Luria bertani

The strains and plasmids used are listed in Table 1. Listeria monocytogenes bacteria were all derived from the serovar 4b human isolate P14 (Ripio et al., 1996; 1997). Listeria and Escherichia coli were grown at 37°C in BHI (Difco‐BD) and Luria–Bertani (Sigma) media, respectively, supplemented with 1.5% agar (w/v) and/or antibiotics as appropriate. Chemicals and oligonucleotides were purchased from Sigma‐Aldrich unless stated otherwise.

Fecal samples and intestinal contents from the duodenum, ileum and cecum were weighed and resuspended in 1 ml of PBS. Tenfold dilutions were plated on Difco Enterococcosel (supplemented with 8 μg/ml vancomycin; Novaplus and 100 μg/ml streptomycin; Fisher) and Luria-Bertani (supplemented with 50 μg/ml neomycin; Sigma-Aldrich and 100 μg/ml carbenicillin; LabScientific) agar plates for the specific detection of VRE and K. pneumoniae, respectively. Mesenteric lymph nodes were mashed through a 40 μm filter, resuspended in PBS and plated directly onto selective plates.

All experiments were carried out in E. coli MG1655 using Luria-Bertani or MHB medium (Sigma-Aldrich). All control and constructed plasmids are listed in Supplementary Table S3. Media was supplemented with colistin (Cayman Chemical) or ampicillin (Sigma-Aldrich) as appropriate to enable selected growth of plasmid carriers and for MIC assays.

The activation of competent E. coli Top 10 cells was carried out in 100 mL of LB medium (Luria–Bertani, Sigma-Aldrich, St. Louis, MO, USA) supplemented with 20 mmol/L of MgCl₂ solution, incubated at 37 °C for 18 h. Komagaetella phaffii X-33 yeast was reactivated in 15 mL YPD liquid medium (Yeast Extract Peptone Dextrose, Sigma-Aldrich) for 24 h/30 °C with shaking at 150 RPM.

Acrylamide, ammonium persulfate, ampicillin, benzamidine, calcium chloride (CaCl₂), dithiothreitol (DTT), ethylenediaminetetraacetic acid (EDTA), glycine, imidazole, isopropyl β-d-1-thiogalactopyranoside (IPTG), Luria-Bertani (LB) broth, magnesium chloride (MgCl₂), nickel chloride, phenylmethylsulfonyl fluoride (PMSF), polyvinylidene fluoride (PVDF) membrane, potassium chloride (KCl), sodium azide (NaN₃), sodium bicarbonate (NaHCO₃), sodium carbonate (Na₂CO₃), sodium chloride (NaCl), sodium dodecyl sulfate, tetramethylethylenediamine (TEMED), tris(hydroxymethyl)aminomethane (Tris) were obtained from Sigma-Aldrich (St. Louis, MO, USA).

Human embryonic kidney cell line 293 (HEK293) was kindly provided by Dr. Joseph Ruiz at Enzerna Biosciences (Raleigh, NC). Media and reagents for growing and maintaining cells were purchased from Life Technologies Corporation (Carlsbad, CA). The cells were maintained in Dulbecco’s Modified Eagle’s medium (DMEM) supplemented with 10% (by volume) fetal bovine serum and 1% penicillin-streptomycin. Cells were sustained in a humidified incubator at 37 °C and 5% CO₂. A 0.25% trypsin-EDTA solution was used for detachment of cells. One Shot Stbl3 Chemically Competent cells of Escherichia coli (Life Technologies Corporation) were used for constructing the mutagenesis plasmid. Bacteria carrying the plasmids were maintained in Luria Bertani (Sigma-Aldrich, St. Louis, MO) agar or broth, and sustained in a humidified incubator at 37 °C.

The bacterial strains used in this study were Sphingomonas sediminicola (DSM-18106) [43 (link)], kanamycin-resistant DH5-α Escherichia coli carrying GusA under the constitutive promoter pNeo in the pOPS0385 plasmid (Addgene ID #133235) [44 (link)], and Rhizobium leguminosarum bv. viciae Rlv3841 [45 (link)]. S. sediminicola was grown in R2A medium (VWR, Fontenay-sous-Bois, France) for 72 h at 30 °C (constant shaking at 150 rpm), R. leguminosarum at 28 °C (150 rpm) in tryptone-yeast extract (TY) medium [46 (link)], and the E. coli strain was grown at 37 °C for 24 h (200 rpm) in LB medium (Luria–Bertani, Sigma Aldrich, St. Louis, MO, USA). Bacterial concentration (CFU mL⁻¹) was determined by the spiral method using an EasySpiral^® automatic plater (Intersciences, Mourjou, France) and counted using a Scan^®500 (Interscience). At DO_{600 nm} = 0.75, the S. sediminicola culture corresponded to 2 10⁶ CFU mL⁻¹, while 1 10⁸ CFU mL⁻¹ was obtained with R. leguminosarum culture.
Mutation of S. sediminicola conferring a resistance to rifampicin was generated spontaneously by plating the bacteria on R2A medium containing rifampicin (25 mg L⁻¹) [46 (link)]. Using bacterial conjugation [47 (link)], the plasmid pOPS0385 carrying GusA was transferred into S. sediminicola^Rif, giving S. sediminicola^Rif [pOPS0385]. This strain was grown in R2A containing 20 mg L⁻¹ kanamycin and 25 mg L⁻¹ rifampicin.