Cadieux Nathalie, Bradbeer Clive, Reeger-Schneider Eva, Köster Wolfgang, Mohanty Arun K, Wiener Michael C, Kadner Robert J
Department of Microbiology, School of Medicine, University of Virginia, Charlottesville, Virginia 22908-0734, USA.
J Bacteriol. 2002 Feb;184(3):706-17. doi: 10.1128/JB.184.3.706-717.2002.
Cells of Escherichia coli take up vitamin B(12) (cyano-cobalamin [CN-Cbl]) and iron chelates by use of sequential active transport processes. Transport of CN-Cbl across the outer membrane and its accumulation in the periplasm is mediated by the TonB-dependent transporter BtuB. Transport across the cytoplasmic membrane (CM) requires the BtuC and BtuD proteins, which are most related in sequence to the transmembrane and ATP-binding cassette proteins of periplasmic permeases for iron-siderophore transport. Unlike the genetic organization of most periplasmic permeases, a candidate gene for a periplasmic Cbl-binding protein is not linked to the btuCED operon. The open reading frame termed yadT in the E. coli genomic sequence is related in sequence to the periplasmic binding proteins for iron-siderophore complexes and was previously implicated in CN-Cbl uptake in Salmonella. The E. coli yadT product, renamed BtuF, is shown here to participate in CN-Cbl uptake. BtuF protein, expressed with a C-terminal His(6) tag, was shown to be translocated to the periplasm concomitant with removal of a signal sequence. CN-Cbl-binding assays using radiolabeled substrate or isothermal titration calorimetry showed that purified BtuF binds CN-Cbl with a binding constant of around 15 nM. A null mutation in btuF, but not in the flanking genes pfs and yadS, strongly decreased CN-Cbl utilization and transport into the cytoplasm. The growth response to CN-Cbl of the btuF mutant was much stronger than the slight impairment previously described for btuC, btuD, or btuF mutants. Hence, null mutations in btuC and btuD were constructed and revealed that the btuC mutant had a strong impairment similar to that of the btuF mutant, whereas the btuD defect was less pronounced. All mutants with defective transport across the CM gave rise to frequent suppressor variants which were able to respond at lower levels of CN-Cbl but were still defective in transport across the CM. These results finally establish the identity of the periplasmic binding protein for Cbl uptake, which is one of few cases where the components of a periplasmic permease are genetically separated.
大肠杆菌细胞通过连续的主动运输过程摄取维生素B12（氰钴胺[CN-Cbl]）和铁螯合物。CN-Cbl跨外膜的运输及其在周质中的积累由TonB依赖性转运蛋白BtuB介导。跨细胞质膜（CM）的运输需要BtuC和BtuD蛋白，它们在序列上与用于铁载体运输的周质通透酶的跨膜和ATP结合盒蛋白最相关。与大多数周质通透酶的基因组织不同，周质Cbl结合蛋白的候选基因与btuCED操纵子不相连。大肠杆菌基因组序列中称为yadT的开放阅读框在序列上与铁载体复合物的周质结合蛋白相关，并且先前已涉及沙门氏菌中CN-Cbl的摄取。此处显示大肠杆菌yadT产物（重新命名为BtuF）参与CN-Cbl的摄取。用C末端His(6)标签表达的BtuF蛋白显示在去除信号序列的同时易位到周质中。使用放射性标记底物或等温滴定量热法进行的CN-Cbl结合试验表明，纯化的BtuF以约15 nM的结合常数结合CN-Cbl。btuF中的无效突变，而不是侧翼基因pfs和yadS中的无效突变，强烈降低了CN-Cbl的利用和向细胞质中的运输。btuF突变体对CN-Cbl的生长反应比先前描述的btuC、btuD或btuF突变体的轻微损害要强得多。因此，构建了btuC和btuD中的无效突变，结果表明btuC突变体具有与btuF突变体相似的强烈损害，而btuD缺陷则不太明显。所有跨CM运输有缺陷的突变体都会产生频繁的抑制变体，这些变体能够在较低水平的CN-Cbl下做出反应，但在跨CM运输方面仍然存在缺陷。这些结果最终确定了用于Cbl摄取的周质结合蛋白的身份，这是周质通透酶成分在基因上分离的少数情况之一。
