Unité des Agents Antibactériens1 and Laboratoire de Génomique des Micro-organismes Pathogènes2, Institut Pasteur, 75724 Paris Cedex 15, France
Author for correspondence: Marc Galimand. Tel: +33 1 45 68 83 18. Fax: +33 1 45 68 83 19. e-mail: galimand{at}pasteur.fr
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ABSTRACT |
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Keywords: Tn1549, glycopeptide resistance, VanB phenotype
The GenBank accession number for the 33803 bp sequence of Tn1549 is AJ192329.
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INTRODUCTION |
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Glycopeptides inhibit peptidoglycan synthesis by binding to the C-terminal dipeptide D-alanyl-D-alanine of the pentapeptide precursor, blocking polymerization of peptidoglycan (Reynolds, 1989 ). Resistance to vancomycin and teicoplanin is due to incorporation of D-alanyl-D-lactate (VanA-, VanB- and VanD-type) or of D-alanyl-D-serine (VanC- and VanE-type) into peptidoglycan precursors that have reduced affinity for glycopeptides (Arthur et al., 1996
; Périchon et al., 1997
; Fines et al., 1999
).
Transfer of antimicrobial resistance determinants among enterococci may occur via transposons or plasmids. The enterococcal conjugative transposons, of which Tn916 and Tn1545 have been the most intensively studied, range in size from 16 kb to more than 50 kb and usually confer tetracycline resistance (Clewell et al., 1995 ). Insertion of transposons into self-transferable plasmids, such as pheromone plasmids pAD1 or pCF10 (Clewell, 1993
), is also an important mechanism for the dissemination of resistance genes among enterococci.
The vanB operon has been described in most cases as part of large conjugative elements integrated into the host chromosome (Quintiliani & Courvalin, 1994 ). These elements were found to contain transposons conferring resistance to vancomycin, such as Tn1547 (Quintiliani & Courvalin, 1996
) or Tn5382 (Carias et al., 1998
). Vancomycin-resistant E. faecalis E93/268 and E. faecium 654 were isolated in 1994 in the United Kingdom. Resistance in these strains was associated with the presence of the vanB gene, borne by conjugative plasmids (Woodford et al., 1995a
, b
). Here we report the characterization of the 34 kb transposon Tn1549 responsible for vancomycin resistance.
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METHODS |
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Recombinant DNA techniques.
DNA isolation, cleavage of DNA with restriction endonucleases (Amersham), purification of DNA fragments from agarose gel, dephosphorylation of vector DNA with calf intestine phosphatase and ligation with T4 DNA ligase (Boehringer Mannheim) were performed by standard methods (Ausubel et al., 1992 ). Gene internal fragments used as probes were labelled with [
-32P]ATP (Amersham) by nick translation using a commercially available kit (Amersham). With Escherichia coli strains JM83 (Yanisch-Perron et al., 1985
) and INV
F' (Invitrogen), the following antibiotics were used: ampicillin (100 mg l-1) for cloning restriction fragments into pUC18 (Vieira & Messing, 1982
) and kanamycin (50 mg l-1) for cloning PCR products into pCR2.1 (Invitrogen).
Plasmid construction.
The plasmids were constructed as follows (Fig. 1
|
pAT772.
To amplify a fragment internal to the xis-Tn1549 and int-Tn1549 genes, two primers, T1 and T2 (Table 2, Fig. 1b
), were designed from the sequence of xis-VB and int-VB from Tn5382 (Carias et al., 1998
) and used with plasmid DNA from strain 268-10 as a template. A PCR product with the 1·4 kb expected size was obtained and cloned into pCR2.1.
|
pAT774.
To amplify the left junction fragment, two primers, TnOUT and P1 (Table 2, Fig. 1a
), were designed from the sequence of Tn5382 (Carias et al., 1998
) and of the traE1 gene (GenBank accession no. M87836) of pAD1, respectively. These primers used with plasmid DNA from strain 268-10 as a template yielded a product with the 250 bp expected size, which was cloned into pCR2.1.
pAT775.
Amplification of the joined extremities of the Tn1549 circular intermediate was obtained by nested PCR (Manganelli et al., 1995 ). Primers T3 and T4 (Table 2
, Fig. 1
) were used with total DNA from strain 268-10 as a template in a first PCR and the product obtained was used as a template in the second PCR using primers TnOUT and TnVAN OUT (Table 2
, Fig. 1
). The 309 bp amplification product was cloned into pCR2.1.
pAT776.
