic acid (2-chloro-DHB), which spontaneously loses Cl- to generate catechol. Dioxygenation of 6CB gives rise to 6-chloro-DHB, which upon dehydrogenation by CbeD is converted into 3CC. The preference of NK8 dioxygenase for 3CB that binds as 5CB (the Cl- substituent is distal to the dioxygenation point) rather than as 3CB, as indicated by the production of greater amount of 4CC than 3CC from 3CB, likewise demonstrates the lack of absolute regioselectivity of the NK8 dioxygenase. Low regiospecificity was also reported for other 2CB-oxidizing enzymes such as those of P. aeruginosa JB2 (Hickey & Focht, 1990
) and Pseudomonas sp. 3CBS (Sylvestre et al., 1989
), which generate 3CC and catechol from 2CB. In contrast, dioxygenases with absolute regiospecificity, as exemplified by the CbdABC of P. cepacia 2CBS (Fetzner et al., 1989
) and by the ortho-halobenzoate 1,2-dioxygenase of P. aeruginosa 142 (Romanov & Hausinger, 1994
; Tsoi et al., 1999
), yield only catechol from the dioxygenation of 2CB.
3CB and 4CB are degraded via chlorocatechols in strain NK8
The complete degradation of benzoate and chlorobenzoates by NK8 apparently proceeds via separate routes as outlined in Fig. 6. Catechol, the CbeABCD-catalysed oxidation intermediate product from benzoate and 2CB, is likely to be converted by the ortho-cleavage pathway enzymes CatA, CatB and CatC, while the 3CC and 4CC generated from 3CB, 4CB and 2CB are processed via the modified ortho-cleavage pathway by the chlorocatechol-oxidizing enzymes. Substantiating this scheme are the following observations. NK8 mutants that are incapable of utilizing 3CB and 4CB, but could grow on benzoate, arose spontaneously after repeated subculture in LB. The chlorocatechol degradation genes of NK8, which have been cloned from its large plasmid, were shown by Southern hybridization to be absent in the mutants (unpublished data). Complementation of one of the spontaneous NK8 mutants (plasmid-, 3CB- and 4CB-) with either the Ralstonia eutropha NH9 chlorocatechol genes cbnRABCD in pEKC1 (Ogawa & Miyashita, 1999
) or the plasmid-borne NK8 chlorocatechol genes enabled the mutant to grow on 3CB and 4CB (unpublished data). These observations indicate that the plasmid-encoded chlorocatechol catabolic genes of the modified ortho pathway are involved in the transformation of 3CC and 4CC. Also, catA could take part in the transformation of 4CC to 3-chloro-cis,cis-muconate in NK8, as in other bacteria (Dorn & Knackmuss, 1978
; Kim et al., 1997
; Sauret-Ignazi et al., 1996
).
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(Chloro)benzoates are inducers of the transcriptional activation of the catA promoter
The transcriptional fusion study in P. putida PRS4020 showed that the transcription of catA and cbeA(BCD) is regulated by CbeR. Results of the ß-galactosidase assay of NBALZ, the cbeA::lacZKmr disruptant strain (Fig. 5), indicate that cbeA is induced by 3CB, 4CB, benzoate and cis,cis-muconate. In the well studied benzoate-degrading bacterium Acinetobacter sp. ADP1, cis,cis-muconate converted from catechol induces the expression of the benzoate dioxygenase (ben) genes and catechol dioxygenase (cat) genes (Collier et al., 1998
). In P. putida, cis,cis-muconate from catechol also activates the cat genes (Parsek et al., 1992
). The amino acid residues in the putative binding region conserved among cis,cis-muconate-responsive regulatory proteins are also conserved in CbeR (from Ile-98 to Glu-152; data not shown). Although the possibility that the degradation product of cis,cis-muconate acts as an effector cannot be excluded, it is probable that cis,cis-muconate binds to CbeR and then activates the coupled transcription of catA and cbeA. The observed increase in ß-galactosidase activity with catechol could be attributed to the cis,cis-muconate rapidly generated from catechol by CatA. Quantitative HPLC analysis showed that benzoate and chlorobenzoates are not degraded by NBALZ. Therefore, the inducers of cbeA expression observed in lacZ assay of NBALZ are benzoate and chlorobenzoates themselves. BenM of Acinetobacter sp. ADP1 also responds to benzoate (Collier et al., 1998
). However, the overall identity of CbeR with BenM (45%) is lower than that with CatR of P. putida RBS2000 (50%) and RB1 (48%), which respond to cis,cis-chloromuconate (Houghton et al., 1995
; Parsek et al., 1992
). NK8 CbeR appears to be the first example of a LysR-type regulator involved in the degradation of (chloro)benzoate that recognizes chlorobenzoates as inducers. The difference among the three monochlorobenzoate isomers in their ability to induce cbeA expression is obvious, 3CB and 4CB being as effective as benzoate and cis,cis-muconate, while 2CB is not an inducer (Fig. 5
). The difference in their ability to support the growth of NK8 is also evident. NK8 grows well on 3CB and 4CB but does not grow on 2CB, notwithstanding the significant transformation by NK8 cell lysate of these three chlorobenzoate isomers (Table 2
). The absence of growth of NK8 on 2CB may be due to the inability of CbeR to recognize 2CB as an effector. The induction of oxidative genes, in addition to the substrate specificity of the encoded enzymes, can be a bottleneck in the degradation of chlorobenzoate. Apparently, regulator recognition of effectors is essential in determining the substrate specificity of chlorobenzoate-degrading bacteria.
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ACKNOWLEDGEMENTS |
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Received 12 April 2000;
revised 7 September 2000;
accepted 14 September 2000.