All mutant strains were confirmed by sequencing Sapitinib in vitro PCR-amplified DNA fragments containing the insertion site. Construction of eGFP translational fusion plasmids To create pJH1, digestion with XbaI/NdeI of pSCrhaB4 resulted in a 784 bp fragment containing eGFP, which was cloned into the same sites in pAP20 [9] such that eGFP is under control of the constitutive

dhfr promoter. E. coli transformants were selected with 20 μg/ml chloramphenicol. The plasmid was conjugated into B. cenocepacia K56-2 by tri-parental mating with E. coli helper strain containing plasmid pRK2013. As B. cenocepacia is intrinsically resistant to Gm, in all conjugations Gm was added to the final transfer to eliminate donor E. coli. To create pJH2, pJH1 was then PCR amplified using divergently

oriented primers (Additional file 1) containing multiple restriction sites on the 5′ ends such that the self-ligated product of the reaction has a multiple cloning site in mTOR inhibitor place of the original promoter. Growth rates for B. cenocepacia K56-2 with or without pJH2 were similar (data not shown). DNA fragments corresponding to paaZ from -420 to +90 (510 bp), paaA from -396 to +84 (480 bp), and paaH from -327 to +72 (399 bp) of B. cenocepacia K56-2 chromosomal DNA were amplified and cloned into pJH2 to create pJH6, pJH7, and pJH8 respectively. Construction of site directed plasmid mutants The plasmids pJH10, pJH11 and pJH12 were constructed by plasmid PCR mutagenesis to contain mutations in the entire, left or right region of the Quisinostat manufacturer conserved IR in the paaA core promoter. Appropriate phosphorylated primers (Additional file 1) were used to divergently amplify template pJH7 (containing the paaA promoter), and each contained mismatch mutations on their 5′ ends.

Plasmids were self-ligated, transformed into E. coli DH5α and then conjugated into B. cenocepacia wild type. Mutations were verified by sequence analysis (The Centre for Applied Genomics, Toronto). Nucleotide accession number The nucleotide sequence of isothipendyl translational fusion vector pJH2 is deposited in GenBank under accession no. FJ607244. Acknowledgements We thank Julian Parkhill and Mathew Holden for allowing us access to the draft annotation of B. cenocepacia J2315, and Ann Karen Brassinga for critically reading the manuscript. JNRH was supported by a graduate scholarship from the Manitoba Health Research Council (MHRC). RAMB is supported by a Manitoba Graduate Scholarship. This study was supported by the NSERC grant N° 327954. Electronic supplementary material Additional file 1: Primers used in this study. (PDF 68 KB) Additional file 2: Position Weight Matrix Calculations. A) The sequences used to generate the matrix of the conserved inverted repeat from the paaA, paaH, paaZ, paaF and BCAL0211 genes. B) The sum the occurrence of nucleotides at each position.