Effect of AraC-type regulator overproduction in K. pneumoniae NCTC 5055 on envelope permeability and gene expression. (a) The accumulation of H33342 dye by K. pneumoniae NCTC 5055 transformants overexpressing ramA, rarA, soxS or marA is represented as a percentage relative to accumulation in the plasmid-only control transformant (set to 100%) over a 30 cycle (45 min) incubation period. Each graph shows the mean data for four biological replicates with eight technical replicates in each, and error bars define the SEM. (b) OMPs were purified from K. pneumoniae NCTC 5055 transformants carrying the control plasmid or those overexpressing ramA, rarA, soxS or marA. The OmpK35 and OmpK36 porins were identified by reference to panel (c), which shows OMPs from E. coli TOP10 and transformants overexpressing ompK35 or ompK36 cloned from K. pneumoniae NCTC 5055. OmpA and TolC are marked by reference to their predicted molecular weight and relatively high abundance. All OMPs were separated using SDS–PAGE and stained as set out in the Materials and methods section. These data are representative of three biological replicates. (d) Fold change in the abundance of selected efflux pump proteins and (e) fold change in the efflux pump gene mRNA levels in K. pneumoniae NCTC 5055 transformants carrying the control plasmid (set to 1) or those overexpressing ramA, rarA or soxS growing in nutrient broth. Data are mean ± SEM; three biological replicates, with four technical replicates for the quantitative RT–PCR.