Sequence Changes in the Ton Box Region of BtuB Affect Its

JOURNAL OF BACTERIOLOGY,0021-9193/00/$04.0010

Nov. 2000, p. 5954–5961 Vol. 182, No. 21

Copyright © 2000, American Society for Microbiology. All Rights Reserved.

Sequence Changes in the Ton Box Region of BtuB Affect ItsTransport Activities and Interaction with TonB Protein

NATHALIE CADIEUX,1 CLIVE BRADBEER,1,2 AND ROBERT J. KADNER1*

Department of Microbiology1 and Department of Biochemistry and Molecular Genetics,2

University of Virginia School of Medicine, Charlottesville, Virginia 22908-0734

Received 17 April 2000/Accepted 2 August 2000

Uptake of cobalamins by the transporter protein BtuB in the outer membrane of Escherichia coli requires theproton motive force and the transperiplasmic protein TonB. The Ton box sequence near the amino terminusof BtuB is conserved among all TonB-dependent transporters and is the only known site of mutations thatconfer a transport-defective phenotype which can be suppressed by certain substitutions at residue 160 inTonB. The crystallographic structures of the TonB-dependent transporter FhuA revealed that the region nearthe Ton box, which itself was not resolved, is exposed to the periplasmic space and undergoes an extensive shiftin position upon binding of substrate. Site-directed disulfide bonding in intact cells has been used to show thatthe Ton box of BtuB and residues around position 160 of TonB approach each other in a highly oriented andspecific manner to form BtuB-TonB heterodimers that are stimulated by the presence of transport substrate.Here, replacement of Ton box residues with proline or cysteine revealed that residue side chain recognition isnot important for function, although replacement with proline at four of the seven Ton box positions impairedcobalamin transport. The defect in cobalamin utilization resulting from the L8P substitution was suppressedby cysteine substitutions in adjacent residues in BtuB or in TonB. This suppression did not restore activetransport of cobalamins but may allow each transporter to function at most once. The uncoupled prolinesubstitutions in BtuB markedly affected the pattern of disulfide bonding to TonB, both increasing the extentof cross-linking and shifting the pairs of residues that can be joined. Cross-linking of BtuB and TonB in thepresence of the BtuB V10P substitution became independent of the presence of substrate, indicating anadditional distortion of the exposure of the Ton box in the periplasmic space. TonB action thus requires aspecific orientation for functional contact with the Ton box, and changes in the conformation of this regionblock transport by preventing substrate release and repeated transport cycles.

TonB function is crucial for the operation of energy-depen-dent transport systems across the outer membrane (OM) ofgram-negative bacteria (reviewed in references 19, 30, and 32).These transport systems carry out the high-affinity uptake ofcorrinoids, such as vitamin B12 (cyano-cobalamin [CN-Cbl]),and of iron complexes with siderophores or host iron-bindingproteins. These nutrients are too large or scarce in naturalenvironments to diffuse effectively through the porin chan-nels, and they require specialized uptake mechanisms com-prising a specific, high-affinity OM transporter and an ATP-dependent periplasmic permease system for transport acrossthe cytoplasmic membrane. Transport across the OM dependson energy coupling through the action of the TonB protein (15,34). TonB spans the periplasmic space to interact with itsaccessory proteins ExbB and ExbD in the cytoplasmic mem-brane and with the OM transporters (23, 35).

The crystallographic structures of the Escherichia coli ferrichydroxamate OM transporter FhuA (12, 25) and the ferricenterobactin OM transporter FepA (7) were very similar. TheC-terminal region of each forms a 22-stranded transmembraneb-barrel with short periplasmic turns and large extracellularloops which partially occlude the top of the channel. TheN-terminal 153 residues of FepA or 160 residues of FhuA forma novel conformation, termed a plug, cork, or hatch, which isheld in the barrel by an extensive series of hydrogen bonds andsalt bridges and which occludes the central channel to prevent

ion fluxes (4, 26). In the FhuA crystal structure, the substrateferrichrome was found to bind to residues on the upper face ofthe central domain and on two external loops of the barrel(25). The binding of ferrichrome to FhuA had little effect onthe conformation of residues around its binding pocket butresulted in a substantial conformational change in the N-ter-minal segment of the protein exposed to the periplasmic open-ing of the barrel.

Part of the N-terminal segment has been implicated in TonBfunction. Called the Ton box, it is one of the conserved motifsthat define the family of TonB-dependent transporters (19).The Ton box in the cobalamin transporter BtuB has the se-quence DTLVVTA between residues 6 and 12. Some muta-tions affecting this region in numerous TonB-dependent trans-porters result in a unique phenotype, characterized by loss ofTonB-dependent functions but without deficit in protein sta-bility, membrane insertion, substrate binding, or TonB-inde-pendent transport activities (3, 10, 16, 22, 33, 37, 38). ThisTonB-uncoupled phenotype can be partially suppressed byamino acid changes elsewhere in the Ton box or at Gln-160 inTonB (3, 13, 17, 37, 38). These results suggested that the Tonbox regions might be sites of interaction with TonB, but someinvestigators claimed that more-direct evidence was needed toshow their contact (20).

Formaldehyde-dependent covalent cross-linking of TonB toFepA and FhuA allowed Postle and colleagues (22, 31, 39) todemonstrate that contact of TonB to both OM proteins oc-curred and was affected by the presence of their transportsubstrate and by the integrity of the Ton box. The sites ofprotein contact could not be deduced owing to the nonspecificaction of this cross-linking agent. Subsequently, we (8) used

* Corresponding author. Mailing address: Department of Microbi-ology, University of Virginia Health System, P.O. Box 800734, Char-lottesville, VA 22908-0734. Phone: (804) 924-2532. Fax: (804) 982-1071. E-mail: [email protected].

5954

on February 19, 2018 by guest

http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

site-directed disulfide bonding to show that disulfide bondsbetween introduced cysteine residues in the Ton box of BtuBand in the vicinity of residue 160 of TonB could be formed.Disulfide-bonded BtuB-TonB heterodimers were formed inintact cells only between Cys residues at specific positions inboth proteins. Thus, TonB with the Q16OC substitution (TonBQ160C) formed cross-links mainly to BtuB L8C and V10C,whereas TonB Q162C reacted with BtuB L8C and A12C andweakly with BtuB Vl0C and T11C, and TonB Y163C reactedonly with BtuB A12C. This high degree of selectivity for cross-linking indicated that this portion of TonB approaches the Tonbox of BtuB in a specific and oriented manner. For most pairs,cross-linking was stimulated by the presence of the transportsubstrate CN-Cbl.

