Mechanisms Of Alky Peroxide Enzyme In Resistance To Multiple Environmental Stresses In Burkholderia Thailandensis

Under the unfavorable environment,the organism will initiate oxidative stress reaction via the production of reactive oxygen species?ROS?which cause the oxidation of intracellular macromolecules.This will break the balance of intracellular redox,causing cell dysfunction,damage and even apoptosis.To cope this oxidative damage,organisms have evolved a set of anti-oxidative defense mechanisms,which mainly include the antioxidant enzyme system and the low molecular mass antioxidant?LMMA?.The mercaptan is the main component of LMMA including branched mercaptan Mycothiolus,cysteine cystatin,vitamin C and glutathione.In this study,the Burkholderia thailandensis was used as the experimental bacterial.To study the role of alkyl hydroperoxide reductase subunit C?AhpC?in the oxidative stress process and the mechanism of its resistance to environmental stress under external physiological phenotypes,the main results are as follows:1.Transcriptome data and real-time fluorescence quantitative?qRT-PCR?assay showed that the expression of AhpC was induced by ROS and negatively regulated by OxyR protein.The EMSA assay showed OxyR binds to ahpC promoter region for transcriptional regulation.2.Thg AhpC deletion and complemented mutants were constructed,the survival rate of these mutants under the treatment of different oxidants,alkylators,nitrite and other stress agents was measured.The results showed that AhpC could significantly enhance the ability of Burkholderia thailandensis to resist environmental stress and revealed the physiological function of AhpC in Burkholderia thailandensis.3.The enzyme activity of AhpC was determined under the treatment of different stress agents.The results showed that AhpC showed different affinity to various substrates,suggesting that AhpD could be the electron donor of AhpC under the treatment of H₂O₂,t-BOOH,CHP and Peroxynitrite.4.By amino acid sequence alignment,Cys57 was found to be a conserved cysteine peroxide residue,which was highly conserved in the ahpC gene of different bacteria.Further studies showed that Cys57 was a cysteine peroxide residue,CPCys171 and Cys173 were regenerated Cys residues.5.The Cys57 Cys171 and Cys173 were mutated?Cys to Ser,namely,C57S,C171S, C171S and C173S?and the AhpC:C171S C173S double mutant was also constructed and the enzyme activity of were detected.The results showed that the three residues of AhpC played different antioxidant role when exposed to different types of oxidative stress.To sum up,this study found that AhpC in Burkholderia thailandensis has the physiological function in resisting environmental stress and the underlying mechanism of its physiological function is to enhance the activity and expression level of intracellular antioxidant enzymes by directly or indirectly eliminating excessive ROS and increasing the intracellular protein reversible mercaptan modification.