Intragenomic diversity of Rhizobium leguminosarum bv. trifolii clover nodule isolates

Andrzej Mazur 0 Grayna Stasiak 0 Jerzy Wielbo 0 Agnieszka Kubik-Komar 1 Monika Marek-Kozaczuk 0 Anna Skorupska 0 0 Department of Genetics and Microbiology, Maria Curie-Skodowska University , Akademicka 19, 20-033 Lublin , Poland 1 Chair of Applied Mathematics and Informatics, Lublin University of Life Sciences , Akademicka 13, 20-950 Lublin , Poland Background: Soil bacteria from the genus Rhizobium are characterized by a complex genomic architecture comprising chromosome and large plasmids. Genes responsible for symbiotic interactions with legumes are usually located on one of the plasmids, named the symbiotic plasmid (pSym). The plasmids have a great impact not only on the metabolic potential of rhizobia but also underlie genome rearrangements and plasticity. Results: Here, we analyzed the distribution and sequence variability of markers located on chromosomes and extrachromosomal replicons of Rhizobium leguminosarum bv. trifolii strains originating from nodules of clover grown in the same site in cultivated soil. First, on the basis of sequence similarity of repA and repC replication genes to the respective counterparts of chromids reported in R. leguminosarum bv. viciae 3841 and R. etli CFN42, chromid-like replicons were distinguished from the pool of plasmids of the nodule isolates studied. Next, variability of the gene content was analyzed in the different genome compartments, i.e., the chromosome, chromid-like and 'other plasmids'. The stable and unstable chromosomal and plasmid genes were detected on the basis of hybridization data. Displacement of a few unstable genes between the chromosome, chromid-like and 'other plasmids', as well as loss of some markers was observed in the sampled strains. Analyses of chosen gene sequences allowed estimation of the degree of their adaptation to the three genome compartments as well as to the host. Conclusions: Our results showed that differences in distribution and sequence divergence of plasmid and chromosomal genes can be detected even within a small group of clover nodule isolates recovered from clovers grown at the same site. Substantial divergence of genome organization could be detected especially taking into account the content of extrachromosomal DNA. Despite the high variability concerning the number and size of plasmids among the studied strains, conservation of the location as well as dynamic distribution of the individual genes (especially replication genes) of a particular genome compartment were demonstrated. The sequence divergence of particular genes may be affected by their location in the given genome compartment. The 'other plasmid' genes are less adapted to the host genome than the chromosome and chromid-like genes. - Background Rhizobia are widely occurring soil bacteria that are able to establish nitrogen-fixing symbioses with legumes. Bacterium-plant interaction is a complex process in which specific plant and bacterial signals are exchanged resulting in formation of nodules, where rhizobia in the form of bacteroids fix nitrogen [1-3]. Rhizobial genomes are large and multipartite, composed of a single circular chromosome and a set of large plasmids [4-6]. The genes responsible for nodulation (nod) and nitrogen-fixation (nif-fix) are either carried by large plasmids (pSym) or are incorporated in the chromosome as symbiotic islands [7,8]. Large genomes of Rhizobiaceae and Bradyrhizobiaceae (above 6-9 Mb) are considered more ecologically advantageous in an environment that is scarce in nutrients but diverse as regards carbon and energy sources. These genomes are disproportionately enriched in regulation and transport genes and in genes involved in secondary metabolism in comparison with medium-and small-size genome containing bacteria [9]. Core and accessory components of Rhizobium genomes can be distinguished. Chromosomes with conserved gene content and order (synteny) are considered as core. Accordingly, plasmids constitute the accessory genome. Plasmids are more flexible than the chromosomes, as defined by more frequent gene gains and losses, even in the same species. They are heterogeneous in size and gene content and lack synteny even in closely related species, except for genes involved in plasmid replication and symbiotic properties [6,10,11]. In some species, such as Rhizobium leguminosarum, plasmids may comprise up to 35% of the total genome [6,7]. Rhizobial plasmids are maintained in the cells via repABC cassettes, comprising genes required for active segregation (repAB) and initiation of replication (repC) [12]. The presence of several repABC operons within a single genome, which are subjected to individual selection pressure and divergence, could be the key element of the existence of different plasmid incompatibility groups in cells and could drive the rearrangement of gene organization and of their functions [11,13-15]. It was proposed that repABC plasmids coexisting in the same strain most probably emerged by separate events of lateral transfer, which required evolution of different incompatibility groups allowing simultaneous residence of plasmids equipped with a similar replication/partition system in a single bacterial species [12]. Thus, the degree of divergence of the plasmid replication apparatus, whose sequence is subject to strong evolutionary pressure and determines the ability to evade incompatibility between plasmids [13], and horizontal gene transfers are potential forces that shaped rhizobial genomes. Recently, some (not only rhizobial) extrachromosomal replicons that have properties distinct from both chromosome and plasmids were reported and named chromids [16]. Chromids are characterized by presence of some important genes essential for growth under all conditions, with nucleotide composition and codon usage similar to the chromosome of the parental strain, and, by contrast, plasmid replication and partition systems [16]. Furthermore, recent analyses of Rhizobium etli strains [11] showed that this species has a pangenomic structure. By definition, a pangenome determines the core genome, which consists of genes shared by all the strains studied and probably encoding functions related to the basic biology and phenotypes of the species [17]. The basis of the pangenome concept emerged from an observation that each newly sequenced genome enriched the pool of species-specific genes with new ones [17,18]. This makes it possible to detect, besides the core genomes, the dispensable genomes composed of both chromosomal and plasmid genes, present only in some of the strains, which contribute to the species diversity and allow adaptation to new ecological niches and a specific environment. Despite the overall genomic divergence, R. etli pangenome comprises a core genome composed of both chromosomal and plasmid sequences, as well as highly conserved symbiosis-related genes on the pSym plasmid. The unusual variability observed in rhizobial genomes may further result from se (...truncated)