The Haemophilus influenzae hFbpABC Fe3� Transporter: Analysis ofthe Membrane Permease and Development of a Gallium-Based
Screen for Mutants�
Damon S. Anderson,1† Pratima Adhikari,1 Katherine D. Weaver,2Alvin L. Crumbliss,2 and Timothy A. Mietzner1*
Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261,1
and Department of Chemistry, Duke University, Durham, North Carolina 27708-03462
Received 29 January 2007/Accepted 27 April 2007
The obligate human pathogen Haemophilus influenzae utilizes a siderophore-independent (free) Fe3� trans-port system to obtain this essential element from the host iron-binding protein transferrin. The hFbpABCtransporter is a binding protein-dependent ABC transporter that functions to shuttle (free) Fe3� through theperiplasm and across the inner membrane of H. influenzae. This investigation focuses on the structure andfunction of the hFbpB membrane permease component of the transporter, a protein that has eluded priorcharacterization. Based on multiple-sequence alignments between permease orthologs, a series of site-directedmutations targeted at residues within the two conserved permease motifs were generated. The hFbpABCtransporter was expressed in a siderophore-deficient Escherichia coli background, and effects of mutations wereanalyzed using growth rescue and radiolabeled 55Fe3� transport assays. Results demonstrate that mutation ofthe invariant glycine (G418A) within motif 2 led to attenuated transport activity, while mutation of theinvariant glycine (G155A/V/E) within motif 1 had no discernible effect on activity. Individual mutations ofwell-conserved leucines (L154D and L417D) led to attenuated and null transport activities, respectively. As acomplement to site-directed methods, a mutant screen based on resistance to the toxic iron analog gallium, anhFbpABC inhibitor, was devised. The screen led to the identification of several significant hFbpB mutations;V497I, I174F, and S475I led to null transport activities, while S146Y resulted in attenuated activity. Significantresidues were mapped to a topological model of the hFbpB permease, and the implications of mutations arediscussed in light of structural and functional data from related ABC transporters.
Pathogenic bacteria employ a number of acquisition strate-gies in competition for host iron (Fe3�). Siderophore-depen-dent iron transport is a widely used strategy that involves thesecretion of organic siderophore molecules that compete foriron bound to the high-affinity host transferrin (Tf) and lacto-ferrin (Lf) proteins (18). Fe3�-siderophore complexes are sub-sequently recovered by the bacteria through the activity ofsiderophore-specific surface receptors and transporters. As analternate strategy, several gram-negative pathogens, includingNeisseria gonorrhoeae, Neisseria meningitidis, and Haemophilusinfluenzae, utilize a siderophore-independent (free) Fe3�
transport system (37). In lieu of siderophores, this system em-ploys surface receptors that bind host iron-binding proteins Tfand Lf directly (17, 45). Fe3� is removed and transportedacross the outer membrane by the Tf/Lf-binding protein com-plex (TbpA/TbpB or LbpA/LbpB) using an energy-dependentmechanism mediated by TonB and associated proteins ExbBand ExbD. Naked (free) Fe3� is transported from theperiplasm to the cytosol by the FbpABC transporter, which iscomposed of a periplasmic ferric ion-binding protein (FbpA)
and an inner membrane ABC transporter consisting of a mem-brane permease (FbpB) and an ATP-binding protein (FbpC)(37).
A fundamental difference between siderophore-associatedand free iron transport involves the chemical nature of thesubstrate. In the former, iron is bound and transported into thecytosol as an intact Fe3�-siderophore complex. Coordinationof iron in this complex serves a dual purpose of assigningmolecular identity to the Fe3�-siderophore for recognition bythe appropriate receptors and transport proteins as well asshielding Fe3� from hydrolysis during transit into the cell (3,11). In contrast, the free iron transport system lacks any knownsiderophore; rather, Fe3� is removed directly from Tf or Lfand transported in free form via direct interaction with specificreceptors and Fe3�-binding proteins. These transport proteinsmust exhibit high specificity and affinity for Fe3� to avoidinsolubility and reactivity, yet they must readily exchange themetal during the process of transport.
Our investigations on the homologous FbpABC transportersfrom H. influenzae hFbpABC (also referred to in the literatureas HitABC) and N. gonorrhoeae (nFbpABC) have focusedlargely on the processes of high-affinity Fe3� binding and re-lease by the FbpA periplasmic binding proteins. X-ray struc-tures of H. influenzae hFbpA, in both Fe3�-bound (holo) andFe3�-free (apo) conformations, have provided insight into theFe3� coordination complex and the structural transitions in-volved in substrate binding and release (13, 14). Thermody-namic and kinetic investigations on N. gonorrhoeae nFbpA
* Corresponding author. Mailing address: Department of MolecularGenetics and Biochemistry, University of Pittsburgh School of Medi-cine, E1240 Biomedical Science Tower, Lothrop Street, Pittsburgh, PA15261. Phone: (412) 648-9244. Fax: (412) 624-1401. E-mail: [email protected].
† Present address: Molecular Cardiology Research Institute, Tufts-New England Medical Center, Boston, MA 02111.
have shed light on the mechanism of Fe3� coordination, par-ticularly with respect to the effects of ternary anions and bind-ing site mutations (10, 22–24, 39, 40, 47). It is clear that theFbpA proteins are structural and functional paralogs of themammalian Tf single iron-binding lobes. In addition to sharinga similar tertiary structure, the FbpA proteins possess a similarset of Fe3�-coordinating residues and undergo a large-scalecentral hinge rotation upon binding Fe3� similar to that of Tf.Although Tf and nFbpA demonstrate similar Fe3� bindingaffinities (nFbpA, 2.4 � 1018 M�1; N-lobe hTf, 1.8 � 1017
M�1), the proteins exhibit important binding site differences,which may be indicative of dissimilar binding and releasemechanisms (47, 51). As evident in the crystal structures ofthese proteins, hFbpA recruits a monodentate PO4 anion anda water to complete the inner coordination sphere of Fe3�,while Tf (and Lf) enlists a bidentate CO3
2� anion (6, 9, 14).This distinction may be the result of slightly different proteinconformations and a larger, more solvent-exposed FbpA Fe3�-binding site. Importantly, binding site differences correlate toincreased exchange and lability of the bound anion and apositive shift in redox potential in nFbpA compared to that ofTf (23, 27, 47). These features have direct influence on thestability of bound Fe3�, with potential implications in themechanism of transport (10, 22, 47).
Recent experiments have demonstrated that the H. influen-zae hFbpABC transporter functions as a bona fide bindingprotein-dependent ABC transporter, employing ATP as anenergy source and exhibiting transport rates similar to those ofother members of this bacterial ABC transporter family (7).However, the exceptionally high FbpA Fe3� binding affinity(approximately 1010 to 1012 higher than typical periplasmicbinding protein affinities) requires further critical evaluation ofFbpABC function and auxiliary processes (anion exchange orredox) that may be involved in the transport process. Clearly,an important event during the transport process is the ex-change of Fe3� from FbpA to the FbpB permease subsequentto transport across the inner membrane. The permease is an�500-amino-acid polypeptide proposed to be a polytopictransmembrane protein, forming both a receptor for FbpA anda channel for the passage of Fe3�. The FbpB homologs possesstwo permease motifs of the template EAA—G———I-LP thatare well conserved among the family of bacterial ABC trans-porter permeases (19, 30, 44). These regions are presumed toreside on cytoplasmically exposed loops that form a mechani-cal coupling between the energy transduction protein (FbpC)and the membrane transport protein (FbpB). The recent crys-tal structure of the vitamin B12 ABC transporter (BtuC2D2)verifies a key role for these “L” loop motifs in mediatingintimate contact between the permease and ATP binding sub-units (34). The hFbpB and nFbpB homologs are highly hydro-phobic and toxic when expressed from recombinant sources;thus, despite rigorous isolation efforts, the permease has re-mained elusive and characterization has been limited to geneticapproaches (1).
As a logical progression in our studies of the FbpABC sys-tem, we have broadened our focus to include the FbpB per-mease and its role in the Fe3� transport process. Expression ofthe H. influenzae hitABC three-gene operon in the sid-erophore-deficient H-1443 aroB Escherichia coli strain hasserved as an important model system with which to investigate
the function of the hFbpABC transporter (2, 7). In this study,we have utilized this system, coupled with quantitative andqualitative assays, to probe the significance of single aminoacids within the hFbpB permease. Multiple-sequence align-ments between FbpB permease homologs and related ABCtransporter permeases served as a basis for a series of site-directed mutations targeted at residues within the conservedpermease motifs. A positive selection screen using the Fe3�
analog gallium (Ga3�) was employed to identify additionalmutants, which were genetically delineated and subjected toFe3� transport analyses. Finally, a topological model of thehFbpB protein is presented, and implications of informativemutations are discussed in light of a hypothetical functionalmechanism of the hFbpB permease protein. These investiga-tions represent an important initial step in probing the struc-ture and function of the heretofore unexplored FbpB per-mease.
