Transcriptional Interference and Repression Modulate the Conjugative Ability of the Symbiotic Plasmid of Rhizobium etli

Abstract

Bacteria of the order Rhizobiales are able to establish nitrogen-fixing symbioses with legumes. Commonly, genes for symbiosis are harbored on large symbiotic plasmids. Although the transfer of symbiotic plasmids is commonly detected in nature, there are few experimentally characterized examples. In Rhizobium etli, the product of rctA inhibits the conjugation of the symbiotic plasmid by reducing the transcription of the virB operon. rctA is transcribed divergently from this operon, and its product is predicted to have a DNA binding domain. In the present study, using DNase I footprinting and binding assays, we demonstrated the specific binding of RctA to the virB operon promoter. A 9-bp motif in the spacer region of this promoter (the rctA binding motif box) and the presence of a functional ؊10 region were critical elements for RctA binding. Transcriptional fusion analyses revealed that the elimination of either element provoked a relief of RctAmediated repression. These data support a model in which RctA inhibits the access of the RNA polymerase to the virB promoter. Interestingly, rctA expression levels were modulated by transcriptional interference from transcripts emanating from the virB promoter. This phenomenon adds another level of regulation for this system, thus revealing a novel mechanism of plasmid transfer regulation in the Rhizobiales.The ability to establish nitrogen-fixing symbioses is prevalent in bacteria of the order Rhizobiales. Commonly, most of the genes needed to establish symbiosis are either harbored on the so-called symbiotic plasmids (pSyms) or restricted to symbiosis islands (SI) located on the bacterial chromosome. As befits a trait that confers niche extension, there is evidence for the mobility of these genomic compartments. Indeed, sequence analyses of pSyms, including pRetCFN42d of Rhizobium etli (17), pNGR234a of Rhizobium sp. strain NGR234 (13), and pSymA of Sinorhizobium meliloti (1, 15), as well as of the SI of Bradyrhizobium japonicum (23,18) and Mesorhizobium loti (22,43), have lead to the identification of conjugation-related genes, mainly the virB1-to-virB11 and traA-traCDG systems, carried by these elements. Moreover, a common feature of these genetic compartments is that the GC contents of these elements differ significantly from those of the rest of the genomes. These data suggest that these gene clusters originated out of, and were transmitted to, other genetic systems. It is likely that these compartments may still be prone to lateral transfer.Evidence for the movement of pSyms among naturally occurring rhizobial populations has been inferred through phylogenetic and/or population genetics analyses of a variety of systems (45, 38). The transfer of SI, initially detected in field experiments investigating the SI of M. loti (41), was recently demonstrated for the SI of B. japonicum (16). Direct experimental evidence for lateral transfers has also been obtained, albeit such transfers have been found to occur at various rates (ranging from 10 Ϫ3 to 10 Ϫ9 transconjugants pe...

Regulation of conjugative transfer of plasmids and integrative conjugative elements

“…RctA binds specifically to a site in the virB promoter, repressing its transcription. Additionally, the divergent localization of rctA and the virB operon generates transcriptional interference between them, modulating the expression of (Pérez-Mendoza et al, 2005;Sepúlveda et al, 2008). Conjugative transfer is achieved in the presence of overexpressed rctB, because this regulator inhibits expression of rctA, allowing induction of the Dtr and Mpf genes.…”

Genetic Characterization of a Novel Rhizobial Plasmid Conjugation System in Rhizobium leguminosarum bv. viciae Strain VF39SM

“…There is considerable evidence for rhizobial plasmid transfer both under laboratory conditions (10)(11)(12)(13), and in natural environments (14)(15)(16). In addition, genome sequences of many rhizobial strains and rhizobial plasmids have revealed potential conjugation genes encoding DNA transfer and replication (Dtr) components and mating pair formation (Mpf) components, as well as putative origins of transfer (oriT).…”

pSymA-Dependent Mobilization of the Sinorhizobium meliloti pSymB Megaplasmid

“…The first class includes diverse plasmids from various species in which plasmid transfer occurs in response to the population density, similar to the Ti plasmid of Agrobacterium tumefaciens. In contrast, transfer of the rctAregulated plasmids, such as Rhizobium etli pRetCFN42d and pSymA of Sinorhizobium meliloti, is repressed by the transcriptional regulator RctA (10,18).…”