Department of Microbiology, Eastman Dental Institute for Oral Health Care Sciences, University College London, University of London, 256 Grays Inn Road, London WC1X 8LD, UK1
Bacterial Pathogenesis Research Group, Department of Microbiology, Monash University, Vic 3800, Australia2
Author for correspondence: Peter Mullany. Tel: +44 20 7915 1223. Fax: +44 20 7915 1127. e-mail: p.mullany{at}eastman.ucl.ac.uk
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ABSTRACT |
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Keywords: conjugative transposons, gene transfer, antibiotic resistance, mobile elements
The GenBank accession numbers for the sequences in this paper are AF333235 (Tn5397) and AF329848 [part of CW459tet(M)].
a These authors contributed equally to the work.
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INTRODUCTION |
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Conjugative transposition of members of the Tn916 family involves excision from the donor genome, circularization of the element, and then transfer to a new host and subsequent insertion into a new target site. Transposition requires the products of the transposon-encoded int and xis genes, which encode Int, a site-specific recombinase of the integrase family, and Xis, respectively (Poyart-Salmeron et al., 1990 ). Both Int and Xis are essential for excision but only Int is required for integration (Senghas et al., 1988
; Poyart-Salmeron et al., 1990
; Lu & Churchward, 1995
; Rudy et al., 1997
; Marra & Scott, 1999
). Int-mediated excision of Tn916 is similar to the excision of
prophage DNA as it utilizes a mechanism that involves staggered cuts at the ends of the element, circularization, and subsequent transfer to a new host. However, the recombination sites of
are homologous, unlike those of Tn916 (Caparon & Scott, 1989
).
Members of the Tn916 family also encode their own conjugative transfer. Analysis of the complete DNA sequence of Tn916 reveals open reading frames (ORFs) that encode putative polypeptides with sequence similarity to proteins known to be involved in conjugation (Flannagan et al., 1994 ). For example, Orf18 has similarity to the anti-restriction protein Ard of plasmid Collb-P9 and Orf23 is related to the MbeA mobilization protein of plasmid ColE1. A functional oriT site is also present between orf21 and orf20 (Jaworski & Clewell, 1995
).
Work in our laboratories has concentrated on determining the molecular basis for tetracycline resistance in the anaerobic pathogens Clostridium difficile and Clostridium perfringens. In C. difficile, resistance is encoded by the conjugative transposon Tn5397, which mediates transfer between C. difficile strains and to and from Bacillus subtilis (Mullany et al., 1990 ). DNA hybridization analysis and partial DNA sequence analysis have shown that Tn5397 contains the tet(M) gene and is closely related to Tn916. However, Tn5397 differs from Tn916 in that it contains a Group II intron inserted into orf14 (Mullany et al., 1996
) and has different sequences at its ends (Wang et al., 2000a
).
In C. perfringens, tetracycline resistance is the most common antimicrobial resistance phenotype (Lyras & Rood, 1996 ). In most strains, this resistance is non-transferable; however, conjugative transfer of tetracycline resistance is not uncommon (Rood, 1983
; Abraham et al., 1985
). Transfer is always associated with large conjugative plasmids that are either identical to, or closely related to, the prototype R-plasmid, pCW3 (Abraham et al., 1985
; Abraham & Rood, 1985
). pCW3 carries the well-characterized tetracycline-resistance determinant Tet(P), which comprises two tetracycline-resistance genes, tetA(P) and tetB(P) (Sloan et al., 1992
). The tetA(P) gene encodes a 46 kDa protein that mediates active efflux of tetracycline from the cell. The tetB(P) gene encodes a putative 72·6 kDa protein that has significant similarity to Tet(M)-like tetracycline-resistance proteins.
Hybridization and PCR analysis of a large number of conjugative and non-conjugative tetracycline-resistant C. perfringens isolates has shown that they all carry the tetA(P) gene. Most (93%) of these isolates carry a second tetracycline-resistance gene, with 53% carrying a tetB(P) gene and 40% carrying a tet(M)-like gene (Lyras & Rood, 1996 ). No isolates have been detected that carry both the tetB(P) and the tet(M)-like genes. Conjugative transfer of the latter gene has not been demonstrated in C. perfringens.
In this paper, we compare the complete nucleotide sequences of Tn5397, Tn916 and the partial sequence of a tet(M)-like element from C. perfringens strain CW459. The results show that all three elements are closely related but have different excision modules. The comparison of these three elements provides valuable insights into the evolution and dissemination of conjugative transposons in Gram-positive bacteria.
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METHODS |
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Molecular techniques.
