(Received for publication, June 8, 1995)
From the
Bacterial iron transport is critical for growth of pathogens in the host environment, where iron is limited as a form of nonspecific immunity. For Gram-negative bacteria such as Haemophilus influenzae, iron first must be transported across the outer membrane and into the periplasmic space, then from the periplasm to the cytosol. H. influenzae express a periplasmic iron-binding protein encoded by the hitA gene. This gene is organized as the first of a three-gene operon purported to encode a classic high affinity iron acquisition system that includes hitA, a cytoplasmic permease (hitB), and a nucleotide binding protein (hitC). In this study we describe the cloning, overexpression, and purification of the H. influenzae hitA gene product. The function of this protein is unambiguously assigned by demonstrating its ability to compete for iron bound to the chemical iron chelator 2,2`-dipyridyl, both in vitro and within the periplasmic space of a siderophore-deficient strain of Escherichia coli. Finally, the importance of a functional hitABC operon for iron acquisition is demonstrated by complementation of this siderophore-deficient E. coli to growth on dipyridyl-containing medium. These studies represent a detailed genetic, biochemical, and physiologic description of an active transport system that has evolved to efficiently transport iron and consequently is widely distributed among Gram-negative pathogenic bacteria.
High affinity acquisition of iron from the host environment is a necessary determinant of virulence for pathogenic bacteria(1, 2, 3, 4, 5, 6, 7, 8, 9) . This acquisition is vital for survival in the human host, where levels of extracellular iron are tightly controlled by the Transferrins (transferrin and lactoferrin), a family of iron-binding proteins that function in the extracellular chelation and transport of host iron(9) . By binding iron with high affinity, Transferrins ensure that all extracellular iron is both efficiently sequestered from pathogenic invaders and mobilized for transport to host tissues. Microorganisms growing in the human host must therefore possess mechanisms for obtaining Transferrin-sequestered iron. For a number of pathogenic members of the Pasteurellaceae (H. influenzae) and Neisseriaceae (Neisseria meningitidis and Neisseria gonorrhoeae), iron acquisition is initiated by cell-surface receptors specific for the Transferrins(10, 11, 12, 13, 14, 15, 16) . Iron is removed from these proteins and transported across the outer membrane, presumably by an energy-dependent TonB-mediated process (17, 18, 19) involving gated-pore properties of the outer membrane receptor (18, 20) . The result is deposition of free iron within the periplasm, where it is separated from the cytosol, its eventual destination, by the cytoplasmic membrane(21) .
Transport of free iron from the periplasmic space into the cytoplasm is proposed to occur by a classic active transport process involving a periplasmic binding protein, a specific cytoplasmic permease, and an energy-supplying nucleotide-binding protein(22) . Much of what is known about the biochemistry of active transport systems has been revealed through the study of model active transport systems for amino acids and sugars in Escherichia coli(22, 23) . Similar systems for siderophore-mediated iron transport have been described for E. coli and related organisms at the genetic level(23) ; however, relatively little is known regarding the basic biochemistry of these iron transport processes.
A genetic locus critical to the transport
of iron in H. influenzae has recently been described by Hansen
and colleagues (24) . This locus was identified through
complementation of a H. influenzae isolate unable to grow on
medium containing protoporphyrin IX and free iron. An 11.5-kb ()genomic DNA fragment from an isolate proficient for growth
on this medium was identified by this analysis(24) . Essential
for this phenotype was a
4-kb operon composed of three genes: hitA, hitB, and hitC (hit for Haemophilus
Iron Transport) proposed to encode a periplasmic iron-binding protein,
a cytoplasmic permease, and a nucleotide-binding protein, respectively.
A homologous three-gene operon was originally described for Serratia marcescens and designated sfu for Serratia Ferric-iron Uptake(25) . The sfu operon was isolated based upon its ability to complement an E.
coli strain (H-1443) deficient in its ability to produce
siderophores for growth on nutrient agar containing 200 µM 2,2`-dipyridyl (dipyridyl), an iron chelator that sequesters free
iron in the medium (25, 26) . The open reading frames
encoded by the hitABC and sfuABC genes were found to
share 38, 37, and 38% identity between respective A, B, and C
components at the predicted amino acid level. The similarities between
the hit and sfu genetic loci suggest a high level of
conservation among two diverse species.
