T cin

Growth curves of strains WT/pIMK2, asnB/pIMK2, and asnB/pIMK2-asnB were established by turbidity measurement (OD₆₃₀) of cultures growing in an automated temperature-controlled microplate reader (Multiskan Ascent®, Thermo Fisher Scientific). Inocula were prepared by adjusting overnight 4-ml BHI broth cultures to the same turbidity (OD₆₀₀≈2) using an Ultrospec™ 10 Cell Density Meter (Biochrom, Cambridge, United Kingdom), and then diluting the suspensions 1,000-fold in BHI or BHI with 3- or 4-mM t-CIN (Acros Organics). Subsequently, 200-μl aliquots were transferred into a 96-well microplate, which was then covered with a transparent adhesive foil (Greiner Bio-One, Frickenhausen, Germany) and incubated at 30 or 37°C in the microplate reader. Every 15min, the plates were shaken at 960rpm and OD630 was measured. The Excel add-in package DMFit (Quadram Institute Bioscience, Norwich, United Kingdom) was used to determine the maximum growth rate (μ_max), the lag phase time (λ), and the maximal cell density (OD_max) value at stationary phase based on the Baranyi and Roberts microbial growth model (Baranyi and Roberts, 1994 (link)).

Bacterial strains and plasmids used in this work are listed in Table 1. L. monocytogenes Scott A was used as the wild-type (WT) strain and acquired from the International Life Sciences Institute (ILSI) North America [17 (link)]. E. coli DH5α [18 (link)] and S17-1 λpir [19 (link)] were employed as the host for cloning constructs and as donor strain for conjugational plasmid transfer, respectively. L. monocytogenes strains were grown at 30 °C in Brain Heart Infusion (BHI; Oxoid, Hampshire, UK). E. coli strains were grown in Luria-Bertani (LB; 10 g/L tryptone, 5 g/L yeast extract, 5 g/L NaCl) at 37 °C. Antibiotics were used when appropriate in the following concentrations: 50 µg/mL erythromycin (Acros Organics, Fair Lawn, NJ, USA) (Ery), 50 µg/mL kanamycin (AppliChem GmbH, Darmstadt, Germany) (Km), 100 μg/mL ampicillin (Thermo Fisher Scientific, Waltham, MA, USA) (Amp), 20 µg/mL polymyxin B sulfate (AppliChem GmbH) and 10 μg/mL chloramphenicol (Acros Organics) (Cm). Other chemicals used in this work include t-CIN (Acros Organics), N-Acetylglucosamine (Sigma-Aldrich, Saint Louis, MO, USA) and isopropyl β-D-1-thiogalactopyranoside (Acros Organics) (IPTG, 1 mM).

The bacterial strains and plasmids used in this work are listed in Table 1. The L. monocytogenes strains were grown at 30°C in brain heart infusion (BHI, Oxoid, Hampshire, United Kingdom) medium, whereas the E. coli and B. subtilis strains were grown in Luria-Bertani (LB; 10 g/L tryptone, 5 g/L yeast extract, 5 g/L NaCl) medium at 37°C. Agar was added at 1.5% for solid media. The media were supplemented with kanamycin (Km; AppliChem, Darmstadt, Germany), erythromycin (Em; Acros Organics, NJ, USA), ampicillin (Amp; Thermo Fisher Scientific), and anhydrotetracycline (ATc; CaymanChem, MI, USA) when appropriate. The antimicrobial essential oil compounds used in this work include t-CIN (Acros Organics), trans-2-hexenal (Sigma-Aldrich, Saint Louis, MO, USA), and 4-hydroxy-2-nonenal (Abcam, Cambridge, GB).