Plasmid DNA was isolated from E. coli using the Qiagen Miniprep kit or the High Pure Plasmid Isolation Kit (Boehringer Mannheim) in accordance with the manufacturers instructions or using a modified small-scale mini alkaline/lysis/PEG precipitation procedure (Applied Biosystems). Genomic DNA was prepared from C. difficile by using the Puregene Gram-positive genomic DNA isolation kit (Flowgen) and from C. perfringens using dye-buoyant gradient ultracentrifugation at 260000 g for 20 h at 20 °C (Abraham & Rood, 1985 ).
A genomic library of C. perfringens strain CW459 (Rood et al., 1978a ) was constructed by digesting purified chromosomal DNA with PstI and ligating it to PstI-digested pSU39 (Bartolome et al., 1991
). After overnight ligation at 14 °C, the DNA was introduced into rubidium chloride competent E. coli DH5
cells (Sambrook et al., 1989
). The library was screened on media containing tetracycline (10 µg ml-1). Recombinant clones that were tetracycline resistant were screened for minocycline resistance (10 µg ml-1). The recombinant plasmid pJIR1470 was found to confer resistance to both tetracycline and minocycline.
DNA sequencing and analysis.
DNA sequencing was carried out using the ABI Prism Big Dye Terminator Cycle Sequencing Ready Reaction Kit on an Applied Biosystems Perkin Elmer 310 Genetic Analyser or an Applied Biosystems 373 DNA Sequencer. DNA sequence analysis was carried out using the DNASIS (Hitachi), NCBI tools (http://www.ncbi.nlm.nih.gov/), European Bioinformatics Institute tools (http://www.ebi.ac.uk/), Seqed (Applied Biosystems), Sequencer 3.0 Software (Gene Codes Corporation) and GeneJockey (Biosoft) programs.
Dot blot hybridization analysis.
Plasmid DNA from E. coli and chromosomal DNA from C. perfringens and C. difficile were applied to Hybond-N+ nylon membranes (Amersham) without prior treatment (Sambrook et al., 1989 ) using a dot blot apparatus (Minifold SRC 96; Schleicher & Schuell). Analysis of dot blots was performed using the DIG DNA Labelling and Detection Kit (Boehringer Mannheim) in accordance with the manufacturers instructions. Hybridization was carried out at 65 °C in 5xSSC (0·75 M NaCl, 0·075 M sodium citrate pH 7·0) with subsequent washes of 2x15 min at 65 °C in 0·1xSSC, 0·1% (w/v) SDS. Digoxigenin-11-dUTP-labelled probes were amplified by PCR from Tn916 template DNA and spanned the length of the Tn916 transposon (Fig. 1
).
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RESULTS AND DISCUSSION |
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Comparison of the overall genetic organization of Tn5397, Tn916 and CW459tet(M)
Previous studies involved the sequence analysis of the first 328 bp of the left end and the last 2442 bp of the right end of Tn5397 (Wang et al., 2000a ). The remaining sequence of Tn5397 was determined on both strands by primer walking on the plasmids pPPM5.3 and pPPM1.6 (Mullany et al., 1996
). Gaps in the sequence were filled by designing PCR primers based on the Tn916 sequence (GenBank accession no. U09422) and the unpublished C. difficile sequence (http://www.sanger.ac.uk/Projects/C_difficile/). To avoid PCR-induced errors, each PCR product was amplified and sequenced at least three times. The results showed that Tn5397 was 20658 bp in length (GenBank accession no. AF333235) and had 21 potential ORFs that had a suitably spaced ribosome-binding site upstream of the putative start codon. The ORF within the Group II intron (Mullany et al., 1996
) and tndX (Wang et al., 2000a
; Wang & Mullany, 2000
) have been described previously. Seventeen of the ORFs were very similar to corresponding ORFs from Tn916 (Flannagan et al., 1994
) and were labelled accordingly (Fig. 2
).
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In Tn916, the int and xis genes, the products of which are essential for excision/insertion, are located to the right of orf5 (Fig. 2). In the clostridial elements, these genes have been replaced with other genes that encode different site-specific recombinases. Database searches with the putative protein encoded at the right end of the CW459tet(M) element revealed 2128% identity to several bacteriophage or transposon-encoded integrases from Gram-positive bacteria (GenBank accession nos AAA85500, AAC48867, A69774 and C55205) and 17·5% identity to Int from Tn916. Therefore, the CW459-derived ORF was designated int459 (integrase CW459). A comparison of Int459 with other members of the integrase family showed that the conserved integrase motif (Abremski & Hoess, 1992
) was present in the putative Int459 protein. This protein may have played a role in the transposition and introduction of the CW459tet(M) element into CW459. However, since conjugative transfer of the CW459tet(M) element is not observed, it appears that either this protein is not produced, is not functional, or is not sufficient on its own for transposition. As observed for the transposition of Tn916 (Jaworski et al., 1996
; Marra & Scott, 1999
), the excision, and therefore transposition, of the CW459tet(M) element may require additional proteins, such as an excisionase, which are not present in this element.
