Involvement of ExbB and TonB in transport across the outer membrane of Escherichia coli: phenotypic complementation of exb mutants by overexpressed tonB and physical stabilization of TonB by ExbB.

The exb locus in Escherichia coli consists of two genes, termed exbB and exbD. Exb functions are related to TonB function in that most TonB-dependent processes are enhanced by Exb. Like tonB mutants, exb mutants were resistant to colicin M and albomycin but, in contrast to tonB mutants, showed only reduced sensitivity to colicins B and D. Overexpressed tonB on the multicopy vector pACYC177 largely restored the sensitivity of exb mutants to colicins B, D, and M but only marginally increased sensitivity to albomycin. Suppression of the btuB451 mutation in the structural gene for the vitamin B12 outer membrane receptor protein by a mutation in tonB occurred only in an exb+ strain. Degradation of the unstable overproduced TonB protein was prevented by overproduced ExbB protein. The ExbB protein also stabilized the ExbD protein. Pulse-chase experiments with radiolabeled ferrichrome revealed release of ferrichrome from exbB, tonB, and fhuC mutants, showing that ferrichrome had not crossed the cytoplasmic membrane. It is concluded that the ExbB and ExbD proteins contribute to the activity of TonB and, like TonB, are involved in receptor-dependent transport processes across the outer membrane.     

Two outer membrane transport systems for vitamin B12 in Salmonella typhimurium.

The involvement of an outer membrane transport component for vitamin B12 uptake in Salmonella typhimurium, analogous to the btuB product in Escherichia coli, was investigated. Mutants of S. typhimurium selected for resistance to bacteriophage BF23 carried mutations at the btuB locus (butBS) (formerly called bfe, at the analogous map position as the E. coli homolog) and were defective in high-affinity vitamin B12 uptake. The cloned E. coli btuB gene (btuBE) hybridized to S. typhimurium genomic DNA and restored vitamin B12 transport activity to S. typhimurium btuBS mutants. An Mr-60,000 protein in the S. typhimurium outer membrane was repressed by growth with vitamin B12 and was eliminated in a btuBS mutant. The btuBS product thus appears to play the same role in vitamin B12 transport by S. typhimurium as does the E. coli btuBE product. A second vitamin B12 transport system that is not present in E. coli was found by cloning a fragment of S. typhimurium DNA that complemented btuB mutants for vitamin B12 utilization. In addition to this plasmid with a 6-kilobase insert of S. typhimurium DNA, vitamin B12 utilization by E. coli btuB strains required the btuC and btuD products, necessary for transport across the cytoplasmic membrane, but not the btuE or tonB product. The plasmid conferred low levels of vitamin B12-binding and energy-dependent transport activity but not susceptibility to phage BF23 or utilization of dicyanocobinamide. The cloned S. typhimurium DNA encoding this new transport system did not hybridize to the btuBE gene or to E. coli chromosomal DNA and therefore does not carry the S. typhimurium btuBS locus. Increased production of an Mr -84,000 polypeptide associated with the outer membrane was seen. The new locus appears to be carried on the large plasmid in most S. typhimurium strains. Thus S. typhimurium possesses both high- and low-affinity systems for uptake of cobalamins across the outer membrane.     

Transport of vitamin B12 in Escherichia coli: cloning of the btuCD region.

The transport of vitamin B12 in Escherichia coli requires a specific vitamin B12 receptor protein in the outer membrane and the tonB gene product. In addition, the btuC gene, located at min 38 on the genetic map, has been found to influence vitamin B12 uptake or utilization. The btuC function is required for the growth response to vitamin B12 when the outer membrane transport process (btuB or tonB function) is defective. However, even in a wild-type strain, btuC is required for proper transport of vitamin B12. Additional mutations in the vicinity of btuC were isolated as lac fusions that produced a phenotype similar to that of a btuC mutant. The btuC region was cloned by selection for complementation of a btuC mutation. Complementation testing with plasmids carrying various deletions or transposon Tn1000 insertions demonstrated that the new mutations defined a separate, independently expressed locus, termed btuD. The coding regions for both genes were identified on a 3.4-kilobase HindIII-HincII fragment and were 800 to 1,000 base pairs in length. They were separated by a 600- to 800-base-pair region. The gene order in this portion of the chromosome map was found to be pps-zdh-3::Tn10-btuD-btuC-pheS. Expression of beta-galactosidase in the btuD-lac fusion-bearing strains, whether proficient or defective in vitamin B12 transport, was not regulated by the presence of vitamin B12 in the growth medium.     

