Functional Characterization Of The ABC Drug Transporters, YbhFSR And YddA, From Escherichia Coli

ATP-binding cassette transporters are ubiquitous distributed in bacteria,fungi and eukaryotes.ABC transporters can transport substrates from small molecules to larger compounds using the energy derived from the hydrolysis of ATP.The ABC transporters is an important factor causing drug resistance of human tumor cells and bacteria resistance.The genomic of Escherichia coli K-12 was predicted to encode 11 of the ABC exporters,among which YbhFSR and YddA were presumed to be drug efflux transporters,but exact functions of them are still lack of experimental research.Functional study of putative ABC drug resistance transporters are important for understanding the resistance of bacteria and even understanding the resistance mechanism of human tumors.In order to study the function of the YbhFSR and YddA transporter,bioinformatics analysis was carried out firstly,the results shown that ybhF,ybhS and ybhR encode the subunits of the YbhFSR transporter.YbhF has two nucleotide-binding domains(NBD),and is the predicted ATP binding component,whereas YbhS and YbhR are predicted membrane components.Amino acid sequence analysis showed that the N-terminus of YddA protein is a transmembrane domain formed by five transmembrane helixes domain(TMD).The C-terminus contained all the conserved domains and is a nucleotide binding domain(NBD).In order to study the function of the YbhFSR and YddA transporter,Knockout strains of E.coli?ybhF,E.coli?ybh R,E.coli?ybh S,E.coli?ybh SR,E.coli?ybhFSR,E.coli?acr B and E.coli?acr B?ybhF were constructed in E.coli K-12 strain using red recombination technology.In addition,the overexpression strain of ybhF E.coli/ pET28a-ybhF was constructed.In order to study the function of the YddA transporter,knockout strains E.coli?ydd A,E.coli?ydd A?acr B and E.coli?ydd B were constructed.Using the malachite green experiment to study the ATPase activity of purified YbhF protein.The results indicated that YbhF protein showed strong ATPase activity compared to the control.This suggested that the YbhF protein belongs to the NBD domain of the ABC family and can transport substrates using the energy derived from the hydrolysis of ATP.The efflux ability of YbhFSR and YddA transporters were tested using EB as a substrate.The results showed the over-expressing strain E.coli/pET28a-ybhF had the ability to enhance EB efflux compared with the control strain E.coli/pET28 a,and the knockout strains E.coli?acr B,E.coli?ybhF and E.coli?acr B?ybhF showed a decrease in EB efflux capacity,especially double transporters knockout strain E.coli?acr B?ybhF had the lowest EB efflux ability.The YddA transporter results were similar to the YbhFSR transporter,E.coli?ydd A significantly reduced EB efflux.EB efflux experiments showed that YbhFSR transporter and YddA transporter have the ability to efflux EB.YbhFSR and YddA transporters are efflux transporters belong to the ABC family.Drug resistance assays was used to screen substrates of YbhFSR and YddA transporters.The results showed that YbhFSR transporter increased sensitivity to tetracyclines such as tetracycline,oxytetracycline,chlortetracycline,doxycycline,EB and Hoechst33342.The above results indicated that the YbhFSR transporter was a single-drug efflux transporter of tetracyclines.However,the MIC results of YddA transporter showed an increased sensitivity to 13 of 20 drugs.YddA transporter is a multi-drug efflux transporter.Using the autofluorescence properties of tetracyclines and quinolone,the accumulation and efflux of drugs in cells were studied.The results showed that the efflux ability of tetracyclines of knockout strains E.coli?acr B,E.coli?ybhF and E.coli?acr B?ybhF were decreased and the accumulation of tetracyclines were increased.The ability to extrude norfloxacin of E.coli?ydd A was significantly reduced,and accumulation of norfloxacin in cells increased.To further study the drug efflux and energy sources of the two transporters,three inhibitors(ortho-vanadate,CCCP and reserpine)and other known multidrug substrates were used in the fluorescence assays.Ortho-vanadate and CCCP can significantly inhibit the efflux of tetracycline regulated by YbhFSR,indicating that the energy source of YbhFSR transporter was ATP.The ability of the YddA transporter to extrude norfloxacin can be inhibited by ortho-vanadate and reserpine,as well as by other known MDR substrates.It shows that the energy source of YddA transporter is ATP,and it can recognize and transport multiple substrates.The plasmid pET28a-ybhF and p ET28 a were transformed into E.coli Na~+ deficient strain KNabc.It was found that the overexpression of ybhF endows KNabc with tolerance to NaCl,LiCl and alkaline p H.The results indicates that the transporter in which Ybh F is located was a Na~+(Li~+)/H~+ antiporter.The results showed that YbhFSR of E.coli was a single drug efflux transporter of ABC family,YbhFSR transporter played a dual role,tetracyclines efflux pump and Na~+(Li~+)/H~+ antiporter.YddA transporter is a multi-drug exporter of ABC family.