Macrophage Infection Assay with E. coli Strains

The RAW 264.7 murine MΦ cell line was maintained in an atmosphere containing 5% CO₂ at 37 °C in RPMI 1640 (Gibco) supplemented with 2 mM L-glutamine (Invitrogen), 1 mM sodium pyruvate (Invitrogen), 10 mM Hepes (Invitrogen), 100 U/ml penicillin/streptomycin (Gibco), 50 µM 2-mercaptoethanol solution (Gibco), 50 µg/ml gentamicin (Sigma) and 10% heat-inactivated fetal calf serum (FCS, standard RPMI complete medium). Infections were performed in RPMI-Strep medium, which is similar to standard RPMI complete medium but contains streptomycin (100 µg/ml) as the sole antibiotic.
Three different strains of Escherichia coli were used, two were the reference/Ancestral strain: MC4100-YFP and MC4100-CFP (MC4100, galK::CFP/YFP, AmpR StrepR), which contain the yellow (YFP) and cyan (CFP) alleles of GFP integrated at the galK locus in MC4100 (E.coli Genetic Stock Center #6152) and differ only by yfp/cfp locus that is constitutively expressed^{59 (link)}. The third strain is M6, a clone of MC4100-CFP that was evolved in the presence of MΦs for 450 generations^{14 (link)}. This strain has three new IS1 element insertions, in the coding regions of potD (1,084,946 nt), yiaW (3,640,515 nt) and in the regulatory region of the yrfF (3,411,601 nt) gene, as described elsewhere^{14 (link)}. Bacteria were grown in RPMI-Strep in similar conditions to the MΦ cell line.