Here we analyzed BtuB variants in which Cys and Pro sub-stitutions were systematically scanned through the Ton box andshow that a Pro substitution at only a few sites confers theTonB-uncoupled phenotype. The L8P and Vl0P substitutions,which strongly interfered with CN-Cbl transport, markedly af-fected the pattern of disulfide binding between Cys residues inTonB and in the Ton box. The altered reactivities of thesePro-Cys double mutants with the TonB Cys variants showedthat the uncoupling mutations did not prevent BtuB-TonBcontact but caused a dramatic change in the positional speci-ficity of their interaction. Finally, the process of suppressionwas found not to restore normal transport function but didallow BtuB to transport substrate in a very limited and energy-uncoupled manner.

MATERIALS AND METHODS

Bacterial strains and plasmids. E. coli strain JM109 (Stratagene) was used asthe host for plasmid construction and maintenance. Phenotypic and cross-linkinganalyses of BtuB and TonB variants used the following derivatives of strainMC4100 [D(argF-lac)U169 araD139 rpsL150 relA1 flbB5301 deoC1 ptsF25 rbsR22non-9 gyrA219] (9). Strain RK8452 is metE70 DbtuB::Km; RK5016 is metE70argH btuB recA; RK5043 is metE70 tonB recA; NC11 is metE70 tonB::Km polA;NC12 is metE70 btuB::Tn10 tonB::Km. The tonB::Km allele was introduced by P1transduction from strain KP1032, which was obtained from K. Postle. Transfer ofmutations to the chromosome of E. coli used strains DHB6521, containing thellnCh1 vector (cl857 Sam7), compatible with pBR vectors (5), DHB6501, thepermissive strain for propagation of the phage, and EMG2 (F9l1), a straincontaining the pKO3 vector for allelic replacement (24).

Isolation and manipulations of DNA followed standard protocols (36). Plas-mid pAG1 carrying the btuB gene in pBR322 was previously described (13).Plasmid pNC1 carries the E. coli tonB and exbBD coding sequences under thecontrol of their native promoters and was constructed by PCR amplification ofboth loci from the chromosome of strain JM109. Each PCR primer introducedunique restriction sites at each end of the fragment; primer sequences areavailable upon request. The resulting amplimers were ligated into the corre-sponding sites of the polylinker region site of plasmid pSU19 (27), tonB as a988-bp PstI-SalI fragment, and exbBD as a 1,321-bp AvaI-SacI fragment. Plas-mids containing separate tonB (pNC2) or exbBD (pNC3) inserts were construct-ed similarly.

Plasmid DNA was introduced into host strains by CaCl2-mediated transfor-mation. Plasmid-bearing cells were maintained in the presence of the appropri-ate antibiotics at the following concentrations: ampicillin, 100 mg/ml; chloram-phenicol, 40 mg/ml; kanamycin, 50 mg/ml. Growth media were Luria-Bertani(LB) broth or minimal A salts medium (29).

Site-directed mutagenesis. A two-step PCR method (18) was used to replacethe single cysteine residue present in wild-type TonB encoded by plasmid pNC1with an alanine residue to yield TonB C18A, encoded in plasmid pNC4 (8). Thesame method was used to create single and double cysteine and proline substi-tutions in the TonB box region of BtuB (residues 6 to 12) encoded by plasmidpAG1. Plasmid pNC4 was modified by PCR mutagenesis to introduce singlecysteine residues at positions 159 to 164 of TonB. All mutational changes wereverified by nucleotide sequencing at the University of Virginia BiomolecularResource Facility.

Genetic techniques. The btuB variants encoding mutants with single cysteineor proline mutations were introduced into the chromosomal btuB locus of strainRK8452 by allelic replacement using the pKO3 system (24). For this purpose, thewild-type btuB gene was first cloned into plasmid pKO3 and the variant alleleswere introduced by replacing a 295-bp HindIII-BsiWI fragment with the corre-sponding mutated coding sequence. The chromosomal btuB::Km gene of RK8452was replaced by the mutant alleles in two steps. The conditionally replication-

deficient plasmids were introduced by transformation, and Campbell-typeintegrants at the btuB locus were obtained by selection for chloramphenicolresistance. The second homologous recombination event to remove plasmidsequences and the btuB::Km allele was obtained by selection for resistance to 5%sucrose, and recombinants sensitive to chloramphenicol and kanamycin werechosen (24). Recombinant clones were verified by PCR with primers that amplifythe entire btuB locus. The amplified fragments were digested with appropriaterestriction enzymes to verify that the kanamycin resistance cassette of strainRK8452 had been replaced by the btuB alleles. The btuB Cys/Pro double muta-tions were introduced into the attl site of strain RK8452 using the llnCh system(5), in which the btuB::Km gene of strain RK8452 is not replaced. Recombinantclones were verified by PCR analysis. The tonB alleles were integrated into thechromosome following transformation into polA tonB strain NC11.

Phenotypic assays. (i) Growth on CN-Cbl. Strains harboring btuB and/or tonBmutations on plasmids or on the chromosome were tested for growth on minimalA salts-agar plates supplemented with 0.02% glucose, 0.01% arginine, and var-ious concentrations of CN-Cbl (0.1 to 5,000 nM) in place of methionine (2).Results are expressed as the lowest CN-Cbl concentration allowing maximalcolony size after a 48-h incubation at 37°C, relative to colony size after growth onmethionine-supplemented medium.

(ii) CN-Cbl uptake assay. CN-[57Co]Cbl (4 mCi/nmol) was prepared by grow-ing cells of Propionibacterium freudenreichii (ATCC 9614) on medium containing1 mCi of 57CoCl2. After growth of cells for 3 days, labeled cobalamins wereextracted with 80% ethanol at 80°C, converted to the CN form, and purified bypaper electrophoresis.

E. coli cells were grown in the minimal medium as described above, harvestedin mid-exponential growth phase, washed, and suspended in 100 mM potassiumphosphate, pH 6.6 (6). Cells were incubated with CN-[57Co]Cbl. Samples (1 ml)were removed at intervals, collected on Millipore filters (0.45 mm, pore size),washed twice with 10 ml of 100 mM LiCl, and dried. Radioactivity in the sampleswas determined by liquid scintillation counting. Results are expressed as pico-moles of CN-Cbl taken up per 109 cells.

(iii) Susceptibility to E colicins and BF23 phase. Onto soft-agar overlays ofeach strain on LB agar plates were spotted 5-ml samples of serial 10-fold dilu-tions of BF23 phage or of culture supernatants containing colicins E1 or E3induced with mitomycin C. After overnight incubation at 37°C, zones of bacterialkilling were seen where the agents had been spotted, and susceptibility is re-ported as the negative logarithm of the highest dilution that gave a zone ofcomplete clearing.

