Characterization of the extrachorosomal replicons present in the extended host range Or191-like strains.

TORRES TEJERIZO G; DEL PAPA MF; GIUSTI MA; LOZANO M; DRAGHI WO; LAGARES A; PISTORIO M

Gansk, Polonia

Conferencia; Internacional Plasmid Biology Conference; 2008

                   A main evolutionary mechanism that challenges our understanding of bacterial genome evolution is the horizontal gene transfer (HGT). Within this context, special attention has been paid to the transfer of genetic material in bacteria via plasmid conjugation as this mechanism is considered one of most efficient means of DNA exchange among prokaryotes. In several rhizobia, bacteria that inhabit the soil in free-living conditions and associate in symbiosis with the root of legumes as nitrogen-fixing organisms, DNA plasmid could constitute a very high percentage 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 (Del Papa, et al., 1999). Such isolates showed to be closely related to the previously described strain R. sp. Or191, isolated from an acid soils in Oregon, USA (Eardly, et al., 1985, Eardly, et al., 1992, Eardly, et al., 1995). 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 (Laguerre et al., 2001; Del Papa et al., 2007). Such observation and evidences from other authors (Sullivan et al., 1995) point HGT as a relevant force driving the symbiotic evolution of rhizobia. 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 rhizobia. Wegener et al. (2001) reported the presence of three plasmids in the Or191-like rhizobia (plasmids pLPU83a-b-c). 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 insertions in the plasmid replicons and conjugation experiments we found that plasmid pLPU83a is self-transmissible with a transfer frequency of approx. 10-9/receptor cell. Plasmid pLPU83c was transferred to S. meliloti and to the Agrobacterium tumefaciens UBAPF2 (plasmid free). At the moment no obvious phenotypic traits could be identified as encoded by 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 (Del Papa et al., 2007). Upon sequencing of 10 kb of the nod region a remakable genetic synteny could be recognized among S. meliloti, S. medicae, and the Or191-like rhizobia. This strongly reinforces the idea that the symbiotic (Sym) region in these rhizobia should arise as a consequence of horizontal gene-transfer events among them. At the DNA sequence level, however, the nod sequences of the Or191-rhizobia showed the highest identity to their homologs in strain Rhizobium mongolense USDA1844. At the nod gene region, R. mongolense appears as 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. A main evolutionary mechanism that challenges our understanding of bacterial genome evolution is the horizontal gene transfer (HGT). Within this context, special attention has been paid to the transfer of genetic material in bacteria via plasmid conjugation as this mechanism is considered one of most efficient means of DNA exchange among prokaryotes. In several rhizobia, bacteria that inhabit the soil in free-living conditions and associate in symbiosis with the root of legumes as nitrogen-fixing organisms, DNA plasmid could constitute a very high percentage 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 (Del Papa, et al., 1999). Such isolates showed to be closely related to the previously described strain R. sp. Or191, isolated from an acid soils in Oregon, USA (Eardly, et al., 1985, Eardly, et al., 1992, Eardly, et al., 1995). 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 (Laguerre et al., 2001; Del Papa et al., 2007). Such observation and evidences from other authors (Sullivan et al., 1995) point HGT as a relevant force driving the symbiotic evolution of rhizobia. 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 rhizobia. Wegener et al. (2001) reported the presence of three plasmids in the Or191-like rhizobia (plasmids pLPU83a-b-c). 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 insertions in the plasmid replicons and conjugation experiments we found that plasmid pLPU83a is self-transmissible with a transfer frequency of approx. 10-9/receptor cell. Plasmid pLPU83c was transferred to S. meliloti and to the Agrobacterium tumefaciens UBAPF2 (plasmid free). At the moment no obvious phenotypic traits could be identified as encoded by 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 (Del Papa et al., 2007). Upon sequencing of 10 kb of the nod region a remakable genetic synteny could be recognized among S. meliloti, S. medicae, and the Or191-like rhizobia. This strongly reinforces the idea that the symbiotic (Sym) region in these rhizobia should arise as a consequence of horizontal gene-transfer events among them. At the DNA sequence level, however, the nod sequences of the Or191-rhizobia showed the highest identity to their homologs in strain Rhizobium mongolense USDA1844. At the nod gene region, R. mongolense appears as 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. A main evolutionary mechanism that challenges our understanding of bacterial genome evolution is the horizontal gene transfer (HGT). Within this context, special attention has been paid to the transfer of genetic material in bacteria via plasmid conjugation as this mechanism is considered one of most efficient means of DNA exchange among prokaryotes. In several rhizobia, bacteria that inhabit the soil in free-living conditions and associate in symbiosis with the root of legumes as nitrogen-fixing organisms, DNA plasmid could constitute a very high percentage 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 (Del Papa, et al., 1999). Such isolates showed to be closely related to the previously described strain R. sp. Or191, isolated from an acid soils in Oregon, USA (Eardly, et al., 1985, Eardly, et al., 1992, Eardly, et al., 1995). 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 (Laguerre et al., 2001; Del Papa et al., 2007). Such observation and evidences from other authors (Sullivan et al., 1995) point HGT as a relevant force driving the symbiotic evolution of rhizobia. 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 rhizobia. Wegener et al. (2001) reported the presence of three plasmids in the Or191-like rhizobia (plasmids pLPU83a-b-c). 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 insertions in the plasmid replicons and conjugation experiments we found that plasmid pLPU83a is self-transmissible with a transfer frequency of approx. 10-9/receptor cell. Plasmid pLPU83c was transferred to S. meliloti and to the Agrobacterium tumefaciens UBAPF2 (plasmid free). At the moment no obvious phenotypic traits could be identified as encoded by 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 (Del Papa et al., 2007). Upon sequencing of 10 kb of the nod region a remakable genetic synteny could be recognized among S. meliloti, S. medicae, and the Or191-like rhizobia. This strongly reinforces the idea that the symbiotic (Sym) region in these rhizobia should arise as a consequence of horizontal gene-transfer events among them. At the DNA sequence level, however, the nod sequences of the Or191-rhizobia showed the highest identity to their homologs in strain Rhizobium mongolense USDA1844. At the nod gene region, R. mongolense appears as 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.