An additional 170 bp incomplete ORF was identified at the right of the sequenced CW459tet(M) region and was designated gmp because the deduced protein product had 70% identity over 47 amino acids to a GMP synthetase protein from B. subtilis (Mantsala & Zalkin, 1992 ). Since this enzyme is a housekeeping protein we concluded that it was outside the CW459tet(M) element. Therefore, the right terminus of the element appeared to be located within the 67 bp region between int459 and gmp (Fig. 2
). Although this region contained several repeat sequences, it did not have any similarity to the ends of Tn916, Tn5397 or any other known conjugative transposons.
Downstream of orf8 in Tn5397 (Fig. 2) is the tndX gene, which encodes a 61·5 kDa protein, TndX (Wang et al., 2000a
), that has 37% identity to TnpX, a large resolvase from the C. perfringens transposon Tn4451 (Bannam et al., 1995
) and the C. difficile transposon Tn4453a (Lyras & Rood, 2000
). Functional analysis has shown that TndX is involved in the excision and integration of Tn5397 in C. difficile. The tndX gene ends 10 bp before the right terminus of Tn5397 (Wang et al., 2000a
; Wang & Mullany, 2000
).
The Tn5397 and CW459tet(M) elements do not have the tet(M) leader peptide orf12
Just upstream of the tet(M) gene in Tn916 there is a small ORF, orf12, which encodes a tet(M) leader peptide that is involved in the regulation of tet(M) expression by a transcriptional attenuation mechanism (Su et al., 1992 ; Celli & Trieu-Cuot, 1998
). In both Tn5397 and the CW459tet(M) element there has been a deletion of all or part of orf12. In Tn5397, the 88 bp deletion is associated with a CCCAGT direct repeat (Fig. 3
). In the CW459tet(M) element, there is a deletion of 155 bp, which is associated with a CCTTTT direct repeat (Fig. 3
). Therefore, both deletions appear to have arisen by homologous recombination. CW459 expresses tetracycline resistance constitutively (J. Rood, unpublished results), but also carries a second tetracycline-resistance gene, tetA(P). Strain 630 is inducible but the mechanism of induction of tetracycline resistance is not known (A. Roberts & P. Mullany, unpublished results). However, although orf12 is partially deleted, this region still contains an inverted repeat (
G=-61·7 kJ mol-1) that has similarity to the repeats which in Tn916 form the upstream transcriptional terminator.
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In both Tn5397 and the CW459tet(M) element there was an intergenic region after orf8, followed by the remnants of orf5. In both elements, the N-terminal encoding region of orf5 is missing. In Tn916, the orf8orf5 intergenic region contains the promoter region for the xis and int genes (Celli & Trieu-Cuot, 1998 ). The intergenic region in the CW459tet(M) element was similar in size and nucleotide sequence identity (79%) to the equivalent region of Tn916. The xisint promoter region was present but there were two single base changes. Two regions of dyad symmetry, which have some similarity to the imperfect inverted repeats palorf8a and palorf8b from Tn916, were present. These repeats are believed to be involved in transcriptional termination from the upstream orf7 promoter (Celli & Trieu-Cuot, 1998
). In contrast, the smaller orf8orf5 intergenic region of Tn5397 has sequence identity (74%) to only part of the equivalent Tn916 region. In Tn5397, a major deletion has occurred at the beginning of this region. This deletion effectively removes the promoter area of the missing xis and int genes and the palorf8 repeats. To the right of the remainder of orf5 the sequence of the clostridial elements diverges dramatically as already discussed.
Tn5397 contains homologues of the Tn916 conjugation genes
As previously described, the 180 bp at the left end of Tn5397 are not related to Tn916 (Wang et al., 2000a ). The first seven nucleotides of orf24 are also missing. Following this region, the two transposons are very closely related (Fig. 2
) although several insertions and deletions are present in Tn5397. The deduced amino acid sequences of Orfs 2316 have 90100% identity to their Tn916 homologues; Orf15 has 80% identity. Following orf21 is a 392 bp intergenic region which contains a sequence that is homologous to the functional oriT region of Tn916 (Jaworski & Clewell, 1995
). Within the oriT sites of Tn916 and the F plasmid there is a conserved nick site, 5'-TGGTGTGG-3'. This site is identical in Tn5397.
Preliminary sequence analysis of the CW459tet(M) element indicated that sequences with similarity to much of the region encompassing the conjugation genes of Tn916 appear to be present. However, its exact genetic organization and precise relationship to the equivalent region in Tn916 remain to be determined. The conserved oriT nick site is present.