Disulfide cross-linking analysis. Cells of btuB strain RK5016 carrying pairs ofcompatible plasmids expressing BtuB and TonB cysteine variants were tested forformation of disulfide-linked species, as previously described (8). Cells weregrown in minimal A salts medium supplemented with 0.02% glucose, 0.01%methionine and arginine, 10 mM MgSO4, 1 mM CaCl2, and 100 mg of ampicillinand 40 mg of chloramphenicol/ml. Overnight cultures were diluted 1:9 into freshmedium and grown to early exponential phase in duplicate tubes. CN-Cbl (5 mMfinal concentration) was added to one series of cultures for 15 min before thecells were harvested, washed, and suspended in phosphate-buffered saline (PBS).The bacteria were adjusted to the same optical density and analyzed by sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Westernimmunoblotting. Each cross-linking assay was repeated at least twice.

SDS-PAGE and Western immunoblot analysis. The bacterial suspensionswere lysed in sample buffer containing 50 mM iodoacetamide to block subse-quent disulfide bond formation. Proteins were resolved by electrophoresis on9.5% (wt/vol) SDS-PAGE gels under nonreducing conditions, using the discon-tinuous buffer system of Laemmli (21). Resolved proteins were transferred to anitrocellulose membrane (Bio-Rad, Hercules, Calif.) by electrophoresis for 1.5 hat 500 mA in buffer consisting of 25 mM Tris-HCl (pH 8.3), 192 mM glycine, and20% (vol/vol) methanol (40). The membranes were blocked in PBS contain-ing 2.5% dried nonfat milk for at least 1 h and were then reacted for 1 hwith monoclonal antibodies directed against BtuB (4B1; 1:10) or TonB (4H4;1:20,000; kindly provided by K. Postle) diluted in the same buffer, as describedpreviously (8). The membranes were washed three times for 10 min in PBS–0.02% Tween 20 before being incubated for 1 h with affinity-purified horseradishperoxidase-conjugated goat anti-mouse immunoglobulin G secondary antibodies(Jackson Immunoresearch) diluted 1:5,000 in PBS with 2.5% dried nonfat milk.After three washes in PBS–0.02% Tween 20, the blots were developed using thechemiluminescent substrate LumiGlo (Kirkegaard & Perry Laboratories) andexposed to X-ray film (Kodak XAR).

RESULTS

Effect of proline substitutions in the BtuB Ton box. Somemutations affecting the Ton box in several transporters conferan energy-uncoupled phenotype (3, 10, 13, 22, 38). Out of 30substitutions in the BtuB Ton box only a few displayed thisphenotype, with the greatest impairment resulting from Pro orGly substitutions for residue Leu-8 or Val-10 (13). To system-atically investigate the sequence requirements of the BtuB Tonbox, residues 6 through 12 were individually changed to Pro or

VOL. 182, 2000 DISTORTION OF BtuB-TonB INTERACTION 5955


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

Cys. When these proteins were expressed from a moderate-copy-number plasmid, their levels were similar to that of thewild-type protein (data not shown). The mutated allele encod-ing each substitution was transferred onto the chromosome byhomologous recombination. The BtuB phenotypes determinedupon expression from the plasmid or chromosome were com-parable, but only the data from single-copy expression arepresented (Table 1).

The Ton box variants carrying proline substitutions weretested for their ability to allow a metE strain to grow on CN-Cbl in place of methionine. The L8P and V10P variants werecompletely defective for growth below 5 mM CN-Cbl (Table 1),which is the same behavior as that shown by a btuB or tonB nullstrain (2). Variants carrying the other five Pro substitutionsshowed the same growth response as the wild-type protein, i.e.,maximal growth with even 0.5 nM CN-Cbl. Thus, Pro substi-tutions at only positions 8 and 10 interfered with CN-Cblutilization in the growth assay. Susceptibility to colicins E1 andE3 and phage BF23, which are TonB-independent BtuB sub-strates, was unaffected in all cases except for the T11P variant,whose susceptibility to all three agents was reduced 10- to100-fold.

The growth response to CN-Cbl is not a good indicator ofBtuB activity because each cell doubling requires the uptake ofas few as 25 molecules of CN-Cbl. Assays of CN-[57Co]Cbluptake provided quantitative information on the effect of thesesubstitutions on BtuB function. The Pro substitutions ex-pressed at single gene copy showed differences in transportactivity which were not apparent in the growth assay (Fig. 1A).The L8P and V10P variants showed no significant CN-Cbluptake subsequent to binding, as expected (13). The L8P mu-tant bound more CN-Cbl than did the V10P mutant. The V9Pand T11P variants showed reduced uptake, and all other vari-ants carrying Pro substitutions flanking positions 8 through 11had uptake within 10% of that of wild-type BtuB.

Effect of Cys and Cys-Pro substitutions in the Ton box. Foruse in site-directed disulfide bonding and spin labeling (8, 28),single Cys substitutions were introduced at each position in theTon box of BtuB. The phenotypes conferred by all seven Cyssubstitutions expressed at single gene copy were indistinguish-able from that of the wild type for utilization of CN-Cbl (Table

2) and for susceptibility to the E colicins and phage BF23.Uptake of labeled CN-Cbl was comparable to that of strainscarrying wild-type BtuB, except for reduced activity of theT11C variant (Fig. 1 B).

Two series of double substitutions, which combined the un-coupled L8P or V10P substitution with Cys residues at theremaining positions in the Ton box, were made. These doublemutants differed substantially in their ability to utilize CN-Cblfor growth in place of methionine (Table 2). Five of the 12double mutants, namely, a mutant with L8P and V9C substi-tutions (L8P V9C), L8P V10C, L8C V10P, V10P T11C, andV10P A12C, showed the complete defect in CN-Cbl utilizationas did the single mutants expressing L8P or V10P alone. Fourdouble mutants, L8P A12C, D6C V10P, T7C V10P, and V9CV10P, showed an intermediate phenotype and grew on 100 to500 nM CN-Cbl. Three double mutants, D6C L8P, T7C L8P,and L8P T11C, grew with CN-Cbl at 0.5 to 5 nM. Similarintragenic suppression of the uncoupled phenotype by othersubstitutions in the Ton box was described previously (13). Alldouble mutants showed wild-type susceptibility to phage BF23and normal or slightly reduced response to the E colicins in thecase of the V10P series (data not shown).

Intragenic suppression. The finding that Cys at positions 6,7, and 11 suppressed the growth defect of the variant with theL8P substitution prompted further analysis of the intragenic-suppression phenotype. CN-Cbl uptake assays showed thatnone of the double mutants containing Cys substitutions in

FIG. 1. CN-Cbl uptake by BtuB variants with Ton box substitutions. Deriv-atives of strain RK8452 carrying the indicated btuB mutations in single gene copywere grown and assayed for rate of uptake of CN-[57Co]Cbl, as described inMaterials and Methods. The strains carried Pro substitutions (A) or Cys substi-tutions (B) at D6 (V), T7 (v), L8 (M), V9 (f), V10 (‚), T11 (Œ), and A12 (e).(r), wild type.

