Acinetobacter sp. Tol 5 and its derivative mutants were grown in

basal salt (BS) medium supplemented with toluene or LB medium at 28°C, as described https://www.selleckchem.com/products/PLX-4720.html previously [28]. E. coli strains were grown in LB medium at 37°C. Antibiotics were used at the following concentrations when required: gentamicin (100 μg/ml) and kanamycin (100 μg/ml) for Tol 5 derivative mutants; gentamicin (10 μg/ml) and kanamycin (50 μg/ml) for E. coli strains. Table 1 Bacterial strains and plasmids used in this study Strain FDA approved Drug Library Description Reference Acinetobacter sp.     Tol 5 Wild type strain [19] G4 A Tol 5 mutant constructed by insertion of a FRT site in the upstream of ataA of Tol 5, Gmr, SacB This study G4K1 A Tol 5 mutant constructed by additional insertion of a FRT site in the downstream of ataA of G4, Gmr, Kmr, SacB This study 4140 Unmarked ΔataA mutant of Tol 5 constructed by FLP/FRT recombination in G4K1 This study E. coli     DH5α Host BMS345541 solubility dmso for routine cloning TaKaRa S17-1 Donor strain for conjugation [4] Plasmid     pJQ200sk Mobile plasmid, SacB, Gmr [32] pK18mob Mobile plasmid, Kmr [33] pLOI2224 Source of FRT sites, Kmr [34] pFT-A Source of FLP recombinase and tetR, Ampr [34] pJQFRT Gene replacement

vector harboring a single FRT sequence, SacB, and Gmr This study pKFRT Mobile plasmid harboring a single FRT sequence, Kmr This study pKFRT/FLP Gene replacement vector harboring a single FRT sequence, FLP recombinase under the control of Ptet promoter, and Kmr This study pJQFRT_AtaAupstream A 1.0-kb fragment containing the upstream region of ataA ligated into the BamHI site of pJQFRT This study pKFRT/FLP_AtaAdownstream A 2.8-kb fragment containing the downstream region of ataA ligated into the BamHI site of pKFRT/FLP This study Genetic manipulation General DNA manipulations, such as PCR, restriction enzyme digestion, and ligation, were performed using standard protocols. The plasmids and primers used in this study are detailed in Table 1 and 2, respectively. Table 2 Primers Erythromycin used in this study Primer Sequence (5′ → 3′) FRT-leftF AATCCATCTTGTTCAATCATGC FRT-rightR

AATTCGAGCTCGGGAAGATC FRT-T7F AAATTAATACGACTCACTATAGG FRT-SP6R TACGATTTAGGTGACACTATAG Inv-pUC118F CAACGTCGTGACTGGGAAAAC Inv-pUC118R TCATGGTCATAGCTGTTTCCTG TetR-FLP2F CGATGGGTGGTTAACTCGAC TetR-FLP2R ACAGGACGGGTGTGGTCG AtaAupstF CGCGGATCCGATCTTCAAAGGTTGTGCTCAG AtaAupstF2 AACGCAAGTTGTTTTACTGC AtaAupstR CGCGGATCCTAGAAGCTGTAGCAGTTGTTCC AtaAdwstF CGCGGATCCACTCGACAGGGAAGATCTTC AtaAdwstR CGCGGATCCAATTGAATCATCAACACCTGCTG AtaAdwstR2 TACGTCGAGCAGCTAAGGTC Underlines indicate BamHI site. Construction of pJQFRT and pKFRT/FLP Two mobile plasmids, pJQ200sk [32] and pK18mob [33], were used as the plasmid backbone. To remove their original multiple cloning sites, inverse-PCR was performed using the primers Inv-pUC118F/Inv-pUC118R.