The obligate human pathogen Haemophilus influenzae utilizes a siderophore-independent (free) Fe3+ transport system to obtain this essential element from the host iron-binding protein transferrin. The hFbpABC transporter is a binding protein-dependent ABC transporter that functions to shuttle (free) Fe3+ through the periplasm and across the inner membrane of H. influenzae. This investigation focuses on the structure and function of the hFbpB membrane permease component of the transporter, a protein that has eluded prior characterization. Based on multiple-sequence alignments between permease orthologs, a series of site-directed mutations targeted at residues within the two conserved permease motifs were generated. The hFbpABC transporter was expressed in a siderophore-deficient Escherichia coli background, and effects of mutations were analyzed using growth rescue and radiolabeled 55Fe3+ transport assays. Results demonstrate that mutation of the invariant glycine (G418A) within motif 2 led to attenuated transport activity, while mutation of the invariant glycine (G155A/V/E) within motif 1 had no discernible effect on activity. Individual mutations of well-conserved leucines (L154D and L417D) led to attenuated and null transport activities, respectively. As a complement to site-directed methods, a mutant screen based on resistance to the toxic iron analog gallium, an hFbpABC 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. Significant residues were mapped to a topological model of the hFbpB permease, and the implications of mutations are discussed in light of structural and functional data from related ABC transporters.
Pathogenic bacteria employ a number of acquisition strategies in competition for host iron (Fe3+). Siderophore-dependent iron transport is a widely used strategy that involves the secretion of organic siderophore molecules that compete for iron bound to the high-affinity host transferrin (Tf) and lactoferrin (Lf) proteins (18). Fe3+-siderophore complexes are subsequently recovered by the bacteria through the activity of siderophore-specific surface receptors and transporters. As an alternate strategy, several gram-negative pathogens, including Neisseria gonorrhoeae, Neisseria meningitidis, and Haemophilus influenzae, utilize a siderophore-independent (free) Fe3+ transport system (37). In lieu of siderophores, this system employs surface receptors that bind host iron-binding proteins Tf and Lf directly (17, 45). Fe3+ is removed and transported across the outer membrane by the Tf/Lf-binding protein complex (TbpA/TbpB or LbpA/LbpB) using an energy-dependent mechanism mediated by TonB and associated proteins ExbB and ExbD. Naked (free) Fe3+ is transported from the periplasm to the cytosol by the FbpABC transporter, which is composed of a periplasmic ferric ion-binding protein (FbpA) and an inner membrane ABC transporter consisting of a membrane permease (FbpB) and an ATP-binding protein (FbpC) (37).
A fundamental difference between siderophore-associated and free iron transport involves the chemical nature of the substrate. In the former, iron is bound and transported into the cytosol as an intact Fe3+-siderophore complex. Coordination of iron in this complex serves a dual purpose of assigning molecular identity to the Fe3+-siderophore for recognition by the appropriate receptors and transport proteins as well as shielding Fe3+ from hydrolysis during transit into the cell (3, 11). In contrast, the free iron transport system lacks any known siderophore; rather, Fe3+ is removed directly from Tf or Lf and transported in free form via direct interaction with specific receptors and Fe3+-binding proteins. These transport proteins must exhibit high specificity and affinity for Fe3+ to avoid insolubility and reactivity, yet they must readily exchange the metal during the process of transport.
Our investigations on the homologous FbpABC transporters from H. influenzae hFbpABC (also referred to in the literature as HitABC) and N. gonorrhoeae (nFbpABC) have focused largely on the processes of high-affinity Fe3+ binding and release by the FbpA periplasmic binding proteins. X-ray structures of H. influenzae hFbpA, in both Fe3+-bound (holo) and Fe3+-free (apo) conformations, have provided insight into the Fe3+ coordination complex and the structural transitions involved in substrate binding and release (13, 14). Thermodynamic and kinetic investigations on N. gonorrhoeae nFbpA have shed light on the mechanism of Fe3+ coordination, particularly with respect to the effects of ternary anions and binding site mutations (10, 22-24, 39, 40, 47). It is clear that the FbpA proteins are structural and functional paralogs of the mammalian Tf single iron-binding lobes. In addition to sharing a similar tertiary structure, the FbpA proteins possess a similar set of Fe3+-coordinating residues and undergo a large-scale central hinge rotation upon binding Fe3+ similar to that of Tf. Although Tf and nFbpA demonstrate similar Fe3+ binding affinities (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 release mechanisms (47, 51). As evident in the crystal structures of these proteins, hFbpA recruits a monodentate PO4 anion and a water to complete the inner coordination sphere of Fe3+, while Tf (and Lf) enlists a bidentate CO32− anion (6, 9, 14). This distinction may be the result of slightly different protein conformations and a larger, more solvent-exposed FbpA Fe3+-binding site. Importantly, binding site differences correlate to increased exchange and lability of the bound anion and a positive shift in redox potential in nFbpA compared to that of Tf (23, 27, 47). These features have direct influence on the stability of bound Fe3+, with potential implications in the mechanism of transport (10, 22, 47).
