Application of multilocus sequence typing to study the genetic structure of megaplasmids in medicago-nodulating rhizobia

Abstract

A multilocus sequence typing (MLST) analysis was used to examine the genetic structure and diversity within the two large extrachromosomal replicons in Medicago-nodulating rhizobia (Sinorhizobium meliloti and Sinorhizobium medicae). The allelic diversity within these replicons was high compared to the reported diversity within the corresponding chromosomes of the same strains (P. van Berkum et al., J. Bacteriol. 188:5570-5577, 2006). Also, there was strong localized linkage disequilibrium (LD) between certain pSymA loci: e.g., nodC and nifD. Although both of these observations could be explained by positive (or diversifying) selection by plant hosts, results of tests for positive selection did not provide consistent support for this hypothesis. The strong LD observed between the nodC and nifD genes could also be explained by their close proximity on the pSymA replicon. Evidence was obtained that some nodC alleles had a history of intragenic recombination, while other alleles of this locus had a history of intergenic recombination. Both types of recombination were associated with a decline in symbiotic competence with Medicago sativa as the host plant. The combined observations of LD between the nodC and nifD genes and intragenic recombination within one of these loci indicate that the symbiotic gene region on the pSymA plasmid has evolved as a clonal segment, which has been laterally transferred within the natural populations.

FIG. 1.

(A) Groups within the population snapshot of the pSymB and pSMED1 extrachromosomal replicons in Medicago-nodulating rhizobia. Founding genotypes that were identified were ST-25, ST-54, ST-83, ST-100, ST-129, and ST-144. Cofounders were ST-26 and ST-104. Singletons are not shown. The size of each circle is associated with the number of strains having the specific pSymB ST. (B) Neighbor-joining tree showing all of the STs of pSymB and pSMED1 within the genomes of 229 strains of Sinorhizobium meliloti and S. medicae. Letters assigned to the groups in panel A are shown in panel B to indicate the approximate placement of the STs on the neighbor-joining tree.

FIG. 2.

(A) Groups within the population snapshot of the pSymA and pSMED2 extrachromosomal replicons in Medicago-nodulating rhizobia. The founding genotypes that were identified were ST-3, ST-19, ST-26, and ST-102. Cofounders were ST-33, ST-37, and ST-40. Singletons are not shown. The size of each circle is associated with the number of strains having the specific pSymA ST. (B) Neighbor-joining tree showing all of the STs of pSymA and pSMED2 within the genomes of 225 strains of Sinorhizobium meliloti and S. medicae. Letters assigned to the groups in panel A are shown in panel B to indicate the approximate placement of the STs on the neighbor-joining tree.

FIG. 3.

Neighbornet of the evolutionary relationships of nifD alleles present in Sinorhizobium meliloti and S. medicae. The nifD alleles within rectangles were present in an S. medicae chromosomal background. The NGR234 sequence NGR_a01120 or nifD2 was used as the outgroup. Bootstrap values of more than 99 are indicated and were derived from an analysis with 1,000 permutations of the data. The nifD alleles present in strains forming an ineffective or effective symbiosis with Medicago sativa are indicated by * or **, respectively.

FIG. 4.

Neighbornet of the evolutionary relationships of nodC alleles present in Sinorhizobium meliloti and S. medicae. The nodC alleles within rectangles were present in an S. medicae chromosomal background. The nodC sequence of R. mongolense strain USDA 1844^T served as the outgroup. Bootstrap values of more than 99 are indicated and were derived from an analysis with 1,000 permutations of the data. Evidence for positive selection by the Z test was obtained for alleles within soft-edged boxes. The nodC alleles present in strains forming an ineffective or effective symbiosis with Medicago sativa are indicated by * or **, respectively.