Bacterial growth was quantified by measuring optical density at 600 nm (OD600) every 3 hours. Antibiotics were used at the following concentrations (in μg ml-1): chloramphenicol (Cm), 30; tetracycline (Tc), 10; kanamycin (Km), 30; gentamicin (Gm), 30; spectinomycin (Sp),

100; nalidixic acid (Nal), 20. E. coli transformants harboring recombinant plasmids (β-galactosidase-positive) were identified by growth on LB plates with 30 μg ml-1 5-bromo-4-chloro-3-indolyl-βP505-15 order -D-galactoside (X-Gal). DNA manipulations Standard techniques described by Sambrook et al. [28] were used for plasmid and total DNA isolation, restriction, cloning, transformations, and agarose gel electrophoresis. Plasmid mobilization from E. coli to Rhizobium was done by conjugation performed on PY plates at 30°C by using overnight cultures grown to stationary phase. Donors (E. coli strain S17-1) and recipients (R. etli CFN42 wild type and mutant strains) selleck inhibitor were mixed at

a 1:2 ratio, and suitable markers were used for transconjugant selection. Mutagenesis of the panC and panB genes and genetic complementation of mutant strains Mutants were generated by site-directed vector integration mutagenesis. Internal 400 bp DNA fragments learn more of panC and panB were amplified by PCR with primers A and B; C and D, respectively (Table 3). PCR fragments of panC and panB were cloned in vector pBC as 400 bp BamHI-XbaI fragments, generating pBC1 and pBC2 respectively, and then subcloned as KpnI-XbaI fragments into suicide vector pK18mob [29] to form plasmids pTV1 and pTV2, respectively. These plasmids were mobilized into R. etli CFN42 by conjugation and single crossover recombinants selected on PY

plates containing Km and Nal. The disruption of the panC and panB genes was confirmed by Southern blot analysis using a 400-bp PCR internal fragment of each gene as a probe (data not shown). The resultant mutants were named ReTV1 and ReTV2 respectively. To complement the phenotype of the panC and panB mutants, plasmids pTV4, pTV5, pTV6 and pTV7 were constructed as follows: a 3.1 kb EcoRI fragment from cosmid vector pCos24, isolated from a genomic library of R. etli CFN42 [30] and containing the panC and panB genes, was subcloned in broad-host-range Angiogenesis inhibitor vector pRK7813, generating plasmid pTV4. To construct plasmid pTV5, a 1.2 kb fragment containing only panC (894 bp) was amplified by PCR with primers E and F and cloned in the KpnI-XbaI sites in the broad-host-range vector pBBRMSC3 so that the gene would be constitutively expressed from the vector’s lacZ promoter. Primers G and H (Table 3) were used to amplify a 1 kb PCR fragment containing only the panB gene (822 bp). This DNA fragment was cloned in plasmid pBBRMSC3 in the KpnI-XbaI restriction sites, generating plasmid pTV6. Plasmid pTV7 contains the second panB gene (RHE_PE00443), encoded on R. etli plasmid p42e, this gene was amplified with primers I and J.