For preparation of total RNA for primer extension assays, overnight cultures were

diluted 100-fold in 30 mL of YESCA medium (per litre: 1 g yeast extract, 10 g Casamino acids) (Pratt & Silhavy, 1998) and MlrA-overproducing E. coli cells were incubated for 5 h at 37 °C until the exponential phase (OD600 nm=0.5–0.6). RNA purification was carried out as described previously (Ogasawara et al., 2007a, b). Primer extension analysis was performed using fluorescently labelled probes according to the protocol of Yamada et al. (1998). In brief, 20 μg of total RNA and 1 pmol of 5′-FITC-labelled primer (csgD-FITC-R: 5′-GCACTGCTGTGTGTAGTAAT-3′) were mixed in 20 μL of 10 mM Tris-HCl (pH 8.3 at 37 °C), 50 mM KCl, 5 mM this website MgCl2, 1 mM

each of dATP, dTTP, dGTP and dCTP, and 20 U of RNase inhibitor. The primer extension reaction was initiated by the addition of 5 U of avian myeloblastosis virus reverse transcriptase. After incubation for 1 h at 50 °C, DNA was extracted with phenol, precipitated with ethanol and subjected to electrophoresis on a 6% polyacrylamide sequencing gel containing 8 M urea. After electrophoresis, gels were dried and subjected to autoradiography using DSQ-500L (Shimadzu). A 438-bp fragment of the csgD promoter, a 489-bp fragment of the csgB promoter and a 355-bp fragment of the dppB promoter upstream Sitaxentan from the respective Sunitinib molecular weight translation start site were prepared by PCR using E. coli K-12 KP7600 genome as a template and a pair of primers, csgD-EcoRI-F

and csgD-BamHI-R2 for csgD, csgB-EcoRI-F and csgB-BamHI-R for csgB, and dppB-EcoRI-F and dppB-BamH1-R for dppB (Table S2). After digestion with EcoRI and BamHI, each fragment was ligated into pRS551 (Simons et al., 1987) at the corresponding sites. The resulting plasmids were transformed into E. coli MC4100 and the transformants were used as hosts for preparation of λRS45. Recombinant λ phages containing csgD–lacZ, csgB–lacZ or dppB–lacZ fusions were infected onto the wild-type, csgD or mlrA mutant E. coli strains (Table S1). Cells were cultured in LB medium or YESCA medium at 37 °C. When necessary, 100 μg mL−1 ampicillin and 50 μg mL−1 kanamycin were added. Escherichia coli transformants were grown in LB medium and subjected to a β-galactosidase assay with o-nitrophenyl-d-galactopyranoside as a substrate (Miller, 1972). For monitoring the influence of MlrA on expression of the lacZ reporter plasmids, an arabinose-inducible MlrA-expression plasmid was constructed using pBAD18 vector (Guzman et al., 1995). In brief, a DNA fragment containing the MlrA-coding frame was prepared by PCR using E. coli KP7600 genome DNA as a template and a set of primer pairs (Table S2).