Transposase-Mediated Excision, Conjugative Transfer, and Diversity of ICE 6013 Elements in Staphylococcus aureus

Abstract

ICE6013 represents one of two families of integrative conjugative elements (ICEs) identified in the pan-genome of the human and animal pathogen Staphylococcus aureus. Here we investigated the excision and conjugation functions of ICE6013 and further characterized the diversity of this element. ICE6013 excision was not significantly affected by growth, temperature, pH, or UV exposure and did not depend on recA. The IS30-like DDE transposase (Tpase; encoded by orf1 and orf2) of ICE6013 must be uninterrupted for excision to occur, whereas disrupting three of the other open reading frames (ORFs) on the element significantly affects the level of excision. We demonstrate that ICE6013 conjugatively transfers to different S. aureus backgrounds at frequencies approaching that of the conjugative plasmid pGO1. We found that excision is required for conjugation, that not all S. aureus backgrounds are successful recipients, and that transconjugants acquire the ability to transfer ICE6013. Sequencing of chromosomal integration sites in serially passaged transconjugants revealed a significant integration site preference for a 15-bp AT-rich palindromic consensus sequence, which surrounds the 3-bp target site that is duplicated upon integration. A sequence analysis of ICE6013 from different host strains of S. aureus and from eight other species of staphylococci identified seven divergent subfamilies of ICE6013 that include sequences previously classified as a transposon, a plasmid, and various ICEs. In summary, these results indicate that the IS30-like Tpase functions as the ICE6013 recombinase and that ICE6013 represents a diverse family of mobile genetic elements that mediate conjugation in staphylococci.IMPORTANCE Integrative conjugative elements (ICEs) encode the abilities to integrate into and excise from bacterial chromosomes and plasmids and mediate conjugation between bacteria. As agents of horizontal gene transfer, ICEs may affect bacterial evolution. ICE6013 represents one of two known families of ICEs in the pathogen Staphylococcus aureus, but its core functions of excision and conjugation are not well studied. Here, we show that ICE6013 depends on its IS30-like DDE transposase for excision, which is unique among ICEs, and we demonstrate the conjugative transfer and integration site preference of ICE6013. A sequence analysis revealed that ICE6013 has diverged into seven subfamilies that are dispersed among staphylococci.

Biofilm Formation Drives Transfer of the Conjugative Element ICE Bs1 in Bacillus subtilis

“…ICE Bs1 is therefore one of the ICEs transferring at the highest rate in the Firmicutes . Indeed, Staphylococcus aureus ICE 6013 , an ICE closely related to ICE Bs1 , has a mating efficiency of around 10 −5 ( 42 ). Tn 916 , an ICE found in a variety of Gram-positive bacteria, was reported to transfer at frequencies ranging from 10 −9 to 10 −4 ( 43 , 44 ), while Streptococcus agalactiae TnGBS1 and TnGBS2 transfer at around 10 −5 ( 45 ).…”

Mobile Genetic Elements Associated with Antimicrobial Resistance

“…ICE6013 represents a family of ICE in staphylococci that are not related to Tn916. ICE6013 was initially identified in human S. aureus ST239 isolates, in which it sometimes carries a Tn552 insertion (443); however, multiple subfamilies have now been identified in different staphylococcal species (444). While ICE6013 displays some sequence similarity to ICEBs1 of Bacillus subtilis (443), its encoded functions are largely uncharacterized.…”

“…While ICE6013 displays some sequence similarity to ICEBs1 of Bacillus subtilis (443), its encoded functions are largely uncharacterized. Interestingly, ORFs encoding an IS30-like DDE transposase were recently shown to be required for excision of ICE6013 prior to conjugative transfer (444). In this regard, the mechanism of recombination is clearly different from that mediated by the tyrosine integrases of Tn916-like elements, which target specific AT-rich sequences (445).…”

Extra-Chromosomal DNA Sequencing Reveals Episomal Prophages Capable of Impacting Virulence Factor Expression in Staphylococcus aureus

“…The other episomal phages we uncovered had chromosomal integration sites that have been described previously ( Bae et al, 2006 ; Goerke et al, 2006 , 2009 ). In addition to these episomal prophage elements, we also uncovered an enriched integrative and conjugative element (ICE 6013 -like) typically located within a predicted membrane protein encoding locus in NRS271, however, its existence as an extra-chromosomal element has been previously reported ( Table 1 ) ( Smyth and Robinson, 2009 ; Sansevere et al, 2017 ). Incidentally, known plasmids, such as pSAS1 in MSSA476, were de novo assembled by using unmapped reads, and while they are not the focus of this current study, they do point to the sensitivity of this exDNA isolation and sequencing method.…”