Using plasmid mutagenesis with primers containing www.selleckchem.com/products/BAY-73-4506.html mismatched mutations on the 5′ ends (Additional file 1) that annealed to plasmid pJH7 containing the P paaA reporter we generated plasmids pJH10, pJH11 and pJH12 (Table 1). The plasmid pJH10 contains 14 mismatch mutations replacing nearly the entire IR within the paaA promoter.

Plasmids pJH11 and pJH12 contain the mutations in the upstream or downstream half of the IR respectively. These plasmids were then transferred to B. cenocepacia K56-2 by triparental mating. Reporter strains were grown in minimal media supplemented with glycerol or PA. Cells harbouring plasmids pJH10, PJH11 and pJH12 exhibited higher levels of relative fluorescence in comparison with K56-2/pJH7 when grown with glycerol, demonstrating that the sequence is indeed required for negative control of paaA promoter activity (Table 2). Table 1 Bacterial Strains and Plasmids Strain or plasmid Features Reference or source B. cenocepacia strains     K56-2 (LMG18863) ET12 clone related to J2315, CF clinical isolate [43] JNRH1 K56-2BCAL0210::pJH9, Tpr This study E. coli strains     DH5α F-, ϕ 80 lacZΔM15 endA1 recA1 hsdR17(rK -mK +)supE44 thi-1 ΔgyrA96 (ΔlacZYA-argF)U169 relA1 Invitrogen SY327 araD

Δ(lac pro) argE (Am) recA56 Rifr nalA λ pir [40] Plasmids     pGPΩTp ori VX-809 chemical structure r6K, ΩTpr mob+ [27] pRK2013 ori colE1, RK2 derivative, Kmr mob + tra + [42] pJH9 pGPΩTp, internal fragment from BCAL0210 This study pJH1 pap20, eGFP [9] pJH2 pJH1, eGFP replaced promoter with multiple cloning site This study pJH5 pJH2, BCAL0211promoter region OSBPL9 (P BCAL0211 ) This study pJH6 pJH2, paaZ promoter region (P paaZ ) This study pJH7 pJH2, paaA promoter region (P paaA ) This study pJH8 pJH2 paaH promoter region (P paaH ) This study

pJH10 pJH7, ACCGACCGGTCGGT → TAGATGTATCTCAG This study pJH11 pJH7, ACCGACCGGTCGGT → TAGATGTGGTCGGT This study pJH12 pJH7, ACCGACCGGTCGGT → ACCGACCATCTCAG This study Cm, chloramphenicol; Km, kanamycin; Tp, trimethoprim. Arrows represent changes introduced to indicated sequences. Because PaaX is involved in the regulation of upstream pathways of PA catabolism in other microorganisms through binding a conserved PaaX box [23, 24] we searched for the consensus IR sequence in the genome of B. cenocepacia. A position weight matrix (PWM) [25] of the conserved IR present in the promoter regions of the paaA, paaH and paaZ plus the divergent promoter of paaF and BCAL0211 was constructed (Additional file 2) and used to search the entire genome sequence of B. cenocepacia J2315. The coordinate positions of sequences detected up to a cut off score of 17.0 are listed (Additional file 3). The top scores for the search were the ones for the paaZ, paaF, paaA and paaH inverted repeats while BCAL0211 IR scored lower at 12.0. Other sequences with scores that ranked from 18.41 to 17.37 did not locate in putative promoters or between -10 and -35 regions, likely representing false positives.