Genetic identification and characterization of genes involved in the 2-C-methyl-D-erythritol 4-phosphate pathway for early isoprenoid biosynthesis in Salmonella typhimurium

Isoprenoid compound are essential for all living organisms. Despite their structural diversity, all isoprenoids are biosynthesized from two five carbon isomers, isopentenyl diphosophate (IPP) and dimethylallyl diphosophate (DMAPP). Until recently, it was thought IPP and DMAPP were biosynthesized exclusively by the mevalonate (MVA) pathway. While this pathway is utilized exclusively in eukaryotes and archae, another pathway for the biosynthesis of IPP and DMAPP exists. The 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway has been recently elucidated and is used exclusively by many gram negative and some gram positive bacteria as well as by some apicomplexan parasites. Plants utilize both pathways. The MVA pathway is utilized for the production of cytosolic isoprenoids, while the orthogonal MEP pathway is used in the plastids. The parallel nature of these two essential pathways in eukaryotes and bacteria makes the MEP pathway an attractive target for the development of antimicrobial agents. While the biochemical steps for enzyme mediated conversion of pyruvate and glyceraldehyde 3-phosphate to IPP and DMAPP via the MEP pathway have been elucidated, little is known about the genetic aspects of the pathway.; Chapter 1 of this dissertation provides a review of the literature and outlines the biochemical steps involved in the MEP pathway. The development of a S. typhimurium strain possessing a synthetic operon for the conversion of MVA to IPP is discussed in Chapter 2. The synthetic operon provides an alternate metabolic route for the biosynthesis of essential isoprenoids. Therefore, mutants with disruptions in the MEP pathway can be isolated and studied. Chapters 3 and 4 describe screens for novel MEP pathway genes using different methods of random mutagenesis. Isolation of mutants using a random Mud-Cam transposon insertion resulted in the identification of the sorbitol phosphoenolpyruvate:phosphotransferase system as the cellular machinery involved in the uptake and phosphorylation of exogenously supplied 2-C-methyl-D-erythritol. Screens using DES as a chemical mutagen were useful in isolating metabolic blocks in the MEP pathway and identifying key amino acids in the catalytic enzymes. Chapter 5 discusses the inconsistencies observed between these two screening methods. Experiments were designed to probe the genomic context and regulation of the MEP pathway genes.