Abstract
Spermidine synthase is an enzyme which catalyzed the biosynthesis of spermidine from putrescine. In the presence of putrescine and spermidine, polyamine deficient mutant E. coli was protected from hydrogen peroxide (H2O2) or oxygen in Vogel-Bonner medium (Chattopadhyay et al., 2003). In addition, recent reports showed that Arabidopsis thaliana with over-expressed spermidine synthase enhance the tolerance to multiple environmental stresses including ROS stress (Kasukabe et al., 2004). According to those report, this study is focused on investigating the relationship between spermidine synthase and oxidative stress.Various concentrations of H2O2 induced ROS and the intracellular contents of spermidine synthase in E. coli were analyzed. After 1 hour treatment with 1, 2, 4 mM H2O2, the growth inhibition based on optical density at 600 nm was down to 80.2, 64.7, and 45.5 % of untreated control, respectively. The survivability based on colony forming unit (CFU) assay, the percentages of viable E. coli colony were detected decreased to 70.4, 46.7, 17.7 % of untreated control, respectively. And the amount of spermidine synthase decreased to 83, 67, and 50 % of untreated control sample. The relationship between spermidine synthase and reactive nitrogen species (RNS) was also investigated in this study. Based on optical density at 600 nm, E. coli after 1 hour treatment with 2, 4, 8 mM sodium nitroprusside (SNP) resulted in growth inhibition to 69.3, 65.6, and 58.3 % of that from untreated control, respectively. Based on CFU assay, the percentages of viable E. coli were reduced to 75, 71, 31 % compared to untreated control, respectively. And the amount of spermidine synthase decreased to 88, 102, and 59 % of untreated control.The response of E. coli after 1 hour treatment with 8, 12, 16 mM of spermidine synthase enzyme inhibitor, dicyclohexylamine (DCHA), was also determined. Based on optical density at 600 nm, 8, 12, 16 mM DCHA treatment resulted in growth inhibition to 82.9, 58.7, and 41.7 % of that from untreated control, respectively. Based on CFU assay, the percentages of viable E. coli were decreased to 72.7, 18.7, 4.9 % compared to untreated control, respectively. And the amount of spermidine synthase was approximately 15% decreased with 8 mM DCHA treatment. However, the amount of spermidine synthase was 60 % increased with 12~16 mM DCHA treatment. Further study investigated that intracellular amount of spermidine synthase at 1, 2, 4 hour incubating time which corresponded to log, late-log and stationary phase was decreased in E. coli culture.Furthermore, 4 mM non-toxic DCHA co-incubated with 2 mM H2O2 treated E. coli for 1 hour, bacteria growth based in O.D.600 reading from DCHA plus H2O2 co-cultured samples reduced approximately 18% of that from H2O2 treated samples. The results suggest the survivability of E. coli co-incubated with H2O2 was highly correlated to the activity and intracellular amount of spermidine synthase.