TABLE 1. Phenotypes of proline substitutions in the BtuB Ton box

btuB allelea CN-Cbl concn (nM)for maximal growthb

Titerc of:

ColE1 ColE3 BF23

Null .5,000 R R RWT 0.5 4 4 6D6P 0.5 4 4 6T7P 0.5 4 4 6L8P .5,000 4 4 6V9P 0.5 4 4 6V10P .5,000 4 4 6T11P 0.5 2 3 4A12P 0.5 4 4 6

a The indicated btuB alleles were present in single gene copy at the chromo-somal btuB locus in strain RK8452. The alleles are designated by the correspond-ing amino acid substitution, numbered from the mature sequence. WT, wild type.

b Strains were streaked on minimal plates supplemented with required nutri-ents and serial 10-fold dilutions of CN-Cbl or L-methionine at 100 mg/ml. Afterthe plates were incubated for 40 h at 37°C, the sizes of colonies were estimated,and the lowest concentrations of CN-Cbl allowing a colony size comparable tothat on methionine-supplemented medium are reported.

c As described in Materials and Methods, serial 10-fold dilutions of the indi-cated colicins or phage were spotted onto cells in soft-agar layers on LB plates.The titers are the negative logs of the highest dilution of the agent that producesa zone of complete clearing. R, resistant to the highest concentration tested.

5956 CADIEUX ET AL. J. BACTERIOL.


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

combination with L8P or V10P showed any detectable CN-Cbltransport beyond the level of binding to BtuB (data notshown), regardless of the ability of the strains to utilize CN-Cblefficiently for growth.

A more sensitive assay for uptake of labeled CN-Cbl mea-sured its intracellular conversion to other metabolic forms.Strains carrying btuB encoding the L8P substitution (btuB L8P)or btuB T7C L8P in single copy were grown to late exponentialphase in 1-liter cultures in minimal medium supplementedwith 1 nM CN-[57Co]Cbl. The cell-associated Cbl was mea-sured, extracted from the cells, and resolved by paper electro-phoresis. The L8P strain bound an average of 264 molecules ofCbl per cell, and roughly 97% of the label was unalteredCN-Cbl, indicating that this strain can bind but not transportCN-Cbl into the periplasm. The suppressed strain T7C L8Ptook up 245 molecules of Cbl per cell, of which 81% wasCN-Cbl, the remainder being converted to aquo-Cbl (11% oftotal), methyl-Cbl (7%), and adenosyl Cbl (3%). Thus, thesuppressed BtuB variant T7C L8P is capable of a small amountof Cbl uptake, corresponding to less than one molecule ofsubstrate transported per BtuB molecule on the cell surface.

To test whether this low level of CN-Cbl uptake was TonBdependent, plasmids expressing the series of BtuB Cys substi-tutions in conjunction with L8P were introduced into tonBstrain RK5043. The growth responses to various concentra-tions of CN-Cbl were compared to those of the same straincarrying the btuB1 plasmid pAG1 and of btuB tonB1 strainRK5016. Strikingly, CN-Cbl utilization by the suppressed BtuBvariants was almost as effective in the absence of TonB func-tion as in its presence, showing a maximal growth response at5 nM CN-Cbl, in contrast to the situation for the btuB1 tonBstrain, which grew well only at 5 mM CN-Cbl. It was possiblethat the low level of CN-Cbl entry allowed by the variant BtuBproteins was the result of nonspecific OM leakage. However,the expression of any of these variants did not result in anychange in the ability to grow on MacConkey agar or in the sizeof the zone of growth inhibition surrounding disks containingSDS, deoxycholate, chloramphenicol, rifampin, or erythromy-cin (data not shown), indicating the retention of the barrierproperties of the OM. Thus, the intragenic suppression of theL8P substitution was mediated by the TonB-independent pas-sage across the OM of less than one molecule of substrate perBtuB molecule.

Phenotype produced by Cys substitutions In TonB. To studythe interaction between BtuB and TonB, Cys residues wereintroduced in TonB at each position between N159 to P164,flanking Q160, the site of suppressors of uncoupled Ton boxmutations (2, 17, 37, 38). Their effect on TonB function and onsuppression of the defective phenotype of the BtuB L8P andV10P variants was determined. The TonB variants were ex-pressed from plasmid pSU19 together with the accessory pro-teins, ExbB and ExbD. pSU19 and derived plasmids encodingthe TonB variants and ExbB and ExbD were introduced intotonB strain RK5043 to test for complementation of the phe-notypic growth defect in CN-Cbl utilization (Table 3). All ofthe Cys-scanning substitutions in TonB complemented thetonB mutation in strain RK5043 to restore the same level ofgrowth on 0.5 nM CN-Cbl as in a strain carrying the wild-typetonB gene. Strains containing the pSU19 vector or pNC3 (Ex-bBD alone) showed the same defective growth phenotype asthe tonB null strain.

To determine the CN-Cbl transport activity of the Cys-sub-stituted TonB variants, each variant gene was transferred tothe chromosome in single copy by integration of the tonBexbBD-carrying plasmids in tonB polA host strain NC11. Evenwhen expressed at single gene copy, all Cys-substituted TonBvariants conferred the same growth response to CN-Cbl as didwild-type TonB. The uptake of CN-[57Co]Cbl was very close(within 10%) to the wild-type activity, whereas the tonB parentand the strain with the integrated exbBD locus showed nodetectable CN-Cbl uptake (data not shown). Thus, the replace-ment of TonB residues from 159 to 164 with Cys had no de-tectable effect on TonB function.

Extragenic suppression. The ability of the TonB Cys vari-ants to suppress the defect in CN-Cbl utilization caused by theBtuB L8P and V10P substitutions was evaluated by growth(Table 3) and transport assays. Plasmids expressing the uncou-pled BtuB variants and the panel of TonB Cys substitutionvariants were transformed pairwise into btuB strain RK5016and btuB tonB strain NC12. In RK5016 the growth pheno-type of the L8P variant, but not that of the V10P variant, wasmodestly reversed by all plasmids expressing any TonB variant.

TABLE 2. CN-Cbl utilization phenotype of cysteine substitutions inthe BtuB Ton box and effect of the L8P and V10P substitutionsa

btuB alleleb

CN-Cbl concn (nM) for maximal growth of a straincarrying BtuB with the indicated substitutionc

Alone L8P V10P

WT 0.5 .5,000 .5,000D6C 0.5 5 500T7C 0.5 0.5 100L8C 0.5 NAd .5,000V9C 0.5 .5,000 500V10C 0.5 .5,000 NAT11C 0.5 5 .5,000A12C 0.5 500 .5,000

a The lowest concentrations of CN-Cbl that allows formation of colonies ofsizes comparable to those on methionine-supplemented medium are reported asin Table 1.

b The indicated btuB alleles were present in single gene copy at the attl locus.Allele designations are as in Table 1. WT, wild type.

c Each btuB allele encoded the cysteine substitution indicated in the leftcolumn along with either no other amino acid substitution (Alone) or the L8P orV10P substitution.

d NA, not applicable.