At the protein level,
Harkness and colleagues (27) originally observed a
quantitatively major, iron-regulated periplasmic protein, subsequently
genetically defined as hitA(24) . The predicted open
reading frame of hitA is nearly 80% homologous with the ferric
iron-binding protein (Fbp) expressed by pathogenic Neisseria(17, 29, 30, 31, 32, 33) .
Similarly, the open reading frame of sfuA predicts a protein
sequence sharing substantial homology (40% identity) with the Neisserial Fbp(17) . Fbp is a periplasmic iron-binding
protein expressed by all pathogenic Neisseria that functions
as the binding component of a high affinity active transport system for
the assimilation of growth-essential iron from the Transferrins.
Purified Fbp binds a single Fe
ion with an affinity
approaching that of the Transferrins (17, 34, 35) and by a mechanism that is
remarkably conserved among this family of proteins, coordinating iron
through two tyrosines, a single histidine, and a bicarbonate
anion(35) . In our study we will refer to these Fbp homologues
as NFbp for Neisseria Fbp derived from the fbp gene
locus (33) , HFbp for Haemophilus Fbp derived from the hitA locus(24) , and SFbp for Serratia Fbp
derived from the sfuA locus(26) . It is clear is that
a common free iron active transport system exists among pathogenic
members of the diverse microbial families Enterobacteriaceae (sfu operon), Pasteurellaceae (hit operon), and Neisseriaceae (fbp operon). The
existence of this common system may reflect its contribution to the
pathogenicity of these organisms.
Studies on the HFbp, NFbp, and SFbp homologues and their respective operons predict that they should function similarly. This report describes the ability of purified HFbp to efficiently compete for dipyridyl-bound iron in vitro. Like the sfu operon, hitABC can complement the siderophore-deficient E. coli strain H-1443 to growth on dipyridyl-containing medium. We further demonstrate that labeled iron from this medium is initially bound to periplasmic HFbp and can only be transported into the cell if a functional permease and nucleotide-binding protein are present. These studies explain why the siderophore-deficient E. coli expressing the hit operon can be complemented to growth on dipyridyl-containing media. Furthermore, they represent the first comprehensive biochemical analysis of a periplasmic iron transport system.
The plasmid pJDS150 was a
generous gift of Dr. Eric Hansen, University of Texas Southwestern.
Plasmid pBR322 was purchased from Promega (Madison, WI).
Oligonucleotides were prepared using an Applied Biosystems
International model 391 DNA Synthesizer (Foster City, CA) and were
deprotected and purified as per the manufacturer's instructions. Taq polymerase used was purchased from either Life
Technologies, Inc. or Boehringer Mannheim. Random hexamers used in
generating labeled PCR probes, T4 DNA ligase, and the restriction
enzymes EcoRI, EcoRV, BamHI, and SmaI were purchased from Boehringer Mannheim. Nutrient broth,
trypticase soy broth, components for Luria-Bertani Broth (LB), NZY
agar, Difco agar, and other media components were purchased from Difco
(Detroit, MI). Cetyltrimethylammonium bromide, 2,2`-dipyridyl,
CM-Sepharose, DEAE-Sepharose, MgS0, tetracycline, and
ampicillin were purchased from Sigma. Low molecular weight protein
standards for SDS-PAGE analysis were purchased from Pharmacia Biotech
Inc. The radioisotopes [
-
P] and
[
Fe
](NO
)
were purchased from DuPont NEN. Eco-Lite scintillation mixture was
purchased from ICN Biomedicals Inc. (Irvine, CA), and samples were
counted using a Packard 1600TR Tri-Carb liquid scintillation analyzer
(Packard Instrument Company, Meridian, CT). Whatman no. 4 filter paper
was purchased from Whatman (Maidstone, United Kingdom). The Amicon
concentration cell and Diaflo ultrafiltration membranes were from
Amicon (Lexington, MA).
Figure 1:
Plasmid map of the HFbp-expressing
plasmid pBSJ1. As described in the text, an 3.5-kb fragment
encoding 1.3 kb upstream and 1.2 kb downstream of the HFbp coding
sequence was excised from a positive
Zap II clone and inserted
into the EcoRI site of the plasmid pBS SK
. Ori = origin of replication, AmpR =
-lactamase gene, lacZ =
-galactosidase
gene.
