To understand what host defenses may induce intracellular bacteriolysis, we examined these mutants for sensitivity to a variety of stresses. MK auxotrophs were not sensitive to cell wall- or cell membrane-targeting antimicrobials (Fig. S6A and B). Although sensitivity to these antimicrobials could be masked by trace amounts of MK in the brain heart infusion (BHI) media, MK auxotrophs were still hypersensitive to oxidative stress in BHI cultures (Fig. S6C). We next examined cellular reactive oxygen species (ROS) production in macrophages infected with the wild-type strain or the ΔmenD mutant but did not detect increases in ROS production during infection (Fig. S6D). Though ROS scavenging with N-acetylcysteine (NAC) did partially lower ROS production in menadione-induced macrophages (positive control), NAC treatment did not rescue MK mutants from bacteriolysis, suggesting that cytosolic ROS is not responsible for the intracellular survival defects of MK-deficient mutants, though additional evidence may be required (Fig. S6D and E). These data leave open the possibility that another unknown cytosolic stress(es) or host defense may be responsible for lysis of MK-deficient L. monocytogenes in macrophages.