The region upstream from the vanRB gene was obtained by inverted PCR (Triglia et al., 1988 ) using primers VB15 and VB36 (Table 2
, Fig. 1b
) (Evers, 1995
) and plasmid DNA from strain 268-10 digested by SspI as a template. The 900 bp PCR product obtained was cloned into pCR2.1.
pAT777.
A strategy similar to that used for construction of pAT773 was followed to clone the right end of Tn1549 from strain 654-6. A fragment internal to int-Tn1549 (Fig. 1b) was used as a probe. Plasmid pAT777 consisted of a 650 bp TaqI insert of pIP835 DNA from strain 654-6 cloned into pUC18.
Nucleotide sequencing.
The inserts in the recombinant plasmids were sequenced by the dideoxynucleotide chain-termination method (Sanger et al., 1977 ) using universal or specific oligodeoxynucleotides as primers, [35S]dCTP
S (Amersham) and the Sequenase version 2.0 DNA sequencing kit (Amersham Life Science). The sequence of the long-PCR product was determined by the shotgun cloning method (Buchrieser et al., 1999
) using an automatic DNA sequencer (model ABI PRISM 377, Perkin Elmer).
Computer analysis.
The sequence was assembled using the PHRED (Ewing & Green, 1998 ; Ewing et al., 1998
) and PHRAP (P. Green, unpublished) programs. CONSED (Gordon et al., 1998
) was used for the editing. Determination of similarity with known proteins involved interrogation of BLASTN, BLASTX and BLASTP (Altschul et al., 1997
) and FASTA (Pearson & Lipman, 1988
) from the Genetics Computer Group (GCG) suite of programs.
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RESULTS |
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PCR mapping with primers (VB, Fig. 1b) complementary to the vanB operon from E. faecalis V583 (Evers, 1995
) showed that the order of the plasmid-borne vanB gene cluster was identical in the two plasmids and similar to that in the chromosome of strain V583; no large insertions or deletions in the noncoding regions were detected. No PCR products were obtained when one of the primers used for amplification was complementary to the region upstream from vanRB or downstream from vanXB. Thus, the genomic environment of the vanB cluster in strains 268-10 and 654-6 differed from that in V583.
The complete nucleotide sequence of the vanB operon in pIP834 was determined. The deduced amino acid sequences of VanRB, VanSB, VanYB, VanW, VanHB and VanXB from pIP834 displayed 96, 96·2, 100, 93, 93·9 and 99% identity with those of the corresponding proteins from V583. The vanB genes from pIP834 and from pIP835 were identical to each other, and were 96·4% similar with that of V583 (Evers, 1995 ) and 99% similar with vanB2 from E. faecalis SF300 (Gold et al., 1993
). Moreover, in the 309 bp region spanning the 175 bp vanSBvanYB intergenic region and adjacent coding sequences of pIP834, the 11 point mutations and the 5 bp deletion described in the vanB2 operon (Dahl et al., 1999
) were also found. These data indicate that resistance to vancomycin in E. faecalis 268-10 and 654-6 was of the vanB2 genotype.
Sequence analysis of the 5·16 kb insert of pAT771 revealed, downstream from vanXB, the presence of two ORFs in the same orientation and the 5' portion of a third ORF. These ORFs were almost identical to orf7-VB, orf8-VB and the 5' end of xis-VB of transposon Tn5382 (Carias et al., 1998 ) (Fig. 1b
). These ORFs are also present with the same organization in Tn916 (Clewell et al., 1995
). Sequencing of the PCR products obtained using primers complementary to the insert of pAT771 and pIP835 DNA as a template indicated an identical gene organization in pIP835 (data not shown). These observations suggest that the vanB operon from pIP834 and pIP835 was part of a transposon designated Tn1549.