MATERIALS AND METHODS
Chemicals, plasmids, and bacterial strains. Ampicillin, 2,2�-dipyridyl (Dip),buffers, glucose, nitrilotriacetic acid (NTA), cetyltrimethylammonium bromide(CTAB), trans-1,2-diamoncyclohexane-N,N,N�,N�-tetraacetic acid (CDTA), phe-nylalanine, tyrosine, tryptophan, ferric nitrate, and gallium nitrate were all pur-chased from Sigma-Aldrich (St. Louis, MO). Nutrient broth (NB), Luria broth(LB), Bacto agar, and sterile supplement disks were purchased from Difco(Detroit, MI). Chelex-100 and sodium dodecyl sulfate-polyacrylamide gel elec-trophoresis (SDS-PAGE) reagents were purchased from Bio-Rad (Hercules,CA). 55Ferric chloride was purchased from New England Nucleides (Boston,MA). Oligonucleotides were purchased from Invitrogen (Carlsbad, CA). Taqpolymerase was purchased from Roche (Basel, Switzerland). Restriction en-zymes were purchased from New England Biolabs (Beverly, MA). Nitrocellulosefilters and scintillation fluid were purchased from Fisher (Pittsburgh, PA). E. colistrains and plasmids were obtained as described (Table 1).
Dip sensitivity, CDTA Fe3�(NTA)2 growth rescue, and radiolabeled irontransport assays. Assays measuring growth sensitivity to the iron chelator Dipwere performed under the following conditions. Single colonies were used toinoculate NB supplemented with 100 �g/ml ampicillin (NBamp100), and cultureswere incubated at 37°C with shaking at 250 rpm. Cells at mid-log growth (opticaldensity at 600 nm, 0.5) were centrifuged, suspended in NBamp100 containing 200�M Dip (NBamp100dip200) top agar (0.7%), and seeded at 106 CFU/plate onNBamp100dip200 agar. Plates were incubated in a water-jacketed incubator at37°C, 5% CO2 for 16 h. Following growth, plates were digitally scanned. Growthwas evaluated as follows: �, no colonies; �, pinpoint colonies.
Assays measuring exogenous Fe3�(NTA)2 growth rescue on media containingthe Fe3�-specific chelator CDTA were performed under the following condi-tions. Single colonies were used to inoculate NBamp100, and cultures were grownat 37°C with shaking at 250 rpm. Mid-log cells were centrifuged, suspended inNBamp100 containing 600 �M CDTA (NBamp100CDTA600) top agar, andseeded at 106 CFU/plate on NBamp100CDTA600 agar. Sterile supplement disks(6 mm) loaded with 100 mM Fe3�(NTA)2 (10 �l) were applied, and the plateswere incubated as described above. Following growth, plates were digitallyscanned and zones of growth were measured.
Radiolabeled iron transport assays were performed as previously described(7). Briefly, M9 transport medium (6 mg/ml Na2HPO4, 3 mg/ml KH2O4, 0.5mg/ml NaCl, 1 mg/ml NH4C, 1 mM MgSO4, 0.1 mM CaCl2) was treated with 100mg/liter Chelex-100 and supplemented with 2 mg/ml glucose, 0.1 mg/ml phenyl-alanine, 0.1 mg/ml tyrosine, and 0.1 mg/ml tryptophan prior to use. Fresh trans-formants were grown in LB supplemented with 100 �g/ml ampicillin(LBamp100), seeded on NBamp100dip75, and grown at 37°C for 18 h. Cells werewashed in transport medium, and following a 10-min preincubation at 37°C,radiolabeled Fe(NTA)2 (3 � 104 cpm/pmol) was added to a final concentrationof 1 �M. At designated time points aliquots were removed, filtered through0.45-�m-pore-size nitrocellulose filters, and washed with 100 mM LiCl. Activity(in counts per minute) was measured using a liquid scintillation counter (Pack-ard, Billerica, MA).
Site-directed mutagenesis. The Gene Editor System (Promega, Madison, WI)was used to perform site-directed mutagenesis. Plasmid DNA was isolated fromsingle putative mutants using standard miniprep techniques and was subjected to
VOL. 189, 2007 MUTATIONAL ANALYSIS OF hFbpABC TRANSPORTER PERMEASE 5131
automated DNA sequencing using an ABI Prism 3100 sequencer operated by theDepartment of Molecular Genetics and Biochemistry Shared Resources Facilityat the University of Pittsburgh.
Selection protocol for gallium-resistant H-1443/pAHIO mutants. H-1443 E.coli cells harboring the pAHIO plasmid were grown to mid-log phase inLBamp100. Cells were centrifuged, suspended in LBamp100, and seeded at 109
CFU/plate in parallel on NBamp100dip75 and NBamp100dip75 containing 100 �M
Ga(NO3) (NBamp100dip75Ga100) agar. Plates were incubated as describedabove. Growth of these cells on NBamp100dip75 resulted in slightly smallercolonies than those of pAHIO/H-1443 grown on NBamp100 (without Dip), whilethe same cells on Ga3�-containing medium (NBamp100dip75Ga100) exhibited ahighly growth-suppressed microcolony phenotype. On NBamp100dip75Ga100
plates, however, approximately 1 � 10�8 colonies (1 to 10 CFU/plate) exhibiteda larger macrocolony phenotype among a lawn of microcolony growth. One of
E. coli strainsH-1443 E. coli aroB 12H-1443/pBR322 H-1443 with pBR322 7H-1443/pAHI�C H-1443 with hitA, hitB, truncated hitC 2H-1443/pAHIO H-1443 with hitABC 2H-1443/pN�MHIO H-1443 with hitABC This studyH-1443/pBE149Q H-1443 with hitAB(E149Q)C This studyH-1443/pBS153A H-1443 with hitAB(S153A)C This studyH-1443/pBL154D H-1443 with hitAB(L154D)C This studyH-1443/pBG155A H-1443 with hitAB(G155A)C This studyH-1443/pBG155V H-1443 with hitAB(G155V)C This studyH-1443/pBG155E H-1443 with hitAB(G155E)C This studyH-1443/pBF162L H-1443 with hitAB(F162L)C This studyH-1443/pBP168A H-1443 with hitAB(P168A)C This studyH-1443/pBV197I H-1443 with hitAB(V497I)C This studyH-1443/pBI497V H-1443 with hitAB(I497V)C This studyH-1443/pBS146Y H-1443 with hitAB(S146Y)C This studyH-1443/pBI174F H-1443 with hitAB(I174F)C This studyH-1443/pBI383N H-1443 with hitAB(I383N)C This studyH-1443/pBI74N-S475I H-1443 with hitAB(I174N-S475I)C This studyH-1443/pBI174N H-1443 with hitAB(I174N)C This studyH-1443/pBS475I H-1443 with hitAB(S475I)C This studyTop10/pB366BLAM Top10 with hitAB(blaM)C This studyTop10/pB366L154D Top10 with hitAB(blaML154D)C This studyTop10/pB366L417D Top10 with hitAB(blaML417D)C This studyTop10/pB366G418A Top10 with hitAB(blaMG418A)C This studyTop10/pB366V497I Top10 with hitAB(blaMV497I)C This study
PlasmidspBR322 4.4-Kb vector; Ampr PromegapAHIO 4.3-Kb SmaI-BamHI fragment containing intact hitABC sequence cloned into corresponding
sites in pBR322; expressing hFbpABC; Ampr2
pAHI�C pAHIO derivative with truncated hitC gene; expressing hFbpAB; Ampr 2pN�MHIO pAHIO with an engineered NsiI site and a deleted MluI site; Ampr This studypACYCHIO 4.3-Kb SmaI-BamHI fragment containing intact hitABC sequence cloned into EcoRV-
BamHI sites in pACYC184; expressing hFbpABC; Camr7
pBE149Q Site-directed mutant derived from pAHIO; expressing hFbpAB(E149Q)C; Ampr This studypBS153A As for pBE149Q; expressing hFbpAB(S153A)C This studypBL154D As for pBE149Q; expressing hFbpAB(L154D)C This studypBG155A As for pBE149Q; expressing hFbpAB(G155A)C This studypBG155V As for pBE149Q; expressing hFbpAB(G155V)C This studypBG155E As for pBE149Q; expressing hFbpAB(G155E)C This studypBF162L As for pBE149Q; expressing hFbpAB(F162L)C This studypBP168A As for pBE149Q; expressing hFbpAB(P168A)C This studypBI497V As for pBE149Q; expressing hFbpAB(I497V) This studypBV497I Gallium selection mutant derived from pAHIO; expressing hFbpAB(V497I)C; Ampr This studypBS146Y Gallium selection mutant derived from pN�MHIO; expressing hFbpAB(S146Y)C; Ampr This studypBI174F As for pBS146Y; expressing hFbpAB(I174F)C This studypBI383N As for pBS146Y; expressing hFbpAB(I383N)C This studypBI174N-S475I As for pBS146Y; expressing hFbpAB(I174NS475I)C This studypBI174N As for pBS146Y; expressing hFbpAB(I174N)C This studypBS475I As for pBS146Y; expressing hFbpAB(S475I)C This studypB366BLAM pACYCHIO with a 1.2-kb EZ-TN5 fragment; blaM gene inserted into hitB (hitB1098::Tn5);
expressing an hFbpB in-frame fusion with BlaM; Camr, Ampr to 100 �g/ml on LBThis study
pB366L154D Site-directed mutant derived from pACYCHIO; expressing hFbpAB(BlaM-L154D)C; Camr,Ampr to 100 �g/ml on LB
This study
pB366L417D As for pB366L154D; expressing hFbpAB(BlaM-L417D)C; Camr, Ampr to 100 �g/ml on LB This studypB366G418A As for pB366L154D; expressing hFbpAB(BlaM-G418A)C; Camr, Ampr to 100 �g/ml on LB This studypB366V497I As for pB366L154D; expressing hFbpAB(BlaM-V497I)C; Camr, Ampr to 100 �g/ml on LB This study
PrimersNdeI-5� Upstream primer used for PCR mutagenesis; 5�-CGCAACTTAAACCCG-3� This studyRevQEc-3� Downstream primer used for PCR mutagenesis; incorporates stop�Gln and EcoRV site in
blaM; 5�-GAGAAAATTGATATCTTGCCAATGCTTAATC-3�This study
ForQEc-5� Upstream primer complementary to RevQEc; 5�-GATTAAGCATTGGCAAGATATCAATTTTCTC-3�
This study
BspHI-3� Downstream primer used for PCR mutagenesis; 5�-CGCAAGGAATGGTGC-3� This studyEcoRV5410-5� Upstream primer used for subcloning; incorporates an EcoRV site in hitB; 5�-CTATTTTTC
these gallium-resistant macrocolonies was chosen as a putative mutant for fur-ther study.
