Laboratorio de Microbiología, Departamento de Genética Molecular y Microbiología, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Alameda 340, 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: chloroaromatics, catabolic plasmid, IS1071 insertion sequence, chlorocatechol pathway
Abbreviations: 3-CB, 3-chlorobenzoate; 2,4-D, 2,4-dichlorophenoxyacetate
The GenBank accession numbers for the 3115 nt BamHI-F and 2833 nt EcoRI-F fragments of pJP4 and the 4037 nt EcoRI-E' fragment of pJP4-F3 are AF225972, AF225973 and AF225974, respectively.
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INTRODUCTION |
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METHODS |
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DNA manipulation.
Screening for plasmid pJP4 was carried out by the procedure described by Kado & Liu (1981) . Plasmid DNA was visualized after electrophoresis on agarose gel (0·8%, w/v, in electrophoresis running buffer containing 40 mM Tris/acetate and 1 mM EDTA, pH 8·0) for 5 h at a constant voltage of 70 V. pJP4 DNA suitable for restriction and Southern analysis was prepared from E. coli XL-1 Blue derivatives obtained after conjugative transfer of the wild-type plasmid or its derivative, pJP4-F3, from R. eutropha JMP134 (Clément et al., 2000
), using the Spin Miniprep Kit (Qiagen). Total cellular DNA preparations were made using the Wizard genomic DNA purification kit (Promega). Restriction, ligation and dephosphorylation reactions and the electroporation of DNA were performed by standard procedures (Ausubel et al., 1992
).
Southern blots of pJP4 DNA fragments (obtained after digestion with HindIII, EcoRI or BamHI and separation on a 0·7% agarose gel) were made using Hybond N+ membranes (Amersham Pharmacia Biotech). Probes were labelled using the BioNick labelling system and hybridization was detected with the PhotoGene nucleic acid detection system (Gibco-BRL), as recommended by the supplier. An 801 bp StyI fragment of the tfdA gene was used as a probe (Holben et al., 1992 ). The remaining probes were prepared from the PCR amplification products cloned in pGem-T Easy (Promega). The IS1071-AB and IS1071-AC probes were obtained with primer pairs IS1071-A (GGGGTCTCCTCGTTTTCAGTGCAA) and IS1071-B (CTTTGAGATATAAAGCTTGCAGCT), and IS1071-A and IS1071-C (GATCCAGAAAGCTGCCAGTTGAAG), respectively (Xia et al., 1998
). Detection of the intergenic region between tfdA and tfdS was carried out by PCR amplification using primer pairs PR33 (GCCGCGCTATTTCTGTCCTTTCCCG, base pairs 9841008; GenBank accession no. S80112) and RC3 (TCGACCCCTGCGGCG, base pairs 783797; GenBank accession no. M16730). The conditions for the PCR were as follows: 95 °C for 5 min, 52 °C for 1·5 min and 72 °C for 3·5 min; 32 cycles of 95 °C for 1·5 min, 55 °C for 1 min and 72 °C for 3 min; and finally 72 °C for 10 min. The final concentrations were 0·5 pM for the primers, 250 µM for the dNTPs, 2 mM MgCl2 and 0·5 U Taq polymerase per 10 µl reaction. PCR amplification for the generation of IS1071 probes was performed as described by Xia et al. (1998)
. The reactions were carried out in a MiniCycler (MJ Research).
Sequence analysis.
Subcloning of fragments for sequencing was carried out in pBlueScript II SK(+/-). Nucleotide sequencing of both DNA strands was carried out using a PRISM sequencing kit (Perkin-Elmer) with double-stranded DNA templates in the presence of 5% DMSO. Samples containing fluorescence-labelled dideoxynucleotide terminators were processed on a 373 stretch automated sequencer (Applied Biosystems). Sequences were compiled and analysed using DNASTAR software (DNAstar). The computational resource of the National Centre of Biotechnology Information was used through the BLASTX software facilities.
Enzyme assays.
