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.     

Characterization of the exbBD operon of Escherichia coli and the role of ExbB and ExbD in TonB function and stability.

TonB protein appears to couple the electrochemical potential of the cytoplasmic membrane to active transport across the essentially unenergized outer membrane of gram-negative bacteria. ExbB protein has been identified as an auxiliary protein in this process. In this paper we show that ExbD protein, encoded by an adjacent gene in the exb cluster at 65', was also required for TonB-dependent energy transduction and, like ExbB, was required for the stability of TonB. The phenotypes of exbB exbD+ strains were essentially indistinguishable from the phenotypes of exbB+ exbD strains. Mutations in either gene resulted in the degradation of TonB protein and in decreased, but not entirely absent, sensitivities to colicins B and Ia and to bacteriophage phi 80. Evidence that the absence of ExbB or ExbD differentially affected the half-lives of newly synthesized and steady-state TonB was obtained. In the absence of ExbB or ExbD, newly synthesized TonB was degraded with a half-life of 5 to 10 min, while the half-life of TonB under steady-state conditions was significantly longer, approximately 30 min. These results were consistent with the idea that ExbB and ExbD play roles in the assembly of TonB into an energy-transducing complex. While interaction between TonB and ExbD was suggested by the effect of ExbD on TonB stability, interaction of ExbD with TonB was detected by neither in vivo cross-linking assays nor genetic tests for competition. Assays of a chromosomally encoded exbD::phoA fusion showed that exbB and exbD were transcribed as an operon, such that ExbD-PhoA levels in an exbB::Tn10 strain were reduced to 4% of the levels observed in an exbB+ strain under iron-limiting conditions. Residual ExbD-PhoA expression in an exbB::Tn10 strain was not iron regulated and may have originated from within the Tn10 element in exbB.     

Microcin-E492-insensitive mutants of Escherichia coli K12

Mutations in three Escherichia coli K12 genes, tonB, exbB and the newly discovered semA, reduce sensitivity to the low Mr polypeptide antibiotic microcin E492. The products of the tonB and exbB genes were previously shown to be involved in the uptake of siderophore-complexed iron and in the action of a number of colicins. Strains mutated at or close to semA (collectively referred to as sem mutations) remained fully sensitive to these colicins, and grew as well as wild-type strains under conditions of iron starvation. Expression of a number of sem-lacZ operon fusions was not affected by iron limitation, and sem mutations did not affect the production of iron-regulated outer membrane proteins which are known or thought to be involved in iron uptake. Hfr conjugation and P1 phage transduction experiments indicated that semA is located close to pabB at 40 min on the E. coli K12 chromosome. This places semA close to the mng locus, wherein mutations result in decreased manganese sensitivity. However, strains carrying the semA mutation exhibited increased manganese sensitivity.     

Vibrio cholerae iron transport: haem transport genes are linked to one of two sets of tonB, exbB, exbD genes

Vibrio cholerae was found to have two sets of genes encoding TonB, ExbB and ExbD proteins. The first set ( tonB1, exbB1, exbD1 ) was obtained by complementation of a V. cholerae tonB mutant. In the mutant, a plasmid containing these genes permitted transport via the known V. cholerae high-affinity iron transport systems, including uptake of haem, vibriobactin and ferrichrome. When chromosomal mutations in exbB1 or exbD1 were introduced into a wild-type V. cholerae background, no defect in iron transport was noted, indicating the existence of additional genes that can complement the defect in the wild-type background. Another region of the V. cholerae chromosome was cloned that encoded a second functional TonB/Exb system ( tonB2, exbB2, exbD2 ). A chromosomal mutation in exbB2 also failed to exhibit a defect in iron transport, but a V. cholerae strain that had chromosomal mutations in both the exbB1 and exbB2 genes displayed a mutant phenotype similar to that of an Escherichia coli tonB mutant. The genes encoding TonB1, ExbB1, ExbD1 were part of an operon that included three haem transport genes ( hutBCD ), and all six genes appeared to be expressed from a single Fur-regulated promoter upstream of tonB1 . A plasmid containing all six genes permitted utilization of haem by an E. coli strain expressing the V. cholerae haem receptor, HutA. Analysis of the hut genes indicated that hutBCD, which are predicted to encode a periplasmic binding protein (HutB) and cytoplasmic membrane permease (HutC and HutD), were required to reconstitute the V. cholerae haem transport system in E. coli. In V. cholerae , the presence of hutBCD stimulated growth when haemin was the iron source, but these genes were not essential for haemin utilization in V. cholerae .     

