Analysis of the vibrioferrin biosynthetic pathway of Vibrio parahