Study On Biological Activity Of YadG Protein Subunits Of The E.coli YadGH Transporter

Escherichia coli(E.coli)is a conditional pathogen,which causes human and animal infection,and brings serious harm to human and animal.Antibiotic is usually used to prevent and treat E.coli infection clinically.However,with the emergence and development of drug-resistant E.coli,there are fewer and fewer drugs available to treat the infection.E.coli drug resistance mediated by the drug efflux transporters is the main mechanism of drug resistance.Drug efflux mediated by transporter is a most important mechanism of E.coli resistance.Among the seven families of E.coli drug efflux transporters,ATP-binding cassette(ABC)transporter superfamily contains the largest number of efflux transporters with the most extensive functions.Whole genome sequencing revealed that the genome of E.coli encodes 80 ABC transporters.However,there are still a few of ABC transporters whose functions have not been verified,and one of them is YadGH transporter.Therefore,in order to confirm the function of the YadGH transporter,we analyzed the transporter through bioinformatics and evaluated the biological activity of its YadG protein by experiment in this study.Analysis of the genes encoding YadGH transporter complex and their deduced peptides through bioinformatics showed that YadGH transporter composed of YadG subunits and YadH subunits.YadG was a hydrophilic subunit with nucleotide-binding domain(NBD)of ABC transporter,including ATP binding sites,Walker A/P-loop,Walker B,Q-loop,H-loop,D-loop,ABC signature motif and other characteristics of conserved domains,indicting that YadG has ATPase activity.Yad H is a hydrophobic subunit with the transmembrane domain(TMD)of ABC transporter,including 6 TMD.The substrate of YadGH transporter driven by YadG is polyketones.According to the ATPase activity and the function of drug transport driven by YadG,we constructed the E.coli expression vector,pET-28a(+)-yadG,and YadG protein was expressed in E.coli BL21.The ATPase activity of YadG protein was detected after purification.The results showed that YadG protein had obvious ATPase activity(3.011 Pi μmol/mgprot/hour).We also constructed yadG gene knockout strains of E.coli K-12,namely E.coli ΔyadG,using Red recombinant technology.At the same time,in order to exclude the effect of Acr AB-Tol C transporter,a both yadG and acr B gene knockout strain,E.coli Δacr BΔyadG,was constructed using an acr B gene knockout K-12 strain.On this basis,E.coli K-12,E.coli ΔyadG,E.coli Δacr B and E.coli Δacr BΔyadG strains were used for the following experimental detection of growth characteristics and minimum inhibitory concentration(MIC)of the drugs.The results showed that there was no significant difference in the growth characteristics of E.coli ΔyadG strain compared with K-12 strain,and the growth rate of E.coli Δacr B and E.coli Δacr BΔyadG strain was slower than that of K-12 strain during the incubation for 5 hours,but there was no significant difference between them after incubation for 6 h.MIC of roxithromycin,tylosin and erythromycin against E.coli ΔyadG strain decreased compared with that against K-12 strain,and MIC of roxithromycin and tylosin against E.coli Δacr BΔyadG strain further decreased compared with that against E.coliΔacr B strain.The results above indicated that YadG has ATPase activity and can drive YadGH to transport macrolide,such as roxithromycin,tylosin and erythromycin,belonging to the polyketone group.