In the original development of the screen, a single putative mutant was se-lected and grown in LBamp100, and plasmid was isolated using standard mini-prep techniques. Purified plasmid was used to transform fresh H-1443 E. coli,and cells were seeded in parallel on NBamp100dip75 and NBamp100dip75Ga100
plates. Upon passage of the gallium resistance phenotype, a single colony wasselected and grown in LBamp100 broth to stationary phase. The culture wasdivided into three aliquots. Aliquot one was used to isolate plasmid DNA; a0.5-�g aliquot of this DNA was run on a 1% agarose electrophoresis gel andcompared to wild-type pAHIO plasmid. The other two aliquots were subjected toFbpA protein analysis. Aliquot two was centrifuged, washed in cold phosphate-buffered saline (PBS), suspended in 30 mM Tris, pH 8.0, 20% sucrose, 1 mMEDTA, and shaken at 25°C for 5 min. The suspensions were pelleted, suspendedin 5 mM ice-cold MgCl2, and shaken for 10 min at 4°C. The suspensions wereagain pelleted, and the supernatant was saved as the periplasmic osmotic shockfluid. Aliquot three was centrifuged, washed with PBS, suspended in 400 mMTris, pH 8.0, 2% CTAB, and shaken at 37°C for 1 h. The suspension was pelleted,and the supernatant was saved as the soluble whole-cell lysate. The solubleperiplasmic fraction and the soluble whole-cell fraction were subjected to SDS-PAGE on a 12% gel. The gels were stained with Coomassie blue and subjectedto densitometry using a Kodak Imagestation 1000 (Rochester, NY).
The initial gallium-resistant mutant was screened using the above methods todetermine whether (i) the phenotype was linked to the pAHIO plasmid, (ii) thephenotype was not due to any major insertions or deletions within pAHIO, or(iii) the mutation was not within or upstream of the hitA gene resulting in lackof expression, truncation, or misprocessing of the hFbpA protein. Upon satisfy-ing these criteria, the hitBC locus within the mutant plasmid was subjected toautomated DNA sequencing as described above.
Subcloning and random mutagenesis for gallium selection of hFbpB mutants.Upon establishing conditions for gallium selection of pAHIO mutants, directedmutations localized to the hitB gene were generated by subcloning putativemutant hitB genes into the pAHIO backbone. This required engineering uniquerestriction sites into the 5� and 3� ends of the hitB gene within the pAHIOplasmid. A unique NsiI 5� hitB site was inserted at 1525 and a MluI site at 3109was deleted, yielding a unique MluI site at 3064 at the 3� end of hitB and resultingin the pN�MHIO plasmid. To enrich the initial pool of putative mutants, aPCR-based random mutagenesis approach was used. The GeneMorph kit (Strat-agene, La Jolla, CA) was used along with primers specific to the 5� and 3� endsof hitB incorporating NsiI and MluI restriction sites under PCR conditionsconsistent with low-mutation frequency (0 to 3 mutations/kb). The mutagenicPCR product was restricted with NsiI and MluI, purified, and subcloned into theidentical sites within pN�MHIO. Ligated plasmid was used to transform freshH-1443 E. coli cells. Putative clones were selected, grown in LBamp100 to mid-logphase, and seeded on NBamp100dip75Ga100 plates. Clones exhibiting positivegrowth on NBamp100dip75Ga100 were selected and grown in LBamp100 to sta-tionary phase, and plasmid was isolated and used to transform fresh H-1443.Upon passage of the gallium resistance phenotype, mutant DNA was isolatedand the hitB locus was subjected to DNA sequencing as described above.
Protein expression analysis. A transposon-mediated -lactamase fusion strat-egy was employed to monitor hFbpB point mutant protein expression levels.Specifically, the EZ-TN5 -lactamase fusion kit (Epicenter, Madison, WI) wasused to generate random insertions within pACYCHIO, a pACYC184-basedconstruct with hitABC inserted between the EcoRV and BamHI sites of the Tetr
gene (7) (Table 1). Following a standardized transposon insertion reaction,TOP10 (recA) E. coli cells were transformed and plated on LB containing 40 to70 �g/ml of ampicillin to select for Ampr insertion clones. Putative clones wereselected and subjected to HindIII restriction analysis. All clones that demon-strated the presence of the 1.2-kb EZ-TN5 blaM insert and appeared to beunique were submitted for sequencing.
One such Ampr clone which possessed an in-frame blaM insertion at nucleo-tide 1098 within the hitB gene (designated pB366) was selected and subjected tooverlap extension PCR mutagenesis using the NdeI-5�, RevQEc-3�, ForQEc-5�,and BspHI-3� primers (Table 1). This method replaced the blaM translationalstop codon with a glutamine codon (TAA�CAA) and introduced an EcoRVrestriction site directly downstream of this missense site. The final 3.7-kb PCRproduct was digested with NdeI-5� and BspHI-3� and ligated into compatiblesites within pACYCHIO to generate pB366QE. The downstream portion of thehitB coding region was PCR amplified from the pB366 plasmid using theEcRV5410-5� and BspHI-3� primers to generate a 1.9-kb product with an intro-duced 5� EcoRV restriction site. This PCR product was digested with EcoRVand BspHI and ligated into a compatible site within pB366QE to generatepB366BLAM. The resulting pB366BLAM plasmid encoded the hFbpB-BlaM
fusion protein, with -lactamase inserted between residues 366 and 374 of thehFbpB permease.
Significant hFbpB point mutants which demonstrated null or attenuated irontransport phenotypes were introduced into both pB366BLAM and pACYCHIO(control) backgrounds and tested for positive growth on ampicillin-containingmedia. Point mutations were generated in pACYCIO using the Gene-Editormutagenesis kit (Stratagene) and then were PCR amplified and subcloned intothe pB366BLAM plasmid. All mutations were verified by sequencing. Cells weregrown in LB containing 30 �g/ml chloramphenicol to mid-log phase, diluted inLB, and then plated on LB agar containing ampicillin ranging in concentrationsfrom 40 to 100 �g/ml. Growth of the point mutants was monitored and comparedto that of both wild-type pB366BLAM and the pACYCHIO negative controls.
