Journal of Bacteriology, September 2004, p . 6220-6229, Vol . 186, No . 18

The hFbpABC Transporter from Haemophilus influenzae Functions as a Binding-Protein-Dependent ABC Transporter with High Specificity and Affinity for Ferric Iron

Damon S . Anderson,¹ Pratima Adhikari,¹ Andrew J . Nowalk,² Cheng Y . Chen,³ and Timothy A . Mietzner^1*

Department of Molecular Genetics and Biochemistry,¹ Department of Pediatrics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania,² Sexually Transmitted Diseases and Tuberculosis Laboratory Research, Division of AIDS, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia³

Received 4 June 2004/ Accepted 9 June 2004

 
Pathogenic Haemophilus influenzae, Neisseria spp . [Neisseria gonorrhoeae and N . meningitidis], Serratia marcescens, and othergram-negative bacteria utilize a periplasm-to-cytosol FbpABCiron transporter . In this study, we investigated the H . influenzaeFbpABC transporter in a siderophore-deficient Escherichia colibackground to assess biochemical aspects of FbpABC transporterfunction . Using a radiolabeled Fe³⁺ transport assay, we establishedan apparent K_m = 0.9 µM and V_max = 1.8 pmol/10⁷cells/min for FbpABC-mediated transport . Complementation experiments showed that hFbpABC is dependent on the FbpA binding protein for transport. The ATPase inhibitor sodium orthovanadate demonstrated dose-dependent inhibition of FbpABC transport, while the protonmotive-force-inhibitor carbonyl cyanide m-chlorophenyl hydrazone had no effect . Metal competition experiments demonstrated that the transporter has high specificity for Fe³⁺ and selectivity for trivalent metals, including Ga³⁺ and Al³⁺, over divalent metals . Metal sensitivityexperiments showed that several divalent metals, including copper,nickel, and zinc, exhibited general toxicity towards E . coli.Significantly, gallium-induced toxicity was specific only toE . coli expressing FbpABC . A single-amino-acid mutation in thegene encoding the periplasmic binding protein, FbpA[Y196I],resulted in a greatly diminished iron binding affinity K_d =5.2 x 10^–4 M^–1, 14 orders of magnitude weaker thanthat of the wild-type protein . Surprisingly, the mutant transporter[FbpA[Y196I]BC] exhibited substantial transport activity, 35% of wild-type transport, with K_m = 1.2 µM and V_max = 0.5pmol/10⁷cells/min . We conclude that the FbpABC complexes possessbasic characteristics representative of the family of bacterialbinding protein-dependent ABC transporters . However, the specificityand high-affinity binding characteristics suggest that the FbpABCtransporters function as specialized transporters satisfyingthe strict chemical requirements of ferric iron [Fe³⁺] bindingand membrane transport.

 
To cause disease, many bacterial pathogens must compete for growth-essential iron within the extracellular environment ofthe human host [30, 33, 45] . The majority of pathogenic bacteriaemploy siderophore-dependent iron acquisition systems in competitionfor host iron [47] . These systems involve the use of nonproteinaceousiron-chelating compounds termed siderophores, which are producedand secreted into the environment [32] . In gram-negative bacteria the uptake of iron-bound siderophores involves the expressionof siderophore-specific outer membrane receptors and specificinner membrane binding protein-dependent ATP-binding cassette[ABC] transporters [14] . These systems offer flexibility in the acquisition of iron from multiple sources; however, the expression of the numerous gene products involved in each specific siderophore-dependent transport pathway may be metabolically demanding.

In contrast, Haemophilus influenzae and pathogenic Neisseria spp . [Neisseria meningitidis and N . gonorrhoeae] utilize a highlyconserved siderophore-independent high-affinity iron acquisitionsystem [31] . This system employs specific surface receptorsthat directly bind host iron-binding proteins, transferrin [Tf]or lactoferrin [Lf] [37] . Iron is extracted from the host proteinsand transported into the periplasm through an energy-dependentTonB-mediated process . Transport of free [naked] iron from theperiplasm to the cytosol is mediated via the FbpABC transporter,which is composed of a ferric ion binding protein [FbpA] andan inner membrane ABC transporter consisting of a membrane permease[FbpB] and an ATP-binding protein [FbpC] [1] . Although bacteriautilizing this system may express several different host protein-specificouter membrane receptors, FbpABC is a convergence point in theacquisition of iron.