Evolutionary relationship of uptake systems for biopolymers in Escherichia coli: cross-complementation between the TonB-ExbB-ExbD and the TolA-TolQ-TolR proteins

Escherichia coli possesses two energy-coupled import systems through which substances of low concentration and of a size too large to permit diffusion through the porins are translocated across the outer membrane. Group B colicins, ferric siderophores and vitamin B₁₂ are taken up via the TonB-ExbB-ExbD, group A colicins via the TolA-TolQ-TolR system. Cross-complementation between the two systems was demonstrated in that tolQ tolR mutants transformed with plasmids carrying exbB exbD became sensitive to group A colicins, and exbB exbD mutants transformed with plasmid-encoded tolQ tolR became sensitive to group B colicins. TolQ-TolR interacted through TonB, and ExbB-ExbD interacted through TolA with the outer membrane receptors and colicins. Activity of ExbB ExbD via TolA was higher in cells laciting TonB, and activity of TolQ TolR via TonB was increased when TolA was missing. The very distinct TolA and TonB proteins mediate exclusive interaction with group A and group B receptors, respectively. ExbB-TolR and ExbD-TolQ mixtures showed little if any complementation of exbB exbD and tolQ tolR mutants indicating coevolution of ExbB with ExbD and TolQ with ToIR. Sequence homology and mutual functional substitution of ExbB-ExbD and TolQ-TolR suggest the evolution of the two import systems from a single import system.     

Unusual structure of the tonB-exb DNA region of Xanthomonas campestris pv. campestris: tonB, exbB, and exbD1 are essential for ferric iron uptake, but exbD2 is not.

The nucleotide sequence of a 3.6-kb HindIII-SmaI DNA fragment of Xanthomonas campestris pv. campestris revealed four open reading frames which, based on sequence homologies, were designated tonB, exbB, exbD1, and exbD2. Analysis of translational fusions to alkaline phosphatase and beta-galactosidase confirmed that the TonB, ExbB, ExbD1, and ExbD2 proteins are anchored in the cytoplasmic membrane. The TonB protein of X. campestris pv. campestris lacks the conserved (Glu-Pro)n and (Lys-Pro)m repeats but harbors a 13-fold repeat of proline residues. By mutational analysis, the tonB, exbB, and exbD1 genes were shown to be essential for ferric iron import in X. campestris pv. campestris. In contrast, the exbD2 gene is not involved in the uptake of ferric iron.     

The structurally related exbB and tolQ genes are interchangeable in conferring tonB-dependent colicin, bacteriophage, and albomycin sensitivity.

Double exbB tolQ mutants of Escherichia coli were completely resistant to bacteriophages T1 and phi 80, in contrast to strains with exbB or tolQ mutations, which were sensitive. Cells carrying mutations in exbB were partially tolerant to colicins B, D, and M and became fully tolerant by the introduction of tolQ mutations. This suggested involvement of both exbB and tolQ in tonB-dependent uptake.     

Characterization of group B colicin-resistant mutants of Escherichia coli K-12: colicin resistance and the role of enterochelin.

Nine classes of group B colicin-resistant mutants were examined to study the role of enterochelin in colicin resistance. Four of the mutants studied (cbt, exbC, exbB, and tonB) hypersecreted enterochelin. Enterochelin hypersecretion was apparently responsible for resistance of the exbC mutant to colicins G and H and for resistance of the exbB mutant to colicins G, H, Ia, Ib, S1, and V. All four mutants scored as colicin B tolerant, even in the absence of enterochelin synthesis. The mutants produced substantially increased amounts of two high-molecular-weight outer membrane polypeptides when grown under limiting iron conditions. The presence of these polypeptides was correlated with increased colicin B-neutralizing activity in the outer membrane preparations.     

Domains of colicin M involved in uptake and activity

Colicin M inhibits murein biosynthesis by interfering with bactoprenyl phosphate carrier regeneration. It belongs to the group B colicins the uptake of which through the outer membrane depends on the Tong, ExbB and ExbD proteins. These colicins contain a sequence, called the Tong box, which has been implicated in transport via Tong. Point mutations were introduced by PCR into the TonB box of the structural gene for colicin M, cma, resulting in derivatives that no longer killed cells. Mutations in the tonB gene suppressed, in an allele-specific manner, some of the cma mutations, suggesting that interaction of colicin M with Tong may be required for colicin M uptake. Among the hydroxylamine-generated colicin M-inactive cma mutants was one which carried cysteine in place of arginine at position 115. This Colicin derivative still bound to the FhuA receptor and killed cells when translocated across the outer membrane by osmotic shock treatment. It apparently represents a new type of transport-deficient colicin M. Additional hydroxylamine-generated inactive derivatives of colicin M carried mutations centered on residues 193–197 and 223–252. Since these did not kill osmotically shocked cells the mutations must be located in a region which is important for colicin M activity. It is concluded that the Tong box at the N-terminal end of colicin M must be involved in colicin uptake via Tong across the outer membrane and that the C-terminal portion of the molecule is likely to contain the activity domain.     

