Department of Genetics, Institute of Molecular and Cell Biology, Estonian Biocentre and Tartu University, 51010 Tartu, Estonia1
Department of Microbiology and Immunology, University of Illinois College of Medicine, Chicago, Illinois, USA2
Author for correspondence: Maia Kivisaar. Tel: +372 7 375 015. Fax: +372 7 420 286. e-mail: maiak{at}ebc.ee
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ABSTRACT |
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Keywords: Pseudomonas putida, transcription activation, CatR, pheBA and catBCA operons, evolution of catabolic pathways
Abbreviations: ABS, activation binding site; CCM, cis,cis-muconate; ß-Gal, ß-galactosidase; IBS, internal binding site; RBS, recognition binding site
a Present address: Department of Microbiology, University of Iowa, Iowa City, IA 52242, USA.
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INTRODUCTION |
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Many micro-organisms metabolize the toxic compound phenol via catechol as an intermediate. The catechol derived from phenol is usually metabolized by a different pathway known as the meta pathway (Herrmann et al., 1988 ; Kukor & Olsen, 1990
; Shingler et al., 1992
; Wigmore et al., 1977
). The pheBA genes specifying phenol degradation originate from multiplasmid Pseudomonas sp. strain EST1001 (Kivisaar et al., 1990
). pheA encodes phenol monooxygenase, which converts phenol to catechol, while pheB encodes catechol 1,2-dioxygenase. When the plasmid-borne pheBA operon (Fig. 1a
) is introduced into a P. putida strain which is normally incapable of utilizing phenol, the bacteria acquire the ability to degrade phenol via the chromosomal cat pathway encoding catechol degradation in concert with the phenol to catechol or CCM conversion mediated by the pheBA operon (Kasak et al., 1993
). Transcription of the chromosomal ortho pathway genes catBCA and of the plasmid-encoded pheBA operon is activated by the same positive regulator, the chromosomally encoded LysR family protein CatR (Kasak et al., 1993
; Rothmel et al., 1990
). Despite the different origins of the catBCA and the pheBA promoters, the CatR-dependent transcriptional activation mechanism of these promoters seems to be similar. This is therefore an example of how evolution took a short-cut to simply evolve a couple of genes for phenol degradation, instead of a whole pathway with its cluster of structural and regulatory genes that normally evolves in micro-organisms.
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To study how similar or dissimilar the pheBA promoter region is to the catBCA promoter region, not just in terms of DNA homology but also in terms of functionality, we conducted site-directed mutagenesis in the RBS, the ABS and the putative IBS of the pheBA promoter. Comparing the effect of these mutations with those introduced into the catBCA promoter region (Parsek et al., 1994 ) allowed us to determine critical nucleotides involved in direct interactions with CatR at both promoters. Data obtained in this study demonstrate that although some interactions of CatR with the RBS seem to be different between these two promoters, both the promoters utilize a very similar mechanism for transcriptional activation.
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METHODS |
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Bacteria were grown on LuriaBertani (LB) medium (Miller, 1992 ) or on M9 minimal medium (Adams, 1959
). Antibiotics were added at the following final concentrations: for E. coli, ampicillin 100 µg ml-1; and for P. putida, carbenicillin at 1500 µg ml-1. P. putida was incubated at 30 °C and E. coli at 37 °C. E. coli was transformed with plasmid DNA as described by Hanahan (1983)
and DNA was electroporated into P. putida by the protocol of Sharma & Schimke (1996)
.
Site-directed mutagenesis of the pheBA promoter region and DNA sequencing.
