Characterization of the depletion of 2-C-methyl-D-erythritol-2,4-cyclodiphosphate synthase in Escherichia coli and Bacillus subtilis

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.

FIG. 1.

Conditional complementation of deletions in ispF and yacN. (A) Arabinose dependence of E. coli ispF deletion strain EB370. E. coli strains EB356 and EB370 were plated on LB/KAN in the presence and absence of arabinose and grown overnight at 37°C. (B) Xylose dependence of B. subtilis yacN deletion strain EB323. B. subtilis strains EB315 and EB323 were plated on LB/CM in the presence or absence of xylose and grown overnight at 30°C.

FIG. 2.

Characterization of E. coli ispF deletion strain by microscopy. E. coli strains EB370 (ispF deletion) and EB356 (wild-type ispF [inset]) were grown in the absence of arabinose and were visualized by light microscopy (A), transmission electron microscopy (B), and scanning electron microscopy (C). Each size bar represents 1 μm. Cells were grown overnight at 37°C on LB/KAN plates with no arabinose.

FIG. 3.

Characterization of B. subtilis yacN deletion strain by microscopy. B. subtilis strains EB323 (yacN deletion [left]) and EB315 (wild-type yacN [right]) were grown in the absence of xylose and were visualized by light microscopy (A), transmission electron microscopy (B), and scanning electron microscopy (C). Each size bar represents 1 μm. Cells (EB323) were grown overnight at 30°C on LB/CM/SPEC plates with no xylose.

FIG. 4.

Growth and morphology of YacN-depleted B. subtilis cells. B. subtilis strain EB323 was grown overnight on LB/CM/SPEC/xylose plates and used to inoculate LB/CM/SPEC with 2% (▾, A), 0.2% (▿, B), 0.063% (▪, C), 0.02% (□), or no (⧫, D) xylose. B. subtilis strain EB315 was grown overnight on LB/CM plates and inoculated into LB/CM with 2% xylose (•) or no xylose (○). Growth was monitored at 30°C for 17 h. For the transmission electron micrographs, cells were grown overnight at 30°C on LB/CM/SPEC plates with 2%, 0.2%, 0.063%, or no xylose. The size bar represents 1 μm and applies to all micrographs.

FIG. 5.

Sensitivity of E. coli and B. subtilis MEC synthase-depleted cells to various antibacterial agents. (A) Change in MIC for the B. subtilis MEC synthase deletion strain (EB323) with various concentrations of inducer to representative antibacterial agents. Fold sensitization in MIC is calculated with respect to the fully complemented deletion strain (2% xylose). From left to right, the amounts of xylose are as follows: 2, 0.2, 0.063, and 0.02%. Cells were grown in a volume of 100 μl in a 96-well microtiter plate for 24 h, and then the A₆₀₀ was read. (B) Change in sensitivity of E. coli MEC synthase deletion strain (EB370) to antibacterial agents with various concentrations of inducer. Fold sensitization in MIC is calculated with respect to the fully complemented deletion strain (0.2% arabinose). From left to right, the amounts of arabinose are as follows: 0.2, 0.02, 0.0063, and 0.002%. Cells were grown in a volume of 150 μl in a 96-well microtiter plate for 16 h, and then the A₆₀₀ was read. In all cases, the MIC was determined as the lowest concentration of drug that resulted in no growth (less than 0.1 absorbance unit at 600 nm).