The Staphylococcal Insertion Sequence IS257Is Active

PLASMID

34, 198–205 (1995)

The Staphylococcal Insertion Sequence IS257 Is Active C. NEEDHAM,* W. C. NOBLE,†

AND

K. G. H. DYKE*,1

*Microbiology Unit, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom; and †Institute of Dermatology, United Medical and Dental Schools, St. Thomas’s Hospital, London, United Kingdom Received May 17, 1995; Revised August 30, 1995 The plasmid pJ3356 confers high-level mupirocin resistance on a strain of Staphylococcus aureus isolated from a hospital. The plasmid also carries two copies of IS257. Recombination of an IS257-containing plasmid conferring erythromycin resistance, pOX7-IS, into either of the IS257s of pJ3356 has been observed. The co-integration of pJ3356 and a small plasmid, pOX7, is also reported and involves duplication of one of the IS257s from pJ3356 together with 8 bp of pOX7 at the site of integration. Thus IS257 has been shown to be an active mobile genetic element. q 1995 Academic Press, Inc.

Staphylococcus aureus is a pathogen, and clinical isolates are often found to be resistant to many antibiotics; hence infections of S. aureus are sometimes difficult to treat. The mechanisms of accumulation of these resistance determinants are therefore of great interest. Mupirocin is a novel, topical antibiotic which has only been available for clinical use in the UK since 1985 (White et al., 1984) yet resistance to high concentrations of mupirocin (MIC ú512 mg/ml) has been observed (Rahman et al., 1987; Cookson et al., 1990). A single resistance determinant, encoded by mupA, confers this high-level resistance. The gene is usually located on a 4.05-kb EcoRI restriction fragment of plasmids of otherwise great dissimilarity (Rahman et al., 1990; Connolly et al., 1993; Rahman et al., 1993). The pJ3356 family of plasmids which bear mupA (Needham et al., 1994) is the first to be described. The family is believed to have arisen relatively recently and hence provides an opportunity to gain insight into the mechanisms of plasmid evolution. The restriction enzyme maps of this family of plasmids (Fig. 1) show that the members of the family are related by gene duplication and co-integration. 1 To whom reprint requests should be addressed. e-mail: [email protected].

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It has been suggested that the IS257s, which flank the novel sections of plasmid which distinguish the family members, were involved in the formation of these rearrangements. Plasmid pJ3358 is the co-integrate of a pT181like plasmid and a pJ3356D-like plasmid with an extra copy of IS257 (Needham et al., 1994). Restriction enzyme mapping and DNA sequencing of pJ3358 demonstrated that 8 bp of the pT181 sequences adjacent to the IS257s flanking the integrated pT181-like plasmid were duplicated relative to the known sequence of pT181 (Khan and Novick, 1983; Guay et al., 1993; Fig. 5). This implied that IS257 transposition had been involved in the evolution of pJ3358 from a pJ3356D-like plasmid. The 8-bp duplication found in pJ3358 is also present flanking the IS257 of pT181-IS, a 5.2-kb plasmid thought to be a deletion product of pJ3358 and comprising a pT181-like plasmid with a single inserted IS257 (Needham et al., 1994). It is not known whether pT181-IS existed prior to pJ3358. pT181-IS may either have been a requisite precursor to pJ3358 or be merely a deletion product of it. IS257 has been cited as an element appearing in many plasmids and chromosomal sites of S. aureus and coagulase-negative staphylococci and is described as an insertion

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Copyright q 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.

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sequence due to this ubiquitous nature and as its primary structure is that expected from an insertion sequence: a putative transposase sequence flanked by inverted repeats (BarberisMaino et al., 1987; Gillespie et al., 1987). However, transposition of IS257 has not been demonstrated, either independently or as part of a composite transposon. The experiments outlined below were designed to discover whether IS257 is active in transposition. MATERIALS AND METHODS

of Chang and Cohen (1979). Protoplasts were made by incubation in 50 mg lysostaphin/ml at 377C for 30 min. DNA Manipulations Restriction enzymes were obtained from Gibco BRL and used according to the manufacturer’s instructions. DNA fragments were separated by electrophoresis in 0.7–1.0% (w/ v) agarose run in Tris/borate buffer, pH8.0, with appropriate size markers.