Sequence and structural topology analysis. Optimal alignment of the per-mease amino acid sequences was performed using ClustalW 2.0 (http://align.genome.jp/), and shading was done using AMAS (33). Set 1 included nFbpB (N.gonorrhoeae GenBank gi, 1098688), nFbpB (N. meningitidis gi, 7379559), hFbpB(H. influenzae gi, 619399), SfuB (Serratia marcescens gi, 1173433), YfuB (Yersiniaenterocolitica gi, 619574), and MhFbpB (Mannheimia haemolytica gi, 3978165).Sequence sets 2 to 4 were obtained by BLAST analysis using hFbpB as the searchsequence and selecting those proteins that fit the classification: Set 2, ironsiderophore; Set 3, thiosulfate/molybdenum/putrescine/glycine-betaine; Set 4,oligosaccharide/glycerophosphate permeases. Hydropathy analysis, topologicalorientation, and secondary structural predictions were performed using thePredictProtein database (43).
RESULTS
Multiple-sequence analysis of the FbpB permeases. As astarting point in our analysis of the hFbpB permease, wesought to compare the primary sequences of FbpB homologsto other permeases of the bacterial ABC transporter family.Tam and Saier demonstrated that high-affinity periplasmicbinding proteins, which bind similar solutes but are derivedfrom evolutionarily diverse bacterial sources, exhibit basic se-quence similarities (48). Based on sequence alignments andsolute binding characteristics, the proteins can be divided intoeight general families or clusters. This classification has re-cently been updated to include a ninth family of manganeseand zinc binding proteins (16). By such criteria, the ferricion-binding proteins (FbpAs) were grouped into cluster 1along with proteins that bind oligosaccharides, phosphate, andglycerol-3-phosphate, among others. By contrast, the sid-erophore-iron and vitamin B12 binding proteins were groupedinto cluster 8. This categorization is consistent with the pre-diction that the FbpA proteins share a common anion-bindingprotein predecessor with phosphate binding protein (14). Thedistinction between the FbpA proteins and the siderophorebinding proteins is also consistent with structural and biochem-ical data that indicate that these proteins exhibit distinct bind-ing mechanisms and that the substrates (Fe3� versus Fe3�-siderophore) are chemically diverse (14, 15). A subsequentstudy by Saurin et al. focused on the consensus motifs of themembrane permeases, the only significantly conserved regionsof these proteins (44). In this work, sequence alignments es-tablished the motifs as signatures for permeases of relatedfunction. Here, the FbpB permeases were grouped with per-meases involved in the uptake of molybdenum, thiosulfate,putrescine, and glycine-betaine. In contrast, the FbpB ho-mologs were distinguished from permeases involved in oligo-saccharide, phosphate, and glycerophosphate uptake as well asthose involved in siderophore-iron uptake.
In accordance with the sequence conservation between per-mease subfamilies and in an attempt to highlight residues ofpotential functional relevance, we performed multiple-se-
VOL. 189, 2007 MUTATIONAL ANALYSIS OF hFbpABC TRANSPORTER PERMEASE 5133
quence alignments of the conserved motifs of the FbpB per-meases (Set 1) with members of the siderophore-iron (Set 2),thiosulfate, molybdenum, putrescine, and glycine-betaine (Set3), and oligosaccharide, phosphate and glycerophosphate (Set4) permease families (Fig. 1). The alignments demonstrate thepresence of two glycine residues that are completely conservedamong all the permeases analyzed and strictly conservedamong all known members of the bacterial ABC transporterpermease family (Fig. 1). Immediately preceding these invari-ant glycines are two leucines that are absolutely conservedamong the FbpB permeases (Fig. 1). These leucines are wellconserved among members of Set 2 and Set 3 as well; theonly exceptions are FhuB (E. coli) (Val in motif 2) and
ProW (E. coli) (phenylalanine in both motifs). Alternatively,in Set 4 permeases these leucines are exchanged with aspar-tates, which are completely conserved among members of thisset (Fig. 1). The leucine-to-aspartate covariance within bothmotifs indicates that these residues may have a definitive rolein permease function.
In addition to the aforementioned residues, the FbpBs (Set1) possess a conserved phenylalanine in motif 1 that is sharedamong Set 4 and Set 3 permeases, with the exception of ProW(E. coli) (Leu; note that this residue covaries with the Phenoted above). This phenylalanine is not at all conserved amongSet 2 permeases. A similar scheme is observed with a con-served proline residue found within motif 1. Motif 2 demon-
FIG. 1. Multiple-sequence alignments of the conserved permease motifs from multiple permeases of the ABC transporter family. Set 1 includesthe free Fe3� transport permeases nFbpB (N. gonorrhoeae [N. gon.]), nFbpB (N. meningitidis [N. men.]), hFbpB (H. influenzae [H. inf.]), SfuB(Serratia marcescens [S. mar.]), YfuB (Yersinia enterocolitica [Y. ent.]), and putative MhFbpB (Mannheimia haemolytica [M. Hae.]) (included in theinitial alignment, excluded in subsequent alignments). Set 2 includes the iron-siderophore permeases FepD/G ferric-enterobactin E. coli, FhuBferric-hydroxymate E. coli, FecC/D ferric-dicitrate E. coli, and FatC/D anguillobactin Vibrio anguillarum (V. ang.). Set 3 includes the thiosulfate/molybdenum/putrescine/glycine-betaine permeases CysT/W thiosulfate E. coli, CysT/W thiosulfate N. meningitidis, ModB molybdenum E. coli,PotB/C putrescine H. influenzae, PotB/C putrescine E. coli, and ProW glycine-betaine E. coli. Set 4 includes the oligosaccharide/glycerophosphatepermeases MalF/G maltose Salmonella enterica serovar Typhimurium (S. typ.), MalF/G maltose E. coli, MalF/G Pseudomonas aeruginosa (P. aer.),MalF/G Vibrio cholerae (V. cho.), UgpA/E glycerophosphate E. coli, MalF/G Thermococcus litoralis (T. lit.), MalC/D maltose Streptococcuspneumoniae (S. pneu.), CymF/G Klebsiella oxytoca (K. oxy.), MsmF/G raffinose-melibiose Streptococcus mutans (S. mut), and putative MalF/G1/G2Streptomyces coelicolor (S. coe.). The numbered sequences of the hFbpB motifs are denoted above the alignments. Asterisks indicate residues inhFbpB that have been targeted for mutagenesis. Homologous residues are enclosed in rectangles, while identical residues are shaded black.
strates an analogous trend regarding conservation of a keyproline, whereas SfuB and YfuB of Set 1 possess arginineresidues at this site.
Mutational analysis of conserved permease residues. Site-directed mutagenesis was performed to probe the functionalrelevance of conserved hFbpB residues. The invariant hFbpBglycine residues, Gly155 of motif 1 and Gly418 of motif 2, wereinitially replaced with the small aliphatic residue alanine, andeffects on transporter function were measured using the radio-labeled Fe3� transport assay as described in Materials andMethods (7). The H-1443/pBR322 (vector-only control) cellsdemonstrated a minimal level of iron uptake, consistent withthe inability of this strain to transport iron under defined assayconditions, with Fe(NTA)2 as a supplement (Fig. 2). TheH-1443/pAHI�C control cells demonstrated a low-level in-crease in signal over time (4.96 0.78 pmol Fe/109 cells at 7min [23.7% of pAHIO results]) (Fig. 2). This result is not dueto functional hFbpABC transport; rather, it is due to bindingof labeled 55Fe(NTA)2 to hFbpA within the periplasm ofH-1443/pAHI�C cells (Fig. 2B), as detailed in a previous study(7). Nonetheless, this control served as the baseline for pro-ductive transport in these experiments. Wild-type controlH-1443/pAHIO cells demonstrated a high-level time-depen-dent increase in signal (20.95 2.69 pmol Fe/109 cells at 7min) (Fig. 2). Transport activities of the pAHIO and pAHI�Ccontrols were similar to those reported previously, and hFbpA
protein expression levels were indistinguishable (data notshown) (7).
Conserved glycine mutations in motifs 1 and 2. The G155Amutant demonstrated transport levels only slightly lower thanthose of wild-type control cells (17.08 0.65 pmol Fe/109 cellsat 7 min) (Fig. 3A; Table 2). This result was unexpected,considering this glycine is completely conserved among all per-meases studied to date. Furthermore, investigations in othertransport systems have demonstrated that mutation of the in-variant glycine on either conserved motif leads to altered trans-port activity (32). To verify this result, several additional mu-tations were tested, including replacement with a largeraliphatic residue (G155V) and a charged residue (G155E).The G155V mutation resulted in transport activity that wassimilar to that of the wild type (19.09 0.83 pmol Fe/109 cellsat 7 min), while the G155E mutation resulted in slightly higherlevels of activity than that of the wild type (22.68 1.40 pmolFe/109 cells at 7 min) (Fig. 3A; Table 2); both levels werewithin standard errors of the wild-type control (Fig. 3A). Theseobservations demonstrate that mutation of Gly155 has no sig-nificant effect on activity, and the identity of the residue haslittle consequence on proper permease function according to
FIG. 2. Radiolabeled 55Fe3� transport assay. (A) Cells grown onNBamp100dip75 were washed and incubated at 37°C in iron-free M9media supplemented with 1 �M 55Fe3�(NTA)2. Samples were re-moved and subjected to filtration, and counts per minute were mea-sured. Radiolabeled iron uptake is plotted versus time. Each strain wastested in triplicate; error bars represent standard errors. (B) Cartoondepiction of the iron transport assay controls shown in panel A. On theleft, H-1443/pBR322 is a vector-only control. In the middle, H-1443/pAHI�C is an FbpA-only control that is missing a functional ABCtransport complex (�FbpC). On the right, H-1443/pAHIO is a wild-type control expressing a functional FbpABC transporter that canmobilize Fe3�(NTA)2 from the periplasm to the cytosol.