TABLE 3. Suppression of btuB L8P and V10P mutationsby Cys substitutions in TonBa

Plasmid orplasmid-encoded

substitution

Concn of CN-Cbl for maximal growth (nM)of strain carrying:

tonBbbtuBc

pBtuB1 pL8P pV10P

pSU19 .5,000 0.5 5,000 5,000pTonB 0.5 0.5 1,000 5,000pExbBD .5,000 0.5 5,000 5,000pTonBExbBD 0.5 0.5 1,000 5,000C18A 0.5 0.5 500 5,000N159C 0.5 0.5 500 50Q160C 0.5 0.5 0.5 0.5P161C 0.5 0.5 1,000 5,000Q162C 0.5 0.5 50 50Y163C 0.5 0.5 1,000 5,000P164C 0.5 0.5 500 5,000

a Concentrations of CN-Cbl affording colony sizes comparable to that onmethionine-supplemented medium are reported as in Table 1.

b The indicated tonB plasmids were introduced into the tonB strain RK5043 toevaluate their ability to complement the defect in CN-Cbl utilization. No btuB-carrying plasmid was present.

c The indicated plasmids were introduced into btuB strain RK5016 in combi-nation with compatible plasmid pAG1 derivatives carrying the wild-type btuBgene or the L8P- or V10P-encoding alleles, as indicated.



http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

The TonB Q160C variant, altered at the site of the originalsuppressors, completely suppressed the growth defect of theBtuB L8P and V10P variants to allow growth on 0.5 nM CN-Cbl. The TonB Q162C variant also showed weaker suppressionand allowed growth on 50 nM CN-Cbl. Other TonB Cys sub-stitutions had a moderate effect on the L8P variant (N159Cand P164C) and on the V10P variant (N159C) to allow growthon 50 to 500 nM CN-Cbl, indicating the existence of allele-specific interactions between TonB and the BtuB Ton box.Essentially identical growth responses to CN-Cbl were seenwhen these plasmids were transferred to tonB host strainNC12, showing that suppression was unaffected by the pres-ence of chromosome-encoded wild-type TonB.

As was seen with intragenic suppression, the suppression byTonB Q160C or Q162C of the BtuB L8P or V10P growthdefects did not result in any detectable restoration of activeaccumulation of CN-Cbl in the transport assay, even thoughthe amount of CN-Cbl binding was elevated in response toplasmid carriage of the btuB genes.

Disulfide cross-linking of BtuB and TonB. We examined thepattern of site-directed disulfide cross-linking between thesecysteine residues in the BtuB Ton box and in TonB. To verifythe identity of cross-linked species, we compared the detectionof dimers formed between TonB Q160C and BtuB V10C whenthe primary monoclonal antibodies against BtuB and TonBwere used separately with duplicate blots or were combined fordetection on a single blot (Fig. 2). The immunoreactive speciesrevealed by anti-BtuB (A) and anti-TonB (B) were identifiedas the 35-kDa TonB monomer, the 66-kDa BtuB monomer,the 100-kDa BtuB-TonB and 90-kDa BtuB-TonB9 hetero-dimers shown previously to contain full-length BtuB and thefull-length and truncated TonB species, respectively, and the130-kDa BtuB dimer (8). A 75-kDa species reactive with an-tibodies to BtuB but not to TonB was present. Probing withboth primary antibodies together (C) detected all species re-active with the individual antibodies and demonstrated thecomigration of the heterodimeric species. Hence, in subse-

quent experiments both antibodies were combined to allowdetection of all cross-linked species and both monomeric spe-cies on the same immunoblot. Note that the addition of CN-Cbl 15 min before cell harvest resulted in substantial increasesin the amounts of the BtuB dimer and the BtuB-TonB hetero-dimers.

All pairwise combinations of cysteine BtuB and TonB vari-ants were examined. Two representative sets of results, inwhich one BtuB substitution, D6C (Fig. 3A) or A12C (Fig.4A), was coexpressed with each TonB Cys substitution fromN159 to P164. The strain expressing BtuB V10C and TonBQ160C was included as a positive control (lanes 1 and 2), andCys-less TonB C18A was a negative control (lanes 3 and 4).BtuB D6C showed only weak cross-linking to TonB cysteinesat residues 159, 160, and 162 (Fig. 3A, lanes 5 to 16), and itformed homodimers mainly in the presence of CN-Cbl. TheBtuB A12C variant formed cross-links to all TonB positionsfrom residues 160 to 164, most strongly to positions 162 and163 (Fig. 4A). Cross-linking between most positions occurredin the absence of CN-Cbl but was stimulated by its presence.Homodimer formation through residue 12 (A12C) was muchweaker than it was through residue 6 (D6C).

The cross-linking patterns observed for the 42 strains ex-pressing all combinations of Cys substitutions in BtuB andTonB are summarized in Fig. 5A. The occurrence of cross-linking between two residues is indicated by a line whose thick-ness is roughly related to the amount of heterodimers. Theseresults confirm and extend our previous observations (8) thatBtuB positions 6 (D6C), 7 (T7C), and 9 (V9C) cross-link veryweakly, if at all, with TonB positions 159 to 164. BtuB cysteines

FIG. 2. Western immunoblots comparing the detection of TonB Q160C andBtuB V10C following their coexpression in strain RK5016 in the absence andpresence of CN-Cbl (5 mM), using for detection anti-BtuB monoclonal antibody4B1 diluted 1:10 (A), anti-TonB monoclonal antibody 4H4 diluted 1:20,000 (B),or both antibodies together (C). The protein species are identified on the right,and the mobilities of molecular weight markers are indicated on the left.

FIG. 3. Western immunoblots showing the cross-linked species producedduring coexpression of each member of the series of TonB Cys substitutions fromresidue 159 to 164, as indicated, with BtuB D6C (A), BtuB D6C L8P (B), andBtuB D6C V10P (C). Cells were grown in the absence or presence of CN-Cbl,added 15 min before harvest, as indicated. Protein detection used a mixture ofthe monoclonal antibodies reactive to Btub and TonB. Included in the first fourlanes are, as a positive control, the pair of BtuB V10C and TonB Q160C, and, asa negative control, the pair of BtuB D6C and TonB C18A lacking a Cys residue.The protein species are identified on the right, and the mobilities of molecularweight markers are indicated on the left.



http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

at positions 8 (L8C), 10 (V10C), and 12 (A12C) reacted pref-erentially with TonB cysteines at positions 160 (Q160C), 162(Q162C), and 163 (Y163C). All cross-linking, except that in-volving TonB Q162C, was stimulated by CN-Cbl.