Recent experiments have demonstrated that the H. influenzae hFbpABC transporter functions as a bona fide binding protein-dependent ABC transporter, employing ATP as an energy source and exhibiting transport rates similar to those of other members of this bacterial ABC transporter family (7). However, the exceptionally high FbpA Fe3+ binding affinity (approximately 1010 to 1012 higher than typical periplasmic binding protein affinities) requires further critical evaluation of FbpABC function and auxiliary processes (anion exchange or redox) that may be involved in the transport process. Clearly, an important event during the transport process is the exchange of Fe3+ from FbpA to the FbpB permease subsequent to transport across the inner membrane. The permease is an ∼500-amino-acid polypeptide proposed to be a polytopic transmembrane protein, forming both a receptor for FbpA and a channel for the passage of Fe3+. The FbpB homologs possess two permease motifs of the template EAA—G———I-LP that are well conserved among the family of bacterial ABC transporter permeases (19, 30, 44). These regions are presumed to reside on cytoplasmically exposed loops that form a mechanical coupling between the energy transduction protein (FbpC) and the membrane transport protein (FbpB). The recent crystal structure of the vitamin B12 ABC transporter (BtuC2D2) verifies a key role for these “L” loop motifs in mediating intimate contact between the permease and ATP binding subunits (34). The hFbpB and nFbpB homologs are highly hydrophobic and toxic when expressed from recombinant sources; thus, despite rigorous isolation efforts, the permease has remained elusive and characterization has been limited to genetic approaches (1).
As a logical progression in our studies of the FbpABC system, we have broadened our focus to include the FbpB permease and its role in the Fe3+ transport process. Expression of the H. influenzae hitABC three-gene operon in the siderophore-deficient H-1443 aroB Escherichia coli strain has served 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 and qualitative assays, to probe the significance of single amino acids within the hFbpB permease. Multiple-sequence alignments between FbpB permease homologs and related ABC transporter permeases served as a basis for a series of site-directed mutations targeted at residues within the conserved permease motifs. A positive selection screen using the Fe3+ analog gallium (Ga3+) was employed to identify additional mutants, which were genetically delineated and subjected to Fe3+ transport analyses. Finally, a topological model of the hFbpB protein is presented, and implications of informative mutations are discussed in light of a hypothetical functional mechanism of the hFbpB permease protein. These investigations represent an important initial step in probing the structure and function of the heretofore unexplored FbpB permease.
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), phenylalanine, tyrosine, tryptophan, ferric nitrate, and gallium nitrate were all purchased 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 electrophoresis (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). Taq polymerase was purchased from Roche (Basel, Switzerland). Restriction enzymes were purchased from New England Biolabs (Beverly, MA). Nitrocellulose filters and scintillation fluid were purchased from Fisher (Pittsburgh, PA). E. coli strains and plasmids were obtained as described (Table 1).
Dip sensitivity, CDTA Fe3+(NTA)2 growth rescue, and radiolabeled iron transport assays.
Assays measuring growth sensitivity to the iron chelator Dip were performed under the following conditions. Single colonies were used to inoculate NB supplemented with 100 μg/ml ampicillin (NBamp100), and cultures were incubated at 37°C with shaking at 250 rpm. Cells at mid-log growth (optical density at 600 nm, 0.5) were centrifuged, suspended in NBamp100 containing 200 μM Dip (NBamp100dip200) top agar (0.7%), and seeded at 106 CFU/plate on NBamp100dip200 agar. Plates were incubated in a water-jacketed incubator at 37°C, 5% CO2 for 16 h. Following growth, plates were digitally scanned. Growth was evaluated as follows: −, no colonies; +, pinpoint colonies.
Assays measuring exogenous Fe3+(NTA)2 growth rescue on media containing the Fe3+-specific chelator CDTA were performed under the following conditions. Single colonies were used to inoculate NBamp100, and cultures were grown at 37°C with shaking at 250 rpm. Mid-log cells were centrifuged, suspended in NBamp100 containing 600 μM CDTA (NBamp100CDTA600) top agar, and seeded at 106 CFU/plate on NBamp100CDTA600 agar. Sterile supplement disks (6 mm) loaded with 100 mM Fe3+(NTA)2 (10 μl) were applied, and the plates were incubated as described above. Following growth, plates were digitally scanned 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, 0.5 mg/ml NaCl, 1 mg/ml NH4C, 1 mM MgSO4, 0.1 mM CaCl2) was treated with 100 mg/liter Chelex-100 and supplemented with 2 mg/ml glucose, 0.1 mg/ml phenylalanine, 0.1 mg/ml tyrosine, and 0.1 mg/ml tryptophan prior to use. Fresh transformants were grown in LB supplemented with 100 μg/ml ampicillin (LBamp100), seeded on NBamp100dip75, and grown at 37°C for 18 h. Cells were washed 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 concentration of 1 μM. At designated time points aliquots were removed, filtered through 0.45-μm-pore-size nitrocellulose filters, and washed with 100 mM LiCl. Activity (in counts per minute) was measured using a liquid scintillation counter (Packard, Billerica, MA).
The Gene Editor System (Promega, Madison, WI) was used to perform site-directed mutagenesis. Plasmid DNA was isolated from single putative mutants using standard miniprep techniques and was subjected to automated DNA sequencing using an ABI Prism 3100 sequencer operated by the Department of Molecular Genetics and Biochemistry Shared Resources Facility at 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 in LBamp100. Cells were centrifuged, suspended in LBamp100, and seeded at 109 CFU/plate in parallel on NBamp100dip75 and NBamp100dip75 containing 100 μM Ga(NO3) (NBamp100dip75Ga100) agar. Plates were incubated as described above. Growth of these cells on NBamp100dip75 resulted in slightly smaller colonies than those of pAHIO/H-1443 grown on NBamp100 (without Dip), while the same cells on Ga3+-containing medium (NBamp100dip75Ga100) exhibited a highly growth-suppressed microcolony phenotype. On NBamp100dip75Ga100 plates, however, approximately 1 × 10−8 colonies (1 to 10 CFU/plate) exhibited a larger macrocolony phenotype among a lawn of microcolony growth. One of these gallium-resistant macrocolonies was chosen as a putative mutant for further study.
