Genetic Manipulation of E. coli K-12 Strains

The strains used in these experiments were derivatives of E. coli K-12 (Table 1). Strain XL-1 Blue was used for routine molecular biology and the fimA-lacZ transcriptional fusion strain VL386, and its derivatives, were used for experiments with the fimS genetic switch. The VL386 Δfis::kan knockout mutant was derived by P1vir-mediated transduction [75, 76 ] using a CSH50 fis::kan mutant lysate. VL386 lrp::cml was also prepared by transduction, using a CSH50 lrp::cml lysate. Complementation of the fis mutation was carried out using plasmid pFIS349, which is a single-copy plasmid based on the mini-F origin plasmid pZC320 [77 (link)]. Bacteria were cultured in lysogeny broth (LB, made from Difco media components) or LB agar (containing agar at 1.5 % w/v) [75 ]. MacConkey lactose agar plates [75 ] were used for Lac phenotype determination. Unless otherwise stated, liquid cultures were grown overnight at 37 °C with aeration at 200 r.p.m in an orbital incubator (New Brunswick). Where appropriate, antibiotics (Sigma-Aldrich) were used at the following concentrations: carbenicillin (100 μg ml⁻¹), chloramphenicol (25 μg ml⁻¹) and kanamycin (20 μg ml⁻¹). Plasmid DNA was introduced to bacterial cells by CaCl₂ transformation [78 ] or electroporation using a Bio-Rad Gene Pulser as described in Hanahan, 1983 [79 (link)].