Study On The Role Of Indole In The Process Of Bacterial SOS Response Promoting Conjugation Transfer

The overuse of antibiotics has led to the development of multidrug resistance in a variety of bacteria.However,in the antibiotic environment,the bacterial DNA damage repair mechanism（SOS response）can be stimulated,and the SOS response can lead to the expression of some resistance genes and the transfer of drug-resistant genes,which must be accompanied by the transmission of signaling molecules.The transfer of genes must be accompanied by the transfer of signaling molecules.In recent years indole has existed in recent years as a new type of molecule,and it has been shown that indole can promote the development of drug resistance in bacteria.In order to confirm and clarify the signal action of indole and its targets,this study first explored the relationship between indole and SOS response,clarified the relationship between indole and the joint transfer of drug-resistant genes,and tried to find the targets of indole action from the two perspectives of bacterial signaling system and type IV secretion system.The purpose is to analyze the action mode and mechanism of indoles,a new signaling molecule,in a more comprehensive and detailed way,clarify the signaling mechanism of SOS response promoting drug-resistant gene transfer,and lay a foundation for fully revealing the mechanism of drug-resistant gene widespread transmission.The experimental results are as follows:1.In this study,cefotaxime sodium was used to induce E.coli to determine the time to establish the SOS response model.The results showed that the levels of lex A and rec A m RNA were high within 20 min after induction with 0.01μg/m L cefotaxime sodium for 1 h,5 h and15 h,and the protein levels at 15 h were basically consistent with their trends,which were consistent with the requirements for SOS response establishment.2.According to the results of conjugation transfer experiment,the conjugation transfer frequency of indole decreased significantly in the achromic acid M9CG medium compared with the Control group,and the conjugation transfer frequency of indole increased significantly after the addition of exogenous indole,indicating that indole can promote conjugation transfer.The number of zygotes increased significantly when the concentration ratio of recipient to donor was1:2 and the indole-induced concentration was 2 m M.Zygote resistance gene bla _CTX-M,fos A3 and plasmid Inc I2 were positive in the electrophoresis.These results indicate that indole can promote conjugation and transfer of drug-resistant genes.3.The mRNA levels of the small heat shock protein ibpA,tryptophanase tnaA,and the indole concentration sensing pump acr EF with the transporter protein mtr were detected to increase downstream of the SOS response after 5 h and 15 h of induction of the SOS response by using fluorescence quantification.Small heat shock protein ibp A is sensitive to temperature.Escherichia coli BL21 was cultured at 20℃,37℃,42℃and 45℃.The m RNA levels of all the above five genes increased at high temperatures.Moreover,when the culture temperature was42℃,the gene levels of acr EF and mtr of the indole concentration induction pump were the highest,indicating that indoles were the most active at this temperature.The results were consistent with the detection of intracellular indole concentration by HPLC-MS at 42℃,and the indole concentration was higher after the addition of cefotaxime sodium than the control group.The conjugation frequency at 20℃,37℃and 42℃also showed that the number of conjugons produced at high temperature was more.The activity of small heat shock protein ibp A was activated or inhibited by high or low temperature,which also resulted in the change of indole concentration.These results indicated that SOS response might regulate the concentration of indoles through ibp A.4.Calculation of the binding energy of indoles to two-component systems using molecular docking.The molecular docking results showed that the two-component system had better binding activity of Uhp A,Pho B,Ato C and Cre C to indole.And the expression levels of the four two component systems increased after the addition of indole,which was in line with the prediction of molecular docking.These results indicate that the four two-component systems may be the indole signal receptors.5.The detection of the second messenger cAMP concentration by HPLC-MS revealed that the c AMP concentration in the culture medium did not change significantly under the induction of cefotaxime sodium,indicating that c AMP was not significantly associated with bacterial SOS response.However,the c AMP content gradually decreased after induction by adding indole,indicating that the signal molecule indole has an antagonistic effect with c AMP,but the exact mechanism is not clear.6.The expression levels of virB2,virB6 and virD4 genes in bacterial type IV secretion system was detected by fluorescence quantification at low levels in M9CG medium without endogenous indole production,and significantly increased after adding 2 m M indole,especially the change of virD4 level.This indicates that the bacterial type IV secretion system is closely related to indole during conjugation transfer.In summary,we reached the following conclusions:Indole does play a role as a signaling molecule in the process of conjugation transfer induced by SOS response,helping bla _CTX-M,fos A3 and other drug-resistant genes to transfer horizontally.Inhibition or activation of small heat shock protein ibp A can cause the simultaneous changes of indole concentration and zygote,indicating that SOS response may regulate indole concentration and zygote transfer through downstream ibp A.In terms of signaling molecules,two-component signaling molecules Uhp A,Pho B,Ato C,Cre C may be indole receptor molecules,which need further verification.Possible antagonistic effect between c AMP and indole,the mechanism is unclear and needs further study.T4SS may co-promote drug resistance gene transfer as an effector of indole signaling.This study provides a new strategy for targeting bacterial signaling systems and blocking the signaling contacts between bacteria to curb bacterial drug resistance.