In the original development of the screen, a single putative mutant was selected and grown in LBamp100, and plasmid was isolated using standard miniprep 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 was selected and grown in LBamp100 broth to stationary phase. The culture was divided into three aliquots. Aliquot one was used to isolate plasmid DNA; a 0.5-μg aliquot of this DNA was run on a 1% agarose electrophoresis gel and compared to wild-type pAHIO plasmid. The other two aliquots were subjected to FbpA protein analysis. Aliquot two was centrifuged, washed in cold phosphate-buffered saline (PBS), suspended in 30 mM Tris, pH 8.0, 20% sucrose, 1 mM EDTA, and shaken at 25°C for 5 min. The suspensions were pelleted, suspended in 5 mM ice-cold MgCl2, and shaken for 10 min at 4°C. The suspensions were again pelleted, and the supernatant was saved as the periplasmic osmotic shock fluid. Aliquot three was centrifuged, washed with PBS, suspended in 400 mM Tris, 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 soluble periplasmic fraction and the soluble whole-cell fraction were subjected to SDS-PAGE on a 12% gel. The gels were stained with Coomassie blue and subjected to densitometry using a Kodak Imagestation 1000 (Rochester, NY).
The initial gallium-resistant mutant was screened using the above methods to determine whether (i) the phenotype was linked to the pAHIO plasmid, (ii) the phenotype 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 lack of expression, truncation, or misprocessing of the hFbpA protein. Upon satisfying these criteria, the hitBC locus within the mutant plasmid was subjected to automated DNA sequencing as described above.
Subcloning and random mutagenesis for gallium selection of hFbpB mutants.
Upon establishing conditions for gallium selection of pAHIO mutants, directed mutations localized to the hitB gene were generated by subcloning putative mutant hitB genes into the pAHIO backbone. This required engineering unique restriction sites into the 5′ and 3′ ends of the hitB gene within the pAHIO plasmid. A unique NsiI 5′ hitB site was inserted at 1525 and a MluI site at 3109 was deleted, yielding a unique MluI site at 3064 at the 3′ end of hitB and resulting in the pNΔMHIO plasmid. To enrich the initial pool of putative mutants, a PCR-based random mutagenesis approach was used. The GeneMorph kit (Stratagene, La Jolla, CA) was used along with primers specific to the 5′ and 3′ ends of hitB incorporating NsiI and MluI restriction sites under PCR conditions consistent with low-mutation frequency (0 to 3 mutations/kb). The mutagenic PCR product was restricted with NsiI and MluI, purified, and subcloned into the identical sites within pNΔMHIO. Ligated plasmid was used to transform fresh H-1443 E. coli cells. Putative clones were selected, grown in LBamp100 to mid-log phase, and seeded on NBamp100dip75Ga100 plates. Clones exhibiting positive growth on NBamp100dip75Ga100 were selected and grown in LBamp100 to stationary phase, and plasmid was isolated and used to transform fresh H-1443. Upon passage of the gallium resistance phenotype, mutant DNA was isolated and the hitB locus was subjected to DNA sequencing as described above.
Protein expression analysis.
A transposon-mediated β-lactamase fusion strategy was employed to monitor hFbpB point mutant protein expression levels. Specifically, the EZ-TN5 β-lactamase fusion kit (Epicenter, Madison, WI) was used to generate random insertions within pACYCHIO, a pACYC184-based construct 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 to 70 μg/ml of ampicillin to select for Ampr insertion clones. Putative clones were selected and subjected to HindIII restriction analysis. All clones that demonstrated the presence of the 1.2-kb EZ-TN5 blaM insert and appeared to be unique were submitted for sequencing.
One such Ampr clone which possessed an in-frame blaM insertion at nucleotide 1098 within the hitB gene (designated pB366) was selected and subjected to overlap extension PCR mutagenesis using the NdeI-5′, RevQEc-3′, ForQEc-5′, and BspHI-3′ primers (Table 1). This method replaced the blaM translational stop codon with a glutamine codon (TAA>CAA) and introduced an EcoRV restriction site directly downstream of this missense site. The final 3.7-kb PCR product was digested with NdeI-5′ and BspHI-3′ and ligated into compatible sites within pACYCHIO to generate pB366QE. The downstream portion of the hitB coding region was PCR amplified from the pB366 plasmid using the EcRV5410-5′ and BspHI-3′ primers to generate a 1.9-kb product with an introduced 5′ EcoRV restriction site. This PCR product was digested with EcoRV and BspHI and ligated into a compatible site within pB366QE to generate pB366BLAM. The resulting pB366BLAM plasmid encoded the hFbpB-BlaM fusion protein, with β-lactamase inserted between residues 366 and 374 of the hFbpB permease.
Significant hFbpB point mutants which demonstrated null or attenuated iron transport phenotypes were introduced into both pB366BLAM and pACYCHIO (control) backgrounds and tested for positive growth on ampicillin-containing media. Point mutations were generated in pACYCIO using the Gene-Editor mutagenesis kit (Stratagene) and then were PCR amplified and subcloned into the pB366BLAM plasmid. All mutations were verified by sequencing. Cells were grown in LB containing 30 μg/ml chloramphenicol to mid-log phase, diluted in LB, and then plated on LB agar containing ampicillin ranging in concentrations from 40 to 100 μg/ml. Growth of the point mutants was monitored and compared to that of both wild-type pB366BLAM and the pACYCHIO negative controls.
Sequence and structural topology analysis.
Optimal alignment of the permease 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 (Yersinia enterocolitica gi, 619574), and MhFbpB (Mannheimia haemolytica gi, 3978165). Sequence sets 2 to 4 were obtained by BLAST analysis using hFbpB as the search sequence and selecting those proteins that fit the classification: Set 2, iron siderophore; Set 3, thiosulfate/molybdenum/putrescine/glycine-betaine; Set 4, oligosaccharide/glycerophosphate permeases. Hydropathy analysis, topological orientation, and secondary structural predictions were performed using the PredictProtein database (43).
