Laboratorio de Microbiología, Departamento de Genética Molecular y Microbiología, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Casilla 114-D Santiago, Chile1
Division of Microbiology, National Research Centre for Biotechnology GBF, Braunschweig, Germany2
Author for correspondence: Bernardo González. Tel: +56 2 6862845. Fax: +56 2 2225515. e-mail: bgonzale{at}genes.bio.puc.cl
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ABSTRACT |
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Keywords: chloroaromatic compounds, maleylacetate reductase, gene acquisition, AraC/XylS regulatory family, gene dosage
Abbreviations: CB, chlorobenzoate; 3-CB, 3-chlorobenzoate; 4-CB, 4-chlorobenzoate; 3,5-DCB, 3,5-dichlorobenzoate; 3-MB, 3-methylbenzoate; 4-MB, 4-methylbenzoate; 2,4-D, 2,4-dichlorophenoxyacetate
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INTRODUCTION |
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Ralstonia eutropha JMP134 is well known for its catabolic properties toward chloroaromatic compounds. This bacterium grows on 2,4-dichlorophenoxyacetate (2,4-D) and 3-chlorobenzoate (3-CB), as well as other chloroaromatic pollutants (Clément et al., 1995 ; Don & Pemberton, 1981; Pieper et al., 1988
). The key catabolic abilities of R. eutropha JMP134 are encoded on the plasmid pJP4 (Don & Pemberton, 1981
), and the catabolic enzymes and genes of this plasmid have been studied extensively (Don et al., 1985
; Kasberg et al., 1995
; Kuhm et al., 1990
; Laemmli et al., 2000
; Leveau et al., 1999
; Matrubutham & Harker, 1994
; Pérez-Pantoja et al., 2000
; Perkins et al., 1990
; Pieper et al., 1988
, 1989
, 1993
; Seibert et al., 1993
; Streber et al., 1987
; Vollmer et al., 1999
). Metabolism of 3-CB is initiated by the chromosomally encoded, low-specificity benzoate dioxygenase and 1,2-dihydro-1,2-dihydroxybenzene dehydrogenase to form 3-chlorocatechol and 4-chlorocatechol (Fig. 1a
, solid arrows). Chlorocatechol metabolism is performed by the enzymes chlorocatechol 1,2-dioxygenase, chloromuconate cycloisomerase, dienelactone hydrolase and maleylacetate reductase (Fig. 1b
), encoded by tfdC, tfdD, tfdE and tfdF, respectively, which are located in the tfdCIDIEIFI and tfdDIICIIEIIFII gene clusters of pJP4 (Fig. 1c
). Neither of the tfd modules is fully iso-functional, because they encode for enzymes whose activity profiles are different (Fig. 1b
) (Laemmli et al., 2000
; Pérez-Pantoja et al., 2000
; Plumeier et al., 2002
).
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METHODS |
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Degradation of CB by resting cells and enzyme-activity assays.
For CB degradation assays, R. eutropha derivatives were grown in minimal medium containing 5 mM fructose. At the late-exponential phase of growth, cells were induced with 3 mM 4-CB or 2 mM 3,5-DCB for at least 4 h or were not induced. About 100 ml of each culture were harvested, and the resulting cell pellet was washed three times with minimal medium. The cells were then resuspended in the same medium to an OD660 value of 2·0, and incubated with 0·3 mM 4-CB or 3,5-DCB. The degradation of CB was monitored in a HP 8452-A UV spectrophotometer (Hewlett Packard). UV spectral profiles (210300 nm) were determined in supernatants from each sample. Under the experimental conditions used here, no spectral changes corresponding to potential metabolic products, i.e. chlorocatechols, chloromuconates or dienelactones, were observed. Abiotic controls were routinely performed by monitoring CB degradation in uninoculated minimal-medium-containing flasks. Degradation rates of 3,5-DCB were calculated from the absorbance decrease at 238 nm (238=3300 M-1 cm-1) after 20 min incubation, and are expressed in µmol 3,5-DCB degraded min-1 (mg protein)-1. Proteins were determined by the Bradford method (Bradford, 1976
), after treatment of cells with 5 M NaOH at 85 °C for 10 min. For activity assays of R. eutropha derivatives, cells were grown in 2 mM benzoate and induced at the late-exponential phase of growth with 1 mM 3-CB. Growth on benzoate and induction with 3-CB was used in these assays because not all of the R. eutropha derivatives grew on 3-CB. The concentration of 3-CB used was chosen to prevent the accumulation of chlorocatechols in strains that had low levels of chlorocatechol 1,2-dioxygenase. Cell extracts were obtained as follows. About 100 ml of each culture were harvested at the end of the exponential phase of growth and centrifuged. The resulting cell pellets were washed twice and resuspended in 5 ml of 50 mM Tris/acetate (pH 7·5) containing 1 mM MnSO4. The cells were then disrupted by sonication (Vibracell; Sonics & Materials). The soluble protein fraction was obtained after 1 h centrifugation at 130000 g, in a Beckman L-80 ultracentrifuge. Cell extracts were used without further purification. Enzyme assays for chlorocatechol 1,2-dioxygenase, maleylacetate reductase and chloromuconate cycloisomerase were performed as described previously (Pérez-Pantoja et al., 2000
).
