In addition to the original 12 bacteriolysis mutants, we isolated a strain with multiple mutations in its chromosome. In this mutant, deletion of shikimate biosynthesis genes (aroED) was responsible for the bacteriolysis phenotype (data not shown) and led to MK auxotrophy since this pathway produces a precursor for MK (30) (see Fig. 6A). This, combined with our isolation of menD and menF mutants, prompted us to examine the role of MK in L. monocytogenes cytosolic survival. Consistent with our data from the transposon mutants as well as with previously published results (30, 31), the ΔmenD and ΔmenF strains can neither efficiently replicate aerobically nor generate a robust membrane potential (Fig. 4A to C). Additionally, these mutants were significantly attenuated for virulence and intracellular survival in macrophages (Fig. 4D and E).