Conserved symbiotic plasmid DNA sequences in the multireplicon pangenomic structure of Rhizobium etli

Abstract

Strains of the same bacterial species often show considerable genomic variation. To examine the extent of such variation in Rhizobium etli, the complete genome sequence of R. etli CIAT652 and the partial genomic sequences of six additional R. etli strains having different geographical origins were determined. The sequences were compared with each other and with the previously reported genome sequence of R. etli CFN42. DNA sequences common to all strains constituted the greater part of these genomes and were localized in both the chromosome and large plasmids. About 700 to 1,000 kb of DNA that did not match sequences of the complete genomes of strains CIAT652 and CFN42 was unique to each R. etli strain. These sequences were distributed throughout the chromosome as individual genes or chromosomal islands and in plasmids, and they encoded accessory functions, such as transport of sugars and amino acids, or secondary metabolism; they also included mobile elements and hypothetical genes. Sequences corresponding to symbiotic plasmids showed high levels of nucleotide identity (about 98 to 99%), whereas chromosomal sequences and the sequences with matches to other plasmids showed lower levels of identity (on average, about 90 to 95%). We concluded that R. etli has a pangenomic structure with a core genome composed of both chromosomal and plasmid sequences, including a highly conserved symbiotic plasmid, despite the overall genomic divergence.

FIG. 1.

Synteny relationships between RetCIAT652 and RetCFN42. (a) Nucmer plots of the chromosomes. (b) Nucmer plots of the concatenated plasmids.

FIG. 2.

Common protein families in R. etli and Rlv3841. A total of 19,085 predicted proteins encoded by the three genomes were clustered into families using the OrthoMCL and MCL algorithms (14, 35) (see Materials and Methods). The total numbers of families are indicated, and the total numbers of proteins are indicated in parentheses. Both single-member families and paralogous families are included. BLASTx searching of the GenBank nonredundant database for homologs of proteins unique to RetCFN42, RetCIAT652, and Rlv3841 resulted in identification of 170, 350, and 291 orphan genes, respectively.

FIG. 3.

Core genome profiles and their distribution in Rhizobium replicons. Single-member protein families were used to construct presence-absence profiles for replicons of the three genomes. Large circles represent the most common profiles, and small circles represent profiles containing few proteins. Plotting of profile distributions was accomplished by using the e-burst program (17, 50). Profile numbers were assigned arbitrarily.

FIG. 4.

DNA common to or unique in R. etli strains. DNA readings from each partial genome sequence assembled into contigs were compared, using BLASTn, with the complete genomic sequences of RetCIAT652 (red bars) and RetCFN42 (orange bars), using the parameters described in Materials and Methods. Contigs without matches to RetCIAT62 or RetCFN42 sequences were compared with the GenBank nonredundant database (dark green and light green bars, respectively). The dark blue bars indicate the remaining DNA in contigs without matches with either RetCIAT652 or the nonredundant database, and the light blue bars indicate the remaining DNA in contigs without matches with RetCFN42 or the nonredundant database. These contigs contain sequences found only in R. etli and orphan genes.

FIG. 5.

Chromosomal islands in R. etli. Readings for every partial genomic sequence were aligned, using BLASTn, with the chromosomes of RetCIAT652 (a) and RetCFN42 (b). The outer blue circles represent sequences from strains 8C-3, Brasil 5, CIAT894, GR56, IE4771, and Kim5, respectively, from the outside toward the center. The inner green circles represent alignments with the chromosomes of Rlv3841 and RetCFN42 (a) or with the chromosome of RetCIAT652 (b). The next circles represent the G+C content, which was either above average (blue peaks) or below average (red peaks). The two black circles represent genes unique to RetCIAT652 (a) or RetCFN42 chromosomes (b), and the distribution of tRNA genes along the chromosomes is also shown (> and <). The numbers outside the circles indicate putative islands in the chromosomes of RetCIAT652 (a) and RetCFN42 (b), predicted by the Alien Hunter program (55), that agree with regions without any sequence representation (red bars) or heterogeneous sequence representation (turquoise bars) in other R. etli strains.

FIG. 6.

Nucleotide identities for the replicons of RetCIAT652 and RetCFN42. Local alignments constructed by BLASTn for the two genomes were computed using individual maximal segment pairs (MSPs). All MSPs more than 200 bp long with levels of nucleotide identity of >79% (the lowest value found) were recorded. The average length of MSPs was 2,234 bp. The MSPs for the pCIAT652b-pCFN42d (pSym) pair were the longest MSPs (average, 3,974 bp; longest match, 42,006 bp) and exhibited the highest levels of nucleotide identity (98 to 100%).

FIG. 7.

Genetic relatedness of R. etli strains. A distance matrix derived from all-versus-all BLASTn alignments was used to estimate the degrees of divergence among R. etli strains and R1v3841. Next, an unrooted tree was constructed using the neighbor-joining method of the PHYLIP program (see Materials and Methods). The numbers at the branch points indicate the numbers of substitutions per site.