An excitation wavelength of 480 nm and an emission wavelength of 520 nm were utilized

ore use. CpxAR Confers b-Lactam Resistance DNA methods Restriction digestion, ligation, transformation, and agarose gel electrophoresis were done according to standard protocols. Plasmids were prepared from E. coli using a QIAprep Spin miniprep kit from Qiagen according to the manufacturer’s protocol. Mobilization of plasmids into K. pneumoniae cells was performed as previously described. Genomic DNA of K. pneumoniae was extracted using the Gene Aid DNA purification kit according to the manufacturer’s instructions. DNA fragments used for cloning were extracted from agarose gels using a QIA quick gel extraction kit from Qiagen. PCR products were purified using a QIA quick PCR purification kit and, when cloned, sequenced to confirm the correct sequences. Primers used in the present study were custom-synthesized. Construction of the cpxAR deletion mutant in K. pneumoniae strain NTUH-K2044 The MisT2 database shows the presence of.466 signaling proteins in the 5,472,672 bp genome sequence of the K1 serotype. The CpxAR operon is located starting from nucleotides 76799 bps to 78867 bps in the genome sequence of K. pneumoniae NTUH-K2044. To construct cpxAR knock out, a 700 bp internal fragment encompassing cpxA and cpxR of the operon was amplified by PCR using DcpxA/cpxR-F and DcpxA/cpxR-R primer from its genomic DNA. The PCR product was ligated into an EcoRI digested plasmid pUT-Km which was blunted by klenow reaction that contained the kanamycin resistance gene, transformed into E. coli S17-1l pir and the resulting recombinant plasmid harbouring the internal fragment of cpxAR was designated as pUT-Km/GR. The plasmid pUT-Km/GR was mobilized into recipient K. pneumoniae NTUH-K2044 from donor E. coli S17-1l pir. Briefly, K. pneumoniae was inoculated into 10 ml LB and was incubated for 23 h ” till OD600 nm reaches 0.2. For matings, recipient and donor culture were mixed in a ratio of 1:2 respectively, pelleted and spotted onto the centre of an LB agar plate. After 3 h of growth at 37uC the cells were plated on Klebsiella selective agar containing Kanamycin 100 mg/ml and 5 mg/ml chlorhexidine to select for colonies. It is expected that colonies that appear on the selective plate would be transconjugants that resulted from one DNA exchange event in which the whole suicidal plasmid gets incorporated in the K. pneumoniae genome. The disruption at cpxAR operon was confirmed with selected transconjugant by PCR and DNA sequencing using gene specific and genome flanking primers and deleted mutant was A-83-01 price denoted as NTUH-K2044DcpxAR. Intact cpxAR genes were amplified along with its promoter using primer NT and primer CT and cloned into a pCRIITOPO-CAT plasmid. The selected recombinant plasmid harbouring the intact cpxAR operon was transformed into the cpxAR isogenic mutant strain by electroporation. The complementation strains were selected on LB agar plates supplemented with 100 mg/mL kanamycin and 100 mg/mL chloramphenicol and transcomplemented strain was designated as NTUH-K2044DcpxARVcpxAR. 12 CpxAR Confers b-Lactam Resistance Primer name DcpxA/cpxR-F DcpxA/cpxR-R Primer NT Primer CT cpxR-F cpxR-R prom ompC-F prom ompC-R eefBnt eefBct acrBnt acrBct acrDnt acrDct kmrAnt kmrAct 15523001” 16sF 16sR Primer sequences inhibition assay was performed as described previously. The efflux pump inhibitors used in this study was carbonyl cyanide 3-chlorophenylhydrazone and reserpine. CCCP is an extremely effective proton motive force inhibitor and used in this study as an active