Large-scale genetic variation of the symbiosis-required megaplasmid pSymA revealed by comparative genomic analysis of Sinorhizobium meliloti natural strains

Abstract

Background: Sinorhizobium meliloti is a soil bacterium that forms nitrogen-fixing nodules on the roots of leguminous plants such as alfalfa (Medicago sativa). This species occupies different ecological niches, being present as a free-living soil bacterium and as a symbiont of plant root nodules. The genome of the type strain Rm 1021 contains one chromosome and two megaplasmids for a total genome size of 6 Mb. We applied comparative genomic hybridisation (CGH) on an oligonucleotide microarrays to estimate genetic variation at the genomic level in four natural strains, two isolated from Italian agricultural soil and two from desert soil in the Aral Sea region.

Results: From 4.6 to 5.7 percent of the genes showed a pattern of hybridisation concordant with deletion, nucleotide divergence or ORF duplication when compared to the type strain Rm 1021. A large number of these polymorphisms were confirmed by sequencing and Southern blot. A statistically significant fraction of these variable genes was found on the pSymA megaplasmid and grouped in clusters. These variable genes were found to be mainly transposases or genes with unknown function.

Conclusion: The obtained results allow to conclude that the symbiosis-required megaplasmid pSymA can be considered the major hot-spot for intra-specific differentiation in S. meliloti.

Figure 1

Number and location of variable ORFs on the three replicons. Genes considered were significantly different in hybridisation from strain Rm1021 at p < 0.001. A), Genes with Log2-ratio > 0; B) genes with Log2-ratio < 0. Asterisks over the columns indicate significant enrichment at p < 0.0001.

Figure 2

Location of variable ORFs along the replicons. Up and down bars indicate ORFs with Log2-ratio > 0 (duplication) or Log2-ratio < 0 (divergence or deletion), respectively. Thickness of bars indicates clusters of variable genes. A, AK58 strain; B, AK83 strain; C, BL225C strain; D, BO21CC strain. Replicon lengths are not in scale.

Figure 3

Functional groups of variable ORFs. A, strain AK58; B, strain AK83; C, strain BL225C; D, strain BO21CC. Classification is as defined by the S. meliloti consortium, subgroups are not reported: I, Small molecule metabolism; II, Macromolecule metabolism; III, Structural elements; IV, Cell processes; V, Elements of external origin; VI, Miscellaneous/unknown function. Groups V and VI were statistically significantly enriched for all strains.