To explicitly identify genes required for cytosolic survival of L. monocytogenes, we designed and executed a novel genetic screen to identify mutants of L. monocytogenes that lyse in the cytosol of host cells. Bacteriolysis of L. monocytogenes at the population level is indirectly measured through delivery of a luciferase-based reporter plasmid (pBHE573) (8) to the host cytosol during infection. Luciferase expression occurs only if the reporter translocates from the bacteria to the host cytosol since the luciferase gene is transcribed from a cytomegalovirus (CMV) promoter (see Fig. S1A in the supplemental material). We performed a nonsaturating screen using approximately 6,500 independent L. monocytogenes transposon mutants carrying pBHE573 and monitored for bacteriolysis (Fig. S1B). Type I interferon receptor-deficient immortalized macrophages (iIFNAR−/−) were used to avoid type I interferon-mediated suppression of translation (20), which would impact luciferase expression. Following secondary screening, we prioritized isolates which induced 2-fold or greater increases in luciferase expression compared to wild-type L. monocytogenes.