The nitrite transport protein NirC from Salmonella typhimurium is a nitrite/proton antiporter

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• 作者：Adriana Rycovska^a ; Lina Hatahet^b ; Klaus Fendler^b ; ^{klaus.fendler@biophys.mpg.de} ; Hartmut Michel^a
• 关键词：His-tag ; polyhistidine tag ; IC50 ; half maximum inhibitory concentration ; IPTG ; isopropyl-尾-d-thiogalactoside ; LB ; Luria-Bertani growth media ; DDM ; n-dodecyl-尾-d-maltoside ; Ni-NTA ; nickel-nitrilotriacetic acid ; ORF ; open reading frame ; SDS-PAGE ; sodium d
• 刊名：Biochimica et Biophysica Acta (BBA)/Biomembranes
• 出版年：2012
• 期刊代码：14_00052736
• 类别：bio
• 出版时间：May, 2012
• 卷：1818
• 期：5
• 页码：1342-1350
• 文件大小：663 K

摘要

In anaerobically grown bacteria, transport of nitrite is catalyzed by an integral membrane protein of the form ate-nitrite transporter family, NirC, which in Salmonella typhimurium plays a critical role in intracellular virulence. We present a functional characterization of the S. typhimurium nitrite transporter StmNirC in native membrane vesicles as well as purified and reconstituted into proteoliposomes. Using an electrophysiological technique based on solid supported membranes, we show nitrite induced translocation of negative charges into proteoliposomes reconstituted with purified StmNirC. These data demonstrate the electrogenicity of StmNirC and its substrate specificity for nitrite. Monitoring changes in 螖pH on everted membrane vesicles containing overexpressed StmNirC using acridine orange as a pH indicator we demonstrate that StmNirC acts as a secondary active transporter. It promotes low affinity transport of nitrite coupled to H⁺ antiport with a pH independent profile in the pH range from 6 to 8. In addition to nitrite also nitrate is transported by StmNirC, but with reduced flux and complete absence of proton antiport activity. Taken together, these data suggest a bispecific anion selectivity of StmNirC with an ion specific transport mode. This may play a role in regulating nitrite transport under physiological conditions.