1 Department of Microbiology, Institute of Biology, University of Bialystok, 15-950 Bialystok, Swierkowa 20 B, Poland; 2 Institut für Tierzucht, Bundesforschungsanstalt für Landwirtschaft (FAL), Höltystr. 10, 31535 Neustadt-Mariensee, Germany
Received 2 March 2005; returned 19 April 2005; revised 29 April 2005; accepted 26 May 2005
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Abstract |
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Methods: Plasmid pSTE2 was transformed into Staphylococcus aureus RN4220. Suitable restriction fragments were cloned into E. coli plasmid vectors and sequenced. In vitro susceptibility testing was performed to confirm the resistance phenotype mediated by this plasmid.
Results: Plasmid pSTE2 consisted of two parts, each of which corresponded closely to previously identified staphylococcal plasmids. The initial 4439 bp represented a pT181-analogous tet(K)-carrying tetracycline resistance plasmid, whereas the remaining 2474 bp represented a pPV141-related erm(C)-carrying macrolide-lincosamide-streptogramin B resistance plasmid. Both putative parental plasmids harboured the staphylococcal recombination site A (RSA) and the pT181-like plasmid also carried the recombinase gene pre whose product acts at RSA. Analysis of the junctions of the pT181-like and the pPV141-like homologous parts in pSTE2 suggested that plasmid pSTE2 developed from pT181- and pPV141-like ancestor plasmids by cointegrate formation at RSA.
Conclusion: Plasmid pSTE2 is the first completely sequenced plasmid from S. lentus and represents the product of an in vivo derived RSA-mediated recombination between two compatible plasmids.
Keywords: site-specific recombination , cointegrate formation , Staphylococcus , recombination sites
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Introduction |
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The aim of this study was to analyse the structure and organization of plasmid pSTE2 to gain insight into the processes that led to its formation.
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Materials and methods |
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Results and discussion |
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The second part of pSTE2 (positions 44406913) exhibited a high degree of similarity to small erm(C)-carrying MLSB resistance plasmids from staphylococci and bacilli with a best match of 98% identity to the 2410 bp plasmid pPV141 from Staphylococcus chromogenes (database accession no. U82607). This segment of pSTE2 contained the erm(C) gene (positions 47815515) which codes for a ribosomal RNA methylase of 244 amino acids. The erm(C) gene was also preceded by a complete translational attenuator which is required for the inducible expression of erm(C).3 In the non-induced state, the original S. lentus strain as well as the S. aureus RN4220::pSTE2 transformant exhibited resistance to the 14-membered macrolide erythromycin, but susceptibility to the non-inducers, e.g. 16-membered macrolides spiramycin and tilmicosin as well as to the lincosamide clindamycin (Table 1). The high MIC (128 mg/L) of tulathromycinwhich is a 1:9 mixture of 13- and 15-membered azalidesindicated that tulathromycin is also an efficient inducer of erm(C) gene expression. When grown in the presence of 1 mg/L erythromycin, the transformant also showed high-level resistance to the 16-membered macrolides and clindamycin (Table 1). This change in the resistance phenotype is indicative of inducible expression of the erm(C) gene.6 An increase in the MIC of erythromycin after induction with erythromycin was not detectable due to the range of concentrations tested. Within the erm(C) regulatory region, a small open reading frame for a 19 amino acid peptide (positions 46624721) and two pairs of inverted repeated sequences were detected. The difference in size between the 2474 bp segment of pSTE2 and the 2410 bp plasmid pPV141 was mainly due to a 58 bp deletion seen in the translational attenuator of the constitutively expressed erm(C) gene of pPV141.3 The repL gene which codes for a 158-amino-acid plasmid replication protein was seen at positions 59636439 and a second RSA site was detected at positions 68906913 in the pSTE2 sequence.
The recombination site RSB is present on many small staphylococcal plasmids.7 RSB-mediated cointegrate formations were seen only after co-transduction of these plasmids and a phage-determined recombination system is believed to play a role in these processes.8 In contrast, RSA is found only rarely on small staphylococcal plasmids and cointegrates were obtained under laboratory conditions with strains carrying different RSA-containing plasmids in the absence of phages.7,9 It has been shown that the plasmid recombination protein Pre mediates a site-specific recombination which involves RSA, but not RSB.9 Heterologous cointegrates between the 4.4 kb tetracycline resistance plasmid pT181 and the 3.7 kb erm(C)-carrying plasmid pE194, both of which carry RSA sites and pre genes, have already been derived in vitro.8,9
In the case of plasmid pSTE2, both partner plasmids had an RSA site, but only the pT181-like partner harboured a pre gene. It is likely that Pre from one such plasmid can mediate cointegrate formation between different co-resident plasmids provided that they have RSA sites. It has been suggested that such heterologous cointegrates might play a role as intermediates in the evolution of larger plasmids carrying multiple antibiotic resistance genes.9 One such model for an RSA-based cointegrate formation between an aadD-encoding kanamycin-neomycin-bleomycin pUB110-like resistance plasmid and a pNS1981-like tet(L)-encoding tetracycline resistance plasmid has been proposed although the intermediate form was not detected.10 Analysis of the sequences flanking the RSA sites in pSTE2 compared with the corresponding sequences in pT181 and pPV141 confirmed that recombination at the RSA sites has led to the cointegrate formation displayed in Figure 1. Moreover, two intact plasmid replication genes, repC and repL, were detected in pSTE2 and their interaction in terms of a coordinated replication and stable maintenance of pSTE2as seen in the original S. lentus as well as in the S. aureus laboratory hostremains to be clarified.
The presence of genes coding for resistance to the two most frequently used classes of antimicrobials in veterinary medicine, tetracyclines and macrolides, on the same plasmid is of relevance with regard to the spread of resistance. Since this plasmid carries two different replication genes, it might be able to replicate in a broader range of bacterial hosts than its parental plasmids. Moreover, a plasmid such as pSTE2 might be able to incorporate other resistance plasmids or parts of them via its RSA and/or RSB sites. Although cointegrates of pT181-like plasmids into larger plasmids via the insertion sequence IS257 have been described,11 this is to the best of our knowledge the first report of a naturally occurring RSA-cointegrate involving tet(K)- and erm(C)-carrying parental plasmids.
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Footnotes |
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Acknowledgements |
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References |
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