Uptake of cyanocobalamin by Escherichia coli B: Some characteristics and evidence for a binding protein

Taylor, Robert T.; Norrell, Stephen A.; Hanna, M.Leslie

doi:10.1016/0003-9861(72)90154-3

Cited by 41 publications

(14 citation statements)

References 40 publications

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“…The observation that spheroplasts of a tonB mutant can transport ferrichrome suggests either an outer cell membrane or periplasmic location for the tonB gene-product (12). The energy-dependent phase of vitamin B-12 transport is sensitive to osmotic shock (13,14), a finding that is consistent with a periplasmic location. However, it has been reported that the tbnB gene product resides in the inner cell membrane on~the basis of Sarkosyl fractionation of the E. coli envelope (15).…”

supporting

confidence: 62%

DNA sequence of the Escherichia coli tonB gene.

Postle¹,

Good²

1983

Proc. Natl. Acad. Sci. U.S.A.

121

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The nucleotide sequence of a cloned section of the Escherichia coli chromosome containing the tonB gene has been determined. Transcription initiation and termination sites for tonB RNA have been determined by SI nuclease mapping. The tonB promoter and terminator resemble other E. coli promoters and terminators; the sequence of the tonB terminator region suggests that it may function bidirectionally. The DNA sequence specifies an open translation reading frame between the 5' and3' RNA termini whose location is consistent with the position of previously isolated tonB::IS1 mutations. The DNA sequence predicts a proline-rich protein with a calculated size of 26.1-26.6 kilodaltons (239-244 amino acids), depending-on.which of three potential initiation codons is utilized. The predicted NH2 terminus of tonB-protein resembles the proteolytically cleaved signal sequences of E coli periplasmic and outer membrane proteins; the overall hydrophilic character of the protein sequence suggests that the bulk of the tonB protein is not embedded within the inner or outer membrane. A significant discrepancy exists between the calculated size of tonB protein and the apparent size of, 36 kilodaltons determined by sodium dodecyl sulfate/polyacrylamide gel electrophoresis.The function of the Escherichia coli gene tonB has been the subject of much speculation due to the pleiotropic nature oftonB mutations; tonB mutants were originally isolated as a class of bacteria surviving exposure to bacteriophage Ti (1). They were subsequently shown to be insensitive to bacteriophage 480 (2) and to the group B colicins (3, 4). In addition, tonB mutants are defective in vitamin B-12 transport (5) and, perhaps most significantly, they are defective in all known high-affinity iron transport systems, including iron transport mediated by enterochelin, ferrichrome, citrate, rhodotorulic acid, and aerobactin (6-9). The tonB gene product appears to be essential for the energy-dependent phase of vitamin B-12 transport (10) and in the energy-dependent, irreversible step of 080 and T1 infection (11).The cellular location of the tonB gene product remains uncertain. The observation that spheroplasts of a tonB mutant can transport ferrichrome suggests either an outer cell membrane or periplasmic location for the tonB gene-product (12). The energy-dependent phase of vitamin B-12 transport is sensitive to osmotic shock (13, 14), a finding that is consistent with a periplasmic location. However, it has been reported that the tbnB gene product resides in the inner cell membrane on~the basis of Sarkosyl fractionation of the E. coli envelope (15). It has also been reported that spheroplasts of tonB mutants are unable to transport enterochelin (16), a finding that suggests an inner cell membrane location for the tonB gene product; it should be noted, however, that other investigators have been unable to confirm this result (12). A number of possible functions for the tonB gene product have been suggested including control of outer membrane permeability (6), to...

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supporting

confidence: 62%

DNA sequence of the Escherichia coli tonB gene.

Postle¹,

Good²

1983

Proc. Natl. Acad. Sci. U.S.A.