Genetic suppression demonstrates interaction of TonB protein with outer membrane transport proteins in Escherichia coli.

Energy-coupled reactions of the Escherichia coli outer membrane transport proteins BtuB and Cir require the tonB product. Some point mutations in a region of btuB and cir that is highly conserved in TonB-dependent transport proteins led to loss of TonB-coupled uptake of vitamin B12 and colicin Ia, whereas binding was unaffected. Most other point mutations in this region had no detectable effect on transport activity. Mutations in tonB that suppressed the transport defect phenotype of these btuB mutations were isolated. All carried changes of glutamine 165 to leucine, lysine, or proline. The various tonB mutations differed markedly in their suppression activities on different btuB or cir mutations. This allele specificity of suppression indicates that TonB interacts directly with the outer membrane transport proteins in a manner that recognizes the local conformation but not specific side chains within this conserved region. An effect of the context of the remainder of the protein was seen, since the same substitution (valine 10----glycine) in btuB and cir responded differently to the suppressors. This finding supports the proposal that TonB interacts with more of the transport proteins than the first conserved domain alone.     

Molecular characterization of the Enterobacter aerogenes tonB gene: identification of a novel type of tonB box suppressor mutant.

The tonB gene of Enterobacter aerogenes was cloned, sequenced, and expressed in Escherichia coli. It complemented an E. coli tonB mutant as efficiently as E. coli tonB, except for colicin B and D sensitivities. However, colicin B and D sensitivities were complemented by a derivative in which the aspartate at position 165 was replaced by a glutamine (TonBD-165-->Q) by site-directed mutagenesis. In E. coli, the corresponding amino acid is a glutamine (Q-160) which is known to be altered in most mutants showing suppression of the btuB451 mutation. Fourteen independent btuB451 suppressor mutations in E. aerogenes tonB which all had suffered the same point mutation resulting in a change from glycine to valine at position 239 (G-239-->V) of the C-terminal end of the protein were isolated. The mutation was located within a region which is nonessential for function of E. aerogenes TonB as well as E. coli TonB. A constructed double mutation, expressing a D-165-->Q/G-239-->V derivative, no longer acted as a btuB451 suppressor. However, it restored colicin B and D sensitivities even more efficiently than the D-165-->Q derivative. Corresponding mutations constructed in E. coli tonB, giving rise to Q-160-->D, G-234-->V, and Q-160-->D/G-234-->V derivatives, showed phenotypes comparable to the E. aerogenes mutations. We take this as evidence that at least a functional interaction between the D-165 (Q-160 in E. coli) and the G-239 (G-234 in E. coli) region is necessary for TonB function. The implications of this interaction for functional instability of TonB are discussed.     

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

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 tonB mutants of Escherichia coli.

It is known that the tonB mutation in Escherichia coli is responsible for a defect in the transport of iron chelates. These are transported by systems that involve outer membrane components. We found that tonB mutants were also deficient in the secondary, energy-dependent phase of vitamin B12 transport, although the mutants have normal levels of B12 receptors on their cell surface. In addition, tonB mutants derived from vitamin B12 auxotrophs required elevated levels of B12 for normal growth. Maltose uptake, mediated by another transport system involving an outer membrane component, was unaffected by the tonB mutation.     

Genetic analysis of components involved in vitamin B12 uptake in Escherichia coli.

The products of three genes are involved in cyanocobalamin (B(12)) uptake in Escherichia coli. btuB (formerly bfe), located at min 88 on the Escherichia coli linkage map, codes for a protein component of the outer membrane which serves as receptor for B(12), the E colicins, and bacteriophage BF23. Four phenotypic classes of mutants varying in response to these agents were found to carry mutations that, based on complementation and reversion analyses, reside in the single btuB cistron. In one mutant class, ligand binding to the receptor appeared to be normal, but subsequent B(12) uptake was defective. The level of receptor and rate of uptake were responsive to btuB gene dosage. Previous studies showed that the tonB product was necessary for energy-dependent B(12) uptake but not for its binding. Other than those in tonB, no mutations that conferred insensitivity to group B colicins affected B(12) utilization. The requirement for the btuB and tonB products could be bypassed by elevated levels of B(12) (>1 muM) or by mutations compromising the integrity of the outer membrane as a permeability barrier. Utilization of elevated B(12) concentrations in strains lacking the btuB-tonB uptake system was dependent on the function of the btuC product. This gene was located at 37.7 min on the linkage map, with the order pps-btuC-pheS. Strains altered in btuC but with an intact btuB-tonB system were only slightly impaired in B(12) utilization, being defective in its accumulation. This defect was manifested as inability to retain B(12), such that intracellular label was almost completely lost by exchange or efflux. It is proposed that btuC encodes a transport system for B(12) in the periplasm.     