Multiple-sequence analysis of the FbpB permeases.
As a starting point in our analysis of the hFbpB permease, we sought to compare the primary sequences of FbpB homologs to other permeases of the bacterial ABC transporter family. Tam and Saier demonstrated that high-affinity periplasmic binding proteins, which bind similar solutes but are derived from evolutionarily diverse bacterial sources, exhibit basic sequence similarities (48). Based on sequence alignments and solute binding characteristics, the proteins can be divided into eight general families or clusters. This classification has recently been updated to include a ninth family of manganese and zinc binding proteins (16). By such criteria, the ferric ion-binding proteins (FbpAs) were grouped into cluster 1 along with proteins that bind oligosaccharides, phosphate, and glycerol-3-phosphate, among others. By contrast, the siderophore-iron and vitamin B12 binding proteins were grouped into cluster 8. This categorization is consistent with the prediction that the FbpA proteins share a common anion-binding protein predecessor with phosphate binding protein (14). The distinction between the FbpA proteins and the siderophore binding proteins is also consistent with structural and biochemical data that indicate that these proteins exhibit distinct binding mechanisms and that the substrates (Fe3+ versus Fe3+-siderophore) are chemically diverse (14, 15). A subsequent study by Saurin et al. focused on the consensus motifs of the membrane permeases, the only significantly conserved regions of these proteins (44). In this work, sequence alignments established the motifs as signatures for permeases of related function. Here, the FbpB permeases were grouped with permeases involved in the uptake of molybdenum, thiosulfate, putrescine, and glycine-betaine. In contrast, the FbpB homologs were distinguished from permeases involved in oligosaccharide, phosphate, and glycerophosphate uptake as well as those involved in siderophore-iron uptake.
In accordance with the sequence conservation between permease subfamilies and in an attempt to highlight residues of potential functional relevance, we performed multiple-sequence alignments of the conserved motifs of the FbpB permeases (Set 1) with members of the siderophore-iron (Set 2), thiosulfate, molybdenum, putrescine, and glycine-betaine (Set 3), and oligosaccharide, phosphate and glycerophosphate (Set 4) permease families (Fig. 1). The alignments demonstrate the presence of two glycine residues that are completely conserved among all the permeases analyzed and strictly conserved among all known members of the bacterial ABC transporter permease family (Fig. 1). Immediately preceding these invariant glycines are two leucines that are absolutely conserved among the FbpB permeases (Fig. 1). These leucines are well conserved among members of Set 2 and Set 3 as well; the only 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 aspartates, which are completely conserved among members of this set (Fig. 1). The leucine-to-aspartate covariance within both motifs indicates that these residues may have a definitive role in permease function.
In addition to the aforementioned residues, the FbpBs (Set 1) possess a conserved phenylalanine in motif 1 that is shared among Set 4 and Set 3 permeases, with the exception of ProW (E. coli) (Leu; note that this residue covaries with the Phe noted above). This phenylalanine is not at all conserved among Set 2 permeases. A similar scheme is observed with a conserved proline residue found within motif 1. Motif 2 demonstrates an analogous trend regarding conservation of a key proline, whereas SfuB and YfuB of Set 1 possess arginine residues at this site.
Mutational analysis of conserved permease residues.
Site-directed mutagenesis was performed to probe the functional relevance of conserved hFbpB residues. The invariant hFbpB glycine residues, Gly155 of motif 1 and Gly418 of motif 2, were initially replaced with the small aliphatic residue alanine, and effects on transporter function were measured using the radiolabeled Fe3+ transport assay as described in Materials and Methods (7). The H-1443/pBR322 (vector-only control) cells demonstrated a minimal level of iron uptake, consistent with the inability of this strain to transport iron under defined assay conditions, with Fe(NTA)2 as a supplement (Fig. 2). The H-1443/pAHIΔC control cells demonstrated a low-level increase in signal over time (4.96 ± 0.78 pmol Fe/109 cells at 7 min [23.7% of pAHIO results]) (Fig. 2). This result is not due to functional hFbpABC transport; rather, it is due to binding of labeled 55Fe(NTA)2 to hFbpA within the periplasm of H-1443/pAHIΔC cells (Fig. 2B), as detailed in a previous study (7). Nonetheless, this control served as the baseline for productive transport in these experiments. Wild-type control H-1443/pAHIO cells demonstrated a high-level time-dependent increase in signal (20.95 ± 2.69 pmol Fe/109 cells at 7 min) (Fig. 2). Transport activities of the pAHIO and pAHIΔC controls were similar to those reported previously, and hFbpA protein expression levels were indistinguishable (data not shown) (7).
Conserved glycine mutations in motifs 1 and 2.
The G155A mutant demonstrated transport levels only slightly lower than those of wild-type control cells (17.08 ± 0.65 pmol Fe/109 cells at 7 min) (Fig. 3A; Table 2). This result was unexpected, considering this glycine is completely conserved among all permeases studied to date. Furthermore, investigations in other transport systems have demonstrated that mutation of the invariant glycine on either conserved motif leads to altered transport activity (32). To verify this result, several additional mutations were tested, including replacement with a larger aliphatic residue (G155V) and a charged residue (G155E). The G155V mutation resulted in transport activity that was similar to that of the wild type (19.09 ± 0.83 pmol Fe/109 cells at 7 min), while the G155E mutation resulted in slightly higher levels of activity than that of the wild type (22.68 ± 1.40 pmol Fe/109 cells at 7 min) (Fig. 3A; Table 2); both levels were within standard errors of the wild-type control (Fig. 3A). These observations demonstrate that mutation of Gly155 has no significant effect on activity, and the identity of the residue has little consequence on proper permease function according to this assay. By contrast, the G418A mutation in motif 2 led to a significant 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 for proper permease function.
