Proteomics Approach For E.coli K-12 Outer Membrane Proteins Related To Disinfectant Tolerance And Antibiotic Resistance

Using proteomics and genomics technologies,we invesigated the outer membrane proteins of Gram-negative bacterium E.coli K-12 in response to disinfectant-tolerance and drug-resistance.This investigation was performed with the use of proteomic methodologies for altered proteome,Western blotting for verification of the altered proteins,and gene-defieient and protein-overexpressed approch for protein function. These studies would help us to understand disinfectant-tolerance and drug-resistance mechanisms of bacteria,find new drug targets,and develop new anti-bacteria agents or vaccine.Seven,seven and five altered outer membrane proteins were characterized in isopropanol-tolerant,ethanol-tolerant and phenol-tolerant E.coli K-12 strains,respectively, in which altered outer membrane proteins in both of isopropanol-tolerance and ethanol-tolerant strains were identical(Tsx,OmpC,OmpF,FadL,OmpT,OmpA and LamB).Out of the seven proteins,altered FadL,OmpT,OmpA,LamB changed were also characterized in phenol-tolerant strain.Following the verification of 2-DE by Western blotting,function of the altered proteins was investigated by gene knockout,gene complementation and gene overexpression experiments.LamB,OmpC and OmpA were determined as the most crucial outer membrane proteins in response to alcohols-tolerance, and LamB,OmpA and OmpT to phenol-tolerance,especially OmpA and OmpT.Further investigation indicated that bacterial tolerance to the threee disinfectants was regulated by EnvZ/OmpR two component signal transduction system.These results showed that outer membrane proteins of Gram-negative bacteria played important role in disinfectant tolerance,in which the characteristic feature of the altered outer membrane proteins was correlated to disinfectant types.Through the sub-proteomics to study functional outer membrane proteins relevant to E.coli K-12 resistant to tetracycline and kanamycin,we found 11 altered spots in tetracycline-resistant strain and 8 of them were identified as outer membrane proteins which belonged to 6 types of outer membrane proteins(LamB,FimD,TolC,OmpC,Tsx and OmpW);11 altered spots in E.coli K-12 resistant to kanamycin and 9 of them were identified as outer membrane proteins which belonged to 7 types of outer membrane proteins(OmpA,TolC,FadL,OstA,Tsx,OmpW and MipA).Furthermore,TolC,Tsx and OmpW were commonly changed outer membrane proteins in these two drug-resistant strains.The reliability of 2-DE was confirmed by Western blotting.On the basis of these results,further functional analysis of these outer membrane proteins showed that LamB,TolC and OmpC were critical in the process of tetracycline-resistance,and MipA and TolC were especially important to kanamycin-resistance.Importantly,MipA was first reported here to be an outer membrane protein in response to bacterial drug-resistance.Moreover,we constructed two mutants,ΔtolC-pET-28a-tolC1 andΔtolC-pET-28a-tolC2,which respectively contained mutated TolC long and short extracellular domains for investigating ability of extracellular domains in response to an antibiotic.Our results indicated that TolC could sense the concentration of environmental drugs,which is a novel finding.