Nucleotide sequence of the gene for the vitamin B12 receptor protein in the outer membrane of Escherichia coli.

The nucleotide sequence of a 2220-base-pair fragment containing the btuB gene of Escherichia coli was determined. There was a single open reading frame which was translated into a 614-amino-acid polypeptide, the first 20 amino acids of which comprised a typical leader sequence. The putative mature or processed form had a molecular weight (66,400) and a composition in close agreement with that determined for the purified receptor. The distribution of amino acids in the receptor protein was similar to that of other outer membrane proteins, showing a fairly even distribution of charged residues and the absence of extensive hydrophobic stretches. The btuB451 mutation appears to alter the receptor to eliminate its ability to function in vitamin B12 uptake without affecting its ligand binding properties. The sequence of the DNA from this mutant was determined and revealed a leucine-to-proline (C-to-T transition) change in the eighth amino acid of the mature form.     

Transport of vitamin B12 in Escherichia coli: common receptor system for vitamin B12 and bacteriophage BF23 on the outer membrane of the cell envelope.

We showed previously that the outer membrane of the Escherichia coli cell envelope normally contains about 200 to 250 B12 receptors, and that these receptors function both in B12 transport and as receptors for the E colicins. This paper shows that this receptor system is also shared with bacteriophage BF23. A strong positive correlation was observed between the number of B12 receptors per cell and the rate of adsorption of BF23. Cells from mutant strains that lacked B12 receptors did not adsorb BF23 particles. The rate of adsorption of BF23 to cells of a merodiploid strain (RK4151), with about 550 B12 receptors per cell, was approximately double that to cells of a normal, haploid strain. The adsorption of BF23 to hole cells, cell envelopes, outer membrane particles, and solubilized outer membranes was inhibited by vitamin B12, with 50% inhibition at B12 concentrations in the range of 0.5 to 2.0 nM. These values are close to the observed KS for B12 binding to the B12 receptors. Vitamin B12 concentrations as high as 100 nM did not inhibit adsorption of bacteriophages T5, T6, and lambdacI to cells of sensitive strains of E. coli. Bacteriophage BF23 inhibited B12 transport by whole cells and was shown to be a competitive inhibitor of B12 binding to isolated cell envelope particles. The B12/BF23 receptors from E. coli strains KBT069 (btuB69) and RK4104 (btuB69) were fully active, but the number per cell was reduced to an average value of about 0.5.     

The wild-type allele of tonB in Escherichia coli is dominant over the tonB1 allele,encoding TonBQ160K,which suppresses the btuB451 mutation

The entire coding sequence of the tonB gene, except for nine codons at the 3′ end, was deleted from the chromosome of Escherichia coli. Introduction of the btuB451 suppressor mutant tonB1 into the chromosome of such a tonB deletion strain showed that the tonB1 allele was active as a suppressor in a single copy at 37° C and 42° C but not at 28° C. No temperature dependence was seen when FepA- or FhuA-dependent activities of the tonB1 gene product (TonB_Q160K) were tested. The btuB451 suppressor activity of tonB1 was inhibited by the simultaneous presence within the cells of the tonB ⁺ allele on a multicopy plasmid. This represents the first case of dominance among different tonB alleles. Inhibition of suppression was abolished by overexpression of the btuB451-encoded receptor protein. Competition for binding of TonB⁺ and TonB_Q150K to ExbB was excluded as the cause of dominance. Based on our data we conclude that competition for binding of TonB ⁺ and TonB_Q160K to the btuB451 gene product is the reason for the observed dominance. The implications of these findings for the mechanism of btuB451 suppression by tonB1 are discussed.     

The wild-type allele of tonB in Escherichia coli is dominant over the tonB1 allele, encoding TonBQ160K, which suppresses the btuB451 mutation

The entire coding sequence of the tonB gene, except for nine codons at the 3 end, was deleted from the chromosome of Escherichia coli. Introduction of the btuB451 suppressor mutant tonB1 into the chromosome of such a tonB deletion strain showed that the tonB1 allele was active as a suppressor in a single copy at 37° C and 42° C but not at 28° C. No temperature dependence was seen when FepA- or FhuA-dependent activities of the tonB1 gene product (TonB_Q160K) were tested. The btuB451 suppressor activity of tonB1 was inhibited by the simultaneous presence within the cells of the tonB ⁺ allele on a multicopy plasmid. This represents the first case of dominance among different tonB alleles. Inhibition of suppression was abolished by overexpression of the btuB451-encoded receptor protein. Competition for binding of TonB⁺ and TonB_Q150K to ExbB was excluded as the cause of dominance. Based on our data we conclude that competition for binding of TonB ⁺ and TonB_Q160K to the btuB451 gene product is the reason for the observed dominance. The implications of these findings for the mechanism of btuB451 suppression by tonB1 are discussed.     