FIG. 3. 55Fe3� transport assay results of conserved permease motifmutations (G155 and G418). (A) The conservative mutation G155Aresults in slightly diminished transport activity compared to that ofwild-type pAHIO. The more severe mutations G155V and G155Eresult in transport activity that is similar to that of the wild type (withinstandard errors). These results indicate that mutation of the invariantglycine on motif 1 has no discernible effect on activity. (B) The con-servative mutation G418A results in an approximately twofold de-crease in iron uptake (53.5% of wild-type uptake at 7 min), indicatingthat mutation of the invariant glycine on motif 2 has a significant effecton activity.
VOL. 189, 2007 MUTATIONAL ANALYSIS OF hFbpABC TRANSPORTER PERMEASE 5135
this assay. By contrast, the G418A mutation in motif 2 led to asignificant reduction of iron transport (12.49 2.97 pmol Fe/109 cells at 7 min) compared to that of wild-type H-1443/pAHIO cells (23.33 2.17 pmol Fe/109 cells at 7 min) (Fig. 3B;Table 2). Thus, this conserved glycine in motif 2 is required forproper permease function.
Conserved leucine mutations in motifs 1 and 2. To probe thesignificance of the conserved leucine residues immediately pre-ceding the glycines, the L154D and L417D mutations wereconstructed and tested for transport. Aspartate substitutionswere made at these sites to directly address the observationthat the conserved leucines among sets 1, 2, and 3 are ex-changed for conserved aspartates among Set 4 permeases. TheL154D mutation in motif 1 demonstrated significantly loweractivity levels than the wild type (10.58 1.36 pmol Fe/109
cells at 7 min) (Fig. 4A; Table 2), indicative of attenuated irontransport. The L417D mutation in motif 2 exhibited dramati-cally lower activity levels than the wild type (6.63 2.04 pmolFe/109 cells at 7 min) (Fig. 4B; Table 2). This latter activity issimilar in magnitude to that of the H-1443/pAHI�C control(5.84 1.44 pmol Fe/109 cells at 7 min) and is indicative of nulliron transport.
Other mutations of conserved residues in motifs 1 and 2.Several other conserved residues were targeted for mutationand had minimal effects on transport activity: E149Q (20.11 1.42 pmol Fe/109 cells at 7 min), S153A (22.92 2.52 pmolFe/109 cells at 7 min), F162L (24.36 1.45 pmol Fe/109 cellsat 7 min), and P168A (26.2 3.59 pmol Fe/109 cells at 7 min).All values were within standard errors of H-1443/pAHIO cells(data not shown).
Growth medium iron transport and phenotype summary ofmutants. To substantiate the results of the radiolabeled irontransport assay, further qualitative and semiquantitative assays
measuring growth on complex media in the presence of Fe3�
chelators were performed. The first such assay measuredgrowth or lack of growth on NB supplemented with 200 �MDip, an avid iron chelator. This assay was used in the molecularcloning of the sfuABC, hitABC, and fbpABC operons in E. colistrain H-1443 and has been used successfully in recent comple-mentation experiments (1, 2, 7, 8). The second assay, devel-oped specifically for the current study, measured growth rescueby supplemental Fe(NTA)2 on media containing the Fe3�-specific chelator CDTA. This assay is a more specific measureof Fe3� uptake as, unlike Dip, which is predominantly an Fe2�
chelator, CDTA coordinates Fe3� with high affinity (FeCDTA1�
log � 28; FeOHCDTA2� log � 19) (42). The results ofthese assays along with results of the radiolabeled transportassay are summarized in Table 2. The combined data indicatethat the L154D mutation results in attenuated iron transport,L417D results in null transport, G418A results in attenuatedtransport, and all others result in wild-type iron transport ac-tivity.
Gallium resistance screen for hFbpB permease mutations.Rather than undertake a large-scale site-directed mutagenesiseffort on a protein lacking significant sequence conservation orhot spots of interest, we sought alternative approaches in pin-pointing further functionally significant residues. In a previousstudy, we demonstrated that several metals inhibited thegrowth of H-1443 E. coli cells (7). Interestingly, gallium (Ga3�)
FIG. 4. 55Fe3� transport assay results of conserved permease motifmutations (L154 and L417). (A) The motif 1 L154D mutation resultsin an approximately twofold decrease in transport activity (50.5% ofwild-type uptake at 7 min). (B) The motif 2 L417D mutation results inan approximately fourfold decrease in activity (28.3% of that at 7 min);this level is similar to that of the pAHI�C control.
TABLE 2. Effects of motif 1 and 2 mutations on transport
a Growth assay of cells plated on NBamp100dip200 (n � 3; averaged).b CDTA Fe(NTA)2 growth rescue assay. Percentage of growth determined as
follows: (X � pAHI�C)/(pAHIO � pAHI�C), where X is the mutant (n � 3;averaged).
c Radiolabeled Fe55(NTA)2 transport assay. Percentage of transport at the7-min time point was determined as follows: (X � pAHI�C)/(pAHIO �pAHI�C), where X is the mutant (n � 3; averaged).
d Each strain was characterized through summation of assay percentages andcomparison to the wild-type (WT) control. Total percentages of �50% of wildtype qualify as attenuated; �25% of the WT qualify as null transport.
toxicity was specific to H-1443/pAHIO cells (H-1443/pBR322and H-1443/pAHI�C cells were unaffected), thus providing acorrelate between hFbpABC transport and gallium-inducedtoxicity. Using a radiolabeled Ga67(NTA)2 transport assay,we subsequently demonstrated direct Ga3� uptake by thehFbpABC transporter (7).
During the course of these metal competition experiments,we made an intriguing observation that warranted further in-vestigation. Upon plating on Ga3�-containing medium(NBamp100dip75Ga100), H-1443/pAHIO cells exhibited agrowth-suppressed microcolony phenotype, indicative ofGa3�-induced toxicity. However, approximately 1 � 10�8 col-onies (1 to 10 CFU/plate) exhibited a larger macrocolony phe-notype among a lawn of microcolony growth. We hypothesizedthat these macrocolonies had developed a mutation(s), per-haps within the hFbpABC transporter, that rendered thecells insensitive to Ga3� toxicity. As detailed in Materials andMethods, an initial putative mutant was taken through thescreening protocol, and the following criteria were satisfied:the gallium-resistant phenotype remained following passage onNBamp100dip75Ga100 plates, the mutation did not result inmajor insertions or deletions within the pAHIO plasmid, andthe mutation did not cause alterations in hFbpA expression orsecretion. Upon sequencing the hitB and hitC genes, a single-
nucleotide missense mutation was identified within hitB, whichtranslated into a single-amino-acid point mutation (V497I). Noother mutations were found, and site-directed mutagenesisback to the wild type (I497V) resulted in complete reversion tothe wild-type gallium-sensitive phenotype (Fig. 5A). Results ofthe radiolabeled iron transport assay demonstrated that theV497I mutation led to a significant effect on iron uptake (10.44 2.39 pmol Fe/109 cells at 7 min) (Fig. 5B; Table 3). The I497Vreverse mutation led to transport activity similar in magnitudeto that of wild-type control cells (23.3 2.5 pmol Fe/109 cellsat 7 min) (Fig. 5B; Table 3).
With proof of concept in hand, we developed a subcloningstrategy to generate a series of further mutations that weredirected to the hitB gene. To limit the extensive screeningprocess involved in authentication of the initial mutation, weimplemented a PCR-based random mutagenesis method whichcreated an enriched starting pool of putative mutants. Thisenrichment, coupled with the subcloning and gallium selectionprotocols described in Materials and Methods, led to the iden-tification of further mutations localized within the hFbpB per-mease. The mutations arose from numerous locations through-out the hitB gene and represented a diverse sampling of aminoacid alterations. The mutants were subjected to the transportassays as shown in Table 3. Subsequently, each mutation wasgenetically reversed to the wild type, which in turn restoredwild-type transport activity (data not shown).