121

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“…We therefore conclude that the three polypeptides comprise cytoplasmic membrane transport system. This conclusion is consistent with the previous observation of an Mr 22,000 periplasmic vitamin B12-binding protein (6,37). The capacity of the BtuE polypeptide to bind vitamin B12 is under investigation.…”

Section: Methodssupporting

confidence: 92%

“…Cellular localization studies indicated that the BtuC and BtuD polypeptides were membrane associated, whereas some of the BtuE polypeptide was released from maxicells by osmotic shock or spheroplast formnation, suggesting a periplasmic location. Previous studies demonstrated that an Mr 22,000 vitamin B12-binding protein was released by osmotic shock (6,37).…”

mentioning

confidence: 99%

Nucleotide sequence of the btuCED genes involved in vitamin B12 transport in Escherichia coli and homology with components of periplasmic-binding-protein-dependent transport systems

Friedrich¹,

Veaux²,

Kadner³

1986

J Bacteriol

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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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“…Uptake of the scarce molecule vitamin B 12 in the milieu by E. coli involves the high affinity outer membrane receptor BtuB (28), the cytoplasmic membrane bound TonB (29), the periplasmic substrate binding protein BtuF (30), and the transmembrane associated BtuCD (31). The TonB-ExbB-ExbD complex is proposed to harness the energy of proton motive force to drive the release of vitamin B 12 from BtuB.…”

Section: Discussionmentioning

confidence: 99%

Transmembrane Gate Movements in the Type II ATP-binding Cassette (ABC) Importer BtuCD-F during Nucleotide Cycle

Joseph

Jeschke

Goetz

et al. 2011

Journal of Biological Chemistry

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ATP-binding cassette (ABC) transporters are ubiquitous integral membrane proteins that translocate substrates across cell membranes. The alternating access of their transmembrane domains to opposite sides of the membrane powered by the closure and reopening of the nucleotide binding domains is proposed to drive the translocation events. Despite clear structural similarities, evidence for considerable mechanistic diversity starts to accumulate within the importers subfamily. We present here a detailed study of the gating mechanism of a type II ABC importer, the BtuCD-F vitamin B 12 importer from Escherichia coli, elucidated by EPR spectroscopy. Distance changes at key positions in the translocation gates in the nucleotide-free, ATP-and ADP-bound conformations of the transporter were measured in detergent micelles and liposomes. The translocation gates of the BtuCD-F complex undergo conformational changes in line with a "two-state" alternating access model. We provide the first direct evidence that binding of ATP drives the gates to an inward-facing conformation, in contrast to type I importers specific for maltose, molybdate, or methionine. Following ATP hydrolysis, the translocation gates restore to an apo-like conformation. In the presence of ATP, an excess of vitamin B 12 promotes the reopening of the gates toward the periplasm and the dislodgment of BtuF from the transporter. The EPR data allow a productive translocation cycle of the vitamin B 12 transporter to be modeled. ATP-binding cassette (ABC)2 transporters couple the energy of ATP hydrolysis to the translocation of substrates across biological membranes. They constitute the largest transmembrane protein family present in all branches of life and mediate the active transport of various substances such as sugars, amino acids, peptides, vitamins, iron siderophores, opines, metals, etc. across the membrane (1-3). Biochemical evidence supports a "two-state, alternating access" mechanistic model for both ABC exporters and importers (4). In this model, an ATP-bound conformation of the transmembrane domains facing the extracellular side of the membrane is converted to an inward-facing conformation via ATP hydrolysis in the nucleotide-binding domains (NBDs). This conformational transition ensures net substrate uptake by the importers or net expulsion by the exporters. Among the canonical ABC importers, which are characterized by the presence of a soluble periplasmic substrate binding protein, two structurally different types exist, namely type I (e.g. maltose, molybdate, and methionine systems) and type II (e.g. vitamin B 12 , heme, and metal systems) (3). For type I ABC importers the details of the alternating access mechanism have been confirmed by a large body of experimental evidence, including the crystal structures showing the maltose transporter in different states during the nucleotide cycle (5-8). The transmembrane domains (TMDs) of type I importers, featuring 10 -14 helices, alternate from an ATP-bound outward-facing conformation where the substrate b...

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Uptake of cyanocobalamin by Escherichia coli B: Some characteristics and evidence for a binding protein

Cited by 41 publications

References 40 publications

DNA sequence of the Escherichia coli tonB gene.

DNA sequence of the Escherichia coli tonB gene.

Nucleotide sequence of the btuCED genes involved in vitamin B12 transport in Escherichia coli and homology with components of periplasmic-binding-protein-dependent transport systems

Transmembrane Gate Movements in the Type II ATP-binding Cassette (ABC) Importer BtuCD-F during Nucleotide Cycle

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