When grown in different media, this is mentioned. In all LY294002 order experiments, the strains were cultured from stocks kept at −80 °C. Double knockout mutants in mutM and mutY were constructed using the Cre-lox system for gene deletion and antibiotic resistance marker recycling. Combined sacB-based negative selection and

cre-lox antibiotic marker recycling for efficient gene deletion in P. aeruginosa were used (Quenee et al., 2005). Upstream and downstream PCR fragments (Primers listed in Table S1) of the wild-type mutM or mutY gene from P. aeruginosa strain PAO1 were digested with HindIII and either BamHI or EcoRI, and cloned by a three way ligation into pEX100Tlink deleted for the HindIII site and opened by EcoRI and BamHI. Eighty-four residues from position 268 were deleted, when the upstream and downstream mutM amplified fragments were joined in pEX100Tlink vector, and 76 residues from position 374 were deleted in mutY, respectively. The resulting plasmids (pEXTMM and pEXTMY) were transformed into E. coli XL1Blue strain, and transformants were Obeticholic Acid ic50 selected in 30 mg L−1 ampicillin LB agar plates. The lox flanked gentamicin resistance cassette (aac1) obtained by HindIII restriction of plasmid pUCGmlox was cloned into the HindIII sites in pEXTMM and pEXTMY

formed by the ligation of the upstream and downstream PCR fragments. The resulting plasmids were transformed into E. coli XL1Blue strain, and transformants were selected on 30 mg L−1 ampicillin–5 mg L−1 gentamicin LB agar plates. The resulting plasmids (pEXTMMGm and pEXTMYgm) Fossariinae were then transformed into the E. coli S17-1 helper strain. Single knockout mutants were generated by conjugation, followed by selection of double recombinants using 5% sucrose-1 mg L−1 cefotaxime-30 mg L−1 gentamicin LB agar plates. Double recombinants were checked by screening for ticarcillin (100 mg L−1)

susceptibility and afterwards by PCR amplification and sequencing. For the recycling of the gentamicin resistance cassettes, plasmid pCM157 was electroporated into different mutants. Transformants were selected in 250 mg L−1 tetracycline LB agar plates. One transformant for each mutant was grown overnight in 250 mg L−1 tetracycline LB broth to allow the expression of the cre recombinase. Plasmid pCM157 was then cured from the strains by successive passages on LB broth. Selected colonies were then screened for tetracycline (250 mg L−1) and gentamicin (30 mg L−1) susceptibility and checked by PCR amplification. The single knockout mutants obtained were named PAOMMgm and PAOMYgm. To obtain the double mutant, the conjugation experiments with pEXMMGm using PAOMY as recipients were performed as described above. MutY-mutM double mutant was named PAOMY-Mgm. The maximum growth rate was found to be the same for PAOMY-Mgm and PAO1 in LB (Philipsen et al., 2008).