Loss of MK biosynthesis in L. monocytogenes disrupts the electron transport chain (ETC) and prevents L. monocytogenes from producing a robust membrane potential (Fig. 4C). To investigate whether the ETC is required for intracellular survival of L. monocytogenes, we characterized transposon mutants with mutations in cydA, qoxA, and atpH (genes encoding ETC components cytochrome bd oxidase, cytochrome aa3 oxidase, and ATP synthase, respectively) (33). Similarly to MK-deficient mutants, the cydA::Tn mutant was deficient for generating a membrane potential and displayed in vitro aerobic growth defects (Fig. 5A and B). qoxA::Tn mutants were partially defective with respect to their ability to generate a membrane potential, whereas atpH::Tn mutants produced a robust membrane potential similar to that seen with the wild-type strain and grew to wild-type levels in vitro. No visible growth of the atpH::Tn mutant was observed under anoxic conditions, likely because ATP synthase is essential for generating a membrane potential during fermentative growth (34) (Fig. S7A). Interestingly, cydA::Tn mutants did not have a growth defect within macrophages but made significantly smaller plaques (Fig. 5C and Fig. S7B). In contrast, qoxA::Tn and atpH::Tn mutants also replicated in macrophages but formed plaques similar to those seen with the wild-type strain, suggesting that neither the cytochrome aa3 oxidase nor the ATP synthase of L. monocytogenes is important for virulence ex vivo (Fig. 5C and Fig. S7B). The atpH::Tn mutant displayed an initial invasion defect in macrophages (Fig. S7B), though this was likely be due to poorer growth in statically grown overnight cultures. Next we examined ETC complex mutants for intracellular survival. cydA::Tn mutants, but not qoxA::Tn or atpH::Tn mutants, lysed in the cytosol of macrophages, albeit not to ΔmenD levels (Fig. 5D), suggesting that the ETC or ability to generate a membrane potential may contribute, at least partially, to L. monocytogenes intracellular survival.