Bacterial Strains and Media

DNA Sequencing

The strains used were S. aureus RN4220 (Novick, 1990) and Escherichia coli DH5a (Yanisch-Perron et al., 1985). Strains used routinely were maintained on CY–agar for S. aureus and 2 x TY–agar for Escherichia coli. Long-term storage was at 0707C in broth containing 30% (v/v) glycerol. Strains containing temperature-sensitive plasmids were grown at 307C unless otherwise stated. Plasmid pOX7 (identical to pOX300, Dyke et al., 1991) is a hybrid plasmid containing both pUC18 (Yanisch-Perron et al., 1985) and pE194 sequences (Horinouchi and Weisblum, 1982); it contains the erythromycin resistance determinant erm, is temperature sensitive for replication in S. aureus, and is thus able to replicate at 307C but not at 427C; it also contains the multiple cloning site of pUC18. pJ3356 carries mupA (Needham et al., 1994), and pOX7-IS was constructed in the course of this study.

DNA sequencing was performed using the method of Sanger et al. (1977) with the Sequenase kit (Amersham) according to the manufacturer’s instructions. The following oligonucleotides used as sequencing primers were made by Valerie Cooper (Dyson Perrins Laboratory, Oxford):

Preparation of Plasmid DNA

The possibility of detecting co-integrate formation between pJ3356 and a small plasmid carrying IS257 was investigated. pOX7IS was created by ligating pOX7 linearized with EcoRI to the 1.3-kb EcoRI fragment of pJ3356D, which contains a complete copy of IS257 (Fig. 1). Selection was for transfer of ampicillin resistance to E. coli DH5a. S. aureus RN4220 (pJ3356) was then transformed with pOX7-IS with selection on 20 mg erythromycin/ml at permissive temperature. Transformants were tested for mupirocin resistance by inoculating on agar containing 5

The alkaline lysis method (Sambrook et al., 1989) was used for both small-scale and fullscale cesium chloride preparation of plasmid DNA, except that for S. aureus lysis was for 30 min at 377C with 50 mg lysostaphin/ml and StrataResin (Stratagene) was used to remove proteins. Transformation of S. aureus S. aureus was transformed using at least 250 ng plasmid DNA by the protoplast method

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1. 5*-CAGATCTACGGATTTTCGCC 3* 2. 5*-CGCAAAAGACATAATCG 3* 3. 5*-GGGTTGCCAGAGTTAAAGG 3* Oligonucleotide (1) was used to sequence out of the DNA specifying the C-terminus region of the IS257 transposase of the EcoRI clones of the co-integrates. Oligonucleotides (2) and (3) were used to sequence out of pOX7 into the flanking IS257. RESULTS

Co-integration of Two Plasmids Each Carrying IS257

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FIG. 1. Maps of the pJ3356 family of plasmids aligned by their mupA genes and the map of pT181-IS. Dashed lines indicate the duplication and the integration events. B, BglII; E, EcoRI; Hp, HpaII; and N, NcoI.

mg mupirocin/ml. Twenty colonies which grew on both the erythromycin and the mupirocin-containing agar were tested for the presence of the two plasmids, pJ3356 and pOX7IS, by restriction enzyme mapping of isolated plasmid DNA. Two of these heteroplasmid isolates, RN4220 (pJ3356/pOX7-IS), were selected and cultures were grown overnight at 307C in broth containing 20 mg erythromycin/ ml. The overnight culture was diluted 10-fold and grown to mid-log phase when known quantities of cells were plated onto agar plates containing 40 mg erythromycin/ml. The plates were incubated at both permissive and restrictive temperatures for 20 h, and then the number of colonies on each plate was counted. The number of colonies at the restrictive temperature was between 8.4 1 1003 and 1.01 1 1002 of those that grew at 307C. That colonies that grew at restrictive temperature in agar containing selective concentrations of erythromycin indicated that the pOX7-based plas-

