The nonmevalonate pathway of isoprenoid biosynthesis in Mycobacterium tuberculosis is essential and transcriptionally regulated by Dxs

Abstract

Mycobacterium tuberculosis synthesizes isoprenoids via the nonmevalonate or DOXP pathway. Previous work demonstrated that three enzymes in the pathway (Dxr, IspD, and IspF) are all required for growth in vitro. We demonstrate the essentiality of the key genes dxs1 and gcpE, confirming that the pathway is of central importance and that the second homolog of the synthase (dxs2) cannot compensate for the loss of dxs1. We looked at the effect of overexpression of Dxr, Dxs1, Dxs2, and GcpE on viability and on growth in M. tuberculosis. Overexpression of dxs1 or dxs2 was inhibitory to growth, whereas overexpression of dxr or gcpE was not. Toxicity is likely to be, at least partially, due to depletion of pyruvate from the cells. Overexpression of dxs1 or gcpE resulted in increased flux through the pathway, as measured by accumulation of the metabolite 4-hydroxy-3-methyl-but-2-enyl pyrophosphate. We identified the functional translational start site and promoter region for dxr and demonstrated that it is expressed as part of a polycistronic mRNA with gcpE and two other genes. Increased expression of this operon was seen in cells overexpressing Dxs1, indicating that transcriptional control is effected by the first enzyme of the pathway via an unknown regulator.

FIG. 1.

DOXP pathway of isoprenoid biosynthesis in M. tuberculosis. The metabolic pathway from Dxs to LytB is shown. Enzyme names are given with the corresponding H37Rv gene numbers in parentheses. IPP, isopentenyl phosphate; DMAPP, dimethylallyl pyrophosphate HMB-PP, (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate is boxed.

FIG. 2.

Genomic organization of the DOXP genes. The chromosomal arrangement is indicated. DOXP pathway genes are in black. The genetic coordinates for the H37Rv strain of M. tuberculosis are given.

FIG. 3.

Overexpression of Dxs1 or Dxs2 is inhibitory to growth in mycobacteria. Transformants overexpressing Dxs1 or Dxs2 were grown in liquid medium under noninduced (S = succinate) or induced (AS = acetamide, succinate) conditions. Symbols: ⧫, control vector pJFR19; ▪, dxs1-overexpressing vector; •, dxs2-overexpressing vector. The results are expressed as the means and standard deviations of three independent transformants.

FIG. 4.

Induction of Dxr expression results in growth inhibition in E. coli, which is partially relieved by pyruvate addition. The growth of transformants expressing Dxr from the arabinose-inducible pBAD plasmid was measured. (A) Inhibition of growth in response to increased Dxr expression. Growth was measured in half-strength LB medium. Various concentrations of arabinose (μg/ml) were used to induce expression from the pBAD promoter, and growth was measured after 4 h. (B) Relief of toxicity by pyruvate. Strains were grown in the presence of various concentrations of arabinose (indicated in μg/ml). □, No sodium pyruvate; ▪, plus 0.5% sodium pyruvate. Growth was measured after 20 h. (C) Truncated Dxr is not toxic. Strains carrying the full-length (“Dxr”) or truncated (“tDxr”) constructs were grown in the presence of no arabinose (□) or 1% arabinose (▪) to induce expression. Growth was measured after 20 h. The results are expressed as the means of three independent transformants.

FIG. 5.

Determination of the correct translational start site for Dxr. (A) The upstream region of Dxr is shown, indicating the region included in the full-length, toxic clone (wDxr) and the shorter clone (tDxr). Potential translational start sites are boxed. The upstream primers used for RT-PCR are indicated. (B) RT-PCR analysis of the dxr transcript. RNA was isolated from wild-type M. tuberculosis, cDNA was synthesized and amplified with the indicated primers plus a common downstream primer: +, plus RT; N, no RT; B, blank (no template); and G, genomic DNA. (C) Functional analysis of the Dxr start codon. Mutations were made in the upstream region as indicated in boldface underline and tested for functionality using gene switching. The potential translated sequence is shown, pOPH encodes the wild-type sequence.

FIG. 6.

Dxr is part of a multicistronic operon containing gcpE. (A) The dxr operon is shown. The upstream regions (∼0.2 kb) of dxr and gcpE tested for promoter activity are indicated. The results are the mean β-galactosidase activities (± the standard deviations) for three independent transformants assayed in duplicate in Miller units. The activity from the control plasmid pSM128 was 2.8 ± 0.2 Miller units. (B) Cotranscription of the dxr operon was confirmed by RT-PCR using primers to amplify the junctions between each gene pair indicate in panel A. Primers were designed to amplify across the gene junctions; the potential amplification products are marked on the operon. +, Plus RT; N, no RT; B, blank (no template); G, genomic DNA.

FIG. 7.

Overexpression of Dxs1 and GcpE leads to increased levels of HMB-PP. M. tuberculosis strains overexpressing Dxs1, Dxs2, Dxr, or GcpE were analyzed for HMB-PP content using an assay for stimulation of γδ T cells. The results are the mean ± the standard deviation of extracts from three individual transformants. The results are expressed relative to levels in the wild-type strain. Dxs1 and Dxs2 were expressed from the acetamidase promoter and assayed under inducing conditions. tDxr (dxr_t) and GcpE were expressed from the constitutive hsp60 promoter. WT, wild-type M. tuberculosis.

FIG. 8.

Quantitation of expression levels of DOXP pathway genes in overexpressor strains. Limiting dilution RT-PCR was carried out with gene specific primers for dxs1, dxs2, dxr_t, and gcpE on cDNA made from the overexpressing strains. Serial 4-fold dilutions (1:1, 1:4, 1:16, 1:64, 1:256, and 1:1,024) of cDNA were used as a template for PCR with gene-specific primers. (A) Gel pictures obtained after PCR, showing product intensity from increasing diluted cDNA; (B) relative expression levels for each gene. RNA input was standardized using sigA expression; values were then normalized to the WT value to give an indication of the relative fold difference. WT, wild-type H37Rv; Dxs1 (NI), strain expressing dxs1 from acetamidase promoter in noninducing conditions (basal level); Dxs1 (I), strain expressing dxs1 from acetamidase promoter in inducing conditions. Dxs2 (NI), strain expressing dxs2 from acetamidase promoter in noninducing conditions (basal level); Dxs2 (I), strain expressing dxs2 from acetamidase promoter in inducing conditions. Dxr, strain constitutively overexpressing dxr_t from the hsp60 promoter; GcpE, strain constitutively overexpressing gcpE from the hsp60 promoter; gDNA, genomic DNA control.