The fragment containing the minimal hitABC from H. influenzae was prepared by PCR amplification as
described in Fig. 2. PCR reactions were performed in 100 µl
volumes using standard conditions previously described (33) and
10 units of Taq polymerase and 10 units of Taq extender. Amplification was achieved by 27 cycles of denaturation
(95 °C for 1.5 min), annealing (60 °C for 2 min), and extension
(72 °C for 3 min). At cycle 17, the reactions were replenished with
an additional 5 units of both Taq polymerase and Taq extender. Specifically, primers were designed to the extreme ends
of the hitABC sequence (24) that included 250 bp
upstream and 230 bp downstream of this operon. For the upstream primer,
hitO-5`, there was an engineered 5` SmaI site; the downstream
primer hitO-3` included a 3` BamHI site (Table 1). Using
these primers and the plasmid pJDS150 as template, a PCR fragment of
approximately 4.2 kb was generated. Following PCR, the amplified
fragment was gel purified and digested simultaneously with BamHI and SmaI for about 4 h at 37 °C. The PCR
fragment was combined with the 4.2-kb EcoRV-BamHI
fragment of pBR322 (gel-purified) at a 3:1 ratio of insert to vector.
Ligation was achieved using standard conditions(33) . This
ligation was used to transform competent E. coli strain H-1443
and the transformants selected on LB agar containing 100 µg/ml
ampicillin. Transformants were screened for tetracycline sensitivity on
LB plates containing 25 µg/ml of this antibiotic. Tetracycline
sensitive clones were screened for plasmid DNA and the presence of hitABC insert verified by PCR amplification as described
above.
Figure 2: SDS-PAGE comparison of HFbp and NFbp. 5 µg of each protein were run on a 12% acrylamide gel as specified under ``Experimental Procedures.'' Numbers on the left refer to molecular weights estimated from a reference curve of standard protein relative mobilities.
A hitC deletion mutant was constructed in order to
demonstrate the essential nature of this gene to the complementation of
H-1443 to growth on nutrient agar containing 200 µM dipyridyl (NA/Dip). A 1.3-kb fragment of DNA was
deleted from the ClaI site (
400 bp from the stop codon in hitC) to the NarI site (at position 1205 in pBR322).
This was achieved by complete digestion of pAHIO with NarI
followed by a partial ClaI digest. From this partial digest
the approximately 7-kb partial product that contains the deleted hitC gene was gel-purified. Subsequently, this fragment was
ligated under standard conditions and used to transform E. coli H-1443 cells to ampicillin resistance. Positive clones were
confirmed by restriction digest analysis and the plasmid expressing
this mutation designated pAHIO
hitC.
Biochemical analyses of purified
HFbp were performed to compare its physical and functional properties
with those of NFbp. The biochemical attributes of NFbp have been
extensively reported (17, 32, 33, 36) and are listed as
part of Table 2. Physical comparison of HFbp with NFbp reveals
that both proteins share similar predicted molecular masses, although
their migration in SDS-PAGE is noticeably different (Table 2, Fig. 2). The isoelectric points of HFbp and NFbp differ by more
than a full pH unit. This difference in charge may explain the
disparity in SDS-PAGE mobility and affinity for ion exchange resins.
However, two other functional indices highlight the similarity that the
proteins share in their coordination of iron. The visible absorbance
maximum of the ferrated protein is nearly identical for the two,
indicating that iron is bound within a very similar ligand field in
both HFbp and NFbp. Secondly, the affinities for Fe are identical, again emphasizing the functional homology between
the two proteins. These and other (37) observations provide
compelling evidence for the functional homology of NFbp and HFbp.
Figure 3:
Iron saturation of HFbp by
Fe(dipyridyl)
. Increasing amounts of
Fe
(dipyridyl)
were added to 60 µM apo-HFbp as described under ``Experimental Procedures.''
Binding of iron by HFbp was monitored by the increase in absorbance at
483 nm, the visible maximum of the ferrated protein. This data
demonstrates that apoHFbp can efficiently compete for dipyridyl-bound
iron in vitro.