Site-directed mutagenesis of the pheBA promoter region and the IBS sequence was carried out using two-step PCR with mutant oligonucleotides containing the specific substitutions. In the first step of the PCR the point mutations were introduced by using the mutant oligonucleotide and oligonucleotide catRylem 5'-GGCATC(EcoRI)GATTGCCTCCCAACTTTTAGTCTTG-3', which is complementary to nucleotides -84 to -113 relative to the transcriptional start site of the pheBA promoter. The first step of the PCR (25 cycles) was: 1 min at 95 °C, 1 min at 54 °C, 1 min at 72 °C. Then 2·5 U ExoI (Amersham) was added to the reaction mixture and the probes were incubated for 30 min at 37 °C to remove the oligonucleotides used in the first step of the PCR. PCR products of ~70 bp were purified and used as oligonucleotides in the second step of the PCR with the oligonucleotide catRall 5'-GGCATC(ClaI)GATTGCCTCCCAACTTTTAGTCTTG-3', which is complementary to nucleotides +14 to +44 relative to the transcriptional start site of the pheBA promoter. The conditions of the second step of the PCR (25 cycles) were: 1 min at 95 °C, 3 min at 54 °C, 1 min at 72 °C. The 158 bp amplified DNA fragments were cloned using EcoRI and BamHI ends into pBluescript SK(+). All mutations were verified by dideoxy-sequencing with a Sequenase version 2.0 kit (Amersham). The pheBA promoter mutants were cloned into the promoter-probe vector pKTlacZ by using the BamHI and XhoI ends.
ß-Galactosidase assay.
Cells of P. putida PaW85 harbouring different pheBA promoter constructs were grown overnight to stationary phase. Sodium benzoate (final concentration 10 mM) was added to the growth medium to induce transcription from the pheBA promoter. The ß-galactosidase (ß-Gal) assay was performed as specified by Miller (1992) . Protein concentration in crude lysates was measured by the method of Bradford (1976)
.
Overproduction of CatR and preparation of CatR lysate.
The CatR protein was overexpressed in E. coli strain BL21(DE3) containing the expression plasmid pET11CatR (see above). Cells were grown in 1 litre M9 minimal medium at 20 °C. IPTG, final concentration 0·4 mM, was added at OD580 0·5, after which growth was allowed to continue for 4 h. Cells were centrifuged at 4600 g for 10 min and suspended in 10 ml CatR lysate buffer (50 mM Tris/HCl pH 7·5; 0·05 mM EDTA; 10 mM MgCl2; 200 mM NaCl; 1 mM DTT; 0·05% Triton X-100; 10%, v/v, glycerol). The cell suspension was sonicated and centrifuged at 25000 g for 10 min.
Gel-mobility-shift assay.
The primer CatRylem, labelled with [-32P]dATP using T4 polynucleotide kinase, and the primer CatRall were used for PCR amplification of the 158 bp DNA fragments containing the wild-type or the mutant pheBA promoter region. The radiolabelled DNA fragments were purified by polyacrylamide gel electrophoresis. The CatR binding reaction was carried out in a volume of 20 µl. DNA probes (200 c.p.m.) were incubated at 20 °C for 20 min with CatR lysate (0·001 mg or 0·01 mg), 1xbinding buffer (50 mM Tris/HCl pH 7·5; 5 mM EDTA; 250 mM KCl; 25 mM MgCl2; 25 mM ß-mercaptoethanol; 0·5% Triton X-100; 20%, v/v, glycerol), 1 µg bovine serum albumin, 5 µg salmon sperm DNA and 100 µM of the inducer CCM. After incubation, the reaction mixture was loaded on a pre-run (20 min) 5% (w/v) nondenaturing polyacrylamide gel. Electrophoresis was carried out at 3 °C in 0·5xTris/borate/EDTA buffer, pH 7·5, at 10 V cm-1 for 2 h. The gel was exposed to a phosphoimager screen.
Determination of the CatR binding activity of the pheBA promoter mutants.
The gel-shift assays were performed three or four times in the presence and absence of 100 µM CCM with the two different concentrations of CatR lysate (as indicated above) which yielded less than 100% bound radioactive probe for the wild-type pheBA promoter region. The bound DNA relative to the unbound DNA was quantified by Phosphoimager (Image Quant 4.2a software, Molecular Dynamics). The relative CatR binding activity for the pheBA promoter mutants was expressed as a percentage of the wild-type pheBA promoter binding.
