The variability of the genome architecture involved not only the number and size of the plasmids, but also the location of specific genes on the particular replicons. GANT61 distribution of repABC operon markers and other genes in the three genome compartments: the chromosome, chromid-like and ‘other plasmids’ was assessed. We found “”stable”" genes that were permanently

located in a specific genome compartment, as well as “”unstable”" ones, which were detected in different replicons of the sampled strains. Sequences of selected chromosome and plasmid genes were subjected to an assessment of adaptation to a particular genome compartment by analyses this website of codon usage and codon adaptation index. A potential evolutionary pathway of Rlt strains was proposed on the basis of gene sequences and their distribution.

Methods R. leguminosarum bv. trifolii (Rlt) strains 129 R. leguminosarum isolates LDN-193189 were obtained from nodules of red clover (Trifolium pratense L. cv. Dajana) growing in sandy loam (N:P:K 0.157:0.014:0.013%). Oxaprozin Plants were grown on 1 m2 plot for six weeks between May and June 2008. Afterwards, ten randomly chosen clover plants growing in each other’s vicinity were harvested, the nodules

were collected, surface-sterilized, crushed and their content plated on 79CA medium [22]. Strains isolated from the nodules were purified by successive streaking of single colonies and pure cultures were used in further experiments. DNA methods Standard techniques were used for labeling of DNA, Southern hybridization and agarose gel electrophoresis [23]. DNA probes for Southern hybridizations were obtained by PCR amplification with RtTA1 genomic DNA as template and appropriate primers (Table 1). The probes were labeled with non-radioactive DIG DNA Labeling and Detection Kit (Roche). Southern blotting, gel pretreatment and capillary transfers were done using standard procedures [23]. Hybridizations were performed at high stringency at 42°C using 50% formamide in pre-hybridization and hybridization solutions. Analyses of the plasmid content of the 129 isolates were performed as described by Eckhardt [24].