ICEMlSym(R7A) of Mesorhizobium loti is an integrative and conjugative element (ICE) that confers the ability to form a nitrogen-fixing symbiosis with Lotus species. Horizontal transfer is activated by TraR and N-acyl-homoserine lactone (AHL), which can stimulate ICE excision in 100% of cells. However, in wild-type cultures, the ICE is excised at low frequency. Here we show that QseM, a widely conserved ICE-encoded protein, is an antiactivator of TraR. Mutation of qseM resulted in TraR-dependent activation of AHL production and excision, but did not affect transcription of traR. QseM and TraR directly interacted in a bacterial two-hybrid assay in the presence of AHL. qseM expression was repressed by a DNA-binding protein QseC, which also activated qseC expression from a leaderless transcript. QseC differentially bound two adjacent operator sites, the lower affinity of which overlapped the -35 regions of the divergent qseC-qseM promoters. QseC homologues were identified on ICEs, TraR/TraM-regulated plasmids and restriction-modification cassettes, suggesting a conserved mode of regulation. Six QseC variants with distinct operators were identified that showed evidence of reassortment between mobile elements. We propose that QseC and QseM comprise a bimodal switch that restricts quorum sensing and ICEMlSym(R7A) transfer to a small proportion of cells in the population.

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

Establishment of the symbiotic relationship that develops between rhizobia and their legume hosts is contingent upon an interkingdom signal exchange. In response to host legume flavonoids, NodD proteins from compatible rhizobia activate expression of nodulation (nod) genes that produce lipochitin oligosaccharide signalling molecules known as Nod factors. Root nodule formation commences upon legume recognition of compatible Nod factor. Rhizobium leguminosarum was previously considered to contain one copy of nodD; here we show that some strains of the Trifolium (clover) microsymbiont R. leguminosarum bv. trifolii (Rlt) contain a second copy designated nodD2. nodD2 genes were present in eight out of 13 strains of Rlt interrogated but were absent from the genomes of 16 R. leguminosarum bv. viciae strains. Analysis of single and double nodD1 and nodD2 mutants in Rlt strain TA1 revealed that NodD2 was functional and enhanced nodule colonisation competitiveness. However, NodD1 showed significantly greater capacity to induce nod gene expression and infection thread (IT) formation. Clover species are either annual or perennial and this phenological distinction is rarely crossed by individual Rlt microsymbionts for effective symbiosis. Of 13 strains with genome sequences available, seven of the eight effective microsymbionts of perennial hosts contained nodD2 whereas the three microsymbionts of annual hosts did not. We hypothesise that NodD2 inducer recognition differs from NodD1, and NodD2 functions to enhance competition and effective symbiosis, which may discriminate in favour of perennial hosts. IMPORTANCE Establishment of the rhizobium-legume symbiosis requires a highly specific and complex signal exchange between both participants. Rhizobia perceive legume flavonoid compounds through LysR-type NodD regulators. Often, rhizobia encode multiple copies of nodD, which is one determinant of host specificity. In some species of rhizobia, the presence of multiple copies of NodD extends their symbiotic host-range. Here we identified and characterised a second copy of nodD present in some strains of the clover microsymbiont Rhizobium leguminosarum bv. trifolii. The second nodD gene contributed to the competitive ability of the strain on white clover, an important forage legume. A screen for strains containing nodD2 could be utilised as one criterion to select strains with enhanced competitive ability for use as inoculants for pasture production.