Conserved leucine mutations in motifs 1 and 2.
To probe the significance of the conserved leucine residues immediately preceding the glycines, the L154D and L417D mutations were constructed and tested for transport. Aspartate substitutions were made at these sites to directly address the observation that the conserved leucines among sets 1, 2, and 3 are exchanged for conserved aspartates among Set 4 permeases. The L154D mutation in motif 1 demonstrated significantly lower activity levels than the wild type (10.58 ± 1.36 pmol Fe/109 cells at 7 min) (Fig. 4A; Table 2), indicative of attenuated iron transport. The L417D mutation in motif 2 exhibited dramatically lower activity levels than the wild type (6.63 ± 2.04 pmol Fe/109 cells at 7 min) (Fig. 4B; Table 2). This latter activity is similar in magnitude to that of the H-1443/pAHIΔC control (5.84 ± 1.44 pmol Fe/109 cells at 7 min) and is indicative of null iron transport.
Other mutations of conserved residues in motifs 1 and 2.
Several other conserved residues were targeted for mutation and had minimal effects on transport activity: E149Q (20.11 ± 1.42 pmol Fe/109 cells at 7 min), S153A (22.92 ± 2.52 pmol Fe/109 cells at 7 min), F162L (24.36 ± 1.45 pmol Fe/109 cells at 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 of mutants.
To substantiate the results of the radiolabeled iron transport assay, further qualitative and semiquantitative assays measuring growth on complex media in the presence of Fe3+ chelators were performed. The first such assay measured growth or lack of growth on NB supplemented with 200 μM Dip, an avid iron chelator. This assay was used in the molecular cloning of the sfuABC, hitABC, and fbpABC operons in E. coli strain H-1443 and has been used successfully in recent complementation experiments (1, 2, 7, 8). The second assay, developed specifically for the current study, measured growth rescue by supplemental Fe(NTA)2 on media containing the Fe3+-specific chelator CDTA. This assay is a more specific measure of Fe3+ uptake as, unlike Dip, which is predominantly an Fe2+ chelator, CDTA coordinates Fe3+ with high affinity (FeCDTA1− log β = 28; FeOHCDTA2− log β = 19) (42). The results of these assays along with results of the radiolabeled transport assay are summarized in Table 2. The combined data indicate that the L154D mutation results in attenuated iron transport, L417D results in null transport, G418A results in attenuated transport, and all others result in wild-type iron transport activity.
Gallium resistance screen for hFbpB permease mutations.
Rather than undertake a large-scale site-directed mutagenesis effort on a protein lacking significant sequence conservation or hot spots of interest, we sought alternative approaches in pinpointing further functionally significant residues. In a previous study, we demonstrated that several metals inhibited the growth of H-1443 E. coli cells (7). Interestingly, gallium (Ga3+) toxicity was specific to H-1443/pAHIO cells (H-1443/pBR322 and H-1443/pAHIΔC cells were unaffected), thus providing a correlate between hFbpABC transport and gallium-induced toxicity. Using a radiolabeled Ga67(NTA)2 transport assay, we subsequently demonstrated direct Ga3+ uptake by the hFbpABC transporter (7).
During the course of these metal competition experiments, we made an intriguing observation that warranted further investigation. Upon plating on Ga3+-containing medium (NBamp100dip75Ga100), H-1443/pAHIO cells exhibited a growth-suppressed microcolony phenotype, indicative of Ga3+-induced toxicity. However, approximately 1 × 10−8 colonies (1 to 10 CFU/plate) exhibited a larger macrocolony phenotype among a lawn of microcolony growth. We hypothesized that these macrocolonies had developed a mutation(s), perhaps within the hFbpABC transporter, that rendered the cells insensitive to Ga3+ toxicity. As detailed in Materials and Methods, an initial putative mutant was taken through the screening protocol, and the following criteria were satisfied: the gallium-resistant phenotype remained following passage on NBamp100dip75Ga100 plates, the mutation did not result in major insertions or deletions within the pAHIO plasmid, and the mutation did not cause alterations in hFbpA expression or secretion. Upon sequencing the hitB and hitC genes, a single-nucleotide missense mutation was identified within hitB, which translated into a single-amino-acid point mutation (V497I). No other mutations were found, and site-directed mutagenesis back to the wild type (I497V) resulted in complete reversion to the wild-type gallium-sensitive phenotype (Fig. 5A). Results of the radiolabeled iron transport assay demonstrated that the V497I 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 I497V reverse mutation led to transport activity similar in magnitude to that of wild-type control cells (23.3 ± 2.5 pmol Fe/109 cells at 7 min) (Fig. 5B; Table 3).
With proof of concept in hand, we developed a subcloning strategy to generate a series of further mutations that were directed to the hitB gene. To limit the extensive screening process involved in authentication of the initial mutation, we implemented a PCR-based random mutagenesis method which created an enriched starting pool of putative mutants. This enrichment, coupled with the subcloning and gallium selection protocols described in Materials and Methods, led to the identification of further mutations localized within the hFbpB permease. The mutations arose from numerous locations throughout the hitB gene and represented a diverse sampling of amino acid alterations. The mutants were subjected to the transport assays as shown in Table 3. Subsequently, each mutation was genetically reversed to the wild type, which in turn restored wild-type transport activity (data not shown).
The initial single-site mutation, I174F, demonstrated null transport activity, while a second mutation, S146Y, demonstrated attenuated activity (Table 3). A third mutant possessed a double mutation, I174N/S475I. Individual single mutations were constructed to discern whether individual alterations contributed to the null iron transport activity or whether one of the mutations was actually a false positive. Indeed, the single 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 previously identified mutation, I174F, resulted in null iron transport activity. This inconsistency is likely due to the difference in substituted residues; phenylalanine introduces a large aromatic moiety which may impart a significant steric or electrostatic effect, while the polar uncharged asparagine may be well tolerated at this site.