Right extremity of Tn1549
Since the gene organization in plasmid pIP834 was identical to that in Tn5382, two primers, T1 and T2 (Table 2, Fig. 1b
), were designed to amplify a fragment internal to the xis-Tn1549 and int-Tn1549 genes and to construct pAT772. Sequence analysis of the insert in pAT772 indicated the presence of genes for an excisase and an integrase which had 98% and 99·9% identity with xis-VB and int-VB from Tn5382, respectively, and 74% and 67% identity with xis-Tn and int-Tn from Tn916-Tn1545. The three conserved residues in domain II (arginine, histidine and tyrosine) of the integrase family of site-specific recombinases (Poyart-Salmeron et al., 1989
) were found (data not shown). To recover the right extremity of Tn1549, the 2·2 kb insert of pAT773 that hybridized with the int-Tn1549 probe was se- quenced. Sequence analysis showed the presence of the 3' end of the int-Tn1549 gene, the right extremity of Tn1549 with a copy of the imperfect inverted repeat of 11 bp identical to that in Tn5382. This 11 bp repeat was found to be adjacent to the 3' part of a gene identical to traE1 (Fig. 2a
) of plasmid pAD1 (Clewell, 1993
). Downstream from traE1, which encodes a positive regulator of the conjugative transfer of pAD1 (Clewell, 1993
), orfy and the 5' extremity of sea1, a gene encoding a segregation protein of pAD1, were present (data not shown) (Clewell, 1993
). A similar approach was used with pIP835 DNA. Sequencing of a 650 bp TaqI insert of pAT777 that hybridized with the int-Tn1549 probe indicated that the right extremity of Tn1549 has disrupted the 3' part of a gene nearly identical to uvrB from plasmid pAD1 (data not shown), which encodes an ultraviolet-resistance protein (Ozawa et al., 1997
). These data indicate that Tn1549 was integrated at different loci of plasmids related to the pheromone-responsive plasmid pAD1.
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Transposition of Tn916-like transposons involves the formation of a nonreplicative circular intermediate (Poyart-Salmeron et al., 1990 ). This was studied for Tn1549 by amplification with primers designed to direct polymerization outward from the ends of the transposon. Sequence analysis of the 309 bp amplification product cloned to form pAT775 confirmed that this fragment corresponded to the joint region (data not shown). This result indicates that Tn1549 can excise and generate a circular intermediate.
Left extremity of Tn1549
A similar PCR strategy was used to complete the sequence of the left part of the transposon. First, the region upstream from the vanRB gene in strain 268-10 was obtained by sequencing the 560 bp insert in pAT776 in which no ORF was found (Fig. 1a). Secondly, two primers, XL1 and XL2, complementary to the left end of Tn1549 and to the region upstream from vanRB (Table 2
, Fig. 1a
) allowed amplification of a 24 kb PCR product from both strains. Restriction profiles of these PCR products obtained after digestion by HincII, SspI or XmnI were identical (data not shown). The 24 kb amplification product obtained from 268-10 DNA was purified and sequenced after shotgun cloning (Buchrieser et al., 1999
).
The mean G+C content of the left part of the transposon (nucleotides 124046) (Fig. 1a) was 55·4 mol%, significantly higher than that reported for Enterococcus chromosomal DNA (38%) or the plasmid traE1 gene (40·7 mol%). Eighteen ORFs were identified by GCG program analysis, with sizes ranging from 213 to 3921 bp (Table 3
). ORFs 13 to 26 were in the same orientation, whereas ORFs 27 to 30 were in the opposite orientation (Fig. 1a
). The start codon of ORFs 16, 20, 23, 24 and 29 overlapped with the end of the previous ORF. Two putative terminator sequences with perfect 8- and 16-base inverted repeats, respectively, were found between ORFs 24 and 25, and between ORFs 26 and 27 (Fig. 1a
). Near-consensus ribosome-binding sites were found for all ORFs (Table 3
), indicating that they were probably coding sequences. The deduced amino acid sequences were compared to those present in the non-redundant protein database at the National Center for Biotechnology Information Web site using the BLASTP program (Table 4
). Eight deduced sequences showed a significant degree of identity with known proteins.
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ORF18 was 39% identical to the MunI methyltransferase of Mycoplasma sp., which recognizes the double-stranded sequence CAATTG and protects DNA from cleavage by MunI endonuclease (Siksnys et al., 1994 ).
ORF20 presented 27% identity with part of the TrsE protein involved in the conjugative transfer of S. aureus plasmid pGO1 (Morton et al., 1993 ).
ORF21 was 45% identical to ORF14 of Tn916 (Flannagan et al., 1994 ).
ORF24 was 38% identical to DNA topoisomerase III from Bacillus subtilis, which catalyses the conversion of one isomer of DNA to another (Beloin et al., 1997 ).