The initial single-site mutation, I174F, demonstrated nulltransport activity, while a second mutation, S146Y, demon-strated attenuated activity (Table 3). A third mutant possesseda double mutation, I174N/S475I. Individual single mutationswere constructed to discern whether individual alterations con-tributed to the null iron transport activity or whether one of themutations was actually a false positive. Indeed, the single
FIG. 5. Growth phenotype of the initial gallium selection mutationV497I. (A) The left side shows that strains plated on NBamp100dip75exhibit similar growth phenotypes. Clockwise from top left are H-1443/pAHIO, H-1443/V497I, H-1443/pAHI�C, and H-1443/I497V. Theright side shows the same strains plated on NBamp100dip75Ga100. TheV497I mutant exhibits an uninhibited growth phenotype, while wild-type (WT) pAHIO and the reverse mutant I497V exhibit growth-suppressed phenotypes in the presence of gallium. (B) 55Fe3� trans-port assay results of the V497I and reverse I497V mutations. TheV497I mutation results in an �2.5-fold decrease in transport activity.The reverse mutation results in activity that is indistinguishable fromthat of wild-type pAHIO.
TABLE 3. Effects of permease mutations on transport
a Growth assay of cells plated on NBamp100dip200 (n � 3; averaged).b CDTA Fe(NTA)2 growth rescue assay. Percentage of growth was determined
as follows: (X � pAHI�C)/(pN�MHIO � pAHI�C), where X is the mutant (n �3; averaged). ND, not determined.
c Radiolabeled Fe55(NTA)2 transport assay. Percentage of transport at the7-min time point was determined as follows: (X � pAHI�C)/(pN�MHIO �pAHI�C), where X is the mutant (n � 3; averaged).
d Each strain was characterized through summation of assay percentages andcomparison to the wild-type (WT) control. Total percentages of �50% of theWT qualify as attenuated; �25% of the WT qualify as null transport.
e Mutations in the pAHIO background tested against the pAHIO wild-typecontrol.
VOL. 189, 2007 MUTATIONAL ANALYSIS OF hFbpABC TRANSPORTER PERMEASE 5137
I174N mutation resulted in wild-type iron transport activity,while the S475I mutation led to a null iron transport phenotype(Table 3). Admittedly, this result was unexpected, as the pre-viously identified mutation, I174F, resulted in null iron trans-port activity. This inconsistency is likely due to the difference insubstituted residues; phenylalanine introduces a large aromaticmoiety which may impart a significant steric or electrostaticeffect, while the polar uncharged asparagine may be well tol-erated at this site.
A fourth point mutation, I383N, exhibited curious activity.Although this mutation led to gallium resistance under selec-tion conditions, iron transport activity was apparently unaf-fected (Table 3). As both Fe3� and Ga3� are trivalent metalsthat share similar ionic radii (Ga3�, 0.62 Å; Fe3�, 0.65 Å),gallium-induced toxicity is normally thought to arise by com-petitive inhibition of Fe3� transport. The I383N result signifiesthat there may actually be subtle differences between themechanisms of Ga3� and Fe3� hFbpABC transport and thatmodest mutations within the permease may create a trans-porter that is selective for one metal over the other. Prelimi-nary experiments suggest that although both metals form sta-ble complexes with hFbpA, the metal-protein interactions aresignificantly different. UV difference spectra suggest weakerbinding for Ga3� to nFbpA in the presence of phosphate (26),and SUPREX analysis of matrix-assisted laser desorption ion-ization–time of flight mass spectra shows that the Ga3�- andFe3�-bound proteins operate in different folding regimens(K. D. Weaver, P. L. Roulhac, M. C. Heymann, M. C. Fitzgerald,T. A. Mietzner, and A. L. Crumbliss, unpublished data). Fur-ther experiments will address whether the Ga3�-bound con-formation of hFbpA is indeed different from the Fe3�-boundform and whether I383N can discriminate between these po-tentially distinct conformations.
Protein expression analysis of FbpB mutants. Clearly, fur-ther investigation of the described residues, through saturationmutagenesis and detailed biochemical assays, is required todelineate their specific roles in the mechanism of transport.Before advancing these experiments further, however, wethought it was essential to develop an assay with which tomeasure permease protein expression levels. As mentionedpreviously, the permease contains an inordinate number ofhydrophobic residues (�65%) and has proven to be particu-larly difficult to track. Recombinant overexpression results incell toxicity preventing isolation by traditional means, and lowexpression coupled with the propensity to adhere to cellularmembranes and to aggregate has precluded reliable visualiza-tion on SDS-PAGE and Western blots. In response to theselimitations, we developed an assay which allowed us to trackfunctional expression through the incorporation of a -lacta-mase (blaM) fusion within the hitB gene.
Using a transposon-based strategy, we generated a -lacta-mase (blaM) gene fusion within pACYCHIO, specifically atnucleotide 1098 within the hitB gene of the hitABC locus.Following the transposon insertion reaction and sequencing toverify the site of insertion, the -lactamase stop codon wasreplaced with a glutamine codon and the noncoding down-stream transposon sequence was deleted. This created an in-frame “sandwich” fusion, with -lactamase inserted betweenhFbpB amino acids 366 and 374. Transposon-mediated inser-tion of the blaM gene included the insertion of upstream mo-
saic sequence, which translated as an 11-residue hydrophobiclinker (LSLIHISTIID) between hFbpB residue 366 and BlaMresidue 1. To accommodate this linker and to negate the effectsof redundant hydrophobic segments, hFbpB residues 367 to373 were removed. The hFbpB-BlaM fusion (expressed fromthe pB366BLAM plasmid) demonstrated resistance to ampi-cillin up to a concentration of 100 �g/ml on LB plates. Thisresult signified that both expression and proper membraneorientation was retained by the hFbpB-BlaM fusion protein,with -lactamase localized to the periplasmic side of the innermembrane. Mutations in hFbpB that resulted in null or atten-uated iron transport phenotypes (as described above) wereintroduced into pACYCHIO (negative control) and pB366BLAM, and growth on LBamp100 was assessed (summarized inTable 1). Results demonstrated that each of the mutant strainstested grew on LBamp100 in a fashion similar to that of wild-typepB366-BLAM, while mutations in the pACYCHIO controlbackground demonstrated no such growth. This indicated thatthe mutations (L154D, L417D, G418A, and Y497I) did notsignificantly alter FbpB permease expression levels comparedto those of the wild type (Table 1). Although this -lactamasefusion assay demonstrated that the point mutations did notsignificantly alter expression or membrane incorporation of thehFbpB-BlaM proteins, this does not rule out the possibilitythat wild-type hFbpB-BlaM expression differs from that ofwild-type hFbpB.
DISCUSSION
A predicted in silica topological model of the permeasedemonstrates the presence of 12 membrane-spanning heliceswith both the N and C termini facing the cytosol (Fig. 6). Thistopology is consistent with previous hydropathy analysis andsatisfies the positive inside rule for polytopic membrane pro-teins (D. S. Anderson and T. A. Mietzner, unpublished data)(50). Four large loops project into the periplasm while severalloops, including two containing the conserved permease mo-tifs, are localized within the cytosol. In accordance with theprimary sequence, there is twofold internal homology betweenthe first and latter halves of the protein (helices 1 to 6 and 7 to12). This twofold homology within a single fused permeasesubunit is similar to that of the FhuB permease from the ferrichydroxymate ABC transporter, which is composed of a singlepermease subunit with 20 putative transmembrane segments(25). These permeases stand in contrast to the majority of ABCtransporter permeases, which are typically composed of twosmaller, similar, or identical subunits that together form afunctional dimer (4, 5, 28). It is reasonable to suggest that thetwo halves of hFbpB are arranged with twofold symmetryaround a centrally located pore.
The recently solved vitamin B12 ABC transporter (BtuC2D2)structure verifies the transmembrane orientation of the per-mease complex and its association with two ATP binding sub-units, as supported by a large body of biochemical evidencefrom multiple ABC transport systems (34). The BtuC per-mease homodimer consists of 20 transmembrane �-helices ar-ranged with pseudo-twofold symmetry around a central poreand periplasmically exposed substrate vestibule. Although thisnumber of transmembrane helices is substantially more thanthe 12 helices predicted for the canonical ABC transporter, the
increased helical content may be required to stabilize a centralchannel associated with B12 translocation that is larger thanthat of smaller substrates (21, 25). Consistent with this, bio-chemical evidence suggests that other well-characterized per-meases, including the maltose (MalFG) and histidine (HisQM)permeases, possess fewer total numbers of membrane-span-ning domains (12 [MalFG] and 10 [HisQM] transmembranehelices) (20, 29). Although the initial topological model of theFbpB permease is consistent overall with this latter group,further biochemical and structural data are needed to verifythis arrangement. Likewise, additional crystal structures willhelp to shed light on the apparent variability of permeasetopologies within the ABC transporter family.