Cloning of the tfd gene modules.
Restrictions, ligations, dephosphorylation reactions, DNA purification, electroporation conditions and Southern analysis were carried out following standard protocols (Ausubel et al., 1992 ). Different chlorocatechol catabolism tfd gene modules were prepared for chromosomal insertions (using vector pUT or pBSL202) or for cloning into a medium-copy-number vector (pBBR1MCS-2). The plasmids constructed and the vectors used in this study are listed in Table 1
. The cloning of the tfdRtfdCIDIEIFI gene module into the pUC18NotI derivative pUCLG2 has been described previously (Pérez-Pantoja et al., 2000
). Plasmid pUCLG2 was digested with NotI and the resulting insert was introduced into pUT, to give pR1TFD. Cloning of tfdRtfdCIDIEIFI into the medium-copy-number vector pBBR1MCS-2 to give pBBR1M-I has also been described previously (Pérez-Pantoja et al., 2000
). To clone tfdRtfdDIICIIEIIFII, pBSDP4 was digested with SacI/KpnI to generate a 5·9 kb fragment containing the tfdRtfdDIICIIEIIFII gene module; this fragment was then introduced into pHRP316 to give pHRPM-II. pHRP316 was only used for intermediate cloning steps. The 6 kb HindIII fragment of pHRPM-II was cloned into pBBR1MCS-2 to yield pBBRHM-II. The tfd gene module was extracted from pBBRHM-II with a SpeI digestion, and then introduced into pBSL202 to give pR2TFDG. Additionally, the SpeI fragment from pBBRHM-II was introduced into pR1TFDG, a pBSL202 derivative in which a 5·1 kb NotI fragment from pUCLG2 containing the tfdRtfdCIDIEIFI gene module had been cloned previously, to produce pR12TFDG. The latter contains tandem, direct copies of each tfd module. Cloning of tfdRtfdDIICIIEIIFII into pBBR1MCS-2, to give pBBR1M-II, has been described previously (Pérez-Pantoja et al., 2000
). The maleylacetate reductase gene encoded on pBBR1M-I was inactivated by a 0·6 kb deletion corresponding to the internal AatII sites in the tfdFI gene, to produce pBBR
FI. To construct a pBBR1M-II derivative lacking the maleylacetate reductase gene, the 8·3 kb EcoRI fragment of pJP4 was cloned in pUC18NotI, and its 4·5 kb SacI fragment containing the tfdRtfdDIICIIEII genes was directly cloned into pBBR1MCS-2, to give pBBR
FII. A 10-fold decrease in maleylacetate reductase activity was determined for strain JMP222(pBBR
FII) with respect to strain JMP222(pBBR1M-II). The determination of this activity in strain JMP222(pBBR
FI) was not performed because JMP222(pBBR1M-I) has a maleylacetate reductase activity level similar to that found in the chromosome of strain JMP222 (Pérez-Pantoja et al., 2000
).
Construction of a xylXYZL gene module.