By a strategy similar to that used in cloning the Serratia marcescens sfuABC operon [5], the H . influenzae hitABC and N . gonorrhoeaefbpABC operons were cloned by complementation of a siderophore-deficient[aroB] Escherichia coli strain for growth on nutrient agar containing 200 µM dipyridyl, an iron chelator [1, 2] . Expressionin this E . coli background has served as a model system withwhich to study the genetic and biochemical basis of FbpABC irontransport . Results of initial studies demonstrated that thetransporter genes from these diverse bacteria exhibit a high level of homology, and a common nomenclature has been devised to designate the genetic and protein components of the transporters: for H . influenzae, the gene name is hitABC and the protein name is hFbpABC; for N . gonorrhoeae, the gene name is fbpABC and the protein name is nFbpABC . The FbpABC transporters are encoded by three-gene operons under negative regulatory control of the ferric uptake regulator [fur] [7, 15] . The gene encoding theferric ion binding protein [FbpA] is separated from the downstreamtwo genes in the operon by a putative stem loop structure indicativeof a rho-independent transcriptional terminator . This is consistentwith the increase in expression of FbpA by several orders ofmagnitude compared with that of FbpB and FbpC [26] . Biochemicalanalyses of nFbpA and hFbpA demonstrate that these proteinsbind a single ferric [Fe³⁺] ion with high affinity and exhibita characteristic spectroscopic profile [13, 34, 35] . Interestingly,X-ray structural analyses of these proteins show that they bindiron in a manner remarkably similar to that of mammalian transferrinby using a common set of amino acid residues and employing asynergistic anion [10, 11] . This FbpA binding mechanism resultsin an extremely high affinity for Fe³⁺, similar in scale tothat of Tf [nFbpA, 2.4 x 10¹⁸ M^–1; N-lobe hTf, 1.8 x 10¹⁷ M^–1] [44, 49] . However, this affinity is 10 to 12 ordersof magnitude greater than the affinities exhibited by typicalbacterial periplasmic binding proteins [PBPs] for their respectivesubstrates [e.g., maltose binding protein for maltose; K_d =1.6 x 10^–6 M] [18] . Based on sequence analysis, the nFbpBand hFbpB proteins are proposed highly hydrophobic proteinsthat function as membrane permeases within the context of theABC transporters . The nFbpC and hFbpC proteins are proposedATP-binding components of these transporters [31] . The largenumber of recent studies on the FbpA ferric binding proteins[3, 8, 10, 16, 19, 22, 38-40, 44, 50] makes a critical investigation of the functional basis of FbpABC transport timely.

This study focuses on functional investigation of the H . influenzae hFbpABC transporter through expression of the hitABC three-gene locus in E . coli . Recombinant expression in this background includes the use of an E . coli strain [H-1443] that has a deletion in the aroB gene, rendering it unable to synthesize the sole E . coli siderophore enterochelin [9] . This background allowsinvestigation of the hFbpABC system while controlling for endogenousiron uptake systems [e.g., FepABCD, FecABCDE, FeoABC, and MntH][17, 25, 28, 41] by the use of high-affinity metal chelators,2,2,-dipyridyl, and nitrilotriacetic acid . Using this modelsystem, we established an assay for radiolabeled iron uptakein intact cells and generated apparent Michaelis-Menten K_m andV_max constants for hFbpABC transport . We then defined the energy requirements and metal specificity of hFbpABC by monitoringthe growth-inhibitory effects of metals competing for hFbpABC-mediated transport . Finally, we investigated the impact of a single-amino-acid hFbpA mutation on hFbpABC-mediated transport and derived the functional basis of this effect . These studies form the basisof continuing investigations aimed at augmenting our understandingof FbpABC iron transport and its contribution to pathogenesisof diverse bacterial species.

 
Biochemicals, plasmids, and bacterial strains. Ampicillin, L-arabinose, 2,2'-dipyridyl, MES [morpholineethanesulfonicacid] and Tris buffers, glucose, carbonyl cyanide m-chlorophenylhydrazone [CCCP], nitrilotriacetic acid [NTA], cetyltrimethylammoniumbromide [CTAB], cupric chloride, aluminum sulfate, zinc chloride,manganese sulfate, nickelous chloride, phenylalanine, tyrosine,tryptophan, and sodium orthovanadate were purchased from Sigma[St . Louis, Mo.] . Nutrient broth [NB], Luria broth [LB], Bactoagar, and sterile supplement disks were purchased from DifcoLaboratories [Detroit, Mich.] . Chelex-100 and sodium dodecylsulfate-polyacrylamide gel electrophoresis reagents were purchasedfrom Bio-Rad [Hercules, Calif.] . Ferric nitrate and galliumnitrate were purchased from Aldrich [Milwaukee, Wis.] . ⁵⁵Ferricchloride was purchased from NEN [Boston, Mass.] . ⁶⁷Gallium citratewas a generous gift from Robert Schork of University of PittsburghMedical Center Presbyterian Nuclear Medicine . Oligonucleotideswere purchased from Gibco/Life Technologies [Gaithersburg, Md.].Taq polymerase was purchased from Boehringer Mannheim [Mannheim,Germany] . Restriction enzymes were purchased from New EnglandBiolabs [Beverly, Mass.] . Nitrocellulose filters and scintillationcocktail were obtained from Fisher [Pittsburgh, Pa.] . E . colistrains and plasmids were obtained as described in Table 1.

 
Molecular cloning. The pAHIB plasmid was constructed by excision of the hitB genefrom the MluI sites of the pAHIO plasmid . The resulting MluI-compatibleends were ligated, and the coding sequence was verified by automatedDNA sequencing [Department of Molecular Genetics and BiochemistryShared Resources Facility, University of Pittsburgh] . The pACYCHIB and pACYCHIO plasmids were made by PCR amplification of the hitABC genes and 250 kb of upstream and downstream sequencefrom the pAHIO plasmid by the use of the HitO-5' and HitO-3'primers described previously [2] . The PCR product was restrictedwith BamHI and SmaI and ligated into compatible BamHI and EcoRVsites of the pACYC184 vector . The pBADHIBC plasmid was constructedby PCR amplification of the hitBC genes from the pAHIO plasmidby the use of the primers HitB5'TA [5'-ATGCCTCGCAGACCGCCATTCTGGCTTAC-3']and HitC3'TA [5'-AGCGTAAAAAAGCCCTTTTTTATGTAAATA-3'] . pBAD-TOPO-TAvector [Invitrogen, Carlsbad, Calif.] was used in direct TAcloning of the PCR product.

