To investigate how MK biosynthesis drives intracellular survival of L. monocytogenes in macrophages, we obtained transposon mutants (from an unpublished genetic screen that identified small-colony variants) with mutations of every gene encoding enzymes in the MK biosynthesis pathway, with the exception of menH and the unknown thioesterase gene. Mutations in genes menF through menB, which are responsible for the first six steps in MK biosynthesis, led to survival defects of L. monocytogenes in macrophages, confirming the findings of our screen (Fig. 6B). Remarkably, the loss of DHNA polyprenyltransferase (MenA) did not lead to intracellular bacteriolysis of L. monocytogenes. The menG::Tn mutant, with a disruption in demethylmenaquinone (DMK) methyltransferase (MenG), displayed only a minor bacteriolysis phenotype, reminiscent of the ΔcydAB mutant results. These data suggest that synthesis of DHNA, but not menaquinone, is required for intracellular survival of L. monocytogenes. Additionally, all of the MK biosynthesis mutants, including the ΔmenA mutant, were sensitive to oxidative stress in vitro (Fig. S6C), further suggesting that cytosolic ROS is not responsible for bacteriolysis of these mutants. Importantly, mutations in menB, menA, or menG abolish the ability of L. monocytogenes to generate a membrane potential (Fig. 6C), highlighting, moreover, that the ETC/membrane potential plays a minor (if any) role in intracellular survival. Curiously, these data also demonstrate that, unlike Staphylococcus aureus (36), L. monocytogenes cannot use demethylmenaquinone (DMK) in the electron transport since the menG::Tn strain, which should accumulate DMK, does not respire (Fig. 6C). ΔmenA and menG::Tn mutants were impaired for plaque formation similarly to the ΔcydAB mutant, though not to the extent seen with other men mutants, further suggesting that, in addition to the function of menaquinone for respiration and generation of a membrane potential, biosynthesis of the DHNA is critical for L. monocytogenes virulence (Fig. 6D). Finally, to pinpoint the exact step of MK biosynthesis critical for cytosolic survival, we generated a double deletion strain (mutant ΔmenA/ΔmenB) and tested it for bacteriolysis in macrophages. ΔmenA/ΔmenB mutants phenocopied a single ΔmenB mutant, lysing significantly in macrophages and being drastically attenuated for virulence in a plaquing assay (Fig. 6E and F). Taken together, our data support the model that DHNA, or a derivative of it, is a critical virulence determinant independently of its role in the synthesis of MK for the ETC. Additionally, synthesis of DHNA, through a yet-to-be-defined mechanism, prevents cytosolic lysis of L. monocytogenes in macrophages.