Genetic and functional characterization of a yet-unclassified rhizobial Dtr (DNA-transfer-and-replication) region from a ubiquitous plasmid conjugal system present in Sinorhizobium meliloti, in Sinorhizobium medicae, and in other nonrhizobial Gram-negative bacteria

Abstract

Rhizobia are Gram-negative bacteria that live in soils and associate with leguminous plantsto establish nitrogen-fixing symbioses. The ability of these bacteria to undergo horizontalgene transfer (HGT) is thought to be one of the main features to explain both the originof their symbiotic life-style and the plasticity and dynamics of their genomes. In ourlaboratory we have previously characterized at the species level the non-pSym plasmidmobilome in Sinorhizobium meliloti, the symbiont of Medicago spp., and have found ahigh incidence of conjugal activity in many plasmids (Pistorio et al., 2008). In this workwe characterized the Dtr (DNA-transfer-and-replication) region of one of those plasmids,pSmeLPU88b. This mobilization region was found to represent a previously unclassifiedDtr type in rhizobia (hereafter type-IV), highly ubiquitous in S. meliloti and found in othergenera of Gram-negative bacteria as well; including Agrobacterium, Ochrobactrum, andChelativorans. The oriT of the type-IV Dtr described here could be located by functionwithin a DNA fragment of 278 bp, between the divergent genes parA and mobC. Thephylogenetic analysis of the cognate relaxase MobZ indicated that this protein groups closeto the previously defined MOBP3 and MOBP4 type of enzymes, but is located in a separateand novel cluster that we have designated MOBP0. Noteworthy, MOBP0 and MOBP4 relaxaseswere frequently associated with plasmids present in rhizospheric soil bacteria.A comparison of the nod-gene locations with the phylogenetic topology of the rhizobialrelaxases revealed that the symbiotic genes are found on diverse plasmids bearing anyof the four Dtr types, thus indicating that pSym plasmids are not specifically associatedwith any particular mobilization system. Finally, we demonstrated that the type-IV Dtrpromoted the mobilization of plasmids from S. meliloti to Sinorhizobium medicae as wellas from these rhizobia to other bacteria by means of their own helper functions. The resultspresent an as-yet-unclassified and seemingly ubiquitous conjugal system that provides amechanistic support for the HGT between sympatric rhizobia of Medicago roots, andbetween other soil and rhizospheric bacteria.