Iron transport assay. NB agar [NBA] and LB agar [LBA] were prepared per manufacturerinstructions and supplemented with ampicillin and/or dipyridyl.M9 minimal medium [M9] was prepared, with the following finalcomposition: 6 mg of Na₂HPO₄/ml, 3 mg of KH₂PO4/ml, 0.5 mg ofNaCl/ml, 1 mg of NH₄Cl/ml, 5 mM MgSO₄, and 0.1 mM CaCl₂ . Priorto sterilization, trace iron was removed from M9 by stirringin the presence of 100 mg of Chelex-100/liter followed by sterile filtration . Transport medium [TM] was prepared by supplementingM9 with 2 mg of glucose/ml, 0.1 mg of phenylalanine/ml, 0.1mg of tyrosine/ml, and 0.1 mg of tryptophan/ml; radiolabeledFe[NTA]₂ or Ga[NTA]₂ was added immediately prior to the assay. Cells were transformed using the heat shock method for chemically competent cells . Fresh transformants were grown to midlog phasein LB supplemented with 100 µg of ampicillin/ml [LBamp₁₀₀], seeded at 10⁶ CFU/plate on NBA supplemented with 100 µgof ampicillin/ml and 75 µM dipyridyl [NBAamp₁₀₀dip₇₅], and then grown at 37°C for 18 h . Cells were suspended inM9 and centrifuged at 4°C for 10 min at 4,000 x g . Pelletedcells were brought up in TM and diluted to an optical densityat 578 nm of 0.5 . The cells were then preincubated at 37°C in 5% CO₂ with shaking in 24-well tissue culture plates in a water-jacketed incubator . Following a 10-min preincubation, radiolabeled Fe[NTA]₂ [3 x 10⁴ cpm/pmol] was added to the cellsuspensions at a final concentration of 1 µM and incubationwas continued . At designated time points, 100-µl aliquotswere removed and filtered through 0.45-µm-pore-size nitrocellulosefilters [filters conditioned with 5 ml of TM] by the use ofa vacuum manifold [Millipore, Billerica, Mass.] . Filters were immediately washed with 5 ml of 100 mM LiCl, removed, and air dried overnight at 25°C . The filters were dissolved in 4ml of scintillation cocktail, and counts per minute were measuredusing a liquid scintillation counter [Packard, Billerica, Mass.].

In complementation experiments, cells were grown as described above except that the growth medium contained 30 µg of chloramphenicol/ml for pACYC vector selection and 0.2% L-arabinose to induce expression of hitBC under the control of the araBAD [pBAD] promoter.

Kinetics of iron transport. Michaelis-Menten constants for the wild-type hFbpABC and mutanthFbpA[Y196I]BC transporters were determined using the above-describediron transport assay with the following conditions . Cells wereused at a concentration of 10⁷ cells per 100 µl, and substrateconcentrations were adjusted over a range of 0.1 to 20 µM.Initial velocity measurements were determined by taking 100-µlsamples at 5 and 120 s and determining counts per minute forthe difference [115-s uptake] . Substrate-dependent uptake wasdetermined by subtracting hFbpA-only background uptake [with pAHIB or pAHIBAY196I] from hFbpABC uptake [with pAHIO or pAHIOAY196I]and plotting initial velocity [in picomoles/10⁷ cells/minute]against substrate concentration [in micromoles] . Results werenot significantly affected by the use of higher cell concentrations[10⁷ to 10⁹ cells/100 µl] . Data were from a single experimentand are representative of data obtained from at least threereplicate assays . Nonlinear regression analysis was performedusing Origin 7 graphing software.

Complementation studies. H-1443/pACYC184-pBADHIBC, H-1443/pACYCHIB-pBADHIBC, and H-1443/pACYCHIO-pBADHIBCwere grown in LBamp₁₀₀cam₃₀ to midlog phase . Cells were seededat 10⁶ CFU/plate on NBAamp₁₀₀cam₃₀dip₇₅ supplemented with 0.2% arabinose and incubated at 37°C for 24 h . The radiolabeled Fe[NTA]₂ transport assay was performed as described above.

Energy utilization, metal competition, and metal sensitivity of hFbpABC. The metabolic inhibitor CCCP or sodium orthovanadate was added 1 or 10 min prior to the addition of radiolabeled Fe[NTA]₂, respectively . In metal competition experiments, metal[NTA]₂ complexes were added 1 min prior to addition of labeled Fe[NTA]₂. Metal sensitivity experiments were performed by growing fresh transformants of H-1443/pBR322, H-1443/pAHIB, and H-1443/pAHIOin LBamp₁₀₀ to midlog phase, diluting, and seeding on NBAamp₁₀₀at 10⁴ CFU/plate . Upon drying, sterile disks were placed onthe plates to which the following metal salts [200 mM] wereapplied: NiCl₂, MnSO₄, ZnCl₂, AlSO₄, CuCl₂, and Ga[NO₃]₃ . Theplates were inverted and incubated at 37°C for 18 h . Following incubation the plates were digitally scanned.

