The E. coli strain Nissle 1917 used in this study was obtained from Dr. Ulrich Dobrindt (University of Münster). The clbA and clbP isogenic mutants were described previously (ref Ollier et Nat Comm). Before infection, the bacteria were grown overnight at 37°C with 240 RPM agitation in 5 mL of Lennox L broth (LB, Invitrogen) then diluted 1/20 in pre-warmed DMEM 25 mM Hepes (Invitrogen) and incubated at 37°C with 240 RPM agitation to reach exponential phase (OD600=0.4 to 0.5).
In vitro DNA crosslinking assay 3x10 6 bacteria or numbers given in the text were inoculated in 100 l of DMEM 25 mM Hepes, incubated at 37°C for 3.5 hours, then EDTA (1 mM) and 400 ng of linearized (BamHI) pUC19
DNA were added and further incubated 40 minutes. As controls, DNA was left uninfected or was treated with 100 or 200 M cisplatin (Sigma). Following a centrifugation to pellet the bacteria, the DNA was purified using Qiagen PCR DNA purification kit before analysis by denaturing gel electrophoresis.
In vitro DNA crosslinking assay 3x10 6 bacteria or numbers given in the text were inoculated in 100 l of DMEM 25 mM Hepes, incubated at 37°C for 3.5 hours, then EDTA (1 mM) and 400 ng of linearized (BamHI) pUC19
DNA were added and further incubated 40 minutes. As controls, DNA was left uninfected or was treated with 100 or 200 M cisplatin (Sigma). Following a centrifugation to pellet the bacteria, the DNA was purified using Qiagen PCR DNA purification kit before analysis by denaturing gel electrophoresis.