Lethal Mutations in the Isoprenoid Pathway of Salmonella enterica

Abstract

Essential isoprenoid compounds are synthesized using the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway in many gram-negative bacteria, some gram-positive bacteria, some apicomplexan parasites, and plant chloroplasts. The alternative mevalonate pathway is found in archaea and eukaryotes, including cytosolic biosynthesis in plants. The existence of orthogonal essential pathways in eukaryotes and bacteria makes the MEP pathway an attractive target for the development of antimicrobial agents. A system is described for identifying mutations in the MEP pathway of Salmonella enterica serovar Typhimurium. Using this system, point mutations induced by diethyl sulfate were found in the all genes of the essential MEP pathway and also in genes involved in uptake of methylerythritol. Curiously, none of the MEP pathway genes could be identified in the same parent strain by transposon mutagenesis, despite extensive searches. The results complement the biochemical and bioinformatic approaches to the elucidation of the genes involved in the MEP pathway and also identify key residues for activity in the enzymes of the pathway.It was recently discovered that bacteria synthesize isoprenoids by a pathway that differs from that found in eukaryotes. In bacteria, pyruvate and glyceraldehyde 3-phosphate are converted, through the 2-C-methyl-D-erythritol phosphate (MEP) pathway, to isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). This has focused recent research on the elucidation of the biosynthetic steps and genes encoding the catalytic enzymes in bacteria. To date, all synthetic steps of the MEP pathway and the genes encoding the biosynthetic enzymes have been identified. Isoprenoid biosynthesis begins with the condensation of pyruvate and glyceraldehyde 3-phosphate by 1-deoxy-D-xylulose 5-phosphate (DXP) synthase, encoded by dxs (26). Next, in the first committed step of the pathway, DXP reductoisomerase, encoded by dxr, converts DXP to MEP (16,32). In the following three steps, a cytidyl monophosphate moiety is attached to the phosphate of MEP; the resulting diphosphodiester is phosphorylated at the C-2 hydroxyl in the methylerythritol moiety and then cyclized to form an eight-membered cyclic diphosphate diester, and these steps are catalyzed by the proteins encoded by ispD (25), ispE (18), and ispF (11), respectively. The enzyme encoded by ispG, 1-hydroxy-2-methyl-2-(E)-butenol 4-diphosphate (HDMAPP) synthase, catalyzes the ring opening and reduction of 2-Cmethyl-D-erythritol-2,4-cyclodiphosphate to yield HDMAPP (10). Finally, HDMAPP is converted to a mixture of IPP and DMAPP by HDMAPP reductase encoded by ispH (1).Mutants blocked in the MEP pathway of Salmonella spp. or Escherichia coli are expected to be lethal, since these organisms are unable to utilize exogenously supplied IPP, DMAPP, or their corresponding alcohols. To allow viability of such mutants, genes of the alternative mevalonate pathway were introduced into bacteria, either on plasmids or in the chromosome (2,5,9,15,24). Strains containing the ...

Structure, function and inhibition of the two- and three-domain 4Fe-4S IspG proteins

“…We then mutated R56, R141, and K204 in TtIspG (corresponding to R55, R128, and K176 in AaIspG) to Ala, finding in each case an approximately 95% reduction in catalytic activity, Table S1. R55, R101, and R128 have been shown to be essential for catalysis in A. aeolicus IspG by Lee et al (7) and R141 (Thermus numbering) is essential for survival in Salmonella enterica (17). So, based on the docking results, their conserved nature (Table S1) and the mutagenesis results, these residues are all likely to be involved in MEcPP, reactive intermediate or HMBPP product binding, via their diphosphate groups, while the reactive intermediate and HMBPP product also interact directly with Fe via Fe-O and/or Fe-C bonds, the HMBPP O-1 Fe interaction being similar to that seen with HMBPP binding to IspH (18).…”

Characterization of the Mycobacterium tuberculosis 4-Diphosphocytidyl-2- C -Methyl- d -Erythritol Synthase: Potential for Drug Development

“…The inability to isolate a nonfunctional mutant using the two-step disruption strategy indicates that Rv3582c is essential for M. tuberculosis survival (33). The essentiality of the IspD enzyme for the growth of two gram-negative organisms, i.e., Salmonella enterica and E. coli, has previously been reported (7,14,34 (31), suggesting subtle differences in the active sites of the enzymes from these organisms. In E. coli IspD, Mg 2ϩ , which is essential for activity, forms coordination bonds with the ␣-, ␤-, and ␥-phosphate oxygens of CTP or the ␣-phosphate oxygen of CDP-ME, as shown for the glycerol 3-phosphate cytidyltransferase of Bacillus subtilis (27,38).…”

Preparation of gem-difluorinated retrohydroxamic-fosmidomycin

“…The mevalonate-independent pathway of isoprenoid biosynthesis is widely found in many microorganisms [1][2][3][4] as well as in higher plants, 5,6 but it is missing in human, which uses mevalonate pathway for isoprenoid biosynthesis. The unique property of 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR), the second enzyme of the DOXP pathway, can therefore be considered as a remarkable and safe target for the discovery of new herbicides.…”