The sorbitol phosphotransferase system is responsible for transport of 2-C-methyl-D-erythritol into Salmonella enterica serovar typhimurium

Abstract

2-C-methyl-D-erythritol 4-phosphate is the first committed intermediate in the biosynthesis of the isoprenoid precursors isopentenyl diphosphate and dimethylallyl diphosphate. Supplementation of the growth medium with 2-C-methyl-D-erythritol has been shown to complement disruptions in the Escherichia coli gene for 1-deoxy-D-xylulose 5-phosphate synthase, the enzyme that synthesizes the immediate precursor of 2-C-methyl-D-erythritol 4-phosphate. In order to be utilized in isoprenoid biosynthesis, 2-C-methyl-D-erythritol must be phosphorylated. We describe the construction of Salmonella enterica serovar Typhimurium strain RMC26, in which the essential gene encoding 1-deoxy-D-xylulose 5-phosphate synthase has been disrupted by insertion of a synthetic mevalonate operon consisting of the yeast ERG8, ERG12, and ERG19 genes, responsible for converting mevalonate to isopentenyl diphosphate under the control of an arabinose-inducible promoter. Random mutagenesis of RMC26 produced defects in the sorbitol phosphotransferase system that prevented the transport of 2-C-methyl-D-erythritol into the cell. RMC26 and mutant strains of RMC26 unable to grow on 2-C-methyl-D-erythritol were incubated in buffer containing mevalonate and deuterium-labeled 2-C-methyl-D-erythritol. Ubiquinone-8 was isolated from these cells and analyzed for deuterium content. Efficient incorporation of deuterium was observed for RMC26. However, there was no evidence of deuterium incorporation into the isoprenoid side chain of ubiquinone Q8 in the RMC26 mutants.

FIG. 1.

MVA (left) and MEP (right) pathways.

FIG. 2.

Diagram of the MVA operon inserted into dxs. araC and P_BAD are upstream of the yeast ERG genes, and the Kan^r cassette is located downstream.

FIG. 3.

Genetic evidence that SrlE, SrlA, and SrlB are involved in the utilization of exogenous ME. Growth patterns of RMC26, CR5, RMC78, and RMC79 on LB-KAN-ME (left) and LB-KAN-MVA-Ara (right) are shown.

FIG. 4.

Q8 derived from MVA (left) and from [1,1-²H₂]ME (right). MW.

FIG. 5.

LSIMS spectra of Q8 isolated from feeding experiments: (A) wild-type LT2 without supplementation; (B) RMC26 supplemented with [1,1-²H₂]ME; (C) RMC26 supplemented with [1,1-²H₂]ME-MVA-Ara; (D) CR5 supplemented with [1,1-²H₂]ME-MVA-Ara.