Clive Bradbeer

Bio: Clive Bradbeer is an academic researcher from University of Virginia. The author has contributed to research in topics: Bacterial outer membrane & Escherichia coli. The author has an hindex of 17, co-authored 20 publications receiving 1482 citations.

Transport of Vitamin B12 in Escherichia coli: Common Receptor Sites for Vitamin B12 and the E Colicins on the Outer Membrane of the Cell Envelope

Abstract: The first step in the transport of cyanocobalamin (CN-B(12)) by cells of Escherichia coli was shown previously to consist of binding of the B(12) to specific receptor sites located on the outer membrane of the cell envelope. In this paper, evidence is presented that these B(12) receptor sites also function as the receptors for the E colicins, and that there is competition between B(12) and the E colicins for occupancy of these sites. The cell strains used were E. coli KBT001, a methionine/B(12) auxotroph, and B(12) transport mutants derived from strain KBT001. Colicins E1 and E3 inhibited binding of B(12) to the outer membrane B(12) receptor sites, and CN-B(12) protected cells against these colicins. Half-maximal protection was given by CN-B(12) concentrations in the range of 1 to 6 nM, depending upon the colicin concentration used. Colicin E1 competitively inhibited the binding of (57)Co-labeled CN-B(12) to isolated outer membrane particles. Functional colicin E receptor sites were found in cell envelopes from cells of only those strains that possessed intact B(12) receptors. Colicin K did not inhibit the binding of B(12) to the outer membrane receptor sites, and no evidence was found for any identity between the B(12) and colicin K receptors. However, both colicin K and colicin E1 inhibited the secondary phase of B(12) transport, which is believed to consist of the energy-coupled movement of B(12) across the inner membrane.

The proton motive force drives the outer membrane transport of cobalamin in Escherichia coli.

Abstract: Cells of Escherichia coli pump cobalamin (vitamin B12) across their outer membranes into the periplasmic space, and it was concluded previously that this process is potentiated by the proton motive force of the inner membrane. The novelty of such an energy coupling mechanism and its relevance to other outer membrane transport processes have required confirmation of this conclusion by studies with cells in which cobalamin transport is limited to the outer membrane. Accordingly, I have examined the effects of cyanide and of 2,4-dinitrophenol on cobalamin uptake in btuC and atp mutants, which lack inner membrane cobalamin transport and the membrane-bound ATP synthase, respectively. Dinitrophenol eliminated cobalamin transport in all strains, but cyanide inhibited this process only in atp and btuC atp mutant cells, providing conclusive evidence that cobalamin transport across the outer membrane requires specifically the proton motive force of the inner membrane. The coupling of metabolic energy to outer membrane cobalamin transport requires the TonB protein and is stimulated by the ExbB protein. I show here that the tolQ gene product can partly replace the function of the ExbB protein. Cells with mutations in both exbB and tolQ had no measurable cobalamin transport and thus had a phenotype that was essentially the same as TonB-. I conclude that the ExbB protein is a normal component of the energy coupling system for the transport of cobalamin across the outer membrane.

Identification of the Periplasmic Cobalamin-Binding Protein BtuF of Escherichia coli

Abstract: Cells of Escherichia coli take up vitamin B12 (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 His6 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.

Point mutations in a conserved region (TonB box) of Escherichia coli outer membrane protein BtuB affect vitamin B12 transport.

Abstract: Uptake of cobalamins and iron chelates in Escherichia coli K-12 is dependent on specific outer membrane transport proteins and the energy-coupling function provided by the TonB protein. The btuB product is the outer membrane receptor for cobalamins, bacteriophage BF23, and the E colicins. A short sequence near the amino terminus of mature BtuB, previously called the TonB box, is conserved in all tonB-dependent receptors and colicins and is the site of the btuB451 mutation (Leu-8----Pro), which prevents energy-coupled cobalamin uptake. This phenotype is partially suppressed by certain mutations in tonB. To examine the role of individual amino acids in the TonB box of BtuB, more than 30 amino acid substitutions in residues 6 to 13 were generated by doped oligonucleotide-directed mutagenesis. Many of the mutations affecting each amino acid did not impair transport activity, although some substitutions reduced cobalamin uptake and the Leu-8----Pro and Val-10----Gly alleles were completely inactive. To test whether the btuB451 mutation affects only cobalamin transport, a hybrid gene was constructed which encodes the signal sequence and first 39 residues of BtuB fused to the bulk of the ferrienterobactin receptor FepA (residues 26 to 723). This hybrid protein conferred all FepA functions but no BtuB functions. The presence of the btuB451 mutation in this fusion gene eliminated all of its tonB-coupled reactions, showing that the TonB box of FepA could be replaced by that from BtuB. These results suggest that the TonB-box region of BtuB is involved in active transport in a manner dependent not on the identity of specific side chains but on the local secondary structure.

