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Background  Tn 5253 , a composite Integrative Conjugative Element (ICE) of  Streptococcus pneumoniae  carrying  tet (M) and  cat  resistance determinants, was found to (i) integrate at specific 83-bp integration site ( att B), (ii) produce circular forms joined by a 84-bp sequence ( att Tn), and (iii) restore the chromosomal integration site. The purpose of this study is to functionally characterize the  att B in  S. pneumoniae  strains with different genetic backgrounds and in other bacterial species, and to investigate the presence of Tn 5253 att B site into bacterial genomes.    Results  Analysis of representative Tn 5253 -carryng transconjugants obtained in  S. pneumoniae  strains with different genetic backgrounds and in other bacterial species, namely  Streptococcus agalactiae ,  Streptococcus gordonii ,  Streptococcus pyogenes , and  Enterococcus faecalis  showed that: (i) Tn 5253  integrates in  rbgA  of  S. pneumoniae  and in orthologous  rbgA  genes of other bacterial species, (ii) integration occurs always downstream of a 11-bp sequence conserved among streptococcal and enterococcal hosts, (iii) length of the  att B site corresponds to length of the duplication after Tn 5253  integration, (iv)  att B duplication restores  rbgA  CDS, (v) Tn 5253  produced circular forms containing the  att Tn site at a concentration ranging between 2.0 × 10 −5  to 1.2 × 10 −2  copies per chromosome depending on bacterial species and strain, (vi) reconstitution of  att B sites occurred at 3.7 × 10 −5  to 1.7 × 10 −2  copies per chromosome. A database search of complete microbial genomes using Tn 5253 att B as a probe showed that (i) thirteen  att B variants were present in the 85 complete pneumococcal genomes, (ii) in 75 pneumococcal genomes (88.3 %), the  att B site was 83 or 84 nucleotides in length, while in 10 (11.7 %) it was 41 nucleotides, (iii) in other 19 bacterial species  att B was located in orthologous  rbgA  genes and its size ranged between 17 and 84 nucleotides, (iv) the 11-bp sequence, which correspond to the last 11 nucleotides of  att B sites, is conserved among the different bacterial species and can be considered the core of the Tn 5253  integration site.    Conclusions  A functional characterization of the Tn 5253 att B integration site combined with genome analysis contributed to elucidating the potential of Tn 5253  horizontal gene transfer among different bacterial species.    Supplementary Information  The online version contains supplementary material available at 10.1186/s13100-021-00253-z.

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Chromosomal integration of Tn5253 occurs downstream of a conserved 11-bp sequence of the rbgA gene in Streptococcus pneumoniae and in all the other known hosts of this integrative conjugative element (ICE)