Plasmid-like replication of a minimal streptococcal integrative and conjugative element

Abstract

Integrative and conjugative elements (ICEs) are mobile genetic elements encoding their own excision from a replicon of their bacterial host, transfer by conjugation to a recipient bacterium and reintegration for maintenance. The conjugation, recombination and regulation modules of ICEs of the ICE<i>St3</i> family are grouped together in a region called the ICE 'core region'. In addition to this core region, elements belonging to this family carry a highly variable region including cargo genes that could be involved in bacterial adaptation or in the maintenance of the element. Although ICEs are a major class of mobile elements through bacterial genomes, the functionality of an element encoding only its excision, transfer, integration and regulation has never been demonstrated experimentally. We engineered MiniICE<i>St3</i>, an artificial ICE derived from ICE<i>St3</i>, devoid of its cargo genes and thus only harbouring the core region. The functionality of this minimal element was assessed. MiniICE<i>St3</i> was found to be able to excise at a rate of 3.1 %, transfer with a frequency of 1.0 × 10^{- 5} transconjugants per donor cell and stably maintain by site-specific integration into the 3' end of the <i>fda</i> gene, the same as ICE<i>St3</i>. Furthermore, MiniICE<i>St3</i> was found in ∼10 copies per chromosome, this multicopy state likely contributing to its stability for >100 generations even in the absence of selection. Therefore, although ICEs were primarily assumed to only replicate along with the chromosome, our results uncovered extrachromosomal rolling-circle replicating plasmid-like forms of MiniICE<i>St3</i>.