Distortion of cross-linking by the BtuB L8P or V10P sub-stitutions. To examine the effect of the uncoupling mutationson the cross-linking of BtuB and TonB, the L8P and V10Psubstitutions were combined with the Cys substitutions at theother positions in BtuB and tested for cross-linking with eachTonB Cys variant (Fig. 3B and C and 4B and C). The presenceof the uncoupling mutations resulted in marked changes incross-linking pattern, indicating a loss of orientation specificity.When combined with the L8P mutation, the weakly reactiveBtuB residue at position 6 (D6C) showed considerably in-creased cross-linking to most TonB positions, particularly po-sitions 159 (N159C), 160 (Q160C), 163 (Y163C), and 164(P164C) (compare Fig. 3A and B). Formation of most of thecross-linked species containing BtuB L8P was stimulated byCN-Cbl, as in the wild-type strain (Fig. 3B). The presence ofthe BtuB L8P substitution reduced the cross-linking activity atposition 12 (A12C), leading to loss of detectable cross-linkingto TonB positions 161 (P161C), 163 (Y163C), and 164 (P164C)(Fig. 4B). As summarized for all pairs of Cys substitutions inFig. 5B, the presence of the L8P substitution caused the weaklyor nonreactive BtuB positions 6 (D6C), 7 (T7C), and 9 (V9C)to participate in cross-linking with numerous TonB positions.Conversely, the TonB residues at positions 161 (P161C), 163(Y163C), and 164 (P164C) showed greatly reduced levels ofcross-linking and change in linking specificity. The nonreactiveresidue at position 159 (N159C) reacted with most BtuB L8Ppositions.

The presence of the V10P substitution also greatly extendedthe range of cross-linking but revealed a different pattern ofreactivities than was seen with the L8P series (Fig. 3C and 4C).When coupled to the V10P substitution, the weakly reactiveBtuB residue at position 6 (D6C) showed increased linkingwith TonB cysteines at 159, 160, 162, and 164 (Fig. 3C). BtuB

residue at position 12 (A12C) cross-linked to every position inthis region of TonB, quite strongly with the residues at posi-tions 159 (N159C) and 160 (Q160C). Indeed, in the presenceof the V10P substitution, almost every Ton box residue reactedwith almost every TonB residue to a degree independent of thepresence of CN-Cbl (summarized in Fig. 5C). Even the forma-tion of BtuB homodimers carrying V10P was independent ofCN-Cbl. Thus, the uncoupling L8P and V10P substitutionsresulted in drastic changes in the cross-linking patterns withTonB, suggesting that the access of the Ton box for contactwith TonB was increased but the orientation of their contactwas decreased in the mutants.

DISCUSSION

Genetic and cross-linking studies have implicated the Tonbox as an important element in the response of OM activetransport proteins to the energy-coupling factor TonB. Contactbetween TonB and its client transporters had been deducedfrom the ability of overexpressed FhuA to stabilize overex-pressed TonB from degradation (14) and from direct demon-stration of formaldehyde-mediated cross-linking by Postle’slaboratory (22, 31, 39) and of site-directed disulfide bonding(8). Results presented here provide information about threemajor aspects of the interaction of BtuB and TonB.

The first result of this study addressed the sequence require-ments for Ton box function, which had not been systematicallyinvestigated. The Ton box is one of the few regions that isconserved among TonB-dependent transporters. Despite thisconservation, the replacement of each residue through theBtuB Ton box (residues 6 to 12) with Cys caused no detectabledefect in function, other than a modest decrease in TonB-dependent activities in the T11C variant, indicating that therecognition of side chains of Ton box residues is not critical forfunction. Substitutions of proline at residues 8 through 11impaired TonB-coupled transport function, with positions 8

FIG. 5. Schematic representation summarizing the cross-linking patterns ob-served upon co expression of the series of TonB Cys substitutions betweenresidues 159 and 164 and the BtuB Cys variants between residues 6 and 12 (A),the BtuB Cys variants in combination with the L8P substitution (B), and theBtuB Cys variants in combination with the V10P substitution (C). The thicknessof the lines is related to the relative amounts of the cross-linked species that wereformed. Black lines, cross-links whose formation was stimulated by the presenceof CN-Cbl; gray lines, cross-links whose formation was not noticeably affected bythe presence of CN-Cbl.

FIG. 4. Western immunoblots showing the cross-linked species producedduring coexpression of each member of the series of TonB Cys substitutions fromresidue 159 to 164, as indicated, with BtuB A12C (A), BtuB A12C L8P (B), andBtuB A12C V10P (C). Conditions were as described in the legend to Fig. 3.



http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

and 10 being most critical. It was noteworthy that T11P exhib-ited a decrease in TonB-independent activities. Prolines intro-duced at flanking residues had no effect on transport. Thesefindings suggest that backbone secondary structure within onlya very small region is important for function, perhaps repre-senting a critical hinge or other structural element. The lack ofeffect of proline or glycine (13) even one residue outside ofthe region from residues 8 to 11 is consistent with the rec-ognition of this region by specific contact, rather than a globalconformational change. Site-directed spin-labeling studies (28)showed that residues in the BtuB Ton box undergo substantialchange in mobility in response to binding of substrate.

The nature of intra- and extragenic suppression of Ton boxvariants of BtuB led to an unexpected view of BtuB action. Thedefect in CN-Cbl utilization owing to the BtuB L8P substitu-tion was substantially suppressed by nearby D6C, T7C, andT11C substitutions, and the V10P defect was partially sup-pressed by the T7C substitution. The defect in CN-Cbl utiliza-tion of the L8P and the V10P variants could also be stronglysuppressed by the TonB Q160C variant and less strongly by theQ162C substitution. These examples of allele-specific suppres-sion could indicate direct interaction of these residues, con-sistent with their participation as the major sites of disulfidecross-linking (see below).

It is clear that the suppression of the growth phenotype isnot accompanied by appreciable restoration of transport activ-ity, and thus suppression must involve the opening of a newpathway of BtuB action. Previous studies of suppression exam-ined mainly the growth response, which requires only uptake ofas few as 25 molecules of CN-Cbl per cell doubling of a metEstrain (11). The suppression of the BtuB L8P variant by TonBQ160K revealed several unexpected properties, namely, thatthe suppressor form of TonB still functioned efficiently withwild-type BtuB and behaved in a manner recessive to wild-typeTonB (1, 17). The transport assays described here showed thateach BtuB L8P molecule, whether suppressed by the T7Cchange or the changes in TonB at Q160 or Q162, gave very lowbut detectable transport of CN-Cbl into the cell. This level oftransport corresponded to no more than one molecule of CN-Cbl per BtuB protein. This transport by BtuB T7C L8P wasTonB independent but was not associated with any detectableincrease in OM leakiness.

