Abstract
The integrative conjugative element (ICE) Tn5253 of Streptococcus pneumoniae, conferring resistance to tetracycline and chloramphenicol, was found integrated at a 83-bp specific target site (attB) located in the rbgA gene of the pneumococcal chromosome. PCR analysis of Tn5253-carrying strains showed evidence of precise excision of Tn5253 from the pneumococcal chromosome with production of (i) circular forms of the ICE in which the ends were joined by a 84-bp sequence (attTn), and (ii) reconstituted chromosomal attB. When integrated into the chromosome, Tn5253 was flanked by attL, identical to attB, and attR, identical to attTn. Circular forms of Tn5253 were present at a concentration of 3.8 × 10-4 copies per chromosome, whereas reconstituted attB sites were at 3.0 × 10-4 copies per chromosome. Deletion of int-xis of Tn5253 abolished production of circular forms (<7.1 × 10-6 copies per chromosome) and was associated to the lack of Tn5253 conjugal transfer suggesting, as expected, that Tn5253 circular form acts as a conjugation intermediate.
Highlights
Horizontal gene transfer, mediated by mobile genetic element (MGE), significantly drives bacterial genome evolution including the acquisition and dissemination of new patterns of antibiotic resistance (Burrus and Waldor, 2004)
Recombination processes of ICEs are catalyzed by Abbreviations: CI: circular intermediate; CDS: coding sequence; CT: conjugative transposon; HGT: horizontal gene transfer; ICE: integrative conjugative element; MGE: mobile genetic element
PCR analysis of cell lysates of Tn5253-carrying pneumococcal strains showed evidence of precise excision of Tn5253 from its specific attachment site in the pneumococcal chromosome. This excision was investigated in liquid cultures of BM6001, the clinical isolate in which Tn5253 was originally found, and four other Tn5253-carrying laboratory strains all deriving from classic type 2 D39 (Table 1)
Summary
Horizontal gene transfer, mediated by MGEs, significantly drives bacterial genome evolution including the acquisition and dissemination of new patterns of antibiotic resistance (Burrus and Waldor, 2004). Deletion of Tn5251 int and xis CDS (orf21 and orf22 of Tn5253) was obtained with a mutagenic construct containing the ami/aad9 spectinomycin resistance cassette flanked at the left by a 496-bp DNA fragment and at the right by a 569-bp fragment corresponding to nucleotides 16,810–17,305 and 18,780–19,348 of Tn5253 (GenBank EU351020), respectively.
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