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RESULTS |
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Effect of mutations in the hinge region
The CatR binding sites RBS and ABS are separated from each other by a hinge region which has been postulated to be involved in DNA bending at the pheBA promoter (Parsek et al., 1995 ). Site-directed mutagenesis in the hinge region, where the G residues were substituted with A residues, was performed in order to generate a more flexible AT-rich region. The 52GA and 51GA mutations increased the efficiency of CatR binding in in vitro binding assays in the presence of CCM approximately 2·5- and 3·5-fold, respectively, and this positive effect was also observed without the inducer (Table 1
). A 2·5-fold increase in CatR binding efficiency was also observed in the case of the double mutation 51/52GA, which generated a stretch of four A residues in the hinge region. However, the effect of these mutations in ß-Gal assays was not so marked. Only the 52GA mutation demonstrated more than a twofold increase in expression of the pheBA promoter in the presence of benzoate; the level of expression of this mutant in the absence of inducer was enhanced 3·5-fold compared to the wild-type promoter (Table 1
). The double mutation 51/52GA resulted in slightly elevated levels of expression and the 51GA mutation resulted in expression levels that were 80% of the wild-type levels in the presence of benzoate. Interestingly, the mutation 50AT, which was neither expected to influence the flexibility of the hinge region nor was previously shown to be involved in the CatR binding on the basis of DNase I footprint analysis (Parsek et al., 1995
), had a negative effect on pheBA promoter activity. This mutation also conferred a reduced CatR-binding efficiency, as observed in the gel-shift assay in the presence of CCM (Table 1
).
Effect of mutations in the ABS region
The CatR ABS overlaps the -35 hexamers of the pheBA promoter and the catBCA promoter. In the absence of CCM, CatR binds to the RBS in the catBCA promoter. Presence of CCM results in the occupation of an additional adjacent binding site, the ABS, and an approximately twofold increase in CatRs DNA binding affinity (Parsek et al., 1994 , 1995
). Binding of CatR to the ABS is cooperative and requires an intact RBS (Parsek et al., 1992
, 1994
). Thus, substituting the nucleotides necessary for the CatR binding to the ABS would result in a decrease in CatRs binding efficiency to the pheBA promoter region to half of that detected with the original promoter sequence in the presence of CCM. As expected, the base substitutions 39AG, 40TC, 41GA, 42AC and 46AC that were introduced upstream of the -35 element of the pheBA promoter led to approximately 50% CatR-binding efficiency to the pheBA promoter region in the presence of CCM as compared with the wild-type promoter (Table 1
). In comparison to these substitutions, the effect of the mutations 45AC, 38TG and 34AC on CatR binding to the ABS was weaker (6075% of the CatR-binding efficiency to the wild-type promoter region was retained) and no negative effect was observed in the case of the mutations 36GC and 35GA (Table 1
).
The in vivo study of the expression of the pheBA promoter carrying different mutations in the ABS region revealed a negative effect of all positions substituted. All mutations except 34AC led to greatly reduced levels of ß-Gal activity in the presence of benzoate (Table 1). The base substitutions in the ABS region also negatively affected the basal level of expression of the pheBA promoter (see the ß-Gal activity in cells grown without benzoate in Table 1
).
Effect of the IBS on expression of the pheBA operon
An additional binding site for CatR downstream of the catBCA promoter, called the internal binding site, IBS, has been identified (Chugani et al., 1998 ). The IBS negatively regulates the expression of the catBCA promoter. Occupation of the IBS by CatR was facilitated in the presence of the RBS and the ABS on the same DNA fragment, and the maintenance of phasing between the promoter and the IBS was important for the IBS-mediated repression (Chugani et al., 1998
). On the basis of these data it was proposed that CatR bound to the DNA at the catBCA promoter, through formation of a DNA loop, could interact with CatR bound to the IBS, and that this interaction could cause impaired transcriptional activation from the catBCA promoter (Chugani et al., 1998
). On the basis of DNase I footprint data a weak CatR-binding site downstream of the transcriptional start site of the pheBA operon (+204 to +221) was found as well (Parsek et al., 1996
). In order to examine the effect of the pheBA IBS (see Fig. 1c
) on the expression of the pheBA operon, this potential CatR binding site was deleted. The resulting construct, pIBS18del, contains an 18 bp deletion of the IBS sequence and is replaced by an 8 bp foreign sequence. The 795 bp Eco47IIIPstI fragment containing the pheBA promoter with the IBS sequence (designated as pIBS) and the deletion variant lacking this sequence were cloned into the plasmid pKTLacZ upstream of the promoterless lacZ gene. The expression of the lacZ transcriptional fusions in P. putida PaW85 grown in the presence of benzoate revealed an approximately twofold higher level of ß-Gal activity in the case of the IBS deletion construct pIBS18del when compared with the original construct pIBS: 621±96 versus 348±38 nmol min-1 (mg protein)-1 (means±SD, n=5). This indicates that the IBS of the pheBA operon could function as the cis-acting repressing element analogous to the IBS of the catBCA operon. However, the effect observed in this study was somewhat less (a twofold effect in comparison with the three- to fourfold effect found in the case of the catBCA system).