The results also showed that the suppressor substitutionsthemselves, BtuB T7C or TonB Q160C, conferred no defect infunction with wild-type BtuB. We propose from these findingsthat BtuB can exist in three transport states. Wild-type BtuB inassociation with TonB can carry out repeated transport cyclesresulting in the accumulation of CN-Cbl in the periplasmicspace. A second state is seen for BtuB L8P or wild-type BtuBin the absence of TonB, in which CN-Cbl binds normally toBtuB but is not released at all into the periplasmic space. Theability of cells carrying BtuB in this state to grow with 5 mMCN-Cbl probably reflects the low-affinity entry of CN-Cbl byway of porin channels, since this growth is independent ofproduction of BtuB or TonB proteins. In the third or sup-pressed state, bound CN-Cbl can be released into the peri-plasm but the transporter is incapable of further transportcycles and no more CN-Cbl can be taken up than there areBtuB proteins in the OM. We propose that interaction withTonB and hence the proper conformation of the Ton box isrequired both for release of CN-Cbl from BtuB and to allowBtuB to carry out repeated transport cycles.

The third aspect of these findings addresses the orientationspecificity of the interaction of the Ton box and TonB. Themost extensive disulfide cross-linking between transport-com-petent proteins occurred between BtuB positions 8, 10, and 12

and TonB positions 160, 162, and 163. Cysteines in adjacentresidues cross-linked weakly, if at all. In all cases except TonBQ162C, cross-linking was stimulated by the presence of CN-Cbl. These cross-linking patterns extended our previous con-clusion (8) that these protein segments approach each other ina highly oriented manner and that access of the Ton box toTonB is substantially increased when substrate is bound.

It has been suggested that the L8P or V10P mutations or theanalogous changes in other transporters prevent interactionwith TonB, based on the effect of the analogous mutants onstabilization of TonB (14) or on formaldehyde-mediated cross-linking (22). In contrast, TonB-BtuB disulfide cross-linkingoccurs even more strongly in these mutants, but the specificorientation of approach of these protein segments was mark-edly deranged. These results are not inconsistent with theother studies since TonB is certain to contact other parts ofBtuB besides the Ton box. The misoriented contact betweenTonB and BtuB in the Ton box mutants may distort the otherprotein contacts so that the formaldehyde-linked residues arenot in proximity and the contacts cannot stabilize TonB fromproteolysis. There is no correlation between cross-linking pat-tern and the level of suppression provided in the various mu-tants, which reflects the situation that the suppression does notrestore normal transport function. The interaction of TonBwith the uncoupled form of the Ton box is still distorted, butthe suppressor forms of BtuB or TonB must allow release ofbound substrate although not recycling of the transport pro-cess.

The binding of substrate to the external face of the centraldomain changes the conformation and exposure of the Tonbox, as indicated by the unfolding of helix 1 in FhuA and theextensive movement of the Ton box attachment region (25).The substrate binding-induced conformational change of theTon box is indicated by the strong stimulation of BtuB D6Chomodimer formation, of disulfide cross-linking to TonB, andof local residue mobility as measured by the electron paramag-netic resonance spin labeling (28). We conclude that the L8Pand V10P substitutions do not prevent contact of BtuB andTonB but distort the orientation of the Ton box to prevent itshighly oriented approach to TonB. These mutations preventthe proper folding of the Ton box into its usual immobilizedconformation in the absence of substrate, as revealed by thehigh mobility of the spin probe attached to all BtuB Ton boxresidues when combined with the uncoupling mutations (C. H.Lin, K. A. Coggshall, N. Cadieux, R. J. Kadner, and D. S.Cafiso, unpublished data). These same mutations result in ageneral increase of cross-linking of BtuB and TonB positionsthat are normally nonreactive and in decreased cross-linking tonormally reactive positions at the C-terminal sides of theseregions. Future studies, particularly determination of the struc-ture of these proteins, are needed to explain why cross-linkingto the V10P-containing Ton box is independent of substrate,whereas cross-linking to the wild-type and L8P proteins isstimulated.

ACKNOWLEDGMENTS

We gratefully acknowledge the receipt of monoclonal antibody toTonB from K. Postle, without which this work could not be done, andhelpful discussions with David Cafiso.

This research was supported by research grant GM19078 from theNational Institute of General Medical Sciences and funds from theUniversity of Virginia.

REFERENCES

1. Anton, M., and K. J. Heller. 1993. The wild-type allele of tonB in Escherichiacoli is dominant over the tonB1 allele, encoding TonBQ160K, which sup-



http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

presses the btuB451 mutation. Mol. Gen. Genet. 239:371–377.2. Bassford, P. J., Jr., and R. J. Kadner. 1977. Genetic analysis of components

involved in vitamin B12 uptake in Escherichia coli. J. Bacteriol. 132:796–805.3. Bell, P. E., C. D. Nau, J. T. Brown, J. Konisky, and R. J. Kadner. 1990.

Genetic suppression demonstrates interaction of TonB protein with outermembrane transport proteins in Escherichia coli. J. Bacteriol. 172:3826–3829.

4. Bonhivers, M., A. Ghazi, P. Boulanger, and L. Letellier. 1996. FhuA, atransporter of the Escherichia coli outer membrane, is converted into achannel upon binding of bacteriophage T5. EMBO J. 15:1850–1856.

5. Boyd, D., D. S. Weiss, J. C. Chen, and J. Beckwith. 2000. Towards single-copygene expression systems making gene cloning physiologically relevant:lambda InCh, a simple Escherichia coli plasmid-chromosome shuttle system.J. Bacteriol. 182:842–847.

6. Bradbeer, C. 1993. The proton motive force drives the outer membranetransport of cobalamin in Escherichia coli. J. Bacteriol. 175:3146–3150.

7. Buchanan, S. K., B. S. Smith, L. Venkatramani, D. Xia, L. Esser, M. Palnit-kar, R. Chakraborty, D. van der Helm, and J. Deisenhofer. 1999. Crystalstructure of the outer membrane active transporter FepA from Escherichiacoli. Nat. Struct. Biol. 6:56–63.

8. Cadieux, N., and R. J. Kadner. 1999. Site-directed disulfide bonding revealsan interaction site between energy-coupling protein TonB and BtuB, the out-er membrane cobalamin transporter. Proc. Natl. Acad. Sci. USA 96:10673–10678.

9. Casadaban, M. J. 1976. Transposition and fusion of the lac genes to selectedpromoters in E. coli using bacteriophage transposons. J. Mol. Biol. 104:541–555.

