CONICET | Buscador de Institutos y Recursos Humanos

The horizontal gene transfer (HGT) is a main evolutionary mechanism that challenges our understanding on bacterial genome evolution. Thus, special attention has been paid to the transfer of genetic material in bacteria via plasmid conjugation as this is considered one of most efficient means of DNA exchange in prokaryotes. In several rhizobia i.e. bacteria that inhabit the soil in free-living conditions and associate with the root of legumes in symbiosis as nitrogen-fixing organisms- DNA plasmid could constitute a very high proportion of the genome. In our laboratory we have characterized acid-tolerant isolates of rhizobia which have an extended host range to nodulate alfalfa, common bean, and Leucena leucocephala (1) . Such isolates showed to be closely related to the previously described strain R. sp. Or191, isolated from an acid soils in Oregon, USA (2). The genetic structure of the Or191-like strains is particularly interesting since they have a chromosome related to bean/peas/clover-nodulating rhizobia, and they present plasmid-encoded symbiotic genes related to those present in Medicago symbionts (3, 4). Such observation together with several evidences from other authors point HGT as a relevant force driving the symbiotic evolution of rhizobia (5). In our group we were interested in the characterization of the plasmid gene pool present in Medicago-nodulating rhizobia in order to unravel plasmid evolutionary relationships within this group of bacteria. Wegener et al. (6) reported the presence of three plasmids in the Or191-like rhizobia (plasmids pLPU83a-b-c). Then, using Ekhardt gel analysis we estimated the size of the plasmids which resulted to be more than 1.5 Mbp for plasmid pSmeLPU83c, ca. 900 kbp for plasmid pLPU83b, and ca. 130 kbp for plasmid pLP83a. By using Tn5 mutagenesis and conjugation experiments we found that plasmid pLPU83a is self-transmissible with a transfer frequency of approx. 10-9/receptor cell. Plasmid pLPU83a could be transferred to S. meliloti and to the plasmid free Agrobacterium tumefaciens UBAPF2. At the moment, no obvious phenotypic traits could be associated to presence of this plasmid. Southern-blot analysis using a nodH probe showed a positive signal hybridizing with pLPU83b suggesting that this is the symbiotic plasmid that carries the nod gene cluster previously identified in a clone of a cosmid library (3). Upon sequencing of 10 kb of the nod region a remakable genetic synteny was recognized in S. meliloti, S. medicae, and the Or191-like rhizobia. This observation reinforces the idea that the symbiotic (Sym) regions of these rhizobia have arise as a consequence of horizontal gene-transfer events. At the DNA sequence level, the nod sequences of the Or191-rhizobia showed the highest identity to their homologs in strain Rhizobium mongolense USDA1844, a rhizobia for which there are not many DNA sequences available. At the nod gene region, R. mongolense appears to be the closer relative of the Or191-like rhizobia. A deeper sequencing approach will be necessary to elucidate the phylogeny of the pSym linage that results in current diversity of the Medicago nodulating bacteria.