A fourth point mutation, I383N, exhibited curious activity. Although this mutation led to gallium resistance under selection conditions, iron transport activity was apparently unaffected (Table 3). As both Fe3+ and Ga3+ are trivalent metals that share similar ionic radii (Ga3+, 0.62 Å; Fe3+, 0.65 Å), gallium-induced toxicity is normally thought to arise by competitive inhibition of Fe3+ transport. The I383N result signifies that there may actually be subtle differences between the mechanisms of Ga3+ and Fe3+ hFbpABC transport and that modest mutations within the permease may create a transporter that is selective for one metal over the other. Preliminary experiments suggest that although both metals form stable complexes with hFbpA, the metal-protein interactions are significantly different. UV difference spectra suggest weaker binding for Ga3+ to nFbpA in the presence of phosphate (26), and SUPREX analysis of matrix-assisted laser desorption ionization-time of flight mass spectra shows that the Ga3+- and Fe3+-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). Further experiments will address whether the Ga3+-bound conformation of hFbpA is indeed different from the Fe3+-bound form and whether I383N can discriminate between these potentially distinct conformations.
Protein expression analysis of FbpB mutants.
Clearly, further investigation of the described residues, through saturation mutagenesis and detailed biochemical assays, is required to delineate their specific roles in the mechanism of transport. Before advancing these experiments further, however, we thought it was essential to develop an assay with which to measure permease protein expression levels. As mentioned previously, the permease contains an inordinate number of hydrophobic residues (∼65%) and has proven to be particularly difficult to track. Recombinant overexpression results in cell toxicity preventing isolation by traditional means, and low expression coupled with the propensity to adhere to cellular membranes and to aggregate has precluded reliable visualization on SDS-PAGE and Western blots. In response to these limitations, we developed an assay which allowed us to track functional expression through the incorporation of a β-lactamase (blaM) fusion within the hitB gene.
Using a transposon-based strategy, we generated a β-lactamase (blaM) gene fusion within pACYCHIO, specifically at nucleotide 1098 within the hitB gene of the hitABC locus. Following the transposon insertion reaction and sequencing to verify the site of insertion, the β-lactamase stop codon was replaced with a glutamine codon and the noncoding downstream transposon sequence was deleted. This created an in-frame “sandwich” fusion, with β-lactamase inserted between hFbpB amino acids 366 and 374. Transposon-mediated insertion of the blaM gene included the insertion of upstream mosaic sequence, which translated as an 11-residue hydrophobic linker (LSLIHISTIID) between hFbpB residue 366 and BlaM residue 1. To accommodate this linker and to negate the effects of redundant hydrophobic segments, hFbpB residues 367 to 373 were removed. The hFbpB-BlaM fusion (expressed from the pB366BLAM plasmid) demonstrated resistance to ampicillin up to a concentration of 100 μg/ml on LB plates. This result signified that both expression and proper membrane orientation was retained by the hFbpB-BlaM fusion protein, with β-lactamase localized to the periplasmic side of the inner membrane. Mutations in hFbpB that resulted in null or attenuated iron transport phenotypes (as described above) were introduced into pACYCHIO (negative control) and pB366BLAM, and growth on LBamp100 was assessed (summarized in Table 1). Results demonstrated that each of the mutant strains tested grew on LBamp100 in a fashion similar to that of wild-type pB366-BLAM, while mutations in the pACYCHIO control background demonstrated no such growth. This indicated that the mutations (L154D, L417D, G418A, and Y497I) did not significantly alter FbpB permease expression levels compared to those of the wild type (Table 1). Although this β-lactamase fusion assay demonstrated that the point mutations did not significantly alter expression or membrane incorporation of the hFbpB-BlaM proteins, this does not rule out the possibility that wild-type hFbpB-BlaM expression differs from that of wild-type hFbpB.
A predicted in silica topological model of the permease demonstrates the presence of 12 membrane-spanning helices with both the N and C termini facing the cytosol (Fig. 6). This topology is consistent with previous hydropathy analysis and satisfies the positive inside rule for polytopic membrane proteins (D. S. Anderson and T. A. Mietzner, unpublished data) (50). Four large loops project into the periplasm while several loops, including two containing the conserved permease motifs, are localized within the cytosol. In accordance with the primary sequence, there is twofold internal homology between the first and latter halves of the protein (helices 1 to 6 and 7 to 12). This twofold homology within a single fused permease subunit is similar to that of the FhuB permease from the ferric hydroxymate ABC transporter, which is composed of a single permease subunit with 20 putative transmembrane segments (25). These permeases stand in contrast to the majority of ABC transporter permeases, which are typically composed of two smaller, similar, or identical subunits that together form a functional dimer (4, 5, 28). It is reasonable to suggest that the two halves of hFbpB are arranged with twofold symmetry around a centrally located pore.
The recently solved vitamin B12 ABC transporter (BtuC2D2) structure verifies the transmembrane orientation of the permease complex and its association with two ATP binding subunits, as supported by a large body of biochemical evidence from multiple ABC transport systems (34). The BtuC permease homodimer consists of 20 transmembrane α-helices arranged with pseudo-twofold symmetry around a central pore and periplasmically exposed substrate vestibule. Although this number of transmembrane helices is substantially more than the 12 helices predicted for the canonical ABC transporter, the increased helical content may be required to stabilize a central channel associated with B12 translocation that is larger than that of smaller substrates (21, 25). Consistent with this, biochemical evidence suggests that other well-characterized permeases, including the maltose (MalFG) and histidine (HisQM) permeases, possess fewer total numbers of membrane-spanning domains (12 [MalFG] and 10 [HisQM] transmembrane helices) (20, 29). Although the initial topological model of the FbpB permease is consistent overall with this latter group, further biochemical and structural data are needed to verify this arrangement. Likewise, additional crystal structures will help to shed light on the apparent variability of permease topologies within the ABC transporter family.
