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

Heptaprenyl diphosphate (C35 -PP) is an isoprenoid intermediate in the synthesis of both menaquinone and the sesquarterpenoids. We demonstrate that inactivation of ytpB, encoding a C35 -PP utilizing enzyme required for sesquarterpenoid synthesis, leads to an increased sensitivity to bacitracin, an antibiotic that binds undecaprenyl pyrophosphate (C55 -PP), a key intermediate in cell wall synthesis. Genetic studies indicate that bacitracin sensitivity is due to accumulation of C35 -PP, rather than the absence of sesquarterpenoids. Sensitivity is accentuated in a ytpB menA double mutant, lacking both known C35 -PP consuming enzymes, and in a ytpB strain overexpressing the HepST enzyme that synthesizes C35 -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 C35 -PP for MenA. Bacitracin sensitivity results from impairment of the BceAB and BcrC resistance mechanisms by C35 -PP: in a bceAB bcrC double mutant disruption of ytpB no longer increases bacitracin sensitivity. These results suggest that C35 -PP inhibits both BcrC (a C55 -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, C55 -PP, rather than the antibiotic across the membrane.