Polysulfides Control The Oxidation State Of OxyR Regulon In Escherichia Coli

In recent years,H₂S has been recognized as the third gaseous signal molecule.In fact,its physiological function is manifested as duality.On one hand,H₂S inhibits the activity of cytochrome oxidase c and affects the electron transport,showing toxicity to organism;on the other hand,appropriate amounts of H₂S have an important function on the regulation of cardiovascular system,central nervous system and inflammatory response,showing signal effects.A lot of eveidence demonstrates that H₂S signals via protein S-sulfhydration?-SSH?;however,the hypothesis is still controversial.The dual effects of H₂S also reflect on its relationship with reactive oxygen species?ROS?.Some studies have shown that the accumulation of H₂S can improve ROS in vivo.Meanwhile,a report shows that E.coli tolerance to antibiotics,which can rise oxidative stress,is related to H₂S.When they knocked out 3-mercapto pyruvate transferase?sseA?,the main H₂S-producing gene in E.coli,the mutant was more sensitive to antibiotics.Based on the close relationship between H₂S and ROS,we selected the major oxidative stress regulatory systems-ArcAB system,SoxRS regulatory system and OxyR regulator in Escherichia coli to carry out the research,in order to uncover the association between H₂S and oxidative stress in E.coli.In this paper,we constructed a series of E.coli strains with different H₂S production by knocking out or overexpressing genes related to the production and consumption of H₂S.Through the survival test and measuring inhibition,we confirmed that H₂S plays an important role in changing the redox state in vivo and responding to external oxidation pressure as well.The results of studies on ArcAB,SoxRS,and OxyR demonstrated that polysulfides can alter the activity of OxyR that further affects the expression of a series of genes in E.coli.For the first time,we found that not only H₂O₂,but also polysulfides can regulate the activity of OxyR.Genes,such as katG,dps,grxA and trxC,which are regulated by OxyR,were selected for further investigation.We amplified the promoter region of these genes by PCR,and linked the promoter region with egfp or mkate reporter gene to construct reporter plasmids.The activity of OxyR was determined by the fluorescence value.Although both oxidized and reduced OxyR can bind to DNA,only oxidized OxyR can activate the expression of downstream genes.Our results showed that the role of polysulfides in the induction of OxyR is similar to H₂O₂.Differently,the lower concentration of polysulfides can change the conformational of OxyR,indicating that OxyR is more sensitive to polysulfides.Furthermore,we found that OxyR treated with polysulfides was more likely to bind to DNA in the form of tetramer,indicating that the regulation of polysulfides on OxyR activity may be achieved by changing the polymerization state of the protein.The conformational changes of OxyR protein were also analyzed.Results showed that'OxyR is modified by H₂O₂ and polysulfides in diverse forms.Moreover,there is no disulfide bond between two cysteines in the oxidized OxyR protein.In addition,the recovery of the OxyR was also studied.Some reports demonstrated that both grxA and trxC genes contribute to the reduction of OxyR,however,our results showed that there is no significant relationship between grxA,trxC and OxyR reduction,indicating there are unknown genes are involved in this process.Our work first demonstrates that polysulfides can induce OxyR,which opened up a new direction for the research of this TF.Our research proves that different signal molecules modify OxyR through different ways,and this difference influence OxyR function.Our research breaks the traditional idea that OxyR transforms to active type through disulfide bond formation.