A pUT derivative, pSPM100, containing the xylSxylXYZL gene module of the TOL plasmid pWW0 and resistance to tellurite as a selective marker was obtained from V. de Lorenzo (S. Panke & V. de Lorenzo, unpublished data). To construct an unregulated xyl gene module, pSPM100 was digested with BstEII, which produces three cuts in this plasmid, to remove a 725 bp internal xylS fragment that included the translation initiation site of the gene. The remaining two fragments of pSPM100 were re-ligated in the proper orientation and designated as pSPMS. The BstEII and NotI restriction profiles of pSPM
S were determined and were in agreement with the deletion of the internal xylS fragment. The absence or presence of an active XylS regulatory protein was verified in E. coli cells containing pSPM
S or pSPM100, respectively, by visual detection of the production of an intense brown colour due to chlorocatechol accumulation when these cells were exposed to 3-CB.
Introduction of the tfd and/or xyl gene modules into R. eutropha.
The introduction of the tfd and/or xyl gene modules was performed by using mini-Tn5-derived vectors (de Lorenzo et al., 1990 ) or pBBR1MCS-2, which were transferred by triparental mating with E. coli CC118
pir containing pBBR1MCS-2 (pBBR1M-I, pBBR1M-II), pUT (pR1TFD, pSPM100, pSPM
S) or pBSL202 (pR2TFDG, pR12TFDG) derivatives as donors (Table 1
), E. coli HB101(pRK600) as the helper and R. eutropha JMP134 or JMP222, or derivatives thereof, as recipients. A donor-to-helper-to-recipient ratio of 1:1:2 was used. After incubation, cells were resuspended and the transconjugants were selected on agar plates containing minimal medium supplemented with 3 mM benzoate plus 50 µg kanamycin ml-1 (pUT and pBBR1MCS-2 derivatives), 3 mM benzoate plus 20 µg gentamicin ml-1 (pBSL202 derivatives) or 2 mM 2,4-D plus 40 µg tellurite ml-1 (pSPM100 derivatives). Six to ten derivatives from each mating were analysed for the absence of plasmid DNA, ampicillin sensitivity and stable maintenance ( 20 generations) of kanamycin, gentamicin or tellurite resistance after growth in LuriaBertani medium without a selective marker. To detect the tnp Tn5 IS50R transposase gene, primer pair TNP-1 (5'-CGGCGGCGCTGGGTGATCCT-3') and TNP-2 (5'-GCCCCAGCTGGCAATTCCGG-3') (nucleotides 148167 and 14141433, respectively, of GenBank accession no. U15573) was used (Ahmed & Podemski, 1995
). Failure to detect this gene was an indication that the vector had been lost and that transposition had occurred (de Lorenzo et al., 1990
). The absence of tnp, sensitivity to ampicillin, stable inheritance of kanamycin, gentamicin or tellurite resistance and the absence of the corresponding plasmid indicated that insertion of cloned genes had occurred. Southern analysis with a biotinylated pR1TFD, pR2TFDG or pSPM100 DNA probe was carried out to confirm that the selected derivatives carried single insertions of the corresponding tfd or xyl gene module. All transconjugants showed single insertions of the tfd or xyl genes, as determined by Southern hybridization, indicating successful establishment of the genes in the transconjugants. Southern analysis performed with a pSPM100 probe showed the presence of single insertions of the xyl genes, into either the chromosome or pJP4. To confirm the presence of the chlorocatechol genes in the transconjugants, PCR was performed using the primer pair BDO-1 and VAL-2 (Pérez-Pantoja et al., 2000
) to amplify a 1·8 kb tfdCIDI fragment. The primer pair RBA-1 (5'-CAACGAAATAGCGAAGCTGTCGA-3') and RDB-1 (5'-ATGAGCACGCTGCTCTGATGCTTG-3') (nucleotides 863885 and 11411164, respectively, of GenBank accession no. M98445) was used to amplify a 302 bp fragment of the tfdRtfdDII intergenic region. Conditions for PCR were: denaturation at 95 °C for 2 min, followed by 35 cycles at 95 °C for 45 s, 55 °C for 30 s and 72 °C for 30 s, with a final extension at 72 °C for 10 min.
Detection of a xylS gene homologue in R. eutropha.
