Characterization of the Depletion of 2- C -Methyl- d -Erythritol-2,4-Cyclodiphosphate Synthase in Escherichia coli and Bacillus subtilis

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Abstract
The ispF gene product in Escherichia coli has been shown to catalyze the formation of 2- C -methyl- d -erythritol 2,4-cyclodiphosphate (MEC) in the deoxyxylulose (DOXP) pathway for isoprenoid biosynthesis. In this work, the E. coli gene ispF and its Bacillus subtilis orthologue, yacN , were deleted and conditionally complemented by expression of these genes from distant loci in the respective organisms. In E. coli , complementation was achieved through integration of ispF at the araBAD locus with control from the arabinose-inducible araBAD promoter, while in B. subtilis , yacN was placed at amyE under control of the xylose-inducible xylA promoter. In both cases, growth was severely retarded in the absence of inducer, consistent with these genes being essential for survival. E. coli cells depleted of MEC synthase revealed a filamentous phenotype. This was in contrast to the depletion of MEC synthase in B. subtilis , which resulted in a loss of rod shape, irregular septation, multicompartmentalized cells, and thickened cell walls. To probe the nature of the predominant deficiency of MEC synthase-depleted cells, we investigated the sensitivity of these conditionally complemented mutants, grown with various concentrations of inducer, to a wide variety antibiotics. Synthetic lethal behavior in MEC synthase-depleted cells was prevalent for cell wall-active antibiotics.