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mids had integrated into temperature-stable replicons, presumably either the chromosome or a plasmid. DNA was prepared from 20 colonies. The colonies grown at 307C contained both plasmid types (pJ3356 and pOX7-IS), giving restriction enzyme fragments like the original heteroplasmid strains. DNA made from 10 colonies taken from plates incubated at restrictive temperature gave EcoRI and BglII restriction patterns containing the same fragments as the original heteroplasmid strain, but all fragments were equimolar, indicating that only one plasmid was present (Fig. 2, lanes 6, 7, 12, and 13). Further mapping demonstrated that pOX7-IS and pJ3356 had co-integrated and that the point of co-integration was the IS257 of pOX7-IS and either of the two IS257s of pJ3356; maps of the two plasmid types found are given in Fig. 3. There are no XhoI sites in pOX7-IS and two in pJ3356 and so the use of this enzyme allowed the determi-

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FIG. 2. Photograph of agarose gel of plasmids and co-integrates.

FIG. 3. Maps of pOX7-IS, pJ3356, and Type I and Type II co-integrates. B, BglII; E, EcoRI; and X, XhoI. The sites for Bc, BclI; C, ClaI; and P, PstI are shown only for within the pOX7 sequences.

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nation of which of the two IS257s had been used. The frequency of co-integration into each IS257 is similar: pOX7-IS had integrated into the small XhoI fragment of pJ3356 in 13 of 28 colonies tested and into the large XhoI fragment in the other 15. It was concluded that recombination between the IS257s on each plasmid had generated the two types of co-integrate plasmids observed, pJ3356::pOX7-IS:I and II. That there was no duplication of either the IS257 or 8 bp of pOX7-IS (as determined by DNA sequencing from primers designed to sequence the junctions of the IS257s with the flanking sequences—data not shown) indicated that the method of co-integrate formation was likely to be different from that which had formed pJ3358 in vivo where the pT181like element is flanked by direct repeats. Co-integration of Plasmids Only One of Which Carried IS257 As it had been possible to generate the cointegrate plasmids pJ3356::pOX7-IS: I and II in the laboratory, the possibility of creating a co-integrate from two plasmids only one of which contained a copy of IS257 was investigated. pOX7 was used to transform RN4220 (pJ3356) as for pOX7-IS. Two examples of a heteroplasmid strain, RN4220 (pJ3356/ pOX7), were selected. These were treated as described for RN4220 (pJ3356/pOX7-IS) strains. The number of colonies on the plates grown at the restrictive temperature was ca. 7 1 1003 of those on the plates at 307C. The BglII restriction maps of plasmids found in eight colonies taken from plates inoculated with RN4220 (pJ3356/pOX7) and incubated at restrictive temperature were identical to those from the similarly treated RN4220 (pJ3356/pOX7-IS); therefore the co-integrate plasmids in the colonies able to grow at restrictive temperature contained an extra BglII site compared to the original starting plasmids (Fig. 4). Indeed further restriction enzyme mapping demonstrated that each colony contained one plasmid which was the co-integrate of pJ3356 with pOX7 but the IS257 had dupli-