Neisseria meningitidis tonB, exbB, and exbD genes: Ton-dependent utilization of protein-bound iron in Neisseriae.

We have recently cloned and characterized the hemoglobin (Hb) receptor gene, hmbR, from Neisseria meningitidis. To identify additional proteins that are involved in Hb utilization, the N. meningitidis Hb utilization system was reconstituted in Escherichia coli. Five cosmids from N. meningitidis DNA library enabled a heme-requiring (hemA), HmbR-expressing mutant of E. coli to use Hb as both porphyrin and iron source. Nucleotide sequence analysis of DNA fragments subcloned from the Hb-complementing cosmids identified four open reading frames, three of them homologous to Pseudomonas putida, E. coli, and Haemophilus influenzae exbB, exbD, and tonB genes. The N. meningitidis TonB protein is 28.8 to 33.6% identical to other gram-negative TonB proteins, while the N. meningitidis ExbD protein shares between 23.3 and 34.3% identical amino acids with other ExbD and TolR proteins. The N. meningitidis ExbB protein was 24.7 to 36.1% homologous with other gram-negative ExbB and TolQ proteins. Complementation studies indicated that the neisserial Ton system cannot interact with the E. coli FhuA TonB-dependent outer membrane receptor. The N. meningitidis tonB mutant was unable to use Hb, Hb-haptoglobin complexes, transferrin, and lactoferrin as iron sources. Insertion of an antibiotic cassette in the 3' end of the exbD gene produced a leaky phenotype. Efficient usage of heme by N. meningitidis tonB and exbD mutants suggests the existence of a Ton-independent heme utilization mechanism. E. coli complementation studies and the analysis of N. meningitidis hmbR and hpu mutants suggested the existence of another Hb utilization mechanism in this organism.     

Protein import into Escherichia coli: colicins A and E1 interact with a component of their translocation system.

Colicins are antibiotic proteins that kill sensitive Escherichia coli cells. Their mode of action involves three steps: binding to specific receptors located in the outer membrane, translocation across this membrane, and action on their targets. A specific colicin domain can be assigned to each of these steps. Colicins have been subdivided into two groups (A and B) depending on the proteins required for them to cross the external membrane. Plasmids were constructed which led to an overproduction of the Tol proteins involved in the import of group A colicins. In vitro binding of overexpressed Tol proteins to either Tol-dependent (group A) or TonB-dependent (group B) colicins was analyzed. The Tol dependent colicins A and E1 were able to interact with TolA but the TonB dependent colicin B was not. The C-terminal region of TolA, which is necessary for colicin uptake, was also found to be necessary for colicin A and E1 binding to occur. Furthermore, only the isolated N-terminal domain of colicin A, which is involved in the translocation step, was found to bind to TolA. These results demonstrate the existence of a correlation between the ability of group A colicins to translocate and their in vitro binding to TolA protein, suggesting that these interactions might be part of the colicin import process.     

Mutational analyses define helix organization and key residues of a bacterial membrane energy-transducing complex

In Gram-negative bacteria, many biological processes are coupled to inner membrane ion gradients. Ions transit at the interface of helices of integral membrane proteins, generating mechanical energy to drive energetic processes. To better understand how ions transit through these channels, we used a model system involved in two different processes, one of which depends on inner membrane energy. The Tol machinery of the Escherichia coli cell envelope is dedicated to maintaining outer membrane stability, a process driven by the proton-motive force. The Tol system is parasitized by bacterial toxins called colicins, which are imported through the outer membrane using an energy-independent process. Herein, we mutated TolQ and TolR transmembrane residues, and we analyzed the mutants for outer membrane stability, colicin import and protein complex formation. We identified residues involved in the assembly of the complex, and a new class of discriminative mutations that conferred outer membrane destabilization identical to a tol deletion mutant, but which remained fully sensitive to colicins. Further genetic approaches revealed transmembrane helix interactions and organization in the bilayer, and suggested that most of the discriminative residues are located in a putative aqueous ion channel. We discuss a model for the function of related bacterial molecular motors.     

The Tol proteins of Escherichia coli and their involvement in the translocation of group A colicins

The Tol proteins are involved in outer membrane stability of Gram-negative bacteria. The TolQRA proteins form a complex in the inner membrane while TolB and Pal interact near the outer membrane. These two complexes are transiently connected by an energy-dependent interaction between Pal and TolA. The Tol proteins have been parasitized by group A colicins for their translocation through the cell envelope. Recent advances in the structure and energetics of the Tol system, as well as the interactions between the N-terminal translocation domain of colicins and the Tol proteins are presented.     

Energy-coupled transport across the outer membrane of Escherichia coli: ExbB binds ExbD and TonB in vitro, and leucine 132 in the periplasmic region and aspartate 25 in the transmembrane region are important for ExbD activity.

