Inhibitory Effect Of Glutathione On The Induction Of λ Prophage

Effect of Glutathione (GSH) on ultraviolet (UV) induction of the λ prophage was studied in the present study. It showed that GSH could inhibit the UV induction of λ prophage in lysogenic cells. The inhibitory rates were in concentration-dependent manner. The inhibitory rate increased at the concentration of GSH from 11.25 mg/mL to 22.5 mg/mL, and the maximal inhibitory rate can reach to 98%.Hypoxanthine - xanthine oxidase (HX-XO) is a typical system for generating free radicals. It was found that HX -XO system could induce lambda prophage from lysogenic state to lytic growth as well as UV irradiation. Along with the increasing of the concentration of HX and XO, the inductive effect was raised straightly upwards. On the contrary, the titers of the bacteriophage decreased quickly with the elongation of the action time of HX and XO. And the cell density of the lysogenic bacteria also greatly affected the inductive effect of the bacteriophage. The most PFUs (plaque forming units) was gotten when the cell density was 10 cells/mL. The inductive effects of HX-XO system were inhibited in the presence of SOD, CAT, GSH and SOD/CAT. SOD/CAT and GSH were showed obvious inhibitory effect with a maximal inhibitory rate of 75 % and 80 %, respectively.By electron spin resonance (ESR) and spin trapping techniques, the signals of free radicals were detected in the suspension of the lysogenic cells induced by UV irradiation. It showed that free radicals could be generated in the suspension of the lysogenic cells by UV irradiation. However, in the presence of GSH (18.75 mg/mL), the signals of free radicals generated by UV irradiation were almost disappeared. It is a direct evidence that free radicals can be generated in the process of UV induction on lysogenic bacteria It also indicated that GSH could scavenge free radicals effectively. When lysogenic cells were induced by HX-XO system, the signals of freeradicals also could be detected.The gene of Y ?glutamylcysteine synthetase was amplified by PCR and inserted into pUCm-T vector. The recombinant plasmid was transformed into lysogenic bacteria Kcoli K12 ( A ) . Recombinants was identified by plasmid DNA electrophoresis, whole cell SDS-PAGE and Southern hybridization. It was found that the recombinants could greatly increase its resistibility to UV irradiation.With previous studies, the free radicals model of UV induction of X prophage was proposed for further elaborating the mechanism of UV induction of prophage in the lysogenic bacteriagsh- gene and pHT-3101 were digested by HincSR and Xbal, respectively, and then linked by DNA ligase. The recombinant plasmids were electrotransformed into B. thwingiensis 250. Compared with the original strain, the survival rate of recombinants was increased approximate 2 times by UV irradiation. When recombinants and the original strain were exposed to sunshine for 3 days, the survival rate of recombinants was 1.7-2 times that of the original strain. It indicated that the recombinant strain carrying gsh-l gene is potentially valuable in practical application.