Integrative and conjugative elements (ICEs) are a newly appreciated group of mobile elements that contribute immensely to bacterial evolution. The symbiosis island ICEMlSym[R7A] of Mesorhizobium loti strain R7A is the largest known mobile ICE (502 kb) and confers the ability to form a nitrogen-fixing symbiosis with plants of the genus Lotus.
Transfer of ICEMlSym[R7A] to non-symbiotic bacteria has been demonstrated both in the laboratory and the environment and involves chromosomal integration of ICEMlSym[R7A] adjacent to the phe-tRNA gene in the recipient bacterium. Integration of ICEMlSym[R7A] requires the ICEMlSym[R7A]-encoded protein IntS which likely catalyses recombination between the attachment sites attP and attB, present on excised ICEMlSym[R7A] and phe-tRNA gene respectively. In this study it was shown that intS was required for both excision and integration and was required in both the donor and recipient bacterium for efficient transfer. A minimal attP region required for integration was defined and a repetitive sequence motif that likely represents the IntS-binding sequence identified. intS is expressed from two alternate promoters, a weak promoter on attL (left ICEMlSym[R7A]-chromosome junction) and a strong promoter formed on attP. Analysis of nested deletions of the attP region demonstrated that an unusually large region of attP, corresponding to that required for integration, was required for full gene expression.
A novel recombination directionality factor RdfS was identified using bioinformatics. A quantitative PCR assay developed to detect the presence of excised ICEMlSym[R7A] in bacterial populations showed that excision of ICEMlSym[R7A] was almost undetectable in an rdfS mutant. Constitutive expression of rdfS resulted in curing of ICEMlSym[R7A] from R7A, creating a non-symbiotic derivative R7ANS.
Bioinformatic analyses of rdfS and associated genes on ICEMlSym[R7A] led to discovery of 26 putative ICEs (ICESym family) in 12 α-proteobacterial species. The ICESym clusters contained homologues of up to 25 ICEMlSym[R7A] proteins, encoding functions involved in mating pore formation, DNA processing and regulation, of which 17 proteins were universally conserved.
Introduction of pJR174 containing a copy of the ICEMlSym[R7A]-encoded quorum sensing (QS) gene traR induced excision in 100% of cells and stimulated a 100-fold increase in ICEMlSym[R7A] transfer. Stable maintenance of ICEMlSym[R7A] in these cells required the DNA relaxase-encoding gene rlxS. Introduction of pJR174 also induced a 1000-fold increase in the production of 3-oxo-C6-homoserine lactone and several other acyl homoserine lactones; this induction required traI1. The induction of QS was accompanied by various growth-inhibitory effects, similar to those observed in other rhizobial QS systems.
The QS system activated ICEMlSym[R7A] excision through expression of msi172 and msi171, genes that are downstream of QS gene traI2. Expression of the adjacent gene msi170 separately repressed both QS and excision. The expression of msi170 was negatively regulated by Msi169, while msi169 was positively autoregulated. msi169 encodes an Xre family DNA-binding protein and homologues of it were identified on the ICESym clusters and on QS-regulated plasmids, suggesting that they have a conserved role as regulators of transfer- and QS-related genes. Overall, the results suggest that ICEMlSym[R7A] excision and transfer are subject to both population-density and cell-cycle-dependent regulation.
