Sequential induction of three recombination directionality factors directs assembly of tripartite integrative and conjugative elements
(A) Schematic of the possible ICEMcSym1271 recombination states and recombination reactions leading to formation of excised ICEMcSym1271 assembled from regions α, β and γ. The recombination targets attP, attB, attL and attR (triangles) and recombination reactions (large arrows) are color-coded for each integrase: cyan for IntS; magenta for IntG; green for IntM. Primers for qPCR measurement of recombination are indicated as block-headed arrows for attB sites and triangle-headed arrows for attP sites (see Materials and Methods and S1 Table for details). Data presented here support a model where RDF-stimulated excisive reactions occur in the order IntS > IntG > IntM (highlighted in yellow) to produce excised ICEMcSym1271. (B) The regulatory model of quorum-sensing mediated stimulation of ICEMcSym1271 assembly and excision. TraR1 and TraR2 bind AHLs produced by TraI1. TraR1/2-AHL complex(es) activate transcription from the traI1 and traI2 promoters. traI2-msi172-msi171 expression leads to production of FseA and transcriptional activation of the rdfS operon. RdfS stimulates excisive IntS-mediated recombination and promotes expression of RdfG and RdfM. RdfG stimulates the excisive IntG-mediated reaction and RdfM stimulates excisive IntM-mediated recombination and excision.