Consistent with previous studies, L. monocytogenes MK-deficient mutants were highly attenuated for virulence ex vivo and in vivo (30, 31) (Fig. 2C and Fig. 4D and E); however, the function of MK itself, MK biosynthetic intermediates, or simply MK-dependent respiration in virulence has not been fully explored. Previous reports have found that MK biosynthesis genes and the cytochrome bd oxidase are upregulated during infection ex vivo (51) and in vivo (52), emphasizing the importance of MK for virulence. Here we uncovered a novel requirement for the MK intermediate DHNA in protecting L. monocytogenes from intracytosolic bacteriolysis (Fig. 6B and E). This phenotype was not due to disruption of the electron transport chain or loss of ATP generation through oxidative phosphorylation (Fig. 5D and F) but rather to the fact that DHNA synthesis is crucial for virulence and intracellular survival (Fig. 6B). Unfortunately, since the enzymatic steps at this stage in the pathway are incompletely understood (Fig. 6A) (35), we cannot determine whether DHNA or DHNA-CoA is the relevant molecule. Inexplicably, cytochrome bd oxidase and menG::Tn mutants displayed intermediate bacteriolysis phenotypes (Fig. 5D and F and Fig. 6B). Our favored hypothesis is that mutants with an incomplete ETC have depleted pools of available DHNA. This could be due either to increased funneling of DHNA into MK biosynthesis caused by incomplete electron transfer or, alternatively, to a transcriptional/posttranslation negative-feedback loop that inhibits DHNA synthesis in the absence of a productive ETC.