Investigating the regulation of the phosphoglycerate mutases gpmA and gpmB in Salmonella enterica serovar typhimurium by means of q-RT-PCR, bioinformatics, and enzymatic assays

Eric C Warrick, Purdue University

Abstract

Bacteria have many choices when it comes to nutrient utilization, particularly when grown in environments with more than one carbon source. The organism must decide which carbon source to use and which pathway to process the carbon through as there are close to 2000 chemical reactions that are interconnected into highly organized processes which we refer to as metabolic pathways. It is of the upmost importance that these pathways be regulated to prevent inefficiency as carbon sources are at a premium in the environment and an energetically inefficient organism is often a dead organism. Several different mechanisms have been discovered to help coordinate and regulate the expression of the various pathways found inside the cell. This distribution of molecular fluxes is regulated by multiple mechanisms including pre- and post-transcriptional gene control, enzyme kinetics, and allosteric regulation. Interestingly, only a few specific pathways are routinely used for carbon flux and are commonly referred to as the central metabolic pathways. The major carbon flux pathways include glycolysis (Embden-Meyerhoff pathway), the pentose phosphate pathway, and the Enter-Doudoroff pathway. Though each pathway is regulated differently, they provide the precursor metabolites to all of the other pathways as they all share the same common theme: to convert glucose to glyceraldehyde-3-phosphate and oxidize glyceraldehyde-3-phosphate to pyruvate. This project was designed with Salmonella typhimurium as the model organism to shed insight on the regulation of the gpmA and gpmB genes which encode phosphoglycerate mutase isoenzymes that are responsible for the reversible interconversion of 3-PGA to 2-PGA in glycolysis and gluconeogenesis. There are two types of phosphoglycerate mutases: the 2,3-BPGA-dependent enzymes (dPGM), encoded by the gpmA gene, which are activated by 2,3-BPGA, and the 2,3-BPGA-independent enzymes (iPGM), encoded by the gpmB (or gpmM) gene, which are fully active in the absence of this metabolite. The Enterobacteriaceae family is unique in that they contain both dPGM and iPGM while other organisms such as Bacillus sp. only contain only the iPGM. Until recently, no one has isolated a gpmA mutant in an enteric organism like S. typhimurium, and as such the factors that regulate this step in glycolysis were unknown. In a hunt for mutants with defects in adaptation to osmotic stress in S. typhimurium, I fortuitously isolated a knockout mutation in the gpmA gene, which specifies the major isoenzyme, GpmA. This mutant has the phenotype that it can grow on gluconeogenic carbon sources but cannot grow on a carbon source that flows through glycolysis. A suppressor mutation in rng , was identified which allows the gpmA mutant the ability to utilize glucose as a carbon source. For reasons that remain unknown, the suppressor is unable to support growth on any osmolarity media containing the osmoprotectant compound GB. Starting with the gpmA single mutant, I constructed a double mutant that also lacks the second isoenzyme GpmB. The gpmA and gpmB double mutant cannot grow on glucose, glycerol, or lactate as a carbon source, but it can grow on the combination of glycerol + lactate (a pair of carbon sources that feed into glycolysis before and after the mutational block). The isolation of the gpmA mutation not only enabled us to characterize the phenotypes of strains lacking this last unexplored reaction of glycolysis, but it also enabled us to study the regulation of the two phosphoglycerate isoenzymes. The goal of this project was to determine the regulation of the PGMs and the effects of a gpmA, gpmB, and combination gpmA and gpmB double mutant on the flux of glycolysis. Approaches used to achieve this goal included genetic analysis, carbon utilization phenotyping, Q-RT-PCR, bioinformatics analysis, and enzymatic assays.

Degree

Ph.D.

Advisors

Csonka, Purdue University.

Subject Area

Molecular biology|Microbiology|Biochemistry|Bioinformatics

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