ORF25 presented 27% identity with a portion of the LtrC-like protein involved in conjugation of plasmid pSK41 from S. aureus (Berg et al., 1998 ).
ORF27 was 28% identical to ORF8 of transposon Tn916, which regulates transcription of the orf7, orf8, xis, int and tra genes (Celli & Trieu-Cuot, 1998 ).
ORF28 was 32% identical to the Rlx protein of S. aureus, which is probably required for relaxation complex formation and plasmid mobilization by conjugative plasmids (Projan et al., 1985 ).
In order to detect related transposons in other organisms, we also performed searches for similar sequences in unfinished microbial genomes. Both BLASTX using the sequence of the Tn1549 and TBLASTN using the predicted amino acid sequence of the 30 proteins as query sequences were carried out. Very similar sequences were identified in E. faecalis (www.tigr.org/) and Clostridium difficile (www.sanger.ac.uk) (Table 4). The vanB operon was found almost identical only in the Enterococcus sequence, as expected from the genotype of the two strains, E. faecalis V583 at TIGR and E. faecalis 268-10, studied. However the vanB operon of V583 did not appear to be linked to sequences in Tn1549.
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DISCUSSION |
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Molecular characterization of the vancomycin-resistance determinant and of its flanking regions indicated that the vanB operon was carried by the 34 kb long transposon Tn1549 (Fig. 1). Sequence comparison of both ends revealed that Tn1549 was highly similar to Tn5382 (Carias et al., 1998
). Analysis of the sequence of the target revealed that, in pIP834, the transposon was inserted into the traE1 gene of the pheromone conjugative plasmid pAD1 (Fig. 2
). The TraE1 protein activates its own transcription from its own promoter, as well as transcription of other genes involved in regulation of the pheromone response (Pontius & Clewell, 1992
). It has been reported that inactivation of traE1 abolishes plasmid transfer (Pontius & Clewell, 1992
). However, transfer of vancomycin resistance between E. faecalis 268-10 and BM4110 occurred at a low frequency (4·4 x10-6). Conjugative transfer of pIP834 could be cis-complemented by proteins encoded by the left part of the transposon, since TrsK and TrsE are involved in conjugation of plasmids from Gram-positive bacteria. Alternatively, transcription of the genes involved in the pheromone response could originate in the right part of the transposon with reading through the attachment site (Celli & Trieu-Cuot, 1998
). By contrast, in pIP835, Tn1549 was inserted into the uvrB gene of pAD1. In this case, conjugal transfer of vancomycin resistance occurred at high frequency (4x10-2) similar to that (10-3 to 10-1) of pAD1 (Pontius & Clewell, 1992
).
In its right extremity, Tn1549 was nearly identical to Tn5382 (Carias et al., 1998 ) and exhibited significant identity to the excisase (74%) and the integrase (67%) genes of transposon Tn916. Interestingly, a 309 bp joint region was amplified by nested PCR (Manganelli et al., 1995
) using total DNA of 268-10 DNA as a template. This result indicated that like Tn5382, Tn1549 was capable of excision to form a circular intermediate.
Tn1549 contained 30 ORFs and appeared to be organized into three distinct functional regions like Tn916: (i) the right end, corresponding to the small HindIII fragment of Tn916 implicated in the excisionintegration process (Celli & Trieu-Cuot, 1998 ) must be functional; (ii) the central portion, in which the vanB2 operon responsible for vancomycin resistance was found instead of the tet(M) gene for tetracycline resistance in Tn916; and (iii) the left extremity, corresponding approximately to the large HindIII fragment of Tn916 (Celli & Trieu-Cuot, 1998
), in which 8 of the 18 identified ORFs could be implicated in the conjugative transfer. The 55·4 mol% G+C content of the left 24 kb of the transposon was significantly different from those of the vanB operon (49 mol%), of the right portion (38 mol%) and of the enterococcal (38 mol%) and C. difficile (2530 mol%) genomes. This indicates that the origin of the left end of the transposon is different from that of the two other functional regions. Analysis of partial genome sequences of E. faecalis and C. difficile indicated that a similar modular organization was present in both species.
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ACKNOWLEDGEMENTS |
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Received 10 February 2000;
revised 21 March 2000;
accepted 24 March 2000.