Evaluation of the Role of gsp, ygiC, and yjfC Genes in Glutathione Metabolism in Escherichia coli by Gene Disruption

Abstract

Glutathione metabolism differs amongst humans and bacteria and has the potential to be a good target for antibacterial agents. Within Escherichia coli, glutathione can form an amide bond with spermidine to produce the conjugate glutathionylspermidine. This reaction is catalyzed by the enzyme glutathionylspermidine synthetase/amidase. Three genes, gsp, ygiC and yjfC have previously been identified and proposed to be associated with glutathionylspermidine metabolism; however, the specific function of ygiC and yjfC has yet to be determined. The objective of this research is to eliminate these genes from the E. coli genome and evaluate the responsiveness of the knockout strains to various stress conditions. Corresponding genes were replaced by antibiotic resistance genes through homologous recombination. Once the gene disruptions had been achieved, the antibiotic resistance gene was eliminated utilizing a helper plasmid pCP20. The techniques used to create gene disruptions, or knockouts, included polymerase chain reaction and electroporation. Seven strains have been created: single knockout (gsp, ygiC, or yjfC), double knockout (gsp/ygiC, gsp/yjfC, or ygiC/yjfC), and triple knockout (gsp/ygiC/yjfC). The sensitivity of the knockout strains were tested by stressors including antibiotics, transition metals, and hydrogen peroxide.