Ferric siderophores, vitamin B12, and group B colicins are taken up through the outer membranes of Escherichia coli cells by an energy-coupled process. Energy from the cytoplasmic membrane is transferred to the outer membrane with the aid of the Ton system, consisting of the proteins TonB, ExbB, and ExbD. In this paper we describe two point mutations which inactivate ExbD. One mutation close to the N-terminal end of ExbD is located in the cytoplasmic membrane, and the other mutation close to the C-terminal end is located in the periplasm. E. coli CHO3, carrying a chromosomal exbD mutation in which leucine at position 132 was replaced by glutamine, was devoid of all Ton-related activities. A plasmid-encoded ExbD derivative, in which aspartate at position 25, the only changed amino acid in the predicted membrane-spanning region of ExbD, was replaced by asparagine, failed to restore the Ton activities of strain CHO3 and negatively complemented ExbD+ strains, indicating an interaction of this mutated ExbD with wild-type ExbD or with another component. This component was shown to be ExbB. ExbB that was labeled with 6 histidine residues at its C-terminal end and that bound to a nickel-nitrilotriacetic acid agarose column retained ExbD and TonB specifically; both were eluted with the ExbB labeled with 6 histidine residues, demonstrating interaction of ExbB with ExbD and TonB. These data further support the concept that TonB, ExbB, and ExbD form a complex in which the energized conformation of TonB opens the channels in the outer membrane receptor proteins.     

Quantification of group A colicin import sites.

Pore-forming colicins are soluble bacteriocins which form voltage-gated ion channels in the inner membrane of Escherichia coli. To reach their target, these colicins first bind to a receptor located on the outer membrane and then are translocated through the envelope. Colicins are subdivided into two groups according to the envelope proteins involved in their translocation: group A colicins use the Tol proteins; group B colicins use the proteins TonB, ExbB, and ExbD. We have previously shown that a double-cysteine colicin A mutant which possesses a disulfide bond in its pore-forming domain is translocated through the envelope but is unable to form a channel in the inner membrane (D. Duché, D. Baty, M. Chartier, and L. Letellier, J. Biol. Chem. 269:24820-24825, 1994). Measurements of colicin-induced K+ efflux reveal that preincubation of the cells with the double-cysteine mutant prevents binding of colicins of group A but not of group B. Moreover, we show that the mutant is still in contact with its receptor and import machinery when it interacts with the inner membrane. From these competition experiments, we conclude that each Escherichia coli cell contains approximately 400 and 1,000 colicin A receptors and translocation sites, respectively.     

Increased production of the outer membrane receptors for colicins B, D and M by Escherichia coli under iron starvation.

Growth of $\text{E. coli}$ K-12 under severe iron stress results in increased production of the outer membrane receptors for colicins B, D, Ib and M. The increase in colicin receptor activity coincides with the appearance of large amounts of two high molecular weight proteins in the outer membrane of the cells. These proteins are identified as the outer membrane receptors for colicins B and D and for colicin M. Mutants lacking a functional outer membrane receptor for colicins B and D are defective in the uptake of iron complexed with the siderochrome enterochelin, and are thus comparable with $\text{tonA}$ mutants which lack a functional receptor for colicin M and are defective in the uptake of iron complexed with ferrichrome (6). The colicin B and D receptor may therefore function in the uptake of ferri-enterochelin.     

A novel protein, TtpC, is a required component of the TonB2 complex for specific iron transport in the pathogens Vibrio anguillarum and Vibrio cholerae

Active transport across the outer membrane in gram-negative bacteria requires the energy that is generated by the proton motive force in the inner membrane. This energy is transduced to the outer membrane by the TonB protein in complex with the proteins ExbB and ExbD. In the pathogen Vibrio anguillarum we have identified two TonB systems, TonB1 and TonB2, the latter is used for ferric-anguibactin transport and is transcribed as part of an operon that consists of orf2, exbB2, exbD2, and tonB2. This cluster was identified by a polar transposon insertion in orf2 that resulted in a strain deficient for ferric-anguibactin transport. Only the entire cluster (orf2, exbB2, exbD2 and tonB2) could complement for ferric-anguibactin transport, while just the exbB2, exbD2, and tonB2 genes were unable to restore transport. This suggests an essential role for this Orf2, designated TtpC, in TonB2-mediated transport in V. anguillarum. A similar gene cluster exists in V. cholerae, i.e., with the homologues of ttpC-exbB2-exbD2-tonB2, and we demonstrate that TtpC from V. cholerae also plays a role in the TonB2-mediated transport of enterobactin in this human pathogen. Furthermore, we also show that in V. anguillarum the TtpC protein is found as part of a complex that might also contain the TonB2, ExbB2, and ExbD2 proteins. This novel component of the TonB2 system found in V. anguillarum and V. cholerae is perhaps a general feature in bacteria harboring the Vibrio-like TonB2 system.