Using an in vitro bacteriolysis assay (28), we also examined each mutant for bacteriolysis during exponential-phase and stationary-phase growth in broth culture (Fig. 1B). As a positive control, we used a conditional mutant of diadenylate cyclase (cΔdacA), a gene essential for growth of L. monocytogenes in nutrient-rich media and for survival in vitro (28, 29). The lmo1602::Tn mutant was the only mutant which significantly lysed in broth culture, suggesting that this gene is essential for survival in vitro. These data highlight the idea that other genes identified in our genetic screen are required for survival in the macrophage cytosolic environment but not during extracellular replication.