An
experimental approach based upon that described for defining the sfuABC operon by Zimmerman et al.(25) was
used to investigate iron transport from the periplasmic space to the
cytoplasm. This approach employs E. coli H-1443, an aroB strain which is deficient in the
synthesis of aromatic compounds (38) including amino acids and
the siderophore enterochelin(39) . The growth of this strain is
inhibited by 200 µM dipyridyl in nutrient agar; however,
at concentrations of 100 µM dipyridyl H-1443 will grow,
presumably due to low affinity iron uptake systems (Table 3). In
order to examine the in vivo competition for dipyridyl-bound
iron by HFbp, H-1443 was grown under conditions in which trace
concentrations of
Fe
-dipyridyl were
incorporated into 75 µM dipyridyl-containing nutrient
agar. This strain and an HFbp-expressing isogenic variant containing
the plasmid pBSJ1 were propagated for 12 h. At this time organisms were
harvested and washed, and periplasmic fractions were extracted from
cells as described under ``Experimental Procedures.'' The
concentrations of labeled iron associated with the periplasm and the
non-periplasmic components were compared for both strains (Fig. 4). The results demonstrate that both strains had
equivalent levels of radioactivity associated with the non-periplasmic
fraction. This is consistent with the observation that these bacteria
share common low affinity systems for iron uptake. In contrast, the
strain expressing HFbp contained 25-fold more
Fe in the
periplasm than did the plasmid-free H-1443. This is consistent with the
prediction that the presence of HFbp in the periplasm would allow
accumulation of free iron from Fe
-dipyridyl at this
site. This demonstrates that, similar to the in vitro ability
of apoHFbp to mobilize iron bound to dipyridyl, periplasmic Hfbp can
effectively liberate Fe
from dipyridyl.
Figure 4:
Competition for dipyridyl-bound labeled
iron by HFbp in the periplasm. E. coli strain H-1443, with and
without the HFbp-producing plasmid pBSJ1, was grown overnight on
nutrient agar containing 75 µM dipyridyl and Fe, as specified under ``Experimental
Procedures.'' Bacteria were scraped from plates, washed, and
separated into periplasmic and non-periplasmic fractions. The amount of
iron in either fraction was determined by scintillation counting. The
results demonstrate the ability of the strain expressing HFbp to
efficiently concentrate iron in the
periplasm.
Based upon the above
observations, we investigated the ability of the hitABC operon
to confer growth upon aroB H-1443 on
NA/Dip
. The plasmid pJDS150 derived by Hansen and
colleagues (24) contains an H. influenzae DNA fragment
encoding the 4.0-kb hitABC operon and an additional 7.5 kb
outside of this operon (Table 1, ``Experimental
Procedures''). This plasmid conferred upon E. coli strain
H-1443 the ability to grow as small colonies on NA/Dip
(data not shown). To demonstrate that only the hitABC genes were required for this functional complementation, a 4.2-kb
PCR fragment containing only hitABC was amplified from pJDS150
and cloned into pBR322. The resulting plasmid pAHIO (Fig. 5)
allowed H-1443 to grow as single colonies on NA/Dip
(Fig. 6, Table 3). Isolated colonies of
H-1443(pJDS150) (data not shown) or H-1443 (pAHIO) were approximately
3-fold smaller than those obtained from plating the aroB
DH5
(data not shown). The negative
controls, untransformed H-1443 (data not shown) and H-1443(pBR322) (Fig. 6), were incapable of growth on NA/Dip
.
Figure 5: Outline of the construction of the hitABC-containing plasmid pAHIO. As described under ``Experimental Procedures,'' a PCR fragment containing the hitABC operon with minimal flanking sequence was amplified from pJDS150. Using SmaI and BamHI ends, the fragment was cloned into the EcoRV and BamHI sites of pBR322.
Figure 6:
Complementation of aroBE. coli for growth on NA/Dip. E. coli strain H-1443 containing either pAHIO or pBR322 were
grown to mid-log and plated to obtain 100-200 colony-forming
units/plate, on either NA or NA/Dip
. Plates A and C represent the growth of H-1443(pBR322) and
H-1443(pAHIO), respectively, on NA/Dip
, while plates
B and D represent their growth on NA. Pinpoint colonies
observed in plate C demonstrate the ability of the hitABC operon to complement H-1443 to growth on dipyridyl-containing
media.
The hitABC iron acquisition operon appears to behave as a
classical active transport system dependent upon a periplasmic binding
protein, a cytoplasmic permease, and a nucleotide-binding protein.
Therefore, disruption of any of these three components should eliminate
the function of the system. In order to demonstrate the requirement for
a functional hitABC operon for growth, a 400-bp C-terminal
deletion of the hitC gene, designated pAHIOhitC,
was constructed (``Experimental Procedures''). Consistent
with the above prediction, H-1443(pAHIO
hitC) was unable
to grow on NA/Dip
(Table 3).