Siderophore production and outer membrane proteins of selected Vibrio vulnificus strains under conditions of iron limitation

Abstract The tonB gene product is necessary for the energy-dependent transport of ferric chelates and vitamin B₁₂ across the Escherichia coli outer membrane. When carried on multicopy plasmids, the cloned tonB gene complemented tonB hosts, restoring transport of ferri-siderophone complexes and vitamin B₁₂, and susceptibility to the group B colicins and phage ф80. The levels of these activities were all markedly lower than when the tonB ⁺ gene was present in single copy. This depression of TonB function occurred even when the chromosome carried the normal tonB ⁺ allele, but plasmids carrying only a portion of the tonB gene, including the 5'-regulatory region, were not inhibitory.     

Outer membrane-dependent transport systems in Escherichia coli: turnover of TonB function.

Recent reports demonstrated that the energy-dependent step of vitamin B12 uptake into cells of Escherichia coli rapidly declines after cessation either of the expression of the tonB gene or of general protein synthesis. It is shown here that inhibition of protein synthesis results in the decline, with similar kinetics, of all tonB-dependent processes, including sensitivity to colicins B and Ia, irreversible adsorption of phage phi80, and siderophore-mediated iron uptake. The role of ongoing TonB-dependent reactions on this lability of TonB function was investigated. Ferrichrome and the enterochelin precursor, 2,3-dihydroxybenzoate, caused both a moderate depression of B12 uptake activity in growing cells (reversed upon removal of the siderophore) and an acceleration of the loss of activity following inhibition of protein synthesis by addition of spectinomycin. Strains lacking the tonB-dependent siderophore uptake systems did not show these responses. The results suggest the consumption of tonB product during its action.     

ExbBD-dependent transport of maltodextrins through the novel MalA protein across the outer membrane of Caulobacter crescentus

Analysis of the genome sequence of Caulobacter crescentus predicts 67 TonB-dependent outer membrane proteins. To demonstrate that among them are proteins that transport nutrients other than chelated Fe(3+) and vitamin B(12)-the substrates hitherto known to be transported by TonB-dependent transporters-the outer membrane protein profile of cells grown on different substrates was determined by two-dimensional electrophoresis. Maltose induced the synthesis of a hitherto unknown 99.5-kDa protein, designated here as MalA, encoded by the cc2287 genomic locus. MalA mediated growth on maltodextrins and transported [(14)C]maltodextrins from [(14)C]maltose to [(14)C]maltopentaose. [(14)C]maltose transport showed biphasic kinetics, with a fast initial rate and a slower second rate. The initial transport had a K(d) of 0.2 microM, while the second transport had a K(d) of 5 microM. It is proposed that the fast rate reflects binding to MalA and the second rate reflects transport into the cells. Energy depletion of cells by 100 microM carbonyl cyanide 3-chlorophenylhydrazone abolished maltose binding and transport. Deletion of the malA gene diminished maltose transport to 1% of the wild-type malA strain and impaired transport of the larger maltodextrins. The malA mutant was unable to grow on maltodextrins larger than maltotetraose. Deletion of two C. crescentus genes homologous to the exbB exbD genes of Escherichia coli abolished [(14)C]maltodextrin binding and transport and growth on maltodextrins larger than maltotetraose. These mutants also showed impaired growth on Fe(3+)-rhodotorulate as the sole iron source, which provided evidence of energy-coupled transport. Unexpectedly, a deletion mutant of a tonB homolog transported maltose at the wild-type rate and grew on all maltodextrins tested. Since Fe(3+)-rhodotorulate served as an iron source for the tonB mutant, an additional gene encoding a protein with a TonB function is postulated. Permeation of maltose and maltotriose through the outer membrane of the C. crescentus malA mutant was slower than permeation through the outer membrane of an E. coli lamB mutant, which suggests a low porin activity in C. crescentus. The pores of the C. crescentus porins are slightly larger than those of E. coli K-12, since maltotetraose supported growth of the C. crescentus malA mutant but failed to support growth of the E. coli lamB mutant. The data are consistent with the proposal that binding of maltodextrins to MalA requires energy and MalA actively transports maltodextrins with K(d) values 1,000-fold smaller than those for the LamB porin and 100-fold larger than those for the vitamin B(12) and ferric siderophore outer membrane transporters. MalA is the first example of an outer membrane protein for which an ExbB/ExbD-dependent transport of a nutrient other than iron and vitamin B(12) has been demonstrated.     