The IBS of the catBCA promoter closely matches the consensus sequence of the CatR-binding site RBS (Chugani et al., 1998 ). The IBS region of the pheBA operon contains the sequence ATACC at positions +207 to +211, which is identical to one half of the interrupted inverted repeat of the RBS sequence (Fig. 1
). The location of an A at position +220 (11 nt from the T of the sequence ATACC) matches the LysR-binding consensus T-N11-A motif. We generated two mutations in the IBS region of the pheBA operon: the 208TC mutation, which substituted the T residue in the IBS to a C residue, and the 220A-GG mutation, which replaced the A residue at position +220 with two G residues (Fig. 1c
). The effect of these mutations was tested using the pKTLacZ reporter system (constructs pIBS208TC and pIBS220A-GG, respectively) with cells grown in the presence of benzoate. Only a slight increase of the ß-Gal activity was observed when the 208TC mutation was compared with the wild-type sequence: 397±35 versus 348±38 nmol min-1 (mg protein)-1. However, the 220A-GG mutation resulted in a twofold increase in the expression of the ß-Gal activity [to 607±54 nmol min-1 (mg protein)-1] in comparison with the wild-type. The twofold positive effect of the IBS deletion and mutation 220A-GG was observed also in cells that were grown without the inducer (data not shown).
Effect of the length of the spacer sequence between the -35 and -10 hexamers of the pheBA promoter on CatR-dependent transcriptional activation of the pheBA promoter
The optimal distance between the -35 and -10 hexamers of the RNA polymerase 70-recognized promoters is usually 17 bp (Stefano & Gralla, 1982
). The spacer sequence between the -35 and -10 hexamers of the pheBA promoter is abnormally long, 19 bp. The 19 bp spacer adds an additional twist angle of at least 34 ° and the two hexamers may be out of phase with respect to each other. This raised the question whether optimizing the distance between the -35 and -10 elements of the pheBA promoter could compensate for the requirement of CatR for transcriptional activation of this promoter. Using PCR, we made deletions in the spacer sequence of the pheBA promoter that reduced the distance between the hexamers from 19 bp to 18, 17 or 16 bp and cloned the mutant promoters into the plasmid pKTLacZ (constructs pDEL18, pDEL17 and pDEL16, respectively; Fig. 2
). Like cells with the wild-type promoter, the deletion mutants exhibited only a low basal level ß-Gal activity both in the wild-type and in the CatR- background when bacteria were grown without the inducer (data not shown). When benzoate was added to the growth medium the 18 bp spacer mutant showed a higher level of ß-Gal activity than the control: 735±10 versus 365±19 nmol min-1 (mg protein)-1. Reducing the distance between the -35 and -10 elements to 17 bp or 16 bp had a negative effect on transcriptional activation of the pheBA promoter: pDEL17 showed a twofold lower level of ß-Gal activity [184±20 nmol min-1 (mg protein)-1] than the construct carrying the wild-type promoter with the 19 bp spacer, and pDEL16 demonstrated only the basal level of activity. Thus, optimizing the distance between the -35 and -10 hexamers of the pheBA promoter is not sufficient for CatR-independent transcriptional activation of this promoter.