Complementation for
growth is a gross measure of the molecular events contributed by the hitABC operon. By examining the distribution of labeled iron
in the cell, the efficiency of iron transport at the molecular level
can be measured. We investigated the movement of iron from the
periplasm into the cytosol using Fe to correlate transport
with the presence or absence of the hitABC components. Using
isogenic variants expressing all or part of the operon, the
distribution of
Fe into the periplasmic or the
non-periplasmic cell-associated fractions was analyzed. As described
above, bacteria were grown on
Fe-dipyridyl/nutrient agar
media, harvested, and separated into the periplasmic and the
non-periplasmic cell-associated fractions. Fig. 7shows the
distribution of
Fe for each of the isogenic variants. The
HFbp-expressing H-1443(pBSJ1) showed a level of radioactivity in the
periplasmic fraction similar to that observed for the hitABC operon-expressing H-1443(pAHIO). This is consistent with the
previous assertion that HFbp is effectively mobilizing
Fe
from dipyridyl and concentrating it in the periplasmic space of these
bacteria. However, the greatly increased amount of non-periplasmic
cell-associated radioactivity in H-1443(pAHIO) compared with
H-1443(pBSJ1) indicates that the effective transport of periplasmic
Fe is dependent on the complete hitABC operon
encoded by pAHIO. The profile of the H-1443(pAHIO
hitC)
bacteria is nearly identical to that of the H-1443 (pBSJ1) strain. This
is consistent with the fact that pAHIO
hitC should produce
comparable amounts of HFbp but be unable to mediate transport of iron
due to the nucleotide-binding protein deletion.
Figure 7:
Comparison of labeled iron uptake by E. coli strain H-1443 isogenic variants containing one of
three plasmids: pBSJ1 (HFbp producing), pAHIO (complete hit operon), or pAHIOhitC (complete hit operon
with deletion in the hitC gene). These analogs were grown
overnight on NA containing 75 µM dipyridyl and
Fe as specified under ``Experimental
Procedures.'' The isogenic bacterial cultures were scraped from
plates, washed, and separated into periplasmic and cell pellet
fractions. The amount of iron in either fraction was determined by
scintillation counting. These results demonstrate that an intact hitABC operon is required for the transport of iron from the
periplasm to the cytosol, forming the basis for the functional
complementation observed in Fig. 6.
This study represents the characterization of a periplasmic
free iron transport system at the genetic and biochemical levels. By
cloning, overexpressing, and purifying the hitA gene product,
we demonstrated that HFbp competes for iron bound to dipyridyl in
vitro. Furthermore, HFbp can compete in vivo for
dipyridyl-bound iron in the E. coli periplasm. Finally, growth
on dipyridyl-containing agar can be conferred upon the E. coli host by expression of a functional operon. The model proposed in Fig. 8illustrates the mechanism by which dipyridyl-bound iron at
sufficient concentrations diffuses across a permeable outer membrane
into the periplasm, where HFbp is able to compete for this iron source.
Subsequent transport of iron across the cytoplasmic membrane can only
be accomplished in the presence of a functional cytoplasmic membrane
permease and a nucleotide-binding protein, as evidenced by growth as
pin-point colonies on NA/Dip (Fig. 6) and the
increased non-periplasmic cell-associated concentration of labeled iron (Fig. 7). The observation that these colonies were small
relative to those produced by aroB
E.
coli indicates that dipyridyl-associated iron is limiting the
growth of the transformed E. coli under these conditions. The
implication of this observation is that studying the hit operon in this E. coli background may provide an
insightful model for detecting subtle alterations in iron-transport
properties of this operon. In toto, these studies elucidate
the component steps in the active transport of free iron from the
periplasm to the cytosol at the biochemical level.
Figure 8:
Model for hitABC-mediated iron
acquisition from dipyridyl in aroBE. coli.
The E. coli strain H-1443 is unable to access dipyridyl-bound
iron since it lacks the production of a periplasmic transport system.
Expression of HFbp (pBSJ1) allows concentration of iron from dipyridyl
in the periplasm, without further transport to the cytosol (Fig. 7). Supplying the intact hitABC operon (pAHIO)
allows both the concentration of iron in the periplasm and subsequent
transport into the cytosol. As such, this transport system represents
the molecular basis for the growth of pAHIO-containing strains on media
containing the iron chelator dipyridyl.
