562015-03-24 , 法政大学大学院理工学・工学研究科
The RND-type xenobiotic efflux system plays an important role in bacterial multidrug resistance. Escherichia coli has five such systems. Among them, only the AcrB-AcrA-TolC complex, consisting of the inner membrane transporter AcrB, the membrane fusion protein AcrA, and the outer membrane channel TolC, is constitutively expressed. Interestingly, AcrA and TolC are shared with another RND-type complex AcrD-AcrA-TolC and the expression of the inner membrane transporter AcrD is induced upon environmental stimuli. Here I visualized AcrD and AcrA in vivo using green fluorescent protein (GFP). Total internal reflection fluorescence microscopy revealed that GFP-labeled AcrD and AcrA are not fixed but diffuse through the membrane unless TolC exists. In the acrA deletion strain, AcrD-GFP was not fixed even in the presence of TolC, suggesting that without AcrA, AcrD cannot form a complex with TolC. In contrast, GFP-AcrA was found to form an AcrA-TolC complex even in the absence of AcrB or AcrD. Next, to estimate the cellular number of each protein, I constructed strains with chromosomal genes encoding 6×His-tagged components (AcrAHis, AcrBHis, AcrDHis, TolCHis). Western blotting revealed that AcrA is by far more abundant than AcrB, AcrD or TolC.