10. Cornelissen, C. N., J. E. Anderson, and P. F. Sparling. 1997. Energy-depen-dent changes in the gonococcal transferrin receptor. Mol. Microbiol. 26:25–35.

11. DiGirolamo, P. M., R. J. Kadner, and C. Bradbeer. 1971. Isolation of vitaminB12 transport mutants of Escherichia coli. J. Bacteriol. 106:751–757.

12. Ferguson, A. D., E. Hofmann, J. W. Coulton, K. Diederichs, and W. Welte.1998. Siderophore-mediated iron transport: crystal structure of FhuA withbound lipopolysaccharide. Science 282:2215–2220.

13. Gudmundsdottir, A., P. E. Bell, M. D. Lundrigan, C. Bradbeer, and R. J.Kadner. 1989. Point mutations in a conserved region (TonB box) of Esche-richia coli outer membrane protein BtuB affect vitamin B12 transport. J. Bac-teriol. 171:6526–6533.

14. Gunter, K., and V. Braun. 1990. In vivo evidence for FhuA outer membranereceptor interaction with the TonB inner membrane protein of Escherichiacoli. FEBS Lett. 274:85–88.

15. Hancock, R. E. W., and V. Braun. 1976. Nature of the energy requirementfor the irreversible adsorption of bacteriophages T1 and f80 to Escherichiacoli. J. Bacteriol. 125:409–415.

16. Heller, K., and R. J. Kadner. 1985. Nucleotide sequence of the gene for thevitamin B12 receptor protein in the outer membrane of Escherichia coli.J. Bacteriol. 161:904–908.

17. Heller, K. J., R. J. Kadner, and K. Gunther. 1988. Suppression of thebtuB451 mutation by mutations in the tonB gene suggests a direct interactionbetween TonB and TonB-dependent receptor proteins in the outer mem-brane of Escherichia coli. Gene 64:147–153.

18. Higuchi, R., B. Krummel, and R. K. Saiki. 1988. A general method of in vitropreparation and specific mutagenesis of DNA fragments: study of proteinand DNA interactions. Nucleic Acids Res. 16:7351–7367.

19. Kadner, R. J. 1990. Vitamin B12 transport in Escherichia coli: energy cou-pling between membranes. Mol. Microbiol. 4:2027–2033.

20. Klebba, P. E., and S. M. C. Newton. 1998. Mechanisms of solute transportthrough outer membrane porins: burning down the house. Curr. Opin. Mi-crobiol. 1:238–248.

21. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of

the head of bacteriophage T4. Nature 227:680–685.22. Larsen, R. A., D. Foster-Hartnett, M. A. McIntosh, and K. Postle. 1997.

Regions of Escherichia coli TonB and FepA proteins essential for in vivophysical interactions. J. Bacteriol. 179:3213–3221.

23. Letain, T. E., and K. Postle. 1997. TonB protein appears to transduce energyby shuttling between the cytoplasmic membrane and the outer membrane inEscherichia coli. Mol. Microbiol. 24:271–283.

24. Link, A. J., D. Phillips, and G. M. Church. 1997. Methods for generatingprecise deletions and insertions in the genome of wild-type Escherichia coli:application to open reading frame characterization. J. Bacteriol. 179:6228–6237.

25. Locher, K. P., B. Rees, R. Koebnik, A. Mitschler, L. Moulinier, J. P. Rosen-busch, and D. Moras. 1998. Transmembrane signaling across the ligand-gated FhuA receptor: crystal structures of free and ferrichrome-bound statesreveal allosteric changes. Cell 95:771–778.

26. Locher, K. P., and J. P. Rosenbusch. 1997. Modeling ligand-gated receptoractivity. FhuA-mediated ferrichrome efflux from lipid vesicles triggered byphage T5. J. Biol. Chem. 272:1448–1451.

27. Martinez, E., B. Bartolome, and F. de la Cruz. 1988. pACYC184-derivedcloning vectors containing the multiple cloning site and lacZ alpha reportergene of pUC8/9 and pUC18/19 plasmids. Gene 68:159–162.

28. Merianos, H. J., N. Cadieux, C. H. Lin, R. J. Kadner, and D. S. Cafiso. 2000.Substrate-induced exposure of an energy-coupling motif of a membranetransporter. Nat. Struct. Biol. 7:205–209.

29. Miller, J. H. 1972. Experiments in molecular genetics. Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.

30. Moeck, G. S., and J. W. Coulton. 1998. TonB-dependent iron acquisition:mechanisms of siderophore-mediated active transport. Mol. Microbiol. 28:675–681.

31. Moeck, G. S., J. W. Coulton, and K. Postle. 1997. Cell envelope signaling inEscherichia coli. Ligand binding to the ferrichrome-iron receptor FhuA pro-motes interaction with the energy-transducing protein TonB. J. Biol. Chem.272:28391–28397.

32. Postle, K. 1999. Active transport by customized b-barrels. Nat. Struct. Biol.6:3–6.

33. Pressler, U., H. Staudenmaier, L. Zimmermann, and V. Braun. 1988. Ge-netics of the iron dicitrate transport system of Escherichia coli. J. Bacteriol.170:2716–2724.

34. Reynolds, P. R., G. P. Mottur, and C. Bradbeer. 1980. Transport of vitaminB12 in Escherichia coli. Some observations on the roles of the gene productsof btuC and tonB. J. Biol. Chem. 255:4313–4319.

35. Roof, S. K., J. D. Allard, K. P. Bertrand, and K. Postle. 1991. Analysis ofEscherichia coli TonB membrane topology using PhoA fusions. J. Bacteriol.173:5554–5557.

36. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: alaboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.

37. Sauer, M., K. Hantke, and V. Braun. 1990. Sequence of the fhuE outer-membrane receptor gene of Escherichia coli K12 and properties of mutants.Mol. Microbiol. 4:427–437.

38. Schoffler, H., and V. Braun. 1989. Transport across the outer membrane ofEscherichia coli via the FhuA receptor is regulated by the TonB protein ofthe cytoplasmic membrane. Mol. Gen. Genet. 217:378–383.

39. Skare, J. T., B. M. M. Ahmer, C. L. Seachord, R. P. Darveau, and K. Postle.1993. Energy transduction between membranes. TonB, a cytoplasmic mem-brane protein, can be chemically cross-linked in vivo to the outer membranereceptor FepA. J. Biol. Chem. 268:16302–16308.

40. Towbin, H., T. Staehlin, and J. Gordon. 1979. Electrophoretic transfer ofproteins from polyacrylamide gels to nitrocellulose sheets: procedure andsome applications. Proc. Natl. Acad. Sci. USA 76:4350–4354.



http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

Documents

Sequence Changes in the Ton Box Region of BtuB Affect Its