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cated relative to the original plasmid. In all the RN4220(pJ3356::pOX7) co-integrates, the additional IS257 flanked the inserted pOX7 plasmid and was in direct repeat with the original IS257s of pJ3356. XhoI restriction enzyme analysis of 26 co-integrates showed that pJ3356 had co-integrated into pOX7 at either of the IS257s of pJ3356 with equal preference: 13 in each. The orientation of pOX7 relative to pJ3356 in the co-integrates varied: of 7 tested, 3 were in one orientation and 4 in the opposite orientation; also the site of co-integration within the pOX7 varied, showing no obvious preference of insertion site within the nonessential sequences. For example, one site was within the amp gene at coordinate 1.4 kb from the EcoRI site. Other sites of insertion given in kilobases from the EcoRI site and proceeding into pUC18 first were 0.6, 0.8, 2.5, 2.6, 2.9, and 3.9. The regions of DNA adjacent to the IS257s flanking the inserted pOX7 were sequenced for two of the co-integrates, and 8 bp of the pOX7 sequence was shown to have duplicated in direct repeat at the point of integration. These two 8-bp repeats were different in the two cases (Fig. 5) and different from the 8 bp of pT181 duplicated in pJ3358. Thus for both the RN4220(pJ3356::pOX7-IS) co-integrates and the RN4220(pJ3356::pOX7) co-integrates, the inserted pOX7 plasmid was flanked by direct repeats of IS257: the original pOX7-IS plasmid had provided this extra IS257 for the RN4220(pJ3356::pOX7-IS) cointegrates, but in the case of the RN4220 (pJ3356::pOX7) co-integrates, duplication of one of the IS257s of pJ3356 must have occurred. DISCUSSION

The co-integrate of pJ3356D-type and pT181-IS-type plasmids, pJ3358, had been recovered from hospital isolates of S. aureus (Needham et al., 1994). IS257s occurred at the site of integration in direct repeat and with 8 bp of the pT181-type plasmid duplicated in direct repeat (Fig. 1), suggesting that the cointegration had occurred, at least in part, as a result of IS257 transposition. The use of a

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FIG. 4. Maps of pOX7, pJ3356, and three examples of pJ3356::pOX7 co-integrates. Symbols are the same as for Fig. 3 and N, NcoI.

pOX7 clone of IS257, pOX7-IS, demonstrated that under appropriate selection conditions in a recombination proficient host, two plasmids containing IS257s can recombine to form a plasmid co-integrate with the IS257s in direct repeat at the junctions. It was not possible to test the role of the host recombination system since no usable rec0 strain was available. Plasmid pOX7, with no homology to pJ3356,

formed co-integrates with pJ3356 at a frequency similar to that of pOX7-IS. Thus cointegration can occur between two plasmids when only one of these plasmids initially contains a copy of IS257. Two sites of specific recombination are known in the pE194 section of pOX7 (Novick et al., 1984) but neither of these was used as the site of co-integration in the various examples studied. It is suggested

FIG. 5. DNA sequences at junctions between pT181 (uppercase) and IS257 (lowercase) in pJ3358 and between pOX7 (uppercase) and IS257 in two examples of pJ3356::pOX7. The 8-bp direct repeats are shown in bold. GenBank Accession Nos. U36910 (pJ3358); U36911 (pJ3356::pOX7;1); U36912 (pJ3356::pOX7;3).

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that the transposase of IS257 catalyzes both the recombination and the transposition activities observed; this is further supported by the fact that the frequency of replicon fusion (ca. 1 1 1003) is much greater than that which would be expected from homologous recombination alone (1 1 1006 to 1 1 1009), which could otherwise explain the co-integration of pOX7-IS with pJ3356. IS257s have been cited in many plasmid and chromosomal locations and are usually found in direct repeat relative to one another. Previously described plasmid families which are related by insertions/deletions flanked by IS257s include the pSK41 family of plasmids (Byrne et al., 1991), which contain the putative transposon Tn4003 (Rouch et al., 1989) in which direct repeats of IS257 flank the trimethroprim resistance determinant, dfrA. The mercury resistance determinants, merA and merB, are also found flanked by direct repeats of IS257 (Gillespie et al., 1987). The aminoglycoside resistance plasmid pUB110 was shown as a co-integrate in a larger plasmid, pSK41 (Byrne et al., 1991); other co-integrates in which the comprising replicons are flanked by IS257s include both pUB110 and pT181 in the mec region of the chromosome of S. aureus (Dubin et al., 1991). These integrated smaller plasmids were all found to have 8-bp direct repeats at the junctions of the small plasmid with the flanking IS257s (Stewart et al., 1994). The sequences of the 8-bp duplications have been shown to be dissimilar (Stewart et al., 1994); the 8-bp sequences duplicated in the experiments reported here differ not only from each other but also from those previously described, further supporting the claim that the target site of transposition of IS257 is nonspecific. Although such previous examples had indicated that IS257 had at one time been active for transposition, leading to the formation of the rearrangements observed, transposition had never been demonstrated. The use of the temperature-sensitive replication plasmid pOX7 in the studies described above has allowed direct demonstration of IS257 transposition. The rearrangement observed between

