Abstract
BackgroundTn5253, 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 (attB), (ii) produce circular forms joined by a 84-bp sequence (attTn), and (iii) restore the chromosomal integration site. The purpose of this study is to functionally characterize the attB in S. pneumoniae strains with different genetic backgrounds and in other bacterial species, and to investigate the presence of Tn5253 attB site into bacterial genomes.ResultsAnalysis of representative Tn5253-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) Tn5253 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 attB site corresponds to length of the duplication after Tn5253 integration, (iv) attB duplication restores rbgA CDS, (v) Tn5253 produced circular forms containing the attTn 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 attB sites occurred at 3.7 × 10−5 to 1.7 × 10−2 copies per chromosome. A database search of complete microbial genomes using Tn5253 attB as a probe showed that (i) thirteen attB variants were present in the 85 complete pneumococcal genomes, (ii) in 75 pneumococcal genomes (88.3 %), the attB site was 83 or 84 nucleotides in length, while in 10 (11.7 %) it was 41 nucleotides, (iii) in other 19 bacterial species attB 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 attB sites, is conserved among the different bacterial species and can be considered the core of the Tn5253 integration site.ConclusionsA functional characterization of the Tn5253 attB integration site combined with genome analysis contributed to elucidating the potential of Tn5253 horizontal gene transfer among different bacterial species.
Highlights
The acquisition of new genetic material by horizontal gene transfer (HGT) significantly drives bacterial genome evolution and is mediated by Mobile Genetic Elements (MGEs)
In order to contribute to mobilome characterization, we first conducted a functional characterization of the Tn5253 integration site, by analyzing attB in Tn5253-carrying transconjugants obtained in S. pneumoniae strains with different genetic backgrounds and in strains belonging to other bacterial species
DNA sequence analysis of Tn5253-chromosome junction fragments showed that: (i) Tn5253 integration occurred at a specific integration site located in rbgA gene of the pneumococcal chromosome [26], (ii) attL was identical to attB and (iii) attR was identical to attTn, as already described for D39 and its derivative strains [26], and that (iv) attB sites among these pneumococcal strains were not identical, with their size varying from 41 nucleotides to 83 nucleotides (Fig. 1)
Summary
The acquisition of new genetic material by horizontal gene transfer (HGT) significantly drives bacterial genome evolution and is mediated by Mobile Genetic Elements (MGEs). We previously characterized Tn5253, a 64,528-bp composite ICE of Streptococcus pneumoniae, containing the ICE Tn5251 of the Tn916-Tn1545 family and the Ωcat(pC194) element carrying tet(M) and cat resistance determinants, respectively [27,28,29]. Tn5253, 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 (attB), (ii) produce circular forms joined by a 84-bp sequence (attTn), and (iii) restore the chromosomal integration site. The purpose of this study is to functionally characterize the attB in S. pneumoniae strains with different genetic backgrounds and in other bacterial species, and to investigate the presence of Tn5253 attB site into bacterial genomes
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