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DISCUSSION |
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Under activating conditions, in the presence of the inducer CCM, CatR binds to the pheBA promoter and the catBCA promoter as a tetramer. One dimer binds to the RBS and the second dimer binds cooperatively to the ABS (Parsek et al., 1994 , 1995
). The CatR binding site ABS encompasses the promoter -35 element. Since the CatR recognition elements are oriented on the opposite face of the DNA helix to the -35 element, both CatR and RNA polymerase may simultaneously interact with the same sequences from opposite sides of the DNA helix. Mutations in the ABS of the pheBA promoter fall into two groups on the basis of their effects: (1) mutations that affect both the promoter activity and CatR binding, and (2) mutations that affect negatively only the expression of the promoter (Fig. 2
, Table 1
). For example, the mutations 36GC and 35GA, which encompass the -35 hexamer sequence TTGGAT of the pheBA promoter, drastically reduced the level of promoter expression in both the presence and the absence of the inducer but did not affect CatR binding. This indicates that these two nucleotides may be involved in interaction of RNA polymerase with the promoter sequence. The -35 hexamer sequence TTGGAT of the pheBA promoter deviates from the -35 consensus sequence TTGACA at three positions. Therefore, it was unexpected that changing the nucleotide from G to A at position -35 relative to the pheBA transcriptional start (mutation 35GA) would inactivate the promoter, since such a change made the promoter sequence similar to the
70-recognized promoter consensus. Comparison of the sequences of the -35 elements of the pheBA promoter and the catBCA promoter reveals that they are highly conserved (sequences TTGGAT and TTGGAC, respectively). The -35 hexamer of the promoter of the chlorocatechol-degradative genes clcABD is identical to that of the catBCA promoter and it was shown that CatR and ClcR activate transcription via a conserved mechanism (McFall et al., 1997
). The elimination of expression from the pheBA promoter as a result of the 35GA mutation and the conservation of a G nucleotide instead of the consensus nucleotide A in these three promoters indicate that this G nucleotide is important for RNA polymerase interactions with the CatR- and ClcR-regulated promoters.
A 19 bp spacer separates the -35 and -10 hexamers of the pheBA promoter and the catBCA promoter. The promoter of the mercury-resistance operon mer also deviates from the 70-recognized promoter consensus in that there is a 19 bp spacer region separating the two hexamers of the promoter (Parkhill & Brown, 1990
). Mercury-dependent activation of this promoter brings the -10 and -35 elements into a better helical alignment through a MerR-mediated untwisting effect at the spacer DNA (Ansari et al., 1992
; OHalloran et al., 1989
). Single and double base-pair deletions in the interhexamer spacer of the mer operon promoter resulted in MerR-independent transcriptional activation of this promoter (Parkhill & Brown, 1990
). In the case of the promoter of the mom gene (encoding a DNA-modification function of the Mu phage), the 19 bp suboptimal spacer region is also known to function (Bolker et al., 1989
). C-protein-mediated torsional changes reorient the -10 and -35 elements to a favourable conformation for RNA polymerase binding at this promoter (Basak & Nagaraja, 1998
). The deletions made by us to optimize the length of the spacer region of the pheBA promoter did not compensate for CCM-dependent CatR-mediated activation. However, these data show that the exact orientation of the CatR-and RNA-polymerase-binding elements on the DNA helical face is important. Deletion of 2 bp or more from the spacer sequence had a negative influence on the promoter activity: the deletion variant with the 16 bp spacer was not activated at all.
CatR induces a DNA bend in the hinge region between the CatR-binding sites RBS and ABS of the pheBA and catBCA promoters (Parsek et al., 1995 ). In the presence of CCM, the DNA bending angle of the catBCA promoter is partially relaxed. In the case of the pheBA promoter, CatR bends the DNA in the presence or absence of inducer to the relaxed bending angle of the catBCA promoter when CCM is present. Although the fixed DNA bending angle may be important for the CatR-mediated transcriptional activation of both the promoters, some other inducer-dependent CatR-mediated conformational changes at the promoterRNA polymerase complex are required as well. The requirement of the carboxy-terminal domain of the
-subunit (
-CTD) of RNA polymerase was demonstrated for the activation of the pheBA and the catBCA promoters (Chugani et al., 1997
). The pattern of activation of these promoters resembles the pattern of activation for upstream enhancer element (UP element)-dependent activation more closely than cyclic AMP receptor protein (CRP)-dependent activation (Murakami et al., 1996
). Therefore, it was suggested that the
-CTD might interact directly not only with CatR but also with the DNA at the putative UP element (Chugani et al., 1997
).