The first ORF of the double-stranded sequenced region of the CW459tet(M) element, orf15, was only partially sequenced. However, the amino acid sequence of the deduced protein had only 21% identity to a corresponding 288-amino-acid region of Orf15 from Tn916 (Flannagan et al., 1994 ) and 23·9% identity to a corresponding region of Orf15 from Tn5397. By comparison with Tn916, Orf15 from both Tn5397 and the CW459tet(M) element contained C-terminal truncations of 25 and 57 amino acids, respectively. In Tn5397, the next ORF, orf14, is disrupted by a Group II intron (Mullany et al., 1996
). Neither the CW459tet(M) element nor Tn916 contains a Group II intron in orf14 (Fig. 2
). Comparative analysis of the deduced amino acid sequences showed that Orf14 (minus the Group II intron) from Tn5397 is most closely related to Orf14 from Tn916.
Evolution of Tn5397 and the CW459tet(M) element
Both Tn5397 and the CW459tet(M) element appear to consist of four distinct modules that may have originated from separate mobile genetic elements. The first module is common although not identical and consists of the Tn916-like conjugation region (orf23orf13), which is present in Tn5397 and most probably in the CW459tet(M) element. The resistance determinant comprises the second module, with the tet(M) genes of Tn916 and CW459 being very closely related and distinct from that of Tn5397, which is more closely related to the tet(M) gene from Tn1545 (Fig. 4). The third module comprises the putative regulatory region (orf12orf8). The regulatory and resistance modules may overlap, as orf12 is involved in sensing tetracycline and consequently regulating tet(M) expression. The fourth major module is responsible for the excision and insertion of the transposons or their precursors and consists of xis and int in Tn916, int459 in CW459tet(M) and tndX in Tn5397. Therefore, the three elements have different excision and integration systems.
Tn916-like sequences have also been found in other mobile genetic elements. The tet(M) genes of the large conjugative plasmids of Neisseria gonorrhoeae and Neisseria meningitidis are associated with Tn916-like sequences that encompass regions upstream and downstream of the resistance determinant (Swartley et al., 1993 ). Other studies involving the non-conjugative multiple antibiotic resistance plasmid pK214 from L. lactis, which carries resistance determinants for streptomycin, tetracycline and chloramphenicol, have revealed that the tetracycline-resistance gene, tet(S), is linked to sequences with similarity to orf7, orf9 and orf6, which have a similar genetic organization to that observed in Tn916. This Tn916-like region is linked to distinct gene regions from three other bacterial species as well as to numerous insertion sequences (Perreten et al., 1997
; Teuber et al., 1999
). Finally, the integrase from a lysogenic Mycoplasma arthriditis bacteriophage has greater similarity to Int from Tn916 than to any other integrase (Voelker & Dybvig, 1998
), suggesting a common evolutionary origin.
There are several hypotheses that may explain the evolution of the non-functional CW459tet(M) element. Firstly, it is possible that a modular conjugative transposon may have been initially formed by recombination events between different mobile genetic elements in another bacterium and then subsequently transferred to C. perfringens. Upon insertion into the chromosome, deletion or rearrangement events may have occurred which resulted in the loss of mobility of the transposon. Similar events, resulting in deletions of sections of Tn916, have been observed in Neisseria (Swartley et al., 1993 ). Altered Tn916-like elements, which are no longer capable of conjugative transfer, have also been detected in the non-conjugative plasmid pKQ1, from Enterococcus faecium (Fletcher & Daneo-Moore, 1992
). Secondly, a composite element may have been formed in C. perfringens strain CW459 by a recombination event between a previously acquired, ancestral Tn916 or Tn916-like transposon and a second mobile genetic element, which carried an Int459-based excision/integration system. Alternatively, the recombination event may have involved a resident element containing int459 and an incoming Tn916-like transposon. Note that the conjugative transposition of Tn916 into the chromosome of C. perfringens from both E. coli and Enterococcus faecalis has been observed (Kaufmann et al., 1996
). Tn916 has also been shown to transfer into C. difficile from B. subtilis (Mullany et al., 1991
) and natural isolates of C. difficile that carry Tn916 have recently been identified (Wang et al., 2000b
).
Although direct transfer of genetic information between C. perfringens and C. difficile has not been demonstrated, closely related transposons have been detected in both species, indicating that genetic exchange whether directly, or indirectly through another bacterial host, can occur (Lyras et al., 1998 ). The TndX protein, which is involved in the excision and integration of Tn5397, is more closely related to the TnpX resolvase that is responsible for the movement of the C. perfringens transposon Tn4451 and the closely related C. difficile Tn4453 transposons than to any other resolvase (Wang et al., 2000a
; Lyras & Rood, 2000
). It has been proposed that one of these elements may have been involved in recombination events with an ancestral Tn916-like element that resulted in the formation of Tn5397 (Wang et al., 2000a
).
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ACKNOWLEDGEMENTS |
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Received 4 January 2001;
revised 2 February 2001;
accepted 5 February 2001.