Functional characterization of the pneumococcal Integrative Conjugative Element Tn5253

Integrative conjugative elements (ICEs) are responsible for pneumococcal genome evolution and more particularly for virulence and drug resistance acquisition. During conjugation, ICEs transfer from a donor cell containing the genetic element to a recipient cell lacking it. ICEs insert into the bacterial genome, excise from it and form a covalently closed circular intermediate which can integrate in a different genomic site and/or can integrate in a new genome upon conjugative transfer. Tn5253 is a composite ICE of Streptococcus pneumoniae conferring resistance to tetracycline and chloramphenicol. The complete nucleotide sequence showed that it is 64,528 bp in size and contains 79 ORFs, 41 of which could not be annotated. Two distinct genetic elements were found integrated in Tn5253: Tn5251 (18,033 bp), of the Tn916-Tn1545 family of CTs, and Ωcat(pC194) (7,627 bp), which could not conjugate, but was capable of intracellular mobility by homologous recombination. Ωcat(pC194) was shown to contain a copy of the staphylococcal plasmid pC194 with a 93-bp deletion in the replication origin, to be flanked by two 1,169-bp direct repeats, and to excise from Tn5253 producing a circular form of the element. The highest transfer frequency of Tn5253 was registered when Streptococcus pyogenes was the donor (6.7 x 10-3 transconjugants/donor). Upon conjugal transfer, Tn5253 was found to always integrate at a single chromosomal site. The target site (attB) was a 83-bp sequence in the rbg gene of S. pneumoniae. Strains carrying Tn5253 all contained circular forms of the ICE in which the ends were joined by a 84-bp sequence (attTn). attB and attTn differed only for an extra nucleotide in attTn. When integrated into the chromosome, Tn5253 was flanked by a copy of attB and a copy of attTn: invariably attB was adjacent to the left end and attTn to the right, suggesting a polarization in the DNA integration process. In order to study the mechanism of integration into pneumococcal chromosome, we constructed an attB mutant in which the first 63 nts were deleted and 5 nucleotide changes introduced in the remaining 20 nts. The resulting attB mutant was used as Tn5253 recipient strain in mating experiments. Conjugation frequency of Tn5253 in the attB mutant recipient was considerably lower when compared to a standard recipient (4.8 x 10-7 vs 1.7 x 10-5 transconjugants per donor cell). Five novel Tn5253 alternative insertion sites were found located in: spr1713, coding for an alpha-galactosidase, spr0540 (cell wall synthesis enzyme), spr1534 (substrate binding protein of an ABC sugar transporter), spr1983 (MFS protein) and spr0546 (nrd, putative nitroreductase). Transfer of Tn5253 from alternative attB sites occurred at lower frequencies than wild type donor ranging from 2 x 10-7 to < 3.6 x 10-8. One transconjugant harboring 3 copies of Tn5253 was able to transfer the ICE at a frequency 100-fold higher than wild type donor. Tn5253 has a strong preference for its primary insertion site, even when it is mutated, but it can also integrate at different sites. The insertion site of Tn5253 affects its transposition rate, which is decreased or abolished when integrated in one of the alternative attB.