The substitution of an A nucleotide with a G nucleotide (52AG mutation) in the catBCA promoter hinge region lowered the activation level of the promoter approximately fivefold but did not affect CatR binding (Parsek et al., 1994 ). It was thought that this change would alter the flexibility of the hinge region of the promoter (Parsek et al., 1994
, 1995
). The in vivo and in vitro effects of the base substitutions that were made in the hinge region of the pheBA promoter were somewhat unexpected (Table 1
). The binding efficiency of CatR as determined by the gel-shift assay was increased approximately threefold by the substitution of G nucleotides with A nucleotides. Concurrently, the increase in the in vivo expression of the promoter (twofold) was seen only in one particular case (mutation 52GA). The reason for such a discrepancy is unclear. According to the model presented by Chugani et al. (1997
, 1998
), both CatR and RNA polymerase may simultaneously interact with the same sequences from opposite sides of the helix and the
-CTD most likely interacts with the nucleotides located between the RBS and ABS. Therefore, it is tempting to speculate that the mutation 52GA improved the binding of
-CTD to the putative UP element sequence of the pheBA promoter for its activation, thereby enhancing the level of transcriptional activation from the promoter.
The third CatR binding site, IBS, was localized within the catB structural gene (Chugani et al., 1998 ). The cooperativity observed in DNase I protection studies and the phasing dependence of IBS function indicated that the CatR dimers, bound to the RBS and ABS, interact with the CatR molecules bound to the IBS, with the looping out of the intervening DNA (Chugani et al., 1998
). This interaction resulted in reduced transcriptional activation from the catBCA promoter. The presence of the CatR low-affinity binding site has also been suggested in the case of the pheBA operon at positions +204 to +221 with respect to the transcriptional start site of the operon (Parsek et al., 1996
). In this study we have examined the possible biological effect of this site on the regulation of the pheBA operon. A study of the effect of base substitutions in the IBS region and the deletion of this site indicated that the IBS of the pheBA operon affects transcription from the pheBA promoter in a similar manner as in the case of IBS-mediated repression of the catBCA promoter.
The genetic organization of the pheBA operon is unusual: besides IS1411, which flanks the pheBA genes downstream, a second IS element, IS1472, is located between these genes and their promoter (Fig. 1a). A very similar IS element, IS1384, which shares approximately 99% homology at the DNA level to IS1472, has been found in phenol-degrading Pseudomonas sp. strain H isolated from soil in Germany (GenBank accession number AF052751). The IBS of the pheBA operon is located between the left IR and the transposase gene tnpA of the IS element IS1472 (Fig. 1c
). A sequence identical to the IBS of the pheBA operon is also present in IS1384. Thus, the location of the IBS of the pheBA operon in IS1472 demonstrates that, in principle, any sequence could play a role of IBS if this sequence can function as a binding site for CatR and has an appropriate location relative to the CatR-regulated promoter. It is also a good example of how mobile DNA elements can modulate the expression of neighbouring genes.
Comparative studies of the interaction of CatR at the promoters of the pheBA and catBCA operons have revealed that the CatR-mediated activation mechanism is well conserved despite the different origins of these operons. The pheBA gene cluster appears to be flanked by transposable elements (Kasak et al., 1993 ; Kallastu et al., 1998
). This in turn could allow a rapid movement of these genes from one DNA molecule to another. After the release of the laboratory P. putida strain carrying the pheBA genes on a plasmid into a phenol-contaminated mining area in Estonia, horizontal transfer of the pheBA operon and its expression in different soil bacteria was observed (Peters et al., 1997
). In all isolates degrading phenol via the ortho pathway and harbouring the pheBA genes integrated into other plasmids, the original pheBA promoter sequence was present as before. Thus, the universal mechanism of CatR-mediated transcriptional activation could be of great importance in enabling catechol-degrading soil bacteria to expand their substrate range via horizontal transfer of the phenol-degradation genes without the need for subsequent extensive genetic rearrangements.
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ACKNOWLEDGEMENTS |
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Received 2 July 1999;
revised 24 September 1999;
accepted 4 October 1999.