Gallium transport assay. The radiolabeled Ga[NTA]₂ transport assay was performed underconditions similar to those for the iron transport assay, using ⁶⁷Ga[NTA]₂ at 3 x 10⁴ cpm/pmol . Dried nitrocellulose filterswere subjected to direct counting using a gamma counter [Packard].

Construction and purification of the hFbpA[Y196I] mutant. Selection of the Tyr196Ile mutation was performed on the basisof the crystal structures of hFbpA and a homologous nFbpA mutant[35] . The mutant was constructed using a Gene Editor system[Promega, Madison, Wis.], with the pAHIO plasmid as the template.The mutation was verified by DNA sequencing as described above.The hFbpA[Y196I] protein was purified using a modification ofa previously reported protocol [2] . Briefly, an overnight cultureof JM109/pAHIOAY196I was used to inoculate 1 liter of NBamp₁₀₀ and the culture was grown at 37°C with shaking for 18 h.Cells were harvested by centrifugation [4,000 x g, 15 min] andwashed in phosphate-buffered saline . The cells were then resuspendedin a 50-ml solution of 400 mM Tris [pH 8.0]-2% [wt/vol] CTABand shaken at 37°C for 2 h . Cell debris was removed by centrifugation,and the soluble lysate was diluted to 500 ml in water . The dilutedlysate was clarified by filtration and applied to a carboxymethyl-SepharoseCL-6B column equilibrated with 10 mM Tris [pH 7.5] . The columnwas washed with 10 volumes of 10 mM Tris [pH 7.5] and subjectedto several step washes: 4 volumes of 10 mM Tris [pH 7.5]-200mM NaCl, 4 volumes of 400 mM NaCl, and 4 volumes of 500 mM NaCl.Purified hFbpA[Y196I] was collected following a gradient elutionof 500 to 1,000 mM NaCl . Fractions were analyzed for protein content by monitoring the A₂₈₀ and assessed for purity usingsodium dodecyl sulfate-polyacrylamide gel electrophoresis . hFbpA[Y196I]-containingfractions were pooled and concentrated in an Amicon concentrationcell with a 10-kDa-cutoff Diaflo ultrafiltration membrane andthen dialyzed using 20 mM MES [pH 6.5]-200 mM NaCl . Wild-typehFbpA was purified according to a previously published protocol[2] . Iron was removed from the hFbpA proteins by incubationwith 1,000-fold-molar-excess sodium citrate [pH 6.0] on icefor 30 min . Protein was then dialyzed using 10 volumes of 10mM sodium citrate followed by exhaustive dialysis using iron-free20 mM MES [pH 6.5]-200 mM NaCl . Concentrated protein was keptat –80°C.

UV/Vis spectroscopy of hFbpA and hFbpA[Y196I]. The visible absorbances of iron-saturated hFbpA and hFbpA[Y196I]were measured using 30 µM solutions of protein in 20 mMMES [pH 6.5]-200 mM NaCl . Protein was incubated with Fe[NTA]₂[1.2 molar equivalents] for 2 days at 4°C . Absorbance spectrawere acquired using an AVIV model 14 UV/Vis spectrophotometer.Three scans of each protein were taken, and the results wereaveraged . Data were plotted using Cricket Graph III.

Iron binding affinity of hFbpA[Y196I]. The effective Fe³⁺ dissociation constant [K_d] of hFbpA[Y196I]was determined using equilibrium binding and ultrafiltrationmethods . Iron-free hFbpA[Y196I] [15 µM] in 20 mM MES [pH6.5]-100 mM NaCl-150 µM NaPO₄ was incubated in the presenceof increasing concentrations of Fe[NTA]₂ labeled with ⁵⁵FeCl₃ [7 x 10⁴ cpm/µl] for 2 days at 4°C . The solutionswere subjected to filtration using BIOMAX ultrafree filter tubeswith 10 NMWL membranes centrifuged at 10,000 x g for 5 min at4°C . Aliquots of both the total protein solution [boundplus free Fe[NTA]₂] and the filtrate [free Fe[NTA]₂] were removedand measured for radioactivity by the use of a liquid scintillationcounter . Bound iron [total minus filtrate] was plotted versusthe total iron concentration, and nonlinear regression analysiswas performed using Origin 7 graphing software.

 
hFbpABC iron transport. Previous work demonstrated that propagation on NBA supplementedwith 200 µM dipyridyl allows the selection of H-1443 E.coli complemented with a functional hitABC operon [2] . In thisstudy, NBAamp₁₀₀ supplemented with 75 µM dip [NBAamp₁₀₀dip₇₅] allowed growth of H-1443 E . coli while forcing upregulationof hitABC derived from the pAHIO plasmid, which is under the transcriptional control of the H . influenzae fur operator . Growth on NBAamp₁₀₀dip₇₅ medium followed by suspension in iron-freeM9 was found to be the optimal condition for measuring iron transport . In this transport assay, ⁵⁵Fe³⁺ was provided as anitrilotriacetate complex for several reasons: [i] previouswork has demonstrated that Fe[NTA]₂ is very efficient in loadingFbpA with Fe³⁺, as NTA can serve as the synergistic anion [16];[ii] once bound, the NTA anion readily exchanges with otherendogenous anions, including PO₄, the preferred anion [16];and [iii] the affinity of NTA for Fe³⁺ is sufficiently highto inhibit competition by E . coli low-affinity iron transport systems . H-1443 E . coli was transformed with pBR322, pAHIB, or pAHIO plasmid [pAHIO and mutants used in this study were derived from the pBR322 background and thus have identical copy numbers] . Fresh transformants were grown on NBAamp₁₀₀dip₇₅, washed with M9, and measured via the transport assay [Fig . 1].