The two consensus motifs of the BtuC2 permease dimer are well resolved in the BtuCD structure, each forming two short helices separated by a hairpin turn. The strong conservation of these motifs throughout the ABC transporter family suggests that these L loops may represent a conserved structural feature among ABC transporter permeases. The conserved glycines allow the sharp bend of the peptide backbone necessary to form the hairpin turn, and examination of the backbone and side-chain contacts between the BtuC permease and the BtuD ATP binding proteins provides a basis for understanding the possible effects of mutations at this and surrounding sites. Moreover, alignment of the BtuC conserved permease motif with the hFbpB motifs permits discussion of hFbpB mutations (Fig. 6). The conserved leucines in hFbpB, Leu154 and Leu417, align with conserved leucines within BtuC (Leu216 from each subunit). In BtuC, these residues make contact with several hydrophobic side chains of the BtuD ATP binding protein, including Leu96. Importantly, Leu96 in BtuD aligns with residue Phe508 in the eukaryotic cystic fibrosis transmembrane conductance regulator (CFTR) (34). Deletion of this residue in CFTR is the molecular basis of 70% of cystic fibrosis cases, pointing out an obvious functional role for this hydrophobic interaction. From these observations, it seems likely that mutation of the hFbpB conserved leucine residues (such as the aspartate substitutions shown in the present work) may destabilize similar hydrophobic interactions, thereby giving rise to altered transport function, perhaps by uncoupling hFbpB Fe3+ permeation from hFbpC ATP hydrolysis.
The conserved glycines in BtuC (G217 in both subunits) do not seem to contribute to specific interactions with the BtuD ATP binding proteins. Rather, it seems the flexibility this residue imparts to the backbone orientation is the most relevant feature. By comparison, mutation of G155 in hFbpB would likely be tolerated as long as the introduced side chain can be accommodated without perturbing surrounding contacts. It is not clear at this time if the backbone flexibility and resulting hairpin turn are preserved through the G155 mutations. Clearly, mutation of the G418 residue abolishes an important structural characteristic of this turn and hence the function of the motif.
Interestingly, several of the gallium mutations localize to sequence directly upstream or downstream of the conserved motifs, namely, within the adjoining transmembrane segments (I174F and I383N) or intervening sequence (S146Y). One explanation for this is that these transmembrane segments serve to anchor the conserved motifs in a productive orientation within the membrane. Furthermore, these domains may serve an important transmembrane signaling function, linking hFbpA binding with hFbpC ATP hydrolysis. The S475I mutation resides on a periplasmic loop segment that may form a portion of the interaction site with the FbpA protein. Future experiments will focus on more clearly delineating the roles of residues within the conserved motifs and adjoining transmembrane domains in the transport mechanism.
The V497I mutation demonstrates that even modest permutation of a permease transmembrane domain (the addition of a methyl group to the Val side chain) can result in a significant effect on transport. Although such a subtle chemical change coupled with a large functional effect is startling, specific aliphatic residues have been shown to play key roles in the gating processes of transporters and channels (35, 38, 41, 49). Furthermore, the Cystic Fibrosis Mutation Database lists four replacements of a valine by an isoleucine in CFTR, one of which (V1147I) is located in transmembrane segment five of MSD2 (31). Whether the hFbpB V497I mutation translates to a long-range effect on the FbpA binding site or affects the permeation pathway through a local structural perturbation awaits further biochemical analysis.
As mentioned in the Results, metal-protein binding studies are consistent with significantly weaker sequestration of Ga3+ than Fe3+ by FbpA. Furthermore, from the vantage point of the protein, the folded protein operates in a different regimen when Ga3+ is bound than when Fe3+ is bound (K. D. Weaver et al., unpublished). These preliminary data suggest that the FbpA-Ga3+ coordination environment is significantly different from that of FbpA-Fe3+. Such differences may translate to important distinctions in the transport pathways of these two metals. Future studies will investigate the physical basis of these interactions and transport mechanisms in more detail.
The gallium selection screen provides a powerful approach to identifying functionally significant residues. We foresee that this technique will be useful in locating further residues within the FbpB permease, a protein that is apparently missing stretches of conserved sequence such as metal binding motifs which have been identified in other metal permeases (36, 46). Furthermore, this approach can be adapted to identify functionally relevant residues within the FbpC and FbpA proteins, the latter of which can be directly mapped to the hFbpA holo and apo crystal structures. With such a screen in place and sensitive functional assays now available, further probing into the structure and function of the transporter will offer a more detailed picture of the transport mechanism.
Growth assay of cells plated on NBamp100dip200 (n = 3; averaged).
CDTA Fe(NTA)2 growth rescue assay. Percentage of growth determined as follows: (X − pAHIΔC)/(pAHIO − pAHIΔC), where X is the mutant (n = 3; averaged).
Radiolabeled Fe55(NTA)2 transport assay. Percentage of transport at the 7-min time point was determined as follows: (X − pAHIΔC)/(pAHIO − pAHIΔC), where X is the mutant (n = 3; averaged).
Each strain was characterized through summation of assay percentages and comparison to the wild-type (WT) control. Total percentages of ≤50% of wild type qualify as attenuated; ≤25% of the WT qualify as null transport.
TABLE 3. Effects of permease mutations on transport
Growth assay of cells plated on NBamp100dip200 (n = 3; averaged).
