This strain was used as receptor to select transconjugants carrying the Tn5mob-labeled pSym of

GR64 (CFN2001-1), the Tn5mob-labeled pSym of GR64 and Tn5-GDYN-labeled pRet42a of R. etli CFN42 (CFN2001-2), and both plasmids of GR64 (CFN2001-3). Other derivatives carried either pSfr64a::Tn5-GDYN or pSfr64b::Tn5mob in Agrobacterium strain GMI9023 genomic background. Plasmid profiles Plasmid profiles were visualized by the Eckhardt technique [38], as modified by Hynes and McGregor [39]. Filter blot hybridization and plasmid visualization For Southern-type hybridizations [40], Eckhardt type gels, or 1% agarose gels where restricted DNA was electrophoresed, were blotted onto nylon membranes, and hybridized under stringent conditions, as previously reported [41], by using Rapid-hyb buffer. Probes were linearized by digesting them with Selleck VX 809 appropriate restriction enzymes and were labeled with [α32P]dCTP by using VEGFR inhibitor a Rediprime DNA labeling system. All restriction endonucleases, [α -32P]dCTP, hybridization buffer, and labeling systems were purchased from Amersham Pharmacia Biotech. Nodulation assays Overnight cultures were used to inoculate surface-sterilized Phaseolus vulgaris cv. Negro Jamapa seeds. Plants were grown in 250-ml Erlenmeyer flasks with Fahraeus agar medium [42], without added nitrogen, at 28°C. Nodulation was scored at day 15 after inoculation. Surface-sterilized nodules were crushed on PY plates,

and the plasmid pattern of single colonies was checked on Eckhardt type gels. Amplification and sequencing of recA, rpoB, and nifH gene fragments Partial nifH, recA and rpoB fragments were amplified with the primer pairs nifH40F/nifH817R, recA41F/recA640R and rpoB454F/rpoB 1364R as previously Sulfite dehydrogenase described [43, 44]. All amplifications were performed with Taq polymerase (USB-Amersham). Amplification products were purified

using Roche’s PCR product purification system. Both strands were commercially sequenced by Macrogen, Korea. Phylogenetic inference Reference nifH, recA, rpoB and repB sequences were retrieved via BLASTP searches from a locally maintained BLAST RG7420 clinical trial database containing all fully sequenced Rhizobiales genomes, and via remote BLASTP searches against NCBI’s non-redundant database. The query sequences for nifH, recA and rpoB used in the BLASTP searches were those obtained from the sequenced PCR amplicons from strain GR64, while that of repB was obtained from the sequence of pSfr64a. Nucleotide sequences were translated and aligned using muscle 3.7 [45]. The resulting protein multiple sequence alignments were used as masks to generate the underlying codon alignments using custom Perl scripts. Models of nucleotide substitution were selected by the Akaike information criterion (AIC), using MODELTEST3.7 [46]. Among-site rate variation was modelled by a gamma distribution, approximated with 4 rate categories, each category being represented by its mean.