R. eutropha JMP134 and its derivatives were grown for 24 h on liquid minimal medium containing 2 mM 2,4-D or 3-CB. A 100 ml aliquot of each culture was harvested at the end of the exponential growth phase by centrifugation for 15 min at 7000 r.p.m. in a Beckman J2-21 centrifuge, washed twice in minimal medium and resuspended in 5 ml 50 mM Tris/acetate, pH 7·5. Cells were disrupted by sonication (Sonics & Materials) four times for 30 s at 90% of the maximum output, and the soluble protein fraction was obtained after 1 h centrifugation at 130000 g in a Beckman L-80 ultracentrifuge. Cell extracts (0·15·0 mg protein ml1) were assayed without further purification. Chlorocatechol 1,2-dioxygenase activity was assayed and quantified as described elsewhere (Pérez-Pantoja et al., 2000 ). Protein determinations were performed as described by Bradford (1976)
.
Nucleotide sequence accession numbers.
The GenBank accession numbers for the 3115 nt BamHI-F and 2833 nt EcoRI-F fragments of pJP4 and the 4037 nt EcoRI-E' fragment of pJP4-F3 are AF225972, AF225973 and AF225974, respectively.
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RESULTS |
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Determination of the ends of the large inverted repeat in pJP4-F3
As mentioned above, a deletion starting in the pJP4 fragment EcoRI-E, spanning fragments EcoRI-C, -H and -I, and ending in the fragment EcoRI-F, occurred to yield pJP4-F3 (for orientation, see double-headed solid arrow in Fig. 2a). In addition, the observation that the EcoRI insert in pEF102 is an inverted repeat shows that a duplication spanning at least the tfdR, tfdDII and tfdCII genes had occurred. This duplication may be significantly longer and may exceed the length of the deletion since pJP4-F3 is larger than the parent pJP4 plasmid. Thus, the duplication may possibly span both the tfdCIDIEIFI module and the tfdDIICIIEIIFII module (see double-headed solid arrow in Fig. 2b
). Besides EcoRI-C', the fragment in which the duplication/deletion starts, the only other new EcoRI fragment visible after pJP4-F3 digestion is EcoRI-E'. Therefore, it may be assumed that the second duplication/deletion junction lies within this fragment (Fig. 2b
). Correspondingly, this junction should also lie within the HindIII-D' fragment of pJP4-F3 (Fig. 2b
). The HindIII-D' fragment from pJP4-F3 was cloned in pBlueScript to give pHF64 (Fig. 2b
), and was used as a probe against pJP4 and pJP4-F3 plasmid DNA digested with HindIII, EcoRI and BamHI. Wild-type pJP4 EcoRI-F and HindIII-D fragments, containing sequences that should be partially deleted in pJP4-F3, hybridized with the probe, suggesting that HindIII-D' contains the deletion/duplication end-point (Fig. 2b
). Hybridization was also observed with the wild-type BamHI-A, BamHI-D and EcoRI-A fragments, indicating that sequences within fragments EcoRI-F and EcoRI-A have some homology and might flank the large inverted repeat (Fig. 2a
). Thus, it can be assumed that the duplication occurring in pJP4-F3 encompasses part of the wild-type EcoRI-E, all of the EcoRI-G and -B fragments and part of the -A fragments, and that the duplication end localized in the EcoRI-A fragment joins the deletion end localized in the EcoRI-F fragment, forming the new HindIII-D' and EcoRI-E' fragments (Fig. 2a
, b
).
To verify this assumption, the HindIII-D fragment of pJP4 was cloned into pBlueScript, to give pH60 (Fig. 2a), and the HindIII-D' fragment of pJP4-F3 was cloned to produce pHF64 (Fig. 2b
). EcoRI subclones from pH60 and pHF64 were prepared in pBlueScript to give pHE30 and pHEF41 (Fig. 2a
, b
). The BamHI-D fragment from the pJP4 plasmid with homology to the HindIII-D' fragment (Fig. 2b
) was also cloned in pBlueScript to give pB54 (Fig. 2a
). This plasmid was digested with HindIII and the smallest fragment (0·6 kb) was also cloned in pBlueScript to give pBH06 (Fig. 2a
). Cloned fragments in plasmids pHE30, pHEF41 and pBH06 were sequenced (GenBank accession nos AF225973 and AF225974 for the first two clones, respectively). Subclones used for the sequencing of pHEF41 and pHE30 are shown in Fig. 2(c)
. As is depicted in Fig. 2(d)
, 2208 bp from the EcoRI-end of pHEF41 (part of the HindIII-D' fragment) are identical to those of pHE30 (part of the HindIII-D fragment); 660 bp at the HindIII-end of pHEF41 are identical in sequence to pBH06 (part of the BamHI-D fragment). The
1·2 kb of pHEF41 between these regions of homology does not match with either of these sequences, and should correspond to the BamHI-A-fragment end, next to the BamHI-D fragment in the wild-type (see Fig. 2a
).
