Characterization of the Depletion of 2-
C
-Methyl-
d
-Erythritol-2,4-Cyclodiphosphate Synthase in
Escherichia coli
and
Bacillus subtilisJournal Articles
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abstract
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.
