| Response to acid stress is a critical mechanism for Escherichia coli to successfully complete its life cycle.This indispensable mechanism allows neutralophilic bacteria,such as E.coli,to survive in the gastrointestinal tract.Bacterial two-component system regulates the expression of downstream genes through the phosphorylation pathways to sense and respond to external environmental changes.There has been little research on the mechanism of the Tor R/Tor S system in acid resistance.And how Tor R/Tor S regulates the expressions of acid-resistant genes is still unclear.In this paper,we found that under the extreme acid culture condition（p H=2.0）,（1）the growth ofΔtor R orΔtor S mutant was inhibited in the ABTGcasa medium;（2）the inhibition of growth was disappeared in the LB medium,indicating that the inhibition of extreme acid to bacterial cells depends on nutritional condition;（3）the cell survival assay further showed that the growth ofΔtor R orΔtor S cells was inhibited under the extreme acid environment.After detecting the phosphorylation levels of Tor R/Tor S,we found that the phosphorylation of regulatory protein Tor R did not entirely depend on the phosphorylation of sensor protein Tor S.This result indicated other kinases might phosphorylate Tor R.RNA-seq and RT-q PCR results suggested that Tor R affected the expressions of acid-resistant genes rpo S,gad B,gad C,hde A,gad E,mdt E,mdt F,gad X,and slp.The results suggested that Tor R might respond to extreme acid environments by specifically regulating the above acid-resistant gene expressions.Further analysis showed that many acid-resistant gene promoter regions contained Tor R binding sites（Tor R-boxes）.Then,we detected the Rpo S protein and rpo S transcription levels in wt,Δtor R,andΔtor S cells under different p H conditions（p H=7.0,4.5,2.0）.These results showed that（1）under the extreme acid condition,both Rpo S levels and rpo S transcription levels were decreased inΔtor R cells;（2）under the neutral condition,the transcription levels of rpo S inΔtor S cells were increased,while the Rpo S levels remained unchanged,indicating that the stability of Rpo S might depend on other proteins.Indeed,the transcription levels of ira M inΔtor R cells were significantly decreased under the extreme acid condition（p H=2.0）but obviously increased under the neutral condition,furthermore,the difference was not significant at p H=4.5.The wild-type rpo Sp promoter and mutant rpo Spmut were respectively inserted before the promoterless lac Z reporter gene in p TAC3953(obtained p TAC3953-rpo Sp and p TAC3953-rpo Spmutplasmids).By electrophoretic mobility shift assay（EMSA）and detection ofβ-galactosidase activity in wt andΔtor R cells in vivo,we found that Tor R-his6 interacted with rpo Sp promoter in vivo and vitro,but this interaction was no longer observed when the Tor R-box in rpo Sp region was mutated.Next,the growth trends of wt andΔtor R cells were measured in ABTGcasa medium containing 20%or 0.5%glucose as a carbon source under different p H conditions,respectively.It was found that after stimulation with extreme acid,the survival rate ofΔtor R cells in ABTGcasa medium with 20%glucose began to decrease in the early stationary phase but began to decrease in the late stationary phase cultured in ABTGcasa medium with 0.5%glucose.The results showed that after extreme acid stimulation the survival rate ofΔtor R cells was inversely proportional to glucose concentration,which indicated that Tor R adapted to extreme acid environments by regulating rpo S expression.Taken together,Tor S phosphorylates Tor R after sensing the external extreme acid stress and the Tor R regulates the expressions of various acid-related genes through interacting with gene promoter regions.Indeed,Tor R interacts with the Tor R-boxes in rpo S promoter region to regulate its expression and then responds to extreme acid stimulation.Among them,Ira M protein may be involved in maintaining the levels of Rpo S protein to cope with extreme acid environments. |
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