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 Several genetic loci in Escherichia coli are required for the high-affinity and irreversible uptake of vitamin B12 (cyanocobalamin) across the outer and cytoplasmic membranes (4). The btuB gene encodes the 66,000-Mr outer membrane receptor protein for cobalamin and cobinamide, the E colicins, and bacteriophage BF23 (6,9,15,18). The tonB product is necessary for the energy-dependent transfer of cobalamin from the receptor into the periplasm (3, 25). This protein is also required for siderophore-mediated iron uptake (reviewed in reference 22). These iron uptake systems also employ specific receptors in the outer membrane as well as other gene products that mediate transport steps after receptor binding (10,24,29).The btuCD locus, located next to himA at min 37.4 on the E. coli chromosome map (2)
The products of the btuCED region of the Escherichia coli chromosome participate in the transport of vitamin B12 across the cytoplasmic membrane. The nucleotide sequence of the 3,410-base-pair HindIH-HincII DNA fragment carrying a portion of the himA gene and the entire btuCED region was determined. (18,38). The tonB product is necessary for the energydependent release of receptor-bound vitamin B12 (3, 31). Iron chelate uptake systems also employ specific outer membrane receptor proteins and tonB function (29). The use of receptors allows these relatively large and scarce growth factors to be translocated across the outer membrane more efficiently than by diffusion through porin channels.Little information is available concerning the passage of vitamin B12 or the iron chelates across the cytoplasmic membrane. Genetic studies of vitamin B12 and siderophore uptake revealed the requirement for additional components acting after the receptors and TonB. Three genes (thuBCD) are necessary for ferric hydroxamate entry (14); ferrienterochelin uptake requires fepBC function in addition to the fepA-coded receptor (30). In the case of vitamin B12, three linked genes, btuCED, have been identified (11,12). In their absence, vitamin B12 is accumulated in an energydependent manner, but, in contrast to the wild-type situation, all of the transported compound is lost from the cell upon chase with excess nonradioactive substrate or disruption of the outer membrane by osmotic shock or treatment with chelators (31). Furthermore, there is little conversion of transported cyanocobalamin into the usual metabolic species encountered in wild-type strains.The btuCED region has been cloned, and the locations of btuC and btuD were defined by further subcloning and transposon mutagenesis. The two genes were separated by * Corresponding author. t Present address:
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