 
In this assay, H-1443/pBR322 demonstrated minimal iron uptake[0.38 pmol Fe/10⁹ cells at time = 6 min [2.1% of pAHIO results]], indicating negligible effects of the E . coli strain under assay conditions with Fe[NTA]₂ as a supplement [Fig . 1A] . The H-1443/pAHIB control demonstrated a low-level time-dependent increase in signal [4.29 pmol Fe/10⁹ cells at time = 6 min [23.3% of pAHIO results]] compared to the background [H-1443/pBR322] [Fig . 1A]. The signal result is not due to functional iron transport via hFbpABC but rather is due to binding of labeled Fe[NTA]₂ to hFbpA within the periplasm of these cells . This is consistent with previous results that showed that pAHIC was unable to mobilizeiron into the cytosol of H-1443 E . coli [2] . Subsequently, theactivities of both pAHIB and pAHIC were measured using the transportassay and shown to be very similar [data not shown] . The pAHIB control was used in the present study to eliminate any interference the presence of the hFbpB permease may exert on transport analyses. Levels of hFbpA expression by both pAHIB and pAHIO were measuredby Western blot and densitometric analysis [Kodak Imagestation1000] and found to be identical [pAHIB:pAHIO, 1.0:0.942], indicatingthat the difference in signal results between pAHIB and pAHIO[see below] was not the result of altered levels of hFbpA expression[Fig . 1B].

To address the observation that pAHIB does not reach time-dependentsaturation, H-1443/pAHIB cells were subjected to chasing with100-fold-excess unlabeled Fe[NTA]₂ at the 6-min time point [Fig.1A] . The chase resulted in a >50% loss of signal, indicatingrelease of Fe[NTA]₂ from hFbpA within the periplasm of the cells.The nonsaturable pAHIB signal may be due to the possibilitythat hFbpA within the H-1443/pAHIB periplasm is nearly saturatedwith unlabeled iron from the growth medium [the concentrationof Fe³⁺ in NBA is approximately 10 µM [personal observation]].Thus, the rate of binding for 1 µM ⁵⁵Fe[NTA]₂ to hFbpAis likely slow and would require increased time to reach saturation.

The H-1443/pAHIB control served as a baseline in the measurementof functional hFbpABC-mediated transport . The full transporterH-1443/pAHIO demonstrated a high-level time-dependent increasein signal [18.37 pmol Fe/10⁹ cells at time = 6 min] [Fig . 1A]. These results demonstrate that the assay is a specific measure of hFbpABC transport and is of sufficient sensitivity to test multiple parameters of hFbpABC transporter function, includingmetal specificity, energy requirements, and the effects of mutations.

Kinetics of hFbpABC transport. Incubation of pAHIO/H-1443 with increasing concentrations of ⁵⁵Fe[NTA]₂ demonstrated saturation of transport characteristicof Michaelis-Menten kinetics . After subtracting the pAHIB controlresults, the pAHIO transport rates were used to calculate estimatedvalues for K_m and V_max . The estimated apparent K_m for wild-typehFbpABC Fe³⁺ transport was 0.9 µM, and the apparent V_maxwas 1.8 pmol/10⁷cells/min . These values are within a close range of those derived for other binding protein-dependent ABC transport systems, including the maltose and histidine transporters [4, 29], and demonstrate that the hFbpABC transporter functionswith a substrate turnover similar to those of other bacterialsmall-ligand transporters.

hFbpABC is a binding protein-dependent ABC transporter. To demonstrate that the hFbpABC transporter is dependent uponthe binding protein for function, complementation experimentswere performed . Initially, the qualitative assay of growth orabsence of growth on NBAamp₁₀₀cam₃₀ containing 200 µM dipyridyl was used to determine whether hFbpA could complementhFbpBC expressed under the control of an exogenous promoter[P_BAD] [data not shown] . Results showed that H-1443 cells expressingthe hFbpBC proteins only [pACYC184-pBADHIBC] exhibited no growthon NBAamp₁₀₀cam₃₀dip₂₀₀ and that cells expressing hFbpBC complementedwith hFbpA [pACYCHIB-pBADHIBC] exhibited growth on this mediumsimilar to control cells [pACYCHIO-pBADHIBC] . The ⁵⁵Fe[NTA]₂transport assay was used as a more sensitive quantitative measureof complementation . Results demonstrated that the hFbpBC-onlycells exhibited a low level of transport similar to backgroundE . coli H-1443 cells observed previously [Fig . 1A and 2] . Cellsexpressing hFbpBC complemented with hFbpA [pACYCHIB-pBADHIBC] exhibited a high level of ⁵⁵Fe[NTA]₂ uptake similar in scaleto cells expressing the full hFbpABC transporter [Fig. 2] . Theseresults verify that the ABC transporter [hFbpBC] is dependentupon the hFbpA binding protein for functional iron transport.