Altered cobalamin metabolism in Escherichia coli btuR mutants affects btuB gene regulation.

Synthesis of the Escherichia coli outer membrane protein BtuB, which mediates the binding and transport of vitamin B12, is repressed when cells are grown in the presence of vitamin B12. Expression of btuB-lacZ fusions was also found to be repressed, and selection for constitutive production of beta-galactosidase in the presence of vitamin B12 yielded mutations at btuR. The btuR locus, at 27.9 min on the chromosome map, was isolated on a 952-base-pair EcoRV fragment, and its nucleotide sequence was determined. The BtuR protein was identified in maxicells as a 22,000-dalton polypeptide, as predicted from the nucleotide sequence. Strains mutant at btuR had negligible pools of adenosylcobalamin but did convert vitamin B12 into other derivatives. Although btuB expression in a btuR strain could not be repressed by cyano- or methylcobalamin, it was repressed by adenosylcobalamin. Growth on ethanolamine as the sole nitrogen source requires adenosylcobalamin. btuR mutants grew on ethanolamine but were affected in the length of the lag period before initiation of growth, which suggested that an alternative route for adenosylcobalamin synthesis might exist. No mutations were found that conferred constitutive btuB expression in the presence of adenosylcobalamin. Other genes near btuR may also be involved in cobalamin metabolism, as suggested from the complementation behavior of strains generated by excision of the Tn10 element in btuR. These results indicated that the btuR product is involved in the metabolism of adenosylcobalamin and that this cofactor, or some derivative, controls btuB expression.     

Energy-coupled colicin transport through the outer membrane of Escherichia coli K-12: mutated TonB proteins alter receptor activities and colicin uptake

Abstract The current model of TonB-dependent colicin transport through the outer membrane of Escherichia coli proposes initial binding to receptor proteins, vectorial release from the receptors and uptake into the periplasm from where the colicins, according to their action, insert into the cytoplasmic membrane or enter the cytoplasm. The uptake is energy-dependent and the TonB protein interacts with the receptors as well as with the colicins. In this paper we have studied the uptake of colicins B and Ia, both pore-forming colicins, into various tonB point mutants. Colicin Ia resistance of the tonB mutant (G186D, R204H) was consistent with a defective Cir receptor-TonB interaction while colicin Ia resistance of E. coli expressing TonB of Serratia marcescens , or TonB of E. coli carrying a C-terminal fragment of the S. marcescens TonB, seemed to be caused by an impaired colicin Ia-TonB interaction. In contrast, E. coli tonB (G174R, V178I) was sensitive to colicin Ia and resistant to colicin B unless TonB, ExbB and ExbD were overproduced which resulted in colicin B sensitivity. The differential effects of tonB mutations indicate differences in the interaction of TonB with receptors and colicins.     

Cloning and expression of the gene for the vitamin B12 receptor protein in the outer membrane of Escherichia coli.

The transport of cyanocobalamin (vitamin B12) in cells of Escherichia coli is dependent on a receptor protein (BtuB protein) located in the outer membrane. A 9.1-kilobase pair BamHI fragment carrying the btuB gene was cloned from a specialized transducing phage into multicopy plasmids. Insertions of transposon Tn1000 which prevented production of the receptor localized btuB to a 2-kilobase pair region. Further subcloning allowed isolation of this region as a 2.3-kilobase pair Sau3A fragment. The BtuB+ plasmids were shown by maxicell analysis to encode a polypeptide with a molecular weight of 66,000 in the outer membrane. This polypeptide was missing in cells with Tn1000 insertions in btuB and was reduced in amount upon growth of plasmid-bearing cells in repressing concentrations of vitamin B12. Several Tn1000 insertions outside the 5' end of the coding region exhibited reduced production of receptor. A deletion at the 3' end of btuB resulted in formation of an altered receptor. Amplified production of this polypeptide was associated with increased levels of binding of the receptor's ligands (vitamin B12 and phage BF23), increased rates of vitamin B12 uptake, and altered susceptibility to the group E colicins. Deficiency in various major outer membrane proteins did not affect production of the btuB product, and the amplified levels of this protein partially reversed the tolerance to E colicins seen in these mutants.