The two consensus motifs of the BtuC2 permease dimer arewell resolved in the BtuCD structure, each forming two shorthelices separated by a hairpin turn. The strong conservation ofthese motifs throughout the ABC transporter family suggeststhat these L loops may represent a conserved structural featureamong ABC transporter permeases. The conserved glycinesallow the sharp bend of the peptide backbone necessary toform the hairpin turn, and examination of the backbone andside-chain contacts between the BtuC permease and the BtuDATP binding proteins provides a basis for understanding thepossible effects of mutations at this and surrounding sites.Moreover, alignment of the BtuC conserved permease motifwith the hFbpB motifs permits discussion of hFbpB mutations(Fig. 6). The conserved leucines in hFbpB, Leu154 and
Leu417, align with conserved leucines within BtuC (Leu216from each subunit). In BtuC, these residues make contact withseveral hydrophobic side chains of the BtuD ATP bindingprotein, including Leu96. Importantly, Leu96 in BtuD alignswith residue Phe508 in the eukaryotic cystic fibrosis transmem-brane conductance regulator (CFTR) (34). Deletion of thisresidue in CFTR is the molecular basis of 70% of cystic fibrosiscases, pointing out an obvious functional role for this hydro-phobic interaction. From these observations, it seems likelythat mutation of the hFbpB conserved leucine residues (suchas the aspartate substitutions shown in the present work) maydestabilize similar hydrophobic interactions, thereby giving riseto altered transport function, perhaps by uncoupling hFbpBFe3� permeation from hFbpC ATP hydrolysis.
The conserved glycines in BtuC (G217 in both subunits) donot seem to contribute to specific interactions with the BtuDATP binding proteins. Rather, it seems the flexibility this res-idue imparts to the backbone orientation is the most relevantfeature. By comparison, mutation of G155 in hFbpB wouldlikely be tolerated as long as the introduced side chain can beaccommodated without perturbing surrounding contacts. It isnot clear at this time if the backbone flexibility and resultinghairpin turn are preserved through the G155 mutations.Clearly, mutation of the G418 residue abolishes an importantstructural characteristic of this turn and hence the function ofthe motif.
Interestingly, several of the gallium mutations localize to
FIG. 6. Topological model of the hFbpB membrane permease. Shaded rectangles represent the 12 putative transmembrane �-helices. Residuesthat are completely conserved among the free Fe3� permeases are indicated with squares. Residues comprising the conserved permease motifs areshaded gray. Residues identified by mutagenesis are shaded black, and those mutations resulting in affected iron transport activity are labeled. Thebottom portion shows alignment between the hFbpB conserved permease motifs and the motif from the single subunit of the vitamin B12 permeaseBtuC, indicating conservation of the leucine and glycine residues as described in Discussion.
VOL. 189, 2007 MUTATIONAL ANALYSIS OF hFbpABC TRANSPORTER PERMEASE 5139
sequence directly upstream or downstream of the conservedmotifs, namely, within the adjoining transmembrane segments(I174F and I383N) or intervening sequence (S146Y). One ex-planation for this is that these transmembrane segments serveto anchor the conserved motifs in a productive orientationwithin the membrane. Furthermore, these domains may servean important transmembrane signaling function, linkinghFbpA binding with hFbpC ATP hydrolysis. The S475I muta-tion resides on a periplasmic loop segment that may form aportion of the interaction site with the FbpA protein. Futureexperiments will focus on more clearly delineating the roles ofresidues within the conserved motifs and adjoining transmem-brane domains in the transport mechanism.
The V497I mutation demonstrates that even modest permu-tation of a permease transmembrane domain (the addition ofa methyl group to the Val side chain) can result in a significanteffect on transport. Although such a subtle chemical changecoupled with a large functional effect is startling, specific ali-phatic residues have been shown to play key roles in the gatingprocesses of transporters and channels (35, 38, 41, 49). Fur-thermore, the Cystic Fibrosis Mutation Database lists fourreplacements of a valine by an isoleucine in CFTR, one ofwhich (V1147I) is located in transmembrane segment five ofMSD2 (31). Whether the hFbpB V497I mutation translates toa long-range effect on the FbpA binding site or affects thepermeation pathway through a local structural perturbationawaits further biochemical analysis.
As mentioned in the Results, metal-protein binding studiesare consistent with significantly weaker sequestration of Ga3�
than Fe3� by FbpA. Furthermore, from the vantage point ofthe protein, the folded protein operates in a different regimenwhen Ga3� is bound than when Fe3� is bound (K. D. Weaveret al., unpublished). These preliminary data suggest that theFbpA-Ga3� coordination environment is significantly differentfrom that of FbpA-Fe3�. Such differences may translate toimportant distinctions in the transport pathways of these twometals. Future studies will investigate the physical basis of theseinteractions and transport mechanisms in more detail.
The gallium selection screen provides a powerful approachto identifying functionally significant residues. We foresee thatthis technique will be useful in locating further residues withinthe FbpB permease, a protein that is apparently missingstretches of conserved sequence such as metal binding motifswhich have been identified in other metal permeases (36, 46).Furthermore, this approach can be adapted to identify func-tionally relevant residues within the FbpC and FbpA proteins,the latter of which can be directly mapped to the hFbpA holoand apo crystal structures. With such a screen in place andsensitive functional assays now available, further probing intothe structure and function of the transporter will offer a moredetailed picture of the transport mechanism.
ACKNOWLEDGMENTS
We thank K. G. Vaughan for technical and editorial assistance.This work was supported in part by The Department of Molecular
Genetics and Biochemistry, the National Institutes of Health grantR29 A132226 (T.A.M.), and the National Science Foundation grantCHE-0418006 (A.L.C.).
REFERENCES
1. Adhikari, P., S. A. Berish, A. J. Nowalk, K. L. Veraldi, S. A. Morse, and T. A.Mietzner. 1996. The fbpABC locus of Neisseria gonorrhoeae functions in theperiplasm-to-cytosol transport of iron. J. Bacteriol. 178:2145–2149.
2. Adhikari, P., S. D. Kirby, A. J. Nowalk, K. L. Veraldi, A. B. Schryvers, andT. A. Mietzner. 1995. Biochemical characterization of a Haemophilus influ-enzae periplasmic iron transport operon. J. Biol. Chem. 270:25142–25149.
3. Albrecht-Gary, A. M., and A. L. Crumbliss. 1998. The coordination chem-istry of siderophores: thermodynamics and kinetics of iron chelation andrelease. In A. Sigel and H. Sigel (ed.), Metal ions in biological systems, vol.35. M. Dekker, Inc., New York, NY.
4. Ames, G. F.-L. 1986. Bacterial periplasmic transport systems: structure,mechanism, and evolution. Annu. Rev. Biochem. 55:397–425.
5. Ames, G. F., C. S. Mimura, S. R. Holbrook, and V. Shyamala. 1992. TrafficATPases: a superfamily of transport proteins operating from Escherichia colito humans. Adv. Enzymol. Relat. Areas Mol. Biol. 65:1–47.
6. Anderson, B. F., H. M. Baker, G. E. Norris, D. W. Rice, and E. N. Baker.1989. Structure of human lactoferrin: crystallographic structure analysis andrefinement at 2.8 Å resolution. J. Mol. Biol. 209:711–734.
7. Anderson, D. S., P. Adhikari, A. J. Nowalk, C. Y. Chen, and T. A. Mietzner.2004. The hFbpABC transporter from Haemophilus influenzae functions as abinding-protein-dependent ABC transporter with high specificity and affinityfor ferric iron. J. Bacteriol. 186:6220–6229.
8. Angerer, A., S. Gaisser, and V. Braun. 1990. Nucleotide sequences of thesfuA, sfuB, and sfuC genes of Serratia marcescens suggest a periplasmic-binding-protein-dependent iron transport mechanism. J. Bacteriol. 172:572–578.
9. Bailey, S., R. W. Evans, R. C. Garratt, B. Gorinsky, S. Hasnain, C. Hors-burgh, H. Jhoti, P. F. Lindley, A. Mydin, R. Sarra, and J. L. Watson. 1988.Molecular structure of serum transferrin at 3.3-Å resolution. Biochemistry27:5804–5812.
10. Boukhalfa, H., D. S. Anderson, T. A. Mietzner, and A. L. Crumbliss. 2003.Kinetics and mechanism of iron release from the bacterial ferric bindingprotein nFbp: exogenous anion influence and comparison with mammaliantransferrin. J. Biol. Inorg. Chem. 8:881–892.
11. Boukhalfa, H., and A. L. Crumbliss. 1998. Chemical aspects of siderophoremediated iron transport. BioMetals 15:325–339.