Common themes in the transport of molecules across membranes have begun to emerge from diverse and detailed studies of prokaryotic and eukaryotic systems. For example, we have recently demonstrated that NFbp functions in the transport of iron across the periplasm in a manner analogous to the transport of iron in serum by the vertebrate Transferrins(35) . This analogy holds not only at the level of function, but also at the level of structure: an identical set of iron-binding ligands is used by NFbp and by transferrin(35) . As a closely related protein homologue, HFbp can be reasonably predicted to function in a similar capacity. Another common theme is the utilization of an ABC (for ATP Binding Cassette) transporter-exporter protein complex(40, 43) . ABC transporter-exporters require energy for transport. This energy is supplied by hydrolysis of a nucleotide triphosphate, which is facilitated by the nucleotide-binding protein. In eukaryotic systems (e.g. the mammalian P-glycoprotein drug exporter, MDR), ATP hydrolysis and membrane permease activities are contained on a single polypeptide. In contrast, the cytoplasmic permease and nucleotide binding activities exist on two separate polypeptides in the bacterial ABC importers(40, 41, 42) . This is consistent with the data presented above for the transport of growth-essential iron by the hitABC operon.
An important aspect of these studies is the correlation of this operon family with bacterial pathogens. The ability to optimally compete for iron from the host environment correlates with pathogenicity of Neisseria species(44, 45) . The microbial pathogens N. gonorrhoeae and N. meningitidis both express antigenically detectable levels of NFbp when propagated under conditions of iron stress, whereas the closely related commensal Neisseria species (e.g. N. sicca and N. perflava) do not(32, 36) . Since we have shown that HFbp requires a functional permease and nucleotide-binding protein to function, it is presumed that these are also present in pathogenic Neisseria but not in commensal Neisseria. Furthermore, the studies of Sanders et al.(24) demonstrated that a functional hitABC operon was associated with a non-typable H. influenzae isolate. This functional operon was used to complement a type b H. influenzae to growth on iron-limited medium. H. influenzae causes a spectrum of disease, ranging from asymptomatic colonization to invasive bacterial meningitis(46) . It is possible that the presence of a functional hit operon may contribute to the differences in pathogenicity of individual H. influenzae isolates.
In addition to the periplasm-to-cytoplasm transport of iron investigated in this report, it has been demonstrated that delivering iron to the periplasmic space is a critical step in obtaining iron from the host environment. We have recently defined a human transferrin-binding protein complex oriented at the surface of H. influenzae(16) . This complex is required as the first step in the process of assimilation of iron from human transferrin. In the current study we circumvent the delivery of iron from transferrin into the periplasm by substituting dipyridyl as an iron source to a E. coli host lacking a functional transferrin receptor complex. Two molecules of dipyridyl bind a single molecule of iron at physiologic pH, giving the complex a molecular mass of less than 400 Da, a size that is freely diffusible through the E. coli porin. Alternatively, since dipyridyl is a hydrophobic compound it may diffuse through the outer membrane by partitioning into the lipid bilayer in a manner similar to what is observed for erythromycin(47) . In either case iron is delivered to the periplasm and the requirement for a functional outer membrane receptor complex is subverted. These studies demonstrate that the hitABC operon can function independent of the transferrin receptor. This correlates with the fact that the sfuABC system from S. marcescens has been demonstrated to be TonB independent, a characteristic which would preclude the involvement of an energy-dependent outer membrane receptor complex. The significance of this mechanism of iron uptake in such a diverse range of microorganisms and its role in pathogenicity is unknown.
HFbp and NFbp are homologous in their biochemical attributes. Because of this relatedness, it is likely that both proteins serve similar functions in these two bacterial species. It has been previously demonstrated that NFbp transiently associates with labeled iron mobilized from transferrin. This evidence has implicated NFbp in the transport of iron from the transferrin-binding complex to the cytoplasm (17) . This study illustrates that, similar to the sfuABC system, the hitABC system is capable of functioning in the absence of Tbp1/2 by acquiring iron directly from sources which diffuse into the periplasm. As a result, the hitABC transporter and the Neisserial equivalent may function in a broader role as a high affinity, periplasmic iron scavenging system. Further characterization of this system will enhance our understanding of this mechanism of iron acquisition and allow a more detailed molecular understanding of how the Fbp family of proteins participates in the process of periplasmic iron transport.