Southern hybridization of R. eutropha JMP134 genomic DNA that had been digested with EcoRI was performed using the 725 bp BstEII-generated fragment from xylS as a biotinylated probe. A 684 bp region from an ORF taken from the genomic sequence of Ralstonia metallidurans CH34, encoding a xylS gene homologue, was amplified by PCR using primers FOR130 (5'-TCCTTCAACCGGCTCAGTTA-3') and REV814 (5'-ATGCGTCATTGAGCAGATCC-3'). R. metallidurans CH34 is a strain closely related to R. eutropha JMP134, whose genome sequence is available at the Department of Energy (DOE) Joint Genome Institute (http://www.jgi.doe.gov/JGI_microbial/html/). Conditions for PCR were: denaturation at 94 °C for 5 min, followed by 28 cycles at 94 °C for 30 s, 55 °C for 1 min and 72 °C for 90 s, with a final extension at 72 °C for 10 min. The resulting PCR product was also used as a probe for the R. eutropha JMP134 genomic DNA that had been digested with EcoRI.
Construction of an R. eutropha JMP134 derivative harbouring a tfdFII-inactivated pJP4.
To inactivate the tfdFII gene in pJP4, the 1·0 kb pBSL202 fragment encoding gentamicin resistance was introduced into the MluI sites of the 1·0 kb EcoRINdeI fragment from the tfdFII gene, and the resulting fragment was cloned into pLITMUS. The resulting plasmid was introduced into R. eutropha JMP134 by electroporation; homologous recombination derivatives were selected on the basis of a gentamicin-resistant, ampicillin-sensitive phenotype. The insertion in the tfdFII gene was verified by Southern analysis of plasmid DNA that had been digested with EcoRI, which showed positive hybridization with the gentamicin-resistance probe only in the pJP4 derivative (pJP4FII) DNA fragment that had altered mobility with respect to the wild-type pJP4 DNA. A sevenfold decrease in maleylacetate reductase activity was found in strain JMP134(pJP4
FII), when compared to the wild-type strain.
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RESULTS AND DISCUSSION |
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Evidence for the presence of a putative AraC/XylS family regulator capable of activating xyl gene expression in R. eutropha JMP134
The ability of R. eutropha JMP134::X to grow on 3,5-DCB suggests that this compound acts as an inducer with the XylS regulatory protein and/or that another regulatory system present in R. eutropha replaces XylS, activating transcription of the xyl operon. The first possibility does not seem correct, because it has been reported that 3,5-DCB is unable to activate the regulator in Pseudomonas putida (Ramos et al., 1986 ). Furthermore, 3,5-DCB can also act as an inhibitor of the XylS-mediated induction of the xylXYZL genes in the presence of other substituted benzoates (Ramos et al., 1986
). To test the second possibility (i.e. that another regulatory system replaces XylS), we constructed a pSPM100 derivative that has a 0·7 kb deletion in xylS and, therefore, lacks the possibility of induction mediated by this regulator. When the xylXYZL gene module was introduced into R. eutropha JMP134, the resulting derivative, R. eutropha JMP134::X
S, was still able to grow on 4-CB and 3,5-DCB to the same extent as the strain harbouring the complete xyl gene module. The degradation of CBs in R. eutropha JMP134 derivatives containing or lacking xylS (strains JMP134::X and JMP134::X
S, respectively) was studied in cells grown on fructose and induced with 4-CB or 3,5-DCB (Table 2
). The results showed that XylXYZ activity in these derivatives is due to the acquisition of the xyl genes, because only negligible activity was observed in the wild-type strain. The results also show that XylXYZ activity is inducible by 4-CB or 3,5-DCB and, more significantly, that XylS was not required for such an induction, indicating the presence of another regulatory system in R. eutropha JMP134. When the rate of CB degradation was determined for R. eutropha JMP222 harbouring the xylSxylXYZL module (R. eutropha JMP222::X), essentially the same pattern of degradation was found (Table 2
), indicating that the activity of a putative XylS element in R. eutropha is located in the chromosome and is not part of the pJP4-encoded genes. Therefore, it is possible that in the chromosome of strain JMP134 a regulatory function is responding to CBs and is cross-activating the expression of the xylXYZL module. This kind of cross-regulation has been shown in P. putida for benzoate catabolism, between the pWW0-encoded proteins XylS and BenR, the regulator of the chromosomal benzoate-degradation pathway and both proteins belonging to the AraC/XylS family of regulators (Cowles et al., 2000
; Jeffrey et al., 1992
). To obtain further evidence for the presence of a putative AraC/XylS family regulator similar to XylS from pWW0 in the chromosome of R. eutropha, Southern hybridization of EcoRI-digested JMP134 and JMP222 genomic DNA was performed using the 725 bp BstEII-generated fragment containing part of xylS. No hybridization signal was observed for either derivative, even under low-stringency conditions. Nevertheless, the use of the 684 bp PCR product from a xylS homologue of R. metallidurans CH34 as a probe revealed a single hybridization band of approximately 4 kb in size in both the R. eutropha JMP134 and the JMP222 genomic DNA that had been digested with EcoRI. It is worth mentioning that the putative protein found in the R. metallidurans genome has only 24% nucleotide identity (30% amino-acid identity) with xylS from pWW0. If it is assumed that the gene from R. eutropha JMP134 is closely related to that of R. metallidurans, such low identity would explain the failure to find the xylS sequence in the R. eutropha genome when using xylS from pWW0 as the probe. Taken together, these results strongly suggest that the chromosome of strain JMP134 has a BenR/XylS-like element with an inducer profile different to that reported for XylS from pWW0.
