de Veaux L C, Clevenson D S, Bradbeer C, Kadner R J
J Bacteriol. 1986 Sep;167(3):920-7. doi: 10.1128/jb.167.3.920-927.1986.
Passage of vitamin B12 across the outer and cytoplasmic membranes of Escherichia coli occurs in two steps, each involving independent transport systems. Since the vitamin accumulated in btuC or btuD mutants is readily released from the cell by chase or osmotic shock and does not undergo the usual metabolic conversions, the products of these genes might participate in transport across the cytoplasmic membrane. Mutations in btuC and btuD are complemented by recombinant plasmids carrying a 3,410-base-pair HindIII-HincII DNA fragment. Transposon Tn1000 mutagenesis and subcloning defined the location of these two genes and showed that they are separated by approximately 800 base pairs. The polypeptides elicited by this fragment and its derivatives were identified by using a maxicell system. The apparent molecular weight of the btuC product was approximately 26,000, that of the btuD product was 29,000. Both polypeptides were associated with the cell membrane. Transposon insertions in the region between btuC and btuD, as well as those in the two genes, conferred a deficiency in vitamin B12 utilization and transport when they were crossed onto the chromosome. This region, termed btuE, encoded a 22,000-Mr polypeptide and lesser amounts of a 20,000-Mr species. A portion of the BtuE protein was released from maxicells by osmotic shock or spheroplast formation. The relative production of BtuE and BtuD in response to plasmids carrying transposon insertions suggested that the three genes are arranged in an operon in the order btuC-btuE-btuD and that internal promoters exist since polarity was incomplete. Substantial elevation of transport activity was engendered by plasmids carrying the intact btu region, but not when any of the btu genes was disrupted. The btuCED region thus may encode a transport system for passage of vitamin B12 across the cytoplasmic membrane. This system bears similarities to periplasmic binding protein-dependent transport systems, although the putative periplasmic component is not required for its function.
维生素B12穿过大肠杆菌外膜和细胞质膜的过程分两步进行，每一步都涉及独立的转运系统。由于在btuC或btuD突变体中积累的维生素很容易通过追踪或渗透休克从细胞中释放出来，并且不会进行通常的代谢转化，因此这些基因的产物可能参与跨细胞质膜的转运。携带3410个碱基对的HindIII - HincII DNA片段的重组质粒可互补btuC和btuD中的突变。转座子Tn1000诱变和亚克隆确定了这两个基因的位置，并表明它们相隔约800个碱基对。通过使用大细胞系统鉴定了由该片段及其衍生物引发的多肽。btuC产物的表观分子量约为26000，btuD产物的表观分子量为29000。两种多肽都与细胞膜相关。当转座子插入btuC和btuD之间的区域以及这两个基因中时，将它们转移到染色体上会导致维生素B12利用和转运缺陷。这个区域称为btuE，编码一种22000道尔顿的多肽和少量20000道尔顿的多肽。一部分BtuE蛋白通过渗透休克或原生质球形成从大细胞中释放出来。响应携带转座子插入的质粒时BtuE和BtuD的相对产量表明，这三个基因以btuC - btuE - btuD的顺序排列在一个操纵子中，并且由于极性不完全，存在内部启动子。携带完整btu区域的质粒可使转运活性大幅提高，但当任何一个btu基因被破坏时则不然。因此，btuCED区域可能编码一种使维生素B12穿过细胞质膜的转运系统。该系统与依赖周质结合蛋白的转运系统有相似之处，尽管其功能不需要假定的周质成分。