A search for sequence homologies in pHE30 and pHEF41 was conducted. A BLASTX analysis of the sequence of pHE30 (Fig. 2d) yielded 99% identity for merA (mercury reductase) of Tn501 (Brown et al., 1983
), 99% identity with merD from Pseudomonas stutzeri (Reniero et al., 1998
), 99% identity with ORF2 of the mercury-resistance transposon from P. stutzeri (Reniero et al., 1998
), and 100% identity with tnpA of IS1071 in Tn5271 (Nakatsu et al., 1991
). Since IS1071 has been localized in catabolic transposons and has been involved in DNA rearrangements, the presence of IS1071 sequences in pJP4 and pJP4-F3 was further studied by Southern analysis. Probes IS1071-AB and IS1071-AC, which correspond to the left and right halves of IS1071, respectively (Xia et al., 1998
), gave the same hybridization pattern. The EcoRI-A, -F, -H and -I fragments from wild-type pJP4, and the EcoRI-A' and -E' fragments from pJP4-F3 hybridized with the IS1071 probe. Fragments BamHI-A and -B, HindIII-A, -D, -E and -H from pJP4, and BamHI-A', BamHI-C', HindIII-A' and -D' from pJP4-F3 also hybridized (see maps in Fig. 2a
, b
for orientation). This evidence, along with sequence data, strongly suggests that there is at least one copy of an IS1071 in pJP4. As the EcoRI-A, HindIII-A and BamHI-A fragments of pJP4 also hybridized with both IS1071 probes, it can be suggested that an IS1071-like element, or part of IS1071, is also present.
During the rearrangement, the part of the IS1071 sequence (containing two EcoRI recognition sites) that corresponds to fragments EcoRI-H, -I and -F was deleted (Fig. 2a). As a consequence, primer IS1071-AC could not anneal; this meant that it was no longer possible to amplify this sequence with primers IS1071A/C. The difference between the wild-type and rearranged plasmids was used to screen isolated colonies, and to quantify the rearrangement frequency (see above).
The data indicate that pJP4-F3 is the result of a duplication event involving a23 kb section that starts in the tfdS gene or the tfdAtfdS intergenic region belonging to the tfdR/tfdS small inverted repeat, spans both the tfdCIDIEIFI module and the tfdDIICIIEIIFII module, and ends 1176 bp into the BamHI-A fragment. Plasmid pJP4 also underwent a deletion that starts in the tfdS gene or the tfdAtfdS intergenic region and ends 637 bp (corresponding to the hatched region of pHE30 shown in Fig. 2d
) into the wild-type EcoRI-F fragment, leaving only the right portion of the IS1071. The absence of tfdA explains the inability of strain JMP134-F3 to grow on 2,4-D. On the other hand, the duplication that forms a 51-kb-long inverted repeat containing two copies of both tfdCD(DC)EF gene clusters is responsible for the increased TfdC activity, and supports the improved growth of this strain on 3-CB.
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DISCUSSION |
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The simplest explanation for the rearrangement reported in pJP4-F3 is a double crossover, homologous recombination between two pJP4 molecules (Fig. 3). A single crossover occurs between the tfdR sequence from one pJP4 molecule and the identical, but opposite, tfdS sequence from the second pJP4 molecule. The second crossover takes place between the IS1071 sequence from one pJP4 molecule and the IS1071-like sequence from the other pJP4 molecule. This recombination gives rise to two rearranged plasmids (Fig. 3
): the first, pJP4-F3, is reported here, while the other one, lacking most of the tfd genes, is selected against during growth in the presence of 3-CB.
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The search for ORFs in the sequences of pJP4 reported here revealed the presence of determinants for mercury resistance. Since DNA hybridization analyses of pJP4 with probes for Tn501 mer genes mapped these determinants to the EcoRI-F and EcoRI-D fragments, it has been proposed that the resistance to mercury in pJP4 is related to the presence of a Tn501 element (Burlage et al., 1990 ; Smith & Thomas, 1987
). Our results show that such ORFs are effectively present in this region of the pJP4 plasmid. The high level of homology suggests that these ORFs are actively involved in the resistance to mercury.
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ACKNOWLEDGEMENTS |
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Received 16 November 2000;
revised 16 March 2001;
accepted 3 April 2001.