12. Braun, V., R. Gross, W. Koster, and L. Zimmermann. 1983. Plasmid andchromosomal mutants in the iron(III)-aerobactin transport system of Esch-erichia coli. Use of streptonigrin for selection. Mol. Gen. Genet. 192:131–139.
13. Bruns, C. M., D. S. Anderson, K. G. Vaughan, P. A. Williams, A. J. Nowalk,D. E. McRee, and T. A. Mietzner. 2001. Crystallographic and biochemicalanalyses of the metal-free Haemophilus influenzae Fe3�-binding protein.Biochemistry 40:15631–15637.
14. Bruns, C. M., A. J. Nowalk, A. S. Arvai, M. A. McTigue, K. G. Vaughan, T. A.Mietzner, and D. E. McRee. 1997. Structure of Haemophilus influenzaeFe(�3)-binding protein reveals convergent evolution within a superfamily.Nat. Struct. Biol. 4:919–924.
15. Clarke, T. E., S. Y. Ku, D. R. Dougan, H. J. Vogel, and L. W. Tari. 2000. Thestructure of the ferric siderophore binding protein FhuD complexed withgallichrome. Nat. Struct. Biol. 7:287–291.
16. Claverys, J. P. 2001. A new family of high-affinity ABC manganese and zincpermeases. Res. Microbiol. 152:231–243.
17. Cornelissen, C. N., and P. F. Sparling. 1994. Identification of receptor-mediated transferrin-iron uptake mechanism in Neisseria gonorrhoeae. Meth-ods Enzymol. 235:356–363.
18. Crosa, J. 1989. Genetics and molecular biology of siderophore-mediatediron transport in bacteria. Microbiol. Rev. 53:517–530.
19. Dassa, E., and M. Hofnung. 1985. Sequence of gene malG in E. coli k12:homologies between integral membrane components from binding protein-dependent transport systems. EMBO J. 4:2287–2293.
20. Dassa, E., and S. Muir. 1993. Membrane topology of MalG, an inner mem-brane protein from the maltose transport system of Escherichia coli. Mol.Microbiol. 7:29–38.
21. Davidson, A. L. 2002. Structural biology. Not just another ABC transporter.Science 296:1038–1040.
22. Dhungana, S., D. S. Anderson, T. A. Mietzner, and A. L. Crumbliss. 2005.Kinetics of iron release from ferric binding protein (FbpA): mechanisticimplications in bacterial periplasm-to-cytosol Fe3� transport. Biochemistry44:9606–9618.
23. Dhungana, S., C. H. Taboy, D. S. Anderson, K. G. Vaughan, P. Aisen, T. A.Mietzner, and A. L. Crumbliss. 2003. The influence of the synergistic anionon iron chelation by ferric binding protein, a bacterial transferrin. Proc. Natl.Acad. Sci. USA 100:3659–3664.
24. Gabricevic, M., D. S. Anderson, T. A. Mietzner, and A. L. Crumbliss. 2004.Kinetics and mechanism of iron(III) complexation by ferric binding protein:the role of phosphate. Biochemistry 43:5811–5819.
25. Groeger, W., and W. Koster. 1998. Transmembrane topology of the twoFhuB domains representing the hydrophobic components of bacterial ABC
transporters involved in the uptake of siderophores, haem and vitamin B12.Microbiology 144:2759–2769.
26. Guo, M., I. Harvey, W. Yang, L. Coghill, D. J. Campopiano, J. A. Parkinson,R. T. MacGillivray, W. R. Harris, and P. J. Sadler. 2003. Synergistic anionand metal binding to the ferric ion-binding protein from Neisseria gonor-rhoeae. J. Biol. Chem. 278:2490–2502.
27. Harris, D. C., A. L. Rinehart, D. Hereld, R. W. Schwartz, F. P. Burke, andA. P. Salvador. 1985. Reduction potential of iron in transferrin. Biochim.Biophys. Acta 838:295–301.
28. Higgins, C. F. 1992. ABC transporters: from microorganisms to man. Annu.Rev. Cell Biol. 8:67–113.
29. Kerppola, R. E., and G. F. Ames. 1992. Topology of the hydrophobic mem-brane-bound components of the histidine periplasmic permease. Compari-son with other members of the family. J. Biol. Chem. 267:2329–2336.
30. Kerppola, R. E., V. K. Shyamala, P. Klebba, and G. F. Ames. 1991. Themembrane-bound proteins of periplasmic permeases form a complex. Iden-tification of the histidine permease HisQMP complex. J. Biol. Chem. 266:9857–9865.
31. Kilinc, M. O., V. N. Ninis, E. Dagli, M. Demirkol, F. Ozkinay, Z. Arikan, O.Cogulu, G. Huner, F. Karakoc, and A. Tolun. 2002. Highest heterogeneityfor cystic fibrosis: 36 mutations account for 75% of all CF chromosomes inTurkish patients. Am. J. Med. Genet. 113:250–257.
32. Koster, W., and B. Bohm. 1992. Point mutations in two conserved glycineresidues within the integral membrane protein FhuB affect iron(III) hydrox-amate transport. Mol. Gen. Genet. 232:399–407.
33. Livingstone, C. D., and G. C. Barton. 1993. Protein sequence alignments: astrategy for the hierarchical analysis of residue conservation. Comput. Appl.Biol. Sci. 9:745–756.
34. Locher, K. P., A. T. Lee, and D. C. Rees. 2002. The E. coli BtuCD structure:a framework for ABC transporter architecture and mechanism. Science296:1091–1098.
35. Lummis, S. C. 2004. The transmembrane domain of the 5-HT3 receptor: itsrole in selectivity and gating. Biochem. Soc. Trans. 32:535–539.
36. Maguire, M. E. 2006. The structure of CorA: a Mg(2�)-selective channel.Curr. Opin. Struct. Biol. 16:432–438.
37. Mietzner, T. A., S. B. Tencza, K. G. Vaughan, P. A. Adhikari, and A. J.Nowalk. 1998. Periplasm-to-cytosol free Fe(III) transporters of pathogenicgram-negative bacteria. Curr. Top. Microbiol. Immunol. 225:113–135.
38. Miyazawa, A., Y. Fujiyoshi, and N. Unwin. 2003. Structure and gating mech-anism of the acetylcholine receptor pore. Nature 423:949–955.
39. Nowalk, A., S. Tencza, and T. Mietzner. 1994. Coordination of iron by theferric-iron binding protein of pathogenic Neisseria is homologous to thetransferrins. Biochemistry 33:12769–12775.
40. Nowalk, A. J., K. J. Vaughan, B. Day, S. B. Tencza, and T. A. Mietzner. 1997.Metal dependant conformers of the periplasmic ferric ion binding protein.Biochemistry 36:13054–13059.
41. Perozo, E., and D. C. Rees. 2003. Structure and mechanism in prokaryoticmechanosensitive channels. Curr. Opin. Struct. Biol. 13:432–442.
42. Ringbom, A. 1963. Complexation in analytical chemistry. A guide for thecritical selection of analytical methods based on complexation reactions.Interscience (Wiley), New York, NY.
43. Rost, B., G. Yachdav, and J. Liu. 2003. The PredictProtein server. NucleicAcids Res. 32:W321–W326.
44. Saurin, W., W. Koster, and E. Dassa. 1994. Bacterial binding protein-de-pendent permeases: characterization of distinctive signatures for functionallyrelated integral cytoplasmic membrane proteins. Mol. Microbiol. 12:993–1004.
45. Schryvers, A. B., and I. Stojiljkovic. 1999. Iron acquisition systems in thepathogenic Neisseria. Mol. Microbiol. 32:1117–1123.
46. Stearman, R., D. S. Yuan, Y. Yamaguchi-Iwai, R. D. Klausner, and A.Dancis. 1996. A permease-oxidase complex involved in high-affinity ironuptake in yeast. Science 271:1552–1557.
47. Taboy, C. H., K. G. Vaughan, T. A. Mietzner, P. Aisen, and A. L. Crumbliss.2001. Fe3� coordination and redox properties of a bacterial transferrin.J. Biol. Chem. 276:2719–2724.
48. Tam, R., and M. H. Saier, Jr. 1993. Structural, functional, and evolutionaryrelationships among extracellular solute-binding receptors of bacteria. Mi-crobiol. Rev. 57:320–346.
49. Van den Berg, B., W. M. Clemons, Jr., I. Collinson, Y. Modis, E. Hartmann,S. C. Harrison, and T. A. Rapoport. 2004. X-ray structure of a protein-conducting channel. Nature 427:36–44.
50. von Heijne, G. 1992. Membrane protein structure prediction. Hydrophobic-ity analysis and the positive-inside rule. J. Mol. Biol. 225:487–494.
51. Zak, O., A. Leibman, and P. Aisen. 1983. Metal-binding properties of asingle-sited transferrin fragment. Biochim. Biophys. Acta 742:490–495.
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