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Journal of Bacteriology, September 2007, p. 6474-6476, Vol. 189, No. 17
0021-9193/07/$08.00+0     doi:10.1128/JB.00384-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

DNA Diversification in Two Sinorhizobium Species{triangledown} ,{dagger}

Xianwu Guo, Margarita Flores, Lucía Morales, Delfino García, Patricia Bustos, Víctor González, Rafael Palacios, and Guillermo Dávila*

Centro de Ciencias Genómicas, Universidad Nacional Autónoma de México, Cuernavaca, Morelos, México

Received 14 March 2007/ Accepted 18 June 2007


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ABSTRACT
 
The comparative analysis of genomic characteristics and single-nucleotide polymorphism patterns from large fragments borne on different replicons of Sinorhizobium spp. genomes clearly demonstrate that DNA recombination among closely related bacteria is a major event in the diversification of this genome, especially in pSymA, resulting in mosaic structure.


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TEXT
 
In nature, some bacteria can recombine the exogenous DNA imported by lateral gene transfer into their genomes (2, 3, 10, 17), leading to portions of a genome containing assorted DNA from different sources, which is called genome mosaicism. The mosaic genome thus reveals the complexity of its evolutionary history (4, 9, 12, 15). The rapid proliferation of sequenced genome data provides an opportunity to explore genome evolution. However, from among several strains belonging to a particular species, in most cases, the genome sequence of only a single model organism is available in the databases so far. Among symbiotic microorganisms, Sinorhizobium meliloti 1021 is an extensively studied model, and its genome sequence, including that of two megaplasmids (pSymA and pSymB), has been reported (8). It is now accepted that some essential nitrogen fixation genes (nif, etc.) were obtained through lateral gene transfer. Some analyses also showed that pSymA could be of exogenous origin (8), and pSymB is a mosaic structure (16). To better understand its microevolution, we compared the general genomic structures and the sequences of orthologous regions between closely related strains of Sinorhizobium. Our results showed that recombination among closely related bacterial strains is a common phenomenon, especially in pSymA, resulting in a mosaic genomic structure, similar to the previous findings observed for the symbiotic plasmid of Rhizobium etli (6) and for some other related bacteria, including a Sinorhizobium sp. and Rhodobacter capusulatus (1, 11).

Genomic structural comparison via genome-wide PCR amplification. A total of 1,329 pairs of primers (see Table S1 in the supplemental material) were designed for genome-wide PCR amplification based on the published genome sequence of S. meliloti 1021 (Table 1). The distance between a pair of primers was about 5 kb. In addition, the primers were designed in such a way that the forward primer of a subsequent pair was upstream of the reverse primer of the preceding pair. Thus, a set of concatenated 5-kb PCR products having the end part of the preceding fragment overlapping with the forward part of the following fragment were generated to cover the whole replicon. In the present study, 269, 334, and 726 pairs of primers were used to amplify each of the three replicons, pSymA (1,354 kb), pSymB (1,683 kb), and the chromosome (3,654 kb), respectively, of S. meliloti 1021. The same sets of primers were also applied to S. meliloti 41 and to Sinorhizobium medicae strain M3, a species closely related to S. meliloti. Figure 1 shows that fewer PCR products were produced using the set of pSymA primers than using the sets of primers of pSymB and the chromosome (Fig. 1). In pSymA of S. medicae M3, only a few PCR products could be generated, with the largest concatenated segment located in the symbiotic region. A 106-kb fragment from Sma0937 to Sma1153, rich in insertion sequences (IS), was found to be absent in S. meliloti 41 and S. medicae M3. It appears that this 106-kb fragment is unique to S. meliloti 1021, as no such PCR product was obtained with the other eight strains tested here (data not shown).


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  		TABLE 1. Bacterial strains


Figure 1
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  		FIG. 1. Genome-wide PCR analysis in two genomes. Three replicons are shown according to their sizes in strain S. meliloti 1021. The inner circle in each replicon corresponds to S. meliloti 41, and the outer circle corresponds to S. medicae M3. Blue segments represent PCR products with the expected size of 5 kb. Red segments represent products with sizes differing from those of S. meliloti 1021; in these cases the length of each product is proportional to the height of the segment. Regions without colored segments indicate that PCR products were not obtained. Black arrowheads point out regions selected for sequencing and SNP analysis.

Sequence analysis revealed that 77 PCR fragments of S. meliloti 1021 harbored IS elements. However, when this group of fragments was amplified from other bacterial strains, we obtained smaller PCR products in most cases, and only a few of the products were of the same size as in S. meliloti 1021 (Fig. 1). This result suggests that IS elements were active at least at some stage of evolutionary history. More interesting is that some incomplete IS elements, such as ISRm7-, ISRm8-, and ISRm25-, present in pSymA of S. meliloti 1021, also existed in most other strains tested. The restriction digestion patterns of these regions showed that these IS elements seem to maintain the same resident sites in various genomes. This implies that the IS elements are either of related origin or a kind of ancient insertion.

