Cultivation and Genetic Manipulation of Listeria monocytogenes

L. monocytogenes mutants were derived from wild type strain 10403S [48 (link), 49 (link)] and cultured in brain heart infusion (BHI) broth at 37°C with shaking (220 rpm), unless otherwise stated. Antibiotics (purchased from Sigma Aldrich) were used at the following concentrations: streptomycin, 200 μg/mL; chloramphenicol, 10 μg/mL (Escherichia coli) and 7.5 μg/mL (L. monocytogenes); and carbenicillin, 100 μg/mL. Porcine bile (Sigma Aldrich) was dissolved in sterile BHI with streptomycin to ensure sterility. In cases where pH adjustments of media were carried out, 1N HCl was used and the pH was determined using VWR sympHony benchtop pH meter. L. monocytogenes strains are listed in Table F in S1 Text and E. coli strains are listed in Table G in S1 Text. Plasmids were introduced in E. coli via chemical competence and heat-shock and introduced into L. monocytogenes wt via trans-conjugation from E. coli SM10 [50 (link)].