cenocepacia strains J2315 CF learn more clinical isolate G. Manno D1 J2315 ΔBCAS0591-BCAS0593 This study D3 J2315 ΔBCAL1672-BCAL1676 This study D4 J2315 ΔBCAL2820-BCAL2822 This study E. coli strains DH5α F- Φ80dlacZΔM15 Δ(lacZYA-argF)U169 endA1 recA1 hsdR17(rK – mK +) supE44 thi-1 ΔgyrA96 relA1 Laboratory stock SY327

araD Δ(lac pro) argE(Am) recA56 nalA λ pir; Rifr [43] Plasmids pGEM-T Easy Vector for PCR cloning, Ampr Promega pGPISce-I PD173074 molecular weight ori R6K, ΩTpr, mob +, containing the ISce-I restriction site [32] pRK2013 ori colE1, RK2 derivative, Kanr, mob +, tra + [44] pDAISce-I pDA12 encoding the ISce-I homing endonuclease [32] pOP1/pGPI-SceI Plasmid for construction of D1 deletion mutant This study pOP3/pGPI-SceI Plasmid for construction of D3 deletion mutant This study pOP4/pGPI-SceI Plasmid for construction of D4 deletion mutant This study pSCR1 Ampr, pQF50 containing PcepI-lacZ and cepR [42] Ampr, ampicillin resistance; Kanr, kanamycin resistance; Rifr, rifampin resistance; Tetr, tetracycline resistance; Tpr, trimethoprim Alvocidib datasheet resistance. Molecular techniques Manipulation of DNA was performed as described previously [39]. Restriction

enzymes and T4 DNA ligase were purchased from GE Healthcare and used following the manufacturer’s instructions. E. coli DH5α and E. coli SY327 cells were transformed by the electroporation method [39]. Plasmids were mobilized into B. cenocepacia J2315 by triparental mating as described previously [40], using E. coli DH5α carrying the helper plasmid pRK2013. Gentamicin was used to counter select against the E. coli donor and helper strains. All PCR reactions used the MJ Mini Personal Thermal Cycler (BioRad). To amplify PCR products Taq DNA polymerase, HotStar HiFidelity Polymerase kit, Hot StarTaq DNA Polymerase or Qiagen LongRange PCR kit (QIAGEN)

were used and each reaction supplemented with Q solution according to the manufacturer’s instructions. DNA fragments were cloned into pGEM-T Easy vector (Promega) and sequenced using the standard M13for and M13rev primers. Southern blot analyses were performed as previously described [39]. MIC determinations Determination of MIC (Minimal Inhibitory Concentration) for B. cenocepacia J2315 and the deletion mutants D1, D3, and D4 was performed pheromone by streaking 1 × 104 cells onto LB agar containing 2-fold dilutions of different drugs. The following compounds were tested to determine the resistance profile: aztreonam, ethidium bromide, chloramphenicol, gentamicin, tobramicin, nalidixic acid, ciprofloxacin, levofloxacin, norfloxacin, sparfloxacin, ampicillin, ceftazidime, erythromycin, meropenem, piperacillin, kanamycin, tetracycline, and trimethoprim. Plates were incubated at 37°C for 3 days and the growth was visually evaluated. The MIC was defined as the lowest drug concentration that prevented visible growth. The results represent the average of three independent replicas.