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pOX7 and pJ3356 was one of replicon fusion: a mechanism predicted to have generated many of the rearrangements in the families cited above. It is of interest to note that the closely related insertion sequence IS15 has been shown to generate co-integrates and that these are suggested to be exclusive end products of the transposition reaction (Trieu-Cuot and Courvalin, 1985). Other homologous insertion sequences also produce co-integrates as products of transposition, e.g., IS26 (Iida et al., 1984) and IS46 (Brown et al., 1984). Rouch and Skurray (1989) have suggested that these structurally similar plasmids represent a superfamily of insertion sequences; the results presented above suggest that this association may also apply to their function and preferred product of transposition: the co-integrate. ACKNOWLEDGMENT C.N. was the recipient of a SERC-CASE award in conjunction with SmithKline Beecham.

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B., AND PHILLIPS, I. (1990). Diversity of staphylococci exhibiting high-level resistance to mupirocin. J. Med. Microbiol. 33, 97–100. RAHMAN, M., NOBLE, W. C., AND DYKE, K. G. H. (1993). Probes for the study of mupirocin resistance in staphylococci. J. Med. Microbiol. 39, 446–449. ROUCH, D. A., AND SKURRAY, R. A. (1989). IS257 from Staphylococcus aureus: Member of an insertion sequence superfamily prevalent among Gram-positive and Gram-negative bacteria. Gene 76, 195–205. ROUCH, D. A., MESSEROTTI, L. J., LOO, L. S. L., JACKSON, C. A., AND SKURRAY, R. A. (1989). Trimethoprim resistance transposon Tn4003 from Staphylococcus aureus encodes genes for a dihydrofolate reductase and thymidylate synthetase flanked by three copies of IS25 7. Mol. Microbiol. 3, 161–175. SAMBROOK, J., FRITSCH, E. F., AND MANIATIS, T. (1989). ‘‘Molecular Cloning: A Laboratory Manual,’’ 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. SANGER, F., NICKLEN, S., AND COULSON, A. R. (1977). DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74, 5463–5467. STEWART, P. R., DUBIN, D. T., CHIKRAMANE, S. G., INGLIS, B., MATTHEWS, P. R., AND POSTON, S. M. (1994). IS257 and small plasmid insertions in the mec region of the chromosome of Staphylococcus aureus. Plasmid 31, 12–20. TRIEU-CUOT, P., AND COURVALIN, P. (1985). Transposition behaviour of IS15 and its progenitor IS15D: Are co-integrates exclusive end products? Plasmid 14, 80– 89. WHITE, A. R., BEALE, A. S., BOON, R. J., GRIFFIN, K. E., MASTERS, P. J., AND SUTHERLAND, R. (1984). Antibacterial activity of mupirocin. In ‘‘Bactroban (mupirocin): Proceedings of an International Symposium’’ (Current Clinical Practice Series 16) (R. L. DOBSON, J. J. LEYDEN, W. C. NOBLE, AND J. D. PRICE, Eds.), pp. 19– 36. Excerpta Medica, Amsterdam. YANISCH-PERRON, C., VIEIRA, J., AND MESSING, J. (1985). Improved M13 phage cloning vectors and host strains: Nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33, 103–108. Communicated by S. A. Khan

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