CDTA Fe(NTA)2 growth rescue assay. Percentage of growth was determined as follows: (X − pAHIΔC)/(pNΔMHIO − pAHIΔC), where X is the mutant (n = 3; averaged). ND, not determined.
Radiolabeled Fe55(NTA)2 transport assay. Percentage of transport at the 7-min time point was determined as follows: (X − pAHIΔC)/(pNΔMHIO − pAHIΔC), where X is the mutant (n = 3; averaged).
Each strain was characterized through summation of assay percentages and comparison to the wild-type (WT) control. Total percentages of ≤50% of the WT qualify as attenuated; ≤25% of the WT qualify as null transport.
Mutations in the pAHIO background tested against the pAHIO wild-type control.
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 grant R29 A132226 (T.A.M.), and the National Science Foundation grant CHE-0418006 (A.L.C.).
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 the periplasm-to-cytosol transport of iron. J. Bacteriol. 178 : 2145-2149.
Adhikari, P., S. D. Kirby, A. J. Nowalk, K. L. Veraldi, A. B. Schryvers, and T. A. Mietzner. 1995. Biochemical characterization of a Haemophilus influenzae periplasmic iron transport operon. J. Biol. Chem. 270 : 25142-25149.
Albrecht-Gary, A. M., and A. L. Crumbliss. 1998. The coordination chemistry of siderophores: thermodynamics and kinetics of iron chelation and release. In A. Sigel and H. Sigel (ed.), Metal ions in biological systems, vol. 35. M. Dekker, Inc., New York, NY.
Ames, G. F., C. S. Mimura, S. R. Holbrook, and V. Shyamala. 1992. Traffic ATPases: a superfamily of transport proteins operating from Escherichia coli to humans. Adv. Enzymol. Relat. Areas Mol. Biol. 65 : 1-47.
Anderson, B. F., H. M. Baker, G. E. Norris, D. W. Rice, and E. N. Baker. 1989. Structure of human lactoferrin: crystallographic structure analysis and refinement at 2.8 Å resolution. J. Mol. Biol. 209 : 711-734.
Anderson, D. S., P. Adhikari, A. J. Nowalk, C. Y. Chen, and T. A. Mietzner. 2004. The hFbpABC transporter from Haemophilus influenzae functions as a binding-protein-dependent ABC transporter with high specificity and affinity for ferric iron. J. Bacteriol. 186 : 6220-6229.
Angerer, A., S. Gaisser, and V. Braun. 1990. Nucleotide sequences of the sfuA, sfuB, and sfuC genes of Serratia marcescens suggest a periplasmic-binding-protein-dependent iron transport mechanism. J. Bacteriol. 172 : 572-578.
Bailey, S., R. W. Evans, R. C. Garratt, B. Gorinsky, S. Hasnain, C. Horsburgh, H. Jhoti, P. F. Lindley, A. Mydin, R. Sarra, and J. L. Watson. 1988. Molecular structure of serum transferrin at 3.3-Å resolution. Biochemistry 27 : 5804-5812.
Boukhalfa, H., D. S. Anderson, T. A. Mietzner, and A. L. Crumbliss. 2003. Kinetics and mechanism of iron release from the bacterial ferric binding protein nFbp: exogenous anion influence and comparison with mammalian transferrin. J. Biol. Inorg. Chem. 8 : 881-892.
Braun, V., R. Gross, W. Koster, and L. Zimmermann. 1983. Plasmid and chromosomal mutants in the iron(III)-aerobactin transport system of Escherichia coli. Use of streptonigrin for selection. Mol. Gen. Genet. 192 : 131-139.
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 biochemical analyses of the metal-free Haemophilus influenzae Fe3+-binding protein. Biochemistry 40 : 15631-15637.
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 influenzae Fe(+3)-binding protein reveals convergent evolution within a superfamily. Nat. Struct. Biol. 4 : 919-924.
Dhungana, S., D. S. Anderson, T. A. Mietzner, and A. L. Crumbliss. 2005. Kinetics of iron release from ferric binding protein (FbpA): mechanistic implications in bacterial periplasm-to-cytosol Fe3+ transport. Biochemistry 44 : 9606-9618.
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 anion on iron chelation by ferric binding protein, a bacterial transferrin. Proc. Natl. Acad. Sci. USA 100 : 3659-3664.
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.
Groeger, W., and W. Koster. 1998. Transmembrane topology of the two FhuB domains representing the hydrophobic components of bacterial ABC transporters involved in the uptake of siderophores, haem and vitamin B12. Microbiology 144 : 2759-2769.
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 anion and metal binding to the ferric ion-binding protein from Neisseria gonorrhoeae. J. Biol. Chem. 278 : 2490-2502.
Kerppola, R. E., and G. F. Ames. 1992. Topology of the hydrophobic membrane-bound components of the histidine periplasmic permease. Comparison with other members of the family. J. Biol. Chem. 267 : 2329-2336.
Kerppola, R. E., V. K. Shyamala, P. Klebba, and G. F. Ames. 1991. The membrane-bound proteins of periplasmic permeases form a complex. Identification of the histidine permease HisQMP complex. J. Biol. Chem. 266 : 9857-9865.
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 heterogeneity for cystic fibrosis: 36 mutations account for 75% of all CF chromosomes in Turkish patients. Am. J. Med. Genet. 113 : 250-257.
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 pathogenic gram-negative bacteria. Curr. Top. Microbiol. Immunol. 225 : 113-135.
Saurin, W., W. Koster, and E. Dassa. 1994. Bacterial binding protein-dependent permeases: characterization of distinctive signatures for functionally related integral cytoplasmic membrane proteins. Mol. Microbiol. 12 : 993-1004.
If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download.