Ribosomal frameshifting and dual-target antiactivation restrict quorum-sensing–activated transfer of a mobile genetic element

Abstract

Symbiosis islands are integrative and conjugative mobile genetic elements that convert nonsymbiotic rhizobia into nitrogen-fixing symbionts of leguminous plants. Excision of the Mesorhizobium loti symbiosis island ICEMlSym R7A is indirectly activated by quorum sensing through TraR-dependent activation of the excisionase gene rdfS. Here we show that a +1 programmed ribosomal frameshift (PRF) fuses the coding sequences of two TraR-activated genes, msi172 and msi171, producing an activator of rdfS expression named Frameshifted excision activator (FseA). Mass-spectrometry and mutational analyses indicated that the PRF occurred through +1 slippage of the tRNA phe from UUU to UUC within a conserved msi172-encoded motif. FseA activated rdfS expression in the absence of ICEMlSym R7A , suggesting that it directly activated rdfS transcription, despite being unrelated to any characterized DNA-binding proteins. Bacterial two-hybrid and gene-reporter assays demonstrated that FseA was also bound and inhibited by the ICEMlSym R7A -encoded quorum-sensing antiactivator QseM. Thus, activation of ICEMlSym R7A excision is counteracted by TraR antiactivation, ribosomal frameshifting, and FseA antiactivation. This robust suppression likely dampens the inherent biological noise present in the quorum-sensing autoinduction circuit and ensures that ICEMlSym R7A transfer only occurs in a subpopulation of cells in which both qseM expression is repressed and FseA is translated. The architecture of the ICEMlSym R7A transfer regulatory system provides an example of how a set of modular components have assembled through evolution to form a robust genetic toggle that regulates gene transcription and translation at both single-cell and cell-population levels.quorum sensing | antiactivator | ribosomal frameshift | ICE | horizontal gene transfer

Assembly and transfer of tripartite integrative and conjugative genetic elements

“…After a 7-y period, only ∼20% of L. corniculatus root nodules contained R7A; the other 80% of nodules contained a diverse mix of native mesorhizobia that had acquired symbiosis genes from R7A (11). Molecular analyses revealed these new symbionts had acquired a 502-kb symbiosis ICE, subsequently named ICEMlSym R7A (12)(13)(14).The mechanism and regulation of ICEMlSym R7A integration and excision have been investigated in detail (12,(15)(16)(17)(18). Integration of ICEMlSym R7A is catalyzed by the integrase IntS, which recombines the attachment (att) site attP located on the circularized ICEMlSym R7A with the attB site located at the 3′ end of the sole phetRNA gene present in Mesorhizobium genomes.…”

“…The mechanism and regulation of ICEMlSym R7A integration and excision have been investigated in detail (12,(15)(16)(17)(18). Integration of ICEMlSym R7A is catalyzed by the integrase IntS, which recombines the attachment (att) site attP located on the circularized ICEMlSym R7A with the attB site located at the 3′ end of the sole phetRNA gene present in Mesorhizobium genomes.…”

Plant nodulation inducers enhance horizontal gene transfer of Azorhizobium caulinodans symbiosis island

“…The ICEMlSym R7A is inserted downstream of a phe-tRNA gene in the M. loti chromosome and can be transferred to nonsymbiotic mesorhizobia both in the laboratory and in the field, converting these strains into symbionts of L. corniculatus (12,13). The transfer of ICEMlSym R7A is mediated by quorum sensing and is controlled by a complex multipartite regulatory system involving an excisionase and an excision activator produced via a programmed ribosomal frameshift (14)(15)(16).…”

Evolutionary persistence of tripartite integrative and conjugative elements

“…Therefore, any mechanisms that can reduce or prevent spurious excision likely stabilize ICEs in the long term. It is clear from our work on the regulation of transfer of ICEMlSym R7A that numerous layers of transcriptional and post-translational regulation are present to prevent spurious excision of this ICE from the M. loti chromosome (Ramsay et al, 2015a). Although there may be unidentified TA modules or other genes facilitating the active stabilization of symbiosis ICEs and ICE 3 s, we suspect that the tripartite configuration of ICE 3 is intrinsically resistant to…”

“…Excision and transfer are positively regulated by quorum-sensing. However, a combination of transcriptional activation, anti-activation and low-frequency ribosomal frameshifting robustly suppress rdfS expression and hence ICEMlSym R7A excision in most cells (Ramsay et al, 2013;Ramsay et al, 2009;Ramsay et al, 2015b). Overexpression of rdfS cures R7A of ICEMlSym R7A , producing the non-symbiotic derivative R7ANS (Ramsay et al, 2006).…”