The conjugative plasmid of a bean-nodulating Sinorhizobium fredii strain is assembled from sequences of two Rhizobium plasmids and the chromosome of a Sinorhizobium strain

Abstract

Background: Bean-nodulating Rhizobium etli originated in Mesoamerica, while soybean-nodulating Sinorhizobium fredii evolved in East Asia. S. fredii strains, such as GR64, have been isolated from bean nodules in Spain, suggesting the occurrence of conjugative transfer events between introduced and native strains. In R. etli CFN42, transfer of the symbiotic plasmid (pRet42d) requires cointegration with the endogenous self-transmissible plasmid pRet42a. Aiming at further understanding the generation of diversity among bean nodulating strains, we analyzed the plasmids of S. fredii GR64: pSfr64a and pSfr64b (symbiotic plasmid).

Results: The conjugative transfer of the plasmids of strain GR64 was analyzed. Plasmid pSfr64a was self-transmissible, and required for transfer of the symbiotic plasmid. We sequenced pSfr64a, finding 166 ORFs. pSfr64a showed three large segments of different evolutionary origins; the first one presented 38 ORFs that were highly similar to genes located on the chromosome of Sinorhizobium strain NGR234; the second one harbored 51 ORFs with highest similarity to genes from pRet42d, including the replication, but not the symbiosis genes. Accordingly, pSfr64a was incompatible with the R. etli CFN42 symbiotic plasmid, but did not contribute to symbiosis. The third segment contained 36 ORFs with highest similarity to genes localized on pRet42a, 20 of them involved in conjugative transfer. Plasmid pRet42a was unable to substitute pSfr64a for induction of pSym transfer, and its own transfer was significantly diminished in GR64 background. The symbiotic plasmid pSfr64b was found to differ from typical R. etli symbiotic plasmids.

Conclusions: S. fredii GR64 contains a chimeric transmissible plasmid, with segments from two R. etli plasmids and a S. fredii chromosome, and a symbiotic plasmid different from the one usually found in R. etli bv phaseoli. We infer that these plasmids originated through the transfer of a symbiotic-conjugative-plasmid cointegrate from R. etli to a S. fredii strain, and at least two recombination events among the R. etli plasmids and the S. fredii genome. As in R. etli CFN42, the S. fredii GR64 transmissible plasmid is required for the conjugative transfer of the symbiotic plasmid. In spite of the similarity in the conjugation related genes, the transfer process of these plasmids shows a host-specific behaviour.

Figure 1

Eckhardt type gel showing the plasmid profile of S. fredii strain GR64 and derivatives, in comparison to R. etli CFN42. Panel A. Ethidium bromide stained Eckhardt gel. Lane 1: CFN42, lane 2: wild type GR64, lane 3: GR64-2, lane 4: GR64-3, lane 5: GR64-4, lane 6: GR64-5, lane 7: GR64-6, lane 8: GMI9023/pSfr64a, lane 9: GMI9023/pSfr64b, lane 10: CFN2001, lane 11: CFN2001-1, lane 12: CFN2001- 2, lane 13: CFN2001-3. Panel B. Ethidium bromide stained Eckhardt gel (lanes 1 and 2), and Southern blot of the plasmid profiles probed with pSfr64a (lanes 3 and 4). Lanes 1 and 3: GR64-1 (GR64/pSfr64a::Tn5-GDYN, pSfr64b::Tn5mob), lanes 2 and 4: GR64-2 (pSfr64a^-, pSfr64b::Tn5mob).

Figure 2

Structure of plasmid pSfr64a. Descriptions are presented from the innermost circle outward: regions with homology to pRet42a (red), pRet42d (green) and the chromosome of NGR234 (blue); ORFs with homology to pRet42a (red), pRet42d (green) and the chromosome of NGR234 (blue); transposon-related ORFs: pSFR64a_00003, pSFR64a_00009, pSFR64a_00084, pSFR64a_00088 (black arrows); transposon-related ORFs on pRet42a (PA00138) and pRet42d (PD00033, PD00041, PD00093, PD00124, PD00101, PD00123, PD00041) located nearby to the ORFs where similarity is interrupted (purple arrows); GC content (blue, low GC; gray, medium GC; red, high GC); predicted ORFs on the forward and reverse strands in color code (the colors are according to their functional category as follows: orange, amino acid biosynthesis; light red, biosynthesis of cofactors; pale green, macromolecule biosynthesis; mild red, central intermediary metabolism; red, energy transfer; magenta, degradation; pink, structural elements or cell processes; dark gray, transport; bright green, transposon-related functions; sky blue, transcriptional regulators; green, transfer functions or replication functions; brown, hypotheticals; bone, orphans; black, function not determined). The locations of the replication genes (R) and of the transfer region (T) are indicated.

Figure 3

Colinearity between pSfr64a and other replicons. Dot matrix view of BLASTN comparisons of pSfr64a vs pRet42a, pRet42d and the chromosome of NGR234.

Figure 4

Phylogeny of S. fredii GR64. Maximum likelihood phylogenetic trees based on chromosomal: (A) recA, (B) rpoB, and plasmid: (C) nifH and (D) repB gene fragments. Arrows indicate the localization of S. fredii GR64, and R.etli CFN42.