Delineation of the integrase-attachment and origin-of-transfer regions of the symbiosis island ICEMlSymR7A

Abstract

Integrative and conjugative elements (ICEs) are chromosomally-integrated mobile genetic elements that excise from their host chromosome and transfer to other bacteria via conjugation. ICEMlSym R7A is the prototypical member of a large family of "symbiosis ICEs" which confer upon their hosts the ability to form a nitrogen-fixing symbiosis with a variety of legume species. Mesorhizobial symbiosis ICEs carry a common core of mobilisation genes required for integration, excision and conjugative transfer. IntS of ICEMlSym R7A enables recombination between the ICEMlSym R7A attachment site attP and the 3' end of the phe-tRNA gene. Here we identified putative IntS attP arm (P) sites within the attP region and demonstrated that the outermost P1 and P5 sites demarcated the minimal region for efficient IntS-mediated integration. We also identified the ICEMlSym R7A origin-of-transfer (oriT) site directly upstream of the relaxase-gene rlxS. The ICEMlSym R7A conjugation system mobilised a plasmid carrying the cloned oriT to Escherichia coli in an rlxS-dependent manner. Surprisingly, an inframe, markerless deletion mutation in the ICEMlSym R7A recombination directionality factor (excisionase) gene rdfS, but not a mutation in intS, abolished mobilisation, suggesting the rdfS deletion tentatively has downstream effects on conjugation or its regulation. In summary, this work defines two critical cis-acting regions required for excision and transfer of ICEMlSym R7A and related ICEs.

Comparative analysis of integrative and conjugative mobile genetic elements in the genus Mesorhizobium

“…All plasmids carrying an ICEMlSym R7A -like conjugation system retained the rdfS gene, which in ICEMlSym R7A and ICEMcSym 1271 stimulates integrase-mediated ICE excision [32,37]. RdfS is also a transcriptional regulator [36,37], so it may have additional regulatory roles in transfer, which may explain its retention on plasmids [9].…”

“…[31]. The regulatory network controlling excision, conjugation and regulation of the ICE has been described in detail [32][33][34][35][36]. ICEMlSym R7A excision and integration is catalysed by the site-specific recombinase IntS.…”

Driving to Safety: CRISPR-Based Genetic Approaches to Reducing Antibiotic Resistance

“…Alternatively, conjugal and phage delivery systems could be combined by placing att phage landing sites in the conjugal element. These approaches may be supplemented with phagemids [19][20][21] carrying minimal anti-AR systems or CRISPRbearing prophages inserted into an ICE carried by the host chromosome [30,77], either of which might offer alternative HGT routes. Having dual modes of carriage should also help break down barriers to accessing different microenvironments.…”

Crystallographic and X-ray scattering study of RdfS, a recombination directionality factor from an integrative and conjugative element

“…The att sites attL and attR flank the ICE (Sullivan & Ronson, 1998). Integration requires the integrase, IntS, a tyrosine recombinase that belongs to the P4 integrase family (Sullivan & Ronson, 1998;Esposito & Scocca, 1997;Ramsay et al, 2006;Verdonk et al, 2019). When excised, the att sites are recombined, producing attP on the extrachromosomal circularized ICEMlSym R7A and attB within the bacterial chromosome.…”