 
Energy utilization by hFbpABC. Pretreatment of cells with increasing concentrations of theATPase inhibitor sodium orthovanadate prior to the additionof ⁵⁵Fe[NTA]₂ resulted in a dose-dependent decrease in ironuptake [Fig. 3] . This inhibition leveled off at an amount of signal similar to that seen with pAHIB, which is indicativeof ⁵⁵Fe[NTA]₂ binding by hFbpA within the periplasm and lossof cytosolic transport [compare Fig . 3 [10 mM vanadate] withFig . 1A [pAHIB]] . These results are consistent with hFbpABCfunctioning as an ATPase-driven transporter, similar to membersof the family of bacterial binding protein-dependent ABC transporters.Interestingly, administration of sodium arsenate under similarconditions had notable effects on transport only at high concentrations[data not shown] . This result is likely due to the fact thatarsenate has previously been shown to function as a suitableternary anion in FbpA Fe³⁺ binding . Thus, the presence of arsenatemay have the side effect of facilitating hFbpA Fe³⁺ loadingand thereby facilitating Fe³⁺ transport . Addition of the protonophore CCCP prior to ⁵⁵Fe[NTA]₂ resulted in no apparent effect on ironuptake [data not shown] . This is in contrast to the MntH metal permease, which functions as a protonmotive-force-driven symporter and is inhibited by CCCP under similar conditions [28] . Theseresults indicate that hFbpABC transport is not driven by protonmotiveforce.

 
Metal specificity of hFbpABC. hFbpABC metal specificity was investigated by testing for competitionby using trivalent and divalent metals for radiolabeled Fe[NTA]₂transport [Fig. 4] . Metal:NTA complexes were added at a 100-fold molar excess to cells 1 min prior to the addition of labeledFe[NTA]₂ . Uptake was quenched at the 6-min time point with 100-fold-excess unlabeled Fe[NTA]₂ . As a control, H-1443/pAHIO to which no competitor was added prior to ⁵⁵Fe[NTA]₂ demonstrated a high-level time-dependentincrease in iron uptake similar to that shown in Fig . 1A . Thecompeting metals were grouped into one of four categories, minimal,low, medium, and high, on the basis of their abilities to inhibitFe³⁺ uptake . The divalent metals Ni[NTA]₂ and Zn[NTA]₂ demonstrated minimal transport competition [13.8 and 22% inhibition of ⁵⁵Fe³⁺ uptake, respectively], while Cu[NTA]₂ and Mn[NTA]₂ demonstratedlow levels of transport competition [34.5 and 36.8% inhibition][Fig . 4A and B] . The trivalent metals Al[NTA]₂ and Ga[NTA]₂demonstrated medium competition [48.1 and 53.7% inhibition][Fig . 4C] . None of the metals, however, exhibited high-levelcompetition on a scale similar to Fe[NTA]₂ [94.5% inhibition][Fig. 4D] . Taken together, these results demonstrate that the hFbpABC transporter exhibits high specificity for ferric iron and has general selectivity for trivalent metals, includinggallium and aluminum, over divalent metals such as copper, manganese,nickel, and zinc.

 
Metal sensitivity of hFbpABC expressed in H-1443 E . coli. Through the course of these studies, several metals tested formetal specificity of hFbpABC [Fig . 4] were identified as inhibiting the growth of H-1443 E . coli expressing hFbpABC . To assess whether hFbpABC was responsible for the toxicity of these metals, the growth-inhibitory effects of specific metals were tested . Theresults indicate that the divalent metals Cu²⁺, Ni²⁺, and Zn²⁺ indeed demonstrate general toxicity to H-1443/pBR322, H-1443/pAHIB, and H-1443/pAHIO, independent of hFbpABC [data not shown] . Interestingly, Ga³⁺ demonstrated significant toxicity toward H-1443/pAHIO only,indicating a possible correlation between hFbpABC-specific transportand gallium-induced toxicity [Fig . 5] . These results, coupledwith previous results showing that Ga[NTA]₂ is able to competefor binding to hFbpA and form a stable complex [unpublisheddata], lead to the hypothesis that gallium may act as an ironanalog and be bound and transported through hFbpABC . To test this, the iron transport assay was adapted for the use of radiolabeled gallium [⁶⁷Ga[NTA]₂] . Results show that the hFbpABC transporterfunctions in the direct uptake of gallium [Fig. 5D] . Presumably,this transport gives rise to the gallium-induced toxicity whichinhibits the growth of H-1443 E . coli expressing hFbpABC.

 
The hFbpA[Y196I]BC mutant transporter. We investigated the effect of a single-amino-acid mutation onhFbpABC transport activity through the construction and analysisof the hFbpA[Y195I]BC mutant transporter . This mutation, selectedthrough assessment of the hFbpA crystal structure, targetedone of the conserved iron-liganding residues in the Fe³⁺ bindingsite of hFbpA [Fig. 6A] . Alteration of this site is predictedto affect the ability of hFbpA to bind iron, as previously observedwith a homologous nFbpA mutant . Using the radiolabeled irontransport assay, we assessed the impact of the hFbpA[Y196I]mutation on hFbpABC transport [Fig . 6B] . The mutant exhibited 35% activity of the hFbpABC wild-type transporter under standard conditions . The difference in activity was not due to altered expression levels of the binding proteins, as densitometricanalysis demonstrated identical amounts of hFbpA and hFbpA[Y196I][1:1.05, respectively] . These results were unexpected in thatone would expect the transport activity of the mutant to becompletely lost, equating to that of the pBR322 vector-onlycontrol in this assay.