Transport of Vitamin B12 in Escherichia coli

Abstract: The uptake of 60Co-labeled cyanocobalamin (vitamin B12) by cells of Escherichia coli K-12λ was shown to consist of an initial rapid phase (complete in <1 min), followed by a slower secondary phase. Methods enabling the measurement of 60Co-B12 uptake after incubation times of 1 to 2 sec were used in studies on the initial rate of B12 uptake. This initial process showed saturation kinetics, with a Vmax of 56 molecules per sec per cell and a Km of 5 nm, and was essentially independent of cellular energy metabolism. No inhibition was obtained with cyanide, fluoride, arsenite, or 2, 4-dinitrophenol, and an energy of activation of 3.8 kcal/mole for this initial phase of uptake was calculated from its response to temperature changes between 15 and 35 C. The inhibition by HgCl2 (50% at 0.1 mm) but not by 1 mmN-ethylmaleimide or 1 mmp-chloromercuribenzoate was consistent with a role for a relatively inaccessible sulfhydryl residue at the initial B12 binding site. The secondary phase of B12 uptake was clearly dependent on the energy metabolism of the cell, and, from its response to temperature, an energy of activation of about 17 kcal/mole was calculated. Cyanide (10 mm), arsenite (10 mm), and 2, 4-dinitrophenol (0.1 mm) gave at least 70% inhibition of the rate of the secondary phase. The formation of other cobalamins, such as 5′-deoxyadenosyl cobalamin, was not an obligate part of B12 transport. The cells were also able to take up 60Co-labeled cobinamide cyanide.

Molecular Basis of Bacterial Outer Membrane Permeability Revisited

Abstract: Gram-negative bacteria characteristically are surrounded by an additional membrane layer, the outer membrane. Although outer membrane components often play important roles in the interaction of symbiotic or pathogenic bacteria with their host organisms, the major role of this membrane must usually be to serve as a permeability barrier to prevent the entry of noxious compounds and at the same time to allow the influx of nutrient molecules. This review summarizes the development in the field since our previous review (H. Nikaido and M. Vaara, Microbiol. Rev. 49:1-32, 1985) was published. With the discovery of protein channels, structural knowledge enables us to understand in molecular detail how porins, specific channels, TonB-linked receptors, and other proteins function. We are now beginning to see how the export of large proteins occurs across the outer membrane. With our knowledge of the lipopolysaccharide-phospholipid asymmetric bilayer of the outer membrane, we are finally beginning to understand how this bilayer can retard the entry of lipophilic compounds, owing to our increasing knowledge about the chemistry of lipopolysaccharide from diverse organisms and the way in which lipopolysaccharide structure is modified by environmental conditions.

Bacterial iron homeostasis

Abstract: Iron is essential to virtually all organisms, but poses problems of toxicity and poor solubility. Bacteria have evolved various mechanisms to counter the problems imposed by their iron dependence, allowing them to achieve effective iron homeostasis under a range of iron regimes. Highly efficient iron acquisition systems are used to scavenge iron from the environment under iron-restricted conditions. In many cases, this involves the secretion and internalisation of extracellular ferric chelators called siderophores. Ferrous iron can also be directly imported by the G protein-like transporter, FeoB. For pathogens, host–iron complexes (transferrin, lactoferrin, haem, haemoglobin) are directly used as iron sources. Bacterial iron storage proteins (ferritin, bacterioferritin) provide intracellular iron reserves for use when external supplies are restricted, and iron detoxification proteins (Dps) are employed to protect the chromosome from iron-induced free radical damage. There is evidence that bacteria control their iron requirements in response to iron availability by down-regulating the expression of iron proteins during iron-restricted growth. And finally, the expression of the iron homeostatic machinery is subject to iron-dependent global control ensuring that iron acquisition, storage and consumption are geared to iron availability and that intracellular levels of free iron do not reach toxic levels.

The E. coli BtuCD structure: a framework for ABC transporter architecture and mechanism.

Abstract: The ABC transporters are ubiquitous membrane proteins that couple adenosine triphosphate (ATP) hydrolysis to the translocation of diverse substrates across cell membranes. Clinically relevant examples are associated with cystic fibrosis and with multidrug resistance of pathogenic bacteria and cancer cells. Here, we report the crystal structure at 3.2 angstrom resolution of the Escherichia coli BtuCD protein, an ABC transporter mediating vitamin B_(12) uptake. The two ATP-binding cassettes (BtuD) are in close contact with each other, as are the two membrane-spanning subunits (BtuC); this arrangement is distinct from that observed for the E. coli lipid flippase MsbA. The BtuC subunits provide 20 transmembrane helices grouped around a translocation pathway that is closed to the cytoplasm by a gate region whereas the dimer arrangement of the BtuD subunits resembles the ATP-bound form of the Rad50 DNA repair enzyme. A prominent cytoplasmic loop of BtuC forms the contact region with the ATP-binding cassette and appears to represent a conserved motif among the ABC transporters.