2-kb fragment containing the otsA region which was cloned in pSKbluescript previously digested with BamHI-XbaI to obtain the plasmid pMotsA4. Subsequently, a BglII recognition site was generated in otsAch gene sequence, using the PCR-based QuickChange Site Directed Mutagenesis Kit (Stratagene) and the primers: otsA R BglII FW (5’-GAAGAGAGGGCATTGGCGAA GATCTCGGCAACGGATTGTTCGATTC-3’), and otsAR check details BglII RV (5’-GAATCGAACAATCCGTTGCCGAGATC TTCGCCAATGCCCTCTCTTC-3’), that were modified (residues underlined) to generate the corresponding restriction site (in bold), to obtain the plasmid pMotsA5. To interrupt the otsA gene, the resulting plasmid was linearized with the enzyme
BglII and ligated to a 2-kb BamHI fragment obtained from pHP45-Ω plasmid [38],
containing the Ω interposon for insertional mutagenesis (Smr). The resulting plasmid was designated pMotsA6. To recombine the otsA mutation into the R. etli chromosome, a 6.1-kb ApaI-XbaI fragment from pMotsA6 was cloned into the suicide vector pJQ200-SK (Gmr) [38] to obtain plasmid pMotsA7, which was mobilized into the R. etli CE3 by triparental mating. Mutant strains resulting from a double homologous recombination event were identified as SpcrGms colonies on TY plates containing 10% sucrose. One of these colonies was purified for further analysis and was designated CMS310 (otsAch). Insertion of the omega cassette in CMS310 was confirmed by PCR and sequencing. Conjugal transfer of plasmids Plasmids were transferred from E. coli to R. etli by triparental Thiazovivin purchase mating on TY medium, using pRK600 as a helper plasmid [37], as described by Vargas et al. [43] but with a 1:2:1:(donor:receptor:helper) ratio. Sequence and phylogenetic analyses The sequence of the R. etli CFN 42 genome is available at NCBI microbial genome database ( http://www.ncbi.nlm.nih.gov/genomes/lproks.cgi; Ac N°: NC_007761), and at http://www.ccg.unam.mx/retlidb/. Sequence data
were analyzed using BLAST (NCBI http://ncbi.nlm.nih.gov/BLAST). ORF assignments of the metabolic pathways more relevant for this work was performed by comparing the information available at the Kyoto Encyclopedia of Genes and Genomes (KEGG) [44] and MetaCyc [45]. Codon preference was analysed at the Kasuza Codon Use Database ( http://www.kazusa.or.jp/codon/). Phylogenetic and molecular evolutionary analyses were conducted using MEGA version 5 [46]. Sequences Reverse transcriptase were aligned with ClustalW (1.6) using a BLOSUM62 matrix, and selleck chemical manually edited. The phylogenetic tree was inferred using the Neighbor-Joining method [47], and the evolutionary distances were computed using the Poisson correction method. The rate variation among sites was modelled with a gamma distribution (shape parameter = 1) and all positions containing gaps and missing data were eliminated only in pairwise sequence comparisons. The robustness of the tree branches was assessed by performing bootstrap analysis of the Neighbour-Joining data based on 1000 resamplings [48].