Identification of the periplasmic cobalamin-binding protein BtuF of Escherichia coli
Identification of the periplasmic cobalamin-binding protein BtuF of Escherichia coli
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DOI：
10.1128/jb.184.3.706-717.2002
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发表时间：
2002-02-01
影响因子：
3.2
通讯作者：
Kadner, RJ
中科院分区：
文献类型：
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作者：
Cadieux, N;Bradbeer, C;Kadner, RJ
大肠杆菌细胞通过连续主动转运过程吸收维生素B-12（氰钴胺素[CN-Cbl]）和铁螯合剂。CN-Cbl通过外膜的运输及其在周质中的积累是由tonb依赖性转运体BtuB介导的。铁铁载体在细胞质膜上的转运需要BtuC和BtuD蛋白，它们在序列上与铁铁载体在质周透膜上的跨膜和atp结合盒蛋白最为相关。与大多数外质渗透膜的遗传组织不同，外质cl结合蛋白的候选基因不与btuCED操纵子相连。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铁载体复合物的质周结合蛋白相关，并且先前与沙门氏菌的CN-Cbl摄取有关。大肠杆菌yadT产物，更名为BtuF，在这里显示参与CN-Cbl摄取。BtuF蛋白，用c端His标签表达，被证明是易位到外周质，同时去除一个信号序列。利用放射性标记底物或等温滴定量热法进行的CN-Cbl结合实验表明，纯化的BtuF与CN-Cbl的结合常数约为15 nM。btuF的零突变，而两侧基因pfs和yadS的零突变，强烈降低了CN-Cbl的利用和向细胞质的转运。btuF突变体对CN-Cbl的生长反应比之前描述的btuC、btuD或btuF突变体的轻微损伤强得多。因此，构建了btuC和btuD的零突变，发现btuC突变体具有与btuF突变体相似的强烈损伤，而btuD缺陷不那么明显。所有跨CM转运缺陷的突变体都会产生频繁的抑制变异体，这些变异体能够对较低水平的CN-Cbl作出反应，但在跨CM转运方面仍然存在缺陷。这些结果最终确定了Cbl摄取的周质结合蛋白的身份，这是周质渗透酶成分遗传分离的少数情况之一。
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, 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.