Balhesteros Heloise, Shipelskiy Yan, Long Noah J, Majumdar Aritri, Katz Benjamin B, Santos Naara M, Leaden Laura, Newton Salete M, Marques Marilis V, Klebba Phillip E
Departamento de Microbiologia, Instituto de Ciencias Biomedicas, Universidade de São Paulo, São Paulo, Brazil.
Department of Biochemistry and Molecular Biophysics, Kansas State University, Manhattan, Kansas, USA.
J Bacteriol. 2017 Feb 28;199(6). doi: 10.1128/JB.00723-16. Print 2017 Mar 15.
Siderophore nutrition tests with strain NA1000 revealed that it utilized a variety of ferric hydroxamate siderophores, including asperchromes, ferrichromes, ferrichrome A, malonichrome, and ferric aerobactin, as well as hemin and hemoglobin. did not transport ferrioxamine B or ferric catecholates. Because it did not use ferric enterobactin, the catecholate aposiderophore was an effective agent for iron deprivation. We determined the kinetics and thermodynamics of [Fe]apoferrichrome and Fe-citrate binding and transport by NA1000. Its affinity and uptake rate for ferrichrome (equilibrium dissociation constant [ ], 1 nM; Michaelis-Menten constant [ ], 0.1 nM; , 19 pMol/10 cells/min) were similar to those of FhuA. Transport properties for Fe-citrate were similar to those of FecA ( , 5.3 nM; , 29 pMol/10 cells/min). Bioinformatic analyses implicated Fur-regulated loci , , , and as TonB-dependent transporters (TBDT) that participate in iron acquisition. We resolved TBDT with elevated expression under high- or low-iron conditions by SDS-PAGE of sodium sarcosinate cell envelope extracts, excised bands of interest, and analyzed them by mass spectrometry. These data identified five TBDT: three were overexpressed during iron deficiency (00028, 02277, and 03023), and 2 were overexpressed during iron repletion (00210 and 01196). CLUSTALW analyses revealed homology of putative TBDT 02277 to FepA and BtuB. A Δ mutant did not transport hemin or hemoglobin in nutrition tests, leading us to designate the structural gene as (for eme/hemoglobin tilization). The physiological roles of the 62 putative TBDT of are mostly unknown, as are their evolutionary relationships to TBDT of other bacteria. We biochemically studied the iron uptake systems of , identified potential iron transporters, and clarified the phylogenetic relationships among its numerous TBDT. Our findings identified the first outer membrane protein involved in iron acquisition by , its heme/hemoglobin transporter (HutA).
用菌株NA1000进行的铁载体营养测试表明，它利用多种高铁异羟肟酸铁载体，包括曲霉色菌素、铁色素、铁色素A、丙二色素和高铁气杆菌素，以及血红素和血红蛋白。它不转运去铁胺B或儿茶酚高铁盐。由于它不使用肠杆菌素铁，儿茶酚脱铁铁载体是一种有效的铁剥夺剂。我们测定了NA1000对[Fe]脱铁铁色素和柠檬酸铁的结合及转运的动力学和热力学。它对铁色素的亲和力和摄取速率（平衡解离常数[Kd]，1 nM；米氏常数[Km]，0.1 nM；Vmax，19 pMol/10⁹细胞/分钟）与FhuA相似。柠檬酸铁的转运特性与FecA相似（Km，5.3 nM；Vmax，29 pMol/10⁹细胞/分钟）。生物信息学分析表明，Fur调控的基因座yfeA、yfeB、yfeC和yfeD是参与铁摄取的TonB依赖性转运蛋白（TBDT）。我们通过对肌氨酸钠细胞包膜提取物进行SDS-PAGE，分离出在高铁或低铁条件下表达升高的TBDT，切下感兴趣的条带，并通过质谱分析它们。这些数据鉴定出5种TBDT：3种在缺铁期间过表达（00028、02277和03023），2种在铁充足时过表达（00210和01196）。CLUSTALW分析显示，假定的TBDT 02277与FepA和BtuB具有同源性。一个ΔyfeB突变体在营养测试中不转运血红素或血红蛋白，这使我们将yfeB结构基因命名为hutA（用于血红素/血红蛋白利用）。大肠杆菌62种假定的TBDT的生理作用大多未知，它们与其他细菌的TBDT的进化关系也不清楚。我们对大肠杆菌的铁摄取系统进行了生化研究，鉴定了潜在的铁转运蛋白，并阐明了其众多TBDT之间的系统发育关系。我们的研究结果鉴定出了大肠杆菌中第一个参与铁摄取的外膜蛋白，即其血红素/血红蛋白转运蛋白（HutA）。
