(2002). In this method, the plasmid pCE37 was integrated into the FRT target sequence immediately downstream of the deleted sbmA gene by the FLP-mediated recombination. The fusion was then transduced into the MC4100 tolC strain. The acrB mutation was generated using the same methodology. In this case, the PFWacrB and PRVacrB primers (Table S2) were used to obtain the PCR product for gene deletion. The ΔsbmA∷lacZY fusion, constructed previously, was then transduced into the MC4100 acrB strain. The degP∷lacZY transcriptional fusion was first constructed in the JW0157 strain using λ Red and FLP-mediated site-specific recombination, which
was described previously (Ellermeier et al., 2002). Finally, the transcriptional fusion was transduced into MC4100 and MC4100 Stem Cell Compound Library tolC strains. We introduced the tolC mutation (tolC∷Tn10) into a rybB fusion strain KMT12000 (Table S1) to obtain the KMT12000 tolC strain. The rseA mutant was obtained by transduction of rseA∷aph from the strain JW2556 into MC4100 strain and the cassette was subsequently removed. The ΔsbmA∷lacZY fusion was transduced into the MC4100 rseA strain, as described previously. The strain CAG22222 contained an uncharacterized mutation that suppresses the σE essentiality (Rouviere et al., 1995). For this reason, the ΔsbmA∷lacZY fusion was transduced into this strain and all assays
with rpoE mutants were performed in this context. The β-galactosidase activities were determined following the method described by Zhou & Gottesman (1998), with a few modifications. The fusion strains L-gulonolactone oxidase were grown to OD600 nm=0.8 and assayed for β-galactosidase activities. Selleck ABT 199 For this, 600-μL
aliquots of these cultures were permeabilized for at least 20 min with 0.1% SDS (24 μL) and chloroform (48 μL). Then, 100 μL of permeabilized cells were placed on 96-well microtiter plate, 100 μL of a 4 mg mL−1 solution of o-nitrophenyl-β-d-galactopyranoside in buffer Z was added and A420 nm were measured for 20 min in a SpectraMax 250 spectrophotometer. Specific activity was calculated by dividing the slope of the line over time by the corresponding OD600 nm and expressed as arbitrary units (AU). The sensitivity to microcin B17 (MccB17) was tested using a spot-on-lawn assay, as follows: doubling dilutions of a partially purified MccB17 were spotted (10 μL) onto M9 plates and dried. To test the sensitivity, stationary-phase culture aliquots (50 μL) were mixed with 3 mL of top agar (M9 containing 0.7% agar) and overlaid onto the plates. After an overnight incubation, the plates were examined for different degrees of inhibition. To compare the ability of MC4100 and MC4100 tolC to produce extracellular MccB17, they were transformed with the pMM39 plasmid carrying the microcin production and immunity genes. The transformed strains were grown on a liquid M9 medium to the stationary phase and MccB17 was partially purified as described previously (Pierrat & Maxwell, 2003).