Abstract
DNA sequencing of whole bacterial genomes has revealed that the entire set of mobile genes (mobilome) represents as much as 25% of the bacterial genome. Despite the huge availability of sequence data, the functional analysis of the mobile genetic elements (MGEs) is rarely reported. Therefore, established laboratory protocols are needed to investigate the biology of this important part of the bacterial genome. Conjugation is a mechanism of horizontal gene transfer which allows the exchange of MGEs among strains of the same or different bacterial species. In streptococci and enterococci, integrative and conjugative elements (ICEs) represent a large part of the mobilome. Here, we describe an efficient and easy-to-perform plate mating protocol for in vitro conjugative transfer of ICEs in streptococci (Streptococcus pneumoniae, Streptococcus agalactiae, Streptococcus gordonii, Streptococcus pyogenes), Enterococcus faecalis, and Bacillus subtilis. Conjugative transfer is carried out on solid media and selection of transconjugants is performed with a multilayer plating. This protocol allows the transfer of large genetic elements with a size up to 81 kb, and a transfer frequency up to 6.7 × 10−3 transconjugants/donor cells.
Highlights
The three major mechanisms of horizontal gene transfer in bacteria are conjugation, transformation, and transduction
We developed a successful plate mating protocol for in vitro transfer of large integrative and conjugative elements (ICEs) with a size up to 81 kb in streptococci (Streptococcus pneumoniae, Streptococcus agalactiae, Streptococcus gordonii, Streptococcus pyogenes), Enterococcus faecalis, and Bacillus subtilis
Tn5253 was successfully transferred from the representative transconjugant FR58 to S. pneumoniae strains with different capsular types, S. pyogenes, S. gordonii, S. agalactiae, and transferred back from representative transconjugants of each bacterial species to S. pneumoniae
Summary
The three major mechanisms of horizontal gene transfer in bacteria are conjugation, transformation, and transduction. Conjugation enables the genetic exchange of MGEs, which provide a major contribution to the spread of antimicrobial resistance and virulence, by recruiting new resistance and virulence genes and facilitating their dissemination [2]. The nature of the mobile elements and their transfer mechanisms have been clarified only in few cases [4,5,6,7]. To elucidate transfer mechanisms and their regulation it is essential to develop an established protocol for efficient conjugal transfer of ICEs from encapsulated clinical bacterial strains. We developed a successful plate mating protocol for in vitro transfer of large ICEs with a size up to 81 kb in streptococci (Streptococcus pneumoniae, Streptococcus agalactiae, Streptococcus gordonii, Streptococcus pyogenes), Enterococcus faecalis, and Bacillus subtilis
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