Single-nucleotide polymorphism (SNP) distribution in orthologous regions. We sequenced 10 5-kb PCR products from each strain to compare the distributions of SNPs (14). The selected fragments were orthologous to two regions borne on pSymA, three on pSymB, and five on the chromosome of S. meliloti 1021. It is worthwhile to mention that, by Southern analyses, we verified that fragments originating from each plasmid or from the chromosome of S. meliloti 1021 hybridized only with the corresponding plasmidic or chromosomal DNA derived from each strain (not shown).

Results of the present study showed that in all the strains of S. meliloti, SNP distribution is definitely not a random phenomenon. The regions with high base substitutions, designated here as hypervariable DNA segments (HYDS), were found to be present as a series of punctuated patches along a genome (as represented in the fragments a1 and a2 in S. meliloti 74B12 and b2, b3, and c3 in S. meliloti 41, etc.) (Fig. 2). In spite of the occurrence of some strain-specific HYDS (e.g., b3 of S. meliloti 41 and part of a1 in S. meliloti 74B12), the different patterns of some HYDS present in certain fragments were found to be shared by different strains; for example, with regard to the patterns of SNPs present in b2, one pattern was shared by S. meliloti 1021 and M119 and another pattern was shared by S. meliloti 41 and 15A5. In addition, patterns of HYDS present in a single strain were also found to be present in various other strains; for example, S. meliloti 15A5 shares a2 with S. meliloti M119, b2 with S. meliloti 41, and part of a1 with S. meliloti 74B12. A close examination of the sequences revealed that HYDS do not correlate with functional genetic elements, as they could start or end within a coding sequence. It is interesting that this mosaicism, although detectable in the different replicons (including the chromosome), is more prominent in the megaplasmids, particularly in pSymA. Furthermore, similar SNP patterns are present among closely related species of S. medicae (Fig. 2, strains M104, M102, and M3). Remarkably, in some strains of S. meliloti, the patterns of SNPs in HYDS are also shared with S. medicae. For instance, the region of the gene nuoE2 present in the fragment a2 of S. meliloti 74B12 had much higher DNA sequence similarity with S. medicae strains than with other S. meliloti strains. In particular, the segment of 156 bp from this strain is shared with all the S. medicae strains but contained nine base substitutions in the consensus sequence derived from S. meliloti. Based on these results, we conclude that the scattered HYDS distribution present in a particular genome can be shared among several genomes, forming a network panorama. The above data cannot be explained by vertical inheritance but evidently indicate that recombination of DNA fragments among closely related bacteria, particularly in pSymA, is a major event in the diversification of genomic sequences.


Figure 2
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  		FIG. 2. Distribution of SNPs of 10 fragments from 10 strains. Strains are reported in Table 1. Species abbreviations: Smel, S. meliloti; Smed, S. medicae. Regions a1, a2, and b1, etc., are the sequenced PCR products. The letters a, b, and c stand for pSymA, pSymB, and the chromosome, respectively. Sequence alignments of each region were performed using Clustal X, and a consensus sequence was obtained for each nucleotide position. The SNPs in the same position were decided by the divergence from consensus. The results of the distribution of SNPs for each fragment are plotted as bars to show the number of nucleotides differing from the consensus in consecutive windows of 300 nucleotides for each region. The rules for the color code for each region are as follows: red, blue, dark green, orange, and yellow represent identical nucleotide variations from the consensus in different strains (with five or more differences); in addition, the divergences shared only by S. medicae strains but different from the consensus are shown in light green; black represents nucleotides differing from the consensus shared by at least two strains in cases where any two strains do not share more than 5 nucleotides differing from the consensus in the whole 5-kb region; gray represents nucleotides differing from the consensus present in only one of the strains. Although the rules of the color code are the same for the different regions, the colors are valid only for each specific DNA region.

Nucleotide sequence accession numbers. The GenBank accession numbers of the nucleotide sequences from regions of the different Sinorhizobium isolates are as follows: DQ898636 to DQ898645 from S. meliloti 41; DQ898566 to DQ898575 from S. meliloti 15A5; DQ898616 to DQ898625 from S. meliloti M119; DQ898576 to DQ898585 from S. meliloti 74B12; DQ898556 to DQ898565 from S. meliloti 102F51; DQ898586 to DQ898595 from S. meliloti CC2013; DQ898606 to DQ898615 from S. medicae M104; DQ898596 to DQ898605 from S. medicae M102; DQ898626 to DQ898635 from S. medicae M3 (see Table S2 in the supplemental material).


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ACKNOWLEDGMENTS
 
We acknowledge the technical assistance of Angeles Moreno, Marisa Rodríguez, Rosa M. Ocampo, Rosa I. Santamaría, and Sara I. Fuentes.

This work was supported in part by grants 046333-Q from CONACYT and IN223005 from PAPIIT-UNAM, México.


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FOOTNOTES
 
* Corresponding author. Mailing address: Centro de Ciencias Genómicas, Universidad Nacional Autónoma de México, Ap. Postal 565-A, Cuernavaca, Morelos, México. Phone: (73) 133881. Fax: (73) 116710. E-mail: davila{at}ccg.unam.mx Back

{triangledown} Published ahead of print on 29 June 2007. Back

{dagger} Supplemental material for this article may be found at http://jb.asm.org/. Back


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Journal of Bacteriology, September 2007, p. 6474-6476, Vol. 189, No. 17
0021-9193/07/$08.00+0     doi:10.1128/JB.00384-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.




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