 
We hypothesized that the mutant protein had lost significantbinding activity but retained diminished affinity for iron andthus might support lower levels of transport activity . To verifythat hFbpA[Y196I] had indeed lost wild-type iron binding activity,the protein was purified and its iron binding characteristicswere assessed . One of the signature characteristics that theiron binding proteins share with the transferrins is the presenceof a strong visible absorption peak in the 480-nm range . Thisis attributable to hard ligand electron donors [phenolate tyrosines]that contribute to ligand-to-metal charge transfer upon interactionwith Fe³⁺ . Comparison of visible spectra for wild-type hFbpAand mutant hFbpA[Y196I] demonstrated the complete loss of thispeak from the mutant [Fig . 6C] . Furthermore, trypsin susceptibility analysis of hFbpA[Y196I] resulted in no discernible Fe³⁺-dependent resistance to trypsin proteolysis [data not shown], in similarity to a result previously observed in the nFbpA mutant [35] . Equilibriumbinding analysis of the mutant hFbpA[Y196I] protein demonstratedan iron dissociation constant in the submillimolar range [K_d= 5.2 x 10^–4 M^–1] [Fig . 6D] . This affinity is approximately 14 orders of magnitude weaker than the affinity of wild-type FbpA for Fe³⁺ . Substrate-dependent kinetic analysis, using conditions similar to those used with wild-type hFbpABC, demonstrated an estimated apparent K_m of 1.2 µM and an apparent V_max of0.5 pmol/10⁷cells/min . Although this K_m is similar to that ofhFbpABC, the V_max is approximately one-third that of the wild-typetransporter . Taken together, these data demonstrate that thehFbpA[Y196I] protein has lost significant iron binding activity,which correlates with a decrease in transport activity-capacity[V_max] . However, this attenuated Fe³⁺ binding activity still supports a reduced level of transport, indicating that the mutant hFbpA[Y196I] protein is able to present the ABC transporterwith Fe³⁺ . These results have interesting implications for themechanism of hFbpABC-mediated transport in that it appears thathigh-affinity Fe³⁺ binding by hFbpA is not an absolute requisitefor transport in this model system.

 
Expression of the fbpABC and hitABC operons in E . coli has providedimportant information regarding the biochemical basis of FbpABCtransporter function . Several previous reports have noted the utility of a model system involving the siderophore-deficient E . coli H-1443 strain complemented with functional FbpABC loci from diverse pathogens, including N . gonorrhoeae, S . marcescens, and H . influenzae [1, 2, 6] . We have employed this approachto investigate the biochemistry of hFbpABC transport in detailto gain information on the functional physiology of this noveltransport system.

The results of this work support the function of hFbpABC asan ATP-dependent transporter with a high specificity for ferriciron . This is consistent with the fact that FbpA is known tobind metals in a manner analogous to that of transferrin andhas a clear preference for ferric iron [11] [unpublished results].The fact that transferrin also binds other metals, includingaluminum, gallium, copper, and zinc [43], is consistent with our observation that these metals compete for hFbpABC transport. FbpABC-mediated transport is a two-step process, the first step involving the binding of metal to FbpA and the second involvingFbpA binding the FbpBC complex and metal transport into thecytosol . It is not clear whether these metals compete for transportat the level of competitive binding to hFbpA or whether theycan indeed gain access to the cytosol and thus compete for cytoplasmiciron uptake . The exception to this is gallium . Metal sensitivityexperiments [Fig. 5] demonstrate that while gallium may impart low-level toxicity to H-1443/pBR322 and H-1443/pAHIB cells [presumablythrough endogenous metal uptake systems], expression of a functionalhFbpABC transporter causes an increased level of toxicity toH-1443/pAHIO . hFbpABC-mediated gallium transport is furtherdemonstrated by the gallium transport assay [Fig. 5D] . Whilegallium has an ionic radius [Ga³⁺, 0.62 Å; Fe³⁺, 0.65Å] and a valence similar to those of ferric iron, it isnot a transition metal and therefore cannot replace iron inthe redox processes essential to many iron-containing proteins.Thus, the hFbpABC-mediated gallium-induced toxicity is likelythe result of the entry of gallium into the cytosol and a generaleffect of cellular iron deprivation . Gallium toxicity has provenuseful in subsequent functional studies of hFbpABC as a selectiontool in the identification of hFbpABC mutants [unpublished data].

The hFbpA protein binds iron utilizing six coordinating ligands. This involves four protein side chains [His 9, Glu 57, Tyr 195,and Tyr 196] and two exogenous molecules, a synergistic anionand a water molecule, to complete the binding site [10] . This repertoire of iron binding residues is highly conserved, asidentical residues are employed by multiple FbpA homologs . Transferrinalso utilizes a set of iron binding residues similar to FbpA,differing by only one side chain [glutamate is replaced withaspartate], consistent with the conservation of a highly specializedmetal binding site . A functional result of this organizationis the remarkable affinity for Fe³⁺ exhibited by both proteins.

In relation to the proposed hFbpA Fe³⁺ binding process [10], mutation of one of the tyrosine residues [Tyr 196 in hFbpA] would likely have a dramatic effect on Fe³⁺ binding, resulting in an altered affinity . Consistent with this, we have measured a large decrease [14 orders of magnitude] in the affinity ofhFbpA[Y196I] for Fe³⁺ compared to that of wild type . Althoughthis is a sizeable decrease, the new affinity value is in theoverall range of affinities exhibited by PBPs from alternatebinding protein-dependent ABC transport systems . This may accountfor the observation that the rate of iron transport is onlymoderately diminished [35% of wild type] in this mutant transporter.

