Accumulation of heptaprenyl diphosphate sensitizes B acillus subtilis to bacitracin: implications for the mechanism of resistance mediated by the BceAB transporter

Summary Heptaprenyl diphosphate ( C 35 ‐ PP ) is an isoprenoid intermediate in the synthesis of both menaquinone and the sesquarterpenoids. We demonstrate that inactivation of ytpB , encoding a C 35 ‐ PP utilizing enzyme required for sesquarterpenoid synthesis, leads to an increased sensitivity to bacitracin, an antibiotic that binds undecaprenyl pyrophosphate ( C 55 ‐ PP ), a key intermediate in cell wall synthesis. Genetic studies indicate that bacitracin sensitivity is due to accumulation of C 35 ‐ PP , rather than the absence of sesquarterpenoids. Sensitivity is accentuated in a ytpB menA double mutant, lacking both known C 35 ‐ PP consuming enzymes, and in a ytpB strain overexpressing the HepST enzyme that synthesizes C 35 ‐ PP . Conversely, sensitivity in the ytpB background is suppressed by mutation of hepT or by supplementation with 1,4‐dihydroxy‐2‐naphthoate, a co‐substrate with C 35 ‐ PP for MenA . Bacitracin sensitivity results from impairment of the BceAB and BcrC resistance mechanisms by C 35 ‐ PP : in a bceAB bcrC double mutant disruption of ytpB no longer increases bacitracin sensitivity. These results suggest that C 35 ‐ PP inhibits both BcrC (a C 55 ‐ PP phosphatase) and BceAB (an ABC transporter that confers bacitracin resistance). These findings lead to a model in which BceAB protects against bacitracin by transfer of the target, C 55 ‐ PP , rather than the antibiotic across the membrane.