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The tfdFII-encoded maleylacetate reductase plays a major role in the metabolism of 3,5-DCB in R. eutropha JMP134
The experiments carried out to study the effect of the presence of multiple copies of the tfd genes showed an additional difference between the two tfd gene modules. Strain JMP222::X(pBBR1M-I) was unable to grow on 3,5-DCB but strain JMP222::X(pBBR1M-II) was able to grow on this substrate, albeit at lower 3,5-DCB concentrations (Fig. 2b). Such behaviour may be due to the different maleylacetate reductase activities that the two tfd gene modules possess (Pérez-Pantoja et al., 2000
; Plumeier et al., 2002
). When strain JMP222(pBBR1M-II) is grown on 3-CB, it expresses about three times more maleylacetate reductase activity than strain JMP222(pBBR1M-I) (Plumeier et al., 2002
). It is worth mentioning that maleylacetate reductase encoded on the chromosome of strain JMP134 is not expressed during growth on 2,4-D or 3-CB (Padilla et al., 2000
; Plumeier et al., 2002
). Therefore, it is improbable that the chromosomal maleylacetate reductase activity is expressed during growth of strain JMP134 on 4-CB or 3,5-DCB. No significant differences between the activity with maleylacetate and 2-chloromaleylacetate (the intermediates from mono- and dichlorinated substrates, respectively) have been reported for maleylacetate reductases (Padilla et al., 2000
; Pérez-Pantoja et al., 2000
; Seibert et al., 1993
). However, maleylacetate reductase is required twice during the metabolism of dichlorinated compounds, because it converts 2-chloromaleylacetate into maleylacetate, and maleylacetate into 3-oxoadipate (Fig. 1b
). Therefore, if TfdFII is more active than TfdFI, although the activity of these two proteins is not as different as initially reported (Pérez-Pantoja et al., 2000
; Plumeier et al., 2002
), its contribution to (chloro)maleylacetate turnover would be more important. Evidence for a significant role of TfdFII in the metabolism of 3,5-DCB came from the analysis of additional R. eutropha derivatives. When strain JMP222::X harboured pBBR
FII, a pBBR1M-II-derived plasmid lacking the tfdFII-encoded maleylacetate reductase gene, the ability of this strain to grow on 3,5-DCB was completely abolished (data not shown). In contrast, its ability to grow on 4-CB was still present, but the ability to grow on 4-CB decreased to a similar extent in strains JMP222::X(pBBR
FI) and JMP222::X(pBBR
FII) (data not shown). More significantly, when tfdFII was inactivated in pJP4 by the insertion of a gentamicin-resistance gene, the corresponding R. eutropha JMP134 derivative, JMP134::X(pJP4
FII), was unable to grow on 3,5-DCB, but still grew on 4-CB (Fig. 3
). These observations strongly suggest that the presence of TfdFI and/or the chromosomal maleylacetate reductase is not enough to allow growth of R. eutropha JMP134 on 3,5-DCB.
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CONCLUSIONS |
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 12 April 2002;
revised 17 June 2002;
accepted 16 July 2002.