In other systems, it has been established that the second stepof the transport process, binding of the PBP to the ABC transporterand substrate transport across the cytoplasmic membrane, isthe rate-limiting step and that the affinity of the PBP forthe substrate [K_d] roughly approximates the K_m of the transporter[36] . Furthermore, mutant PBPs with decreased substrate bindingaffinities [K_d] do not necessarily correlate with a proportionaldecrease in transport [K_m] [48] . These observations are likelyexplained by fact that the PBP is present at concentrationsseveral orders of magnitude greater than the ABC transporterprotein concentrations; PBP concentrations can reach the millimolarrange within the periplasm [36] . This is consistent with the results seen with the hFbpA[Y196I]BC transporter; the hFbpA[Y196I] protein with a K_d of 500 µM gives rise to a transportrate 35% that of the wild type . By extrapolation, it is plausiblethat an hFbpA mutant with a K_d of 1 µM could support arate of transport similar in scale to that of the wild type,although further investigations are required to verify this.If a binding protein with micromolar affinity is indeed sufficientfor wild-type transport [in consistency with other systems],why then has FbpA evolved such a high natural Fe³⁺ binding affinity?This may have to do with several possible factors, includinga role in [i] contributing to a thermodynamic driving force in the import of Fe³⁺ [from transferrin or lactoferrin], [ii] maintaining an available pool of Fe³⁺ within the periplasm for utilization in times of iron stress, or [iii] satisfying the requirements of maintaining ferric iron in a controlled protein environment to prevent reactivity-toxicity upon transit intothe cell [16].

The FbpAs are novel PBPs in that they utilize a ternary anionin binding substrate [iron], they utilize a highly conservedset of iron binding residues, and they exhibit iron bindingaffinities 10 to 12 orders of magnitude greater than the affinitiesexhibited by typical PBPs for their respective substrates . Thesecharacteristics may be the result of the strict requirementsthat ferric iron places on proteins that must bind it, perhapsdue to the propensity of free [naked] Fe³⁺ in aqueous, aerobicconditions to undergo hydrolysis and precipitate . The abilityof ferric iron to induce cellular damage through hydroxyl-radicalcatalysis places additional constraints on transport and management.Whether unique characteristics, based on the strict requirementsof Fe³⁺ binding and transport, extend to the membrane permeaseand ATP binding protein components of the FbpABC transportersis an area of present study.

Although FbpABC is the first identified ferric Fe³⁺-specific ABC transporter, several alternate systems that have free-iron transport activity, including the ferrous iron transport system feoABC, specific for ferrous iron under anaerobic conditions,and the CorA magnesium transport system, transporting Fe²⁺ with low affinity under low-magnesium conditions, have been identified [24, 25] . Both these systems require free iron in the reducedsoluble form, Fe²⁺ . An additional metal transporter, MntH, isa bacterial homolog of the NRAMP [natural resistance againstmicrobial pathogens] family of eukaryotic metal permeases [12,28] and demonstrates specificity towards manganese and lowerselectivity towards ferrous iron, zinc, and other metals . However,it functions as an individual permease independent of a bindingprotein and is energized through protonmotive force . Recently,a stimulator of Fe transport system has been identified, withhomologs thus far identified in humans [23] and yeast [S . cerevisiae] [42] and a putative stimulator of Fe transport system identifiedin bacteria [B . subtilis] [42] . Although the function of thesetransporters remains largely undescribed, they appear to bespecific for ferric iron . Generally speaking, these systemsappear to be driven by individual membrane permeases that functionin both ATP hydrolysis and metal transport without the interplayof a separate PBP and ATP binding protein . Additionally, theymay require the presence of both an iron oxidase and reductasefor function . Further detailed investigations of the FbpB andFbpC proteins are required for fuller understanding of the novelstructural and functional aspects of ferric iron-specific FbpABCtransport.

In conclusion, the FbpABC iron transporters have several characteristics common to the family of bacterial binding protein-dependentABC transporters, including similar kinetics and the use ofATP hydrolysis as the energy source driving transport . The transporters are highly selective for Fe³⁺ and have a preference for trivalent metals, including Ga³⁺ . Interestingly, the transporters possess characteristics which may be shared by other iron transport systems and may have additional attributes specifically relatedto the transport of free ferric iron . Homologs of FbpABC andindividual components thereof have recently been identifiedin numerous bacteria, including Yersinia spp . [21], Campylobacterjejuni [20], Pasteurella haemolytica [27], and Actinobacillus actinomycetemcomitans [46]; ongoing genome projects will undoubtedlyuncover further homologs . Therefore, the FbpABC transportersmay comprise a widely employed iron acquisition system withimplications for virulence among many diverse bacterial species.

 
We thank K.G . Vaughan, C . B . Bahr, and S . M . Phadke for technicaland editorial assistance.

This work was supported by National Institutes of Health grantR29 A132226 [T.A.M.].

 
* Corresponding author . Mailing address: Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Room E1240 Biomedical Science Tower, Lothrop St., Pittsburgh, PA 15261 . Phone: [412] 648-9244 . Fax: [412] 624-1401 . E-mail: mietzner@pitt.edu .

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