To exclude the possibility of secondary mutations, we transduced the transposon mutations into clean wild-type backgrounds and reexamined these strains for survival in macrophages (Fig. 1A). Holin-lysin, a L. monocytogenes strain expressing inducible bacteriophage holin and lysin proteins, and ΔyvcK mutants were used as positive bacteriolysis controls (8). Δhly mutants which cannot escape the host vacuole (27) were used as negative controls (8, 18). L. monocytogenes bacteriolysis mutants displayed bacteriolysis that was 4-fold to 8-fold higher than that seen with the wild-type strain, results similar to those obtained with ΔyvcK mutants (Fig. 1A). Importantly, while the mutants that we identified lysed more frequently than the wild-type strain, lysis of these strains was still a relatively rare event compared to lysis of the ~100% lysis control, holin-lysin. We also constructed markerless deletions and genetic complements for every gene identified in the genetic screen and tested them for survival in macrophages (Fig. S2A). With the exception of a ΔpdhC strain which we were unable to construct, the deletion strains reliably phenocopied their transposon mutant counterparts. Additionally, complementation of clean deletions and transposon mutants of the majority of mutants was successful, with the exception of ΔnrdD and ΔyvcJ mutants. Furthermore, to test whether mutants undergo complete bacteriolysis and not simply plasmid secretion, we created a chromosomal CMV-luciferase reporter and assayed L. monocytogenes mutants for intracellular survival. pdhC::Tn, menD::Tn, menF::Tn, and yvcJ::Tn mutants underwent complete bacteriolysis (Fig. S2B). The level of luciferase production from the chromosomal lysis reporter was significantly reduced compared to that seen in the plasmid-based assay, and as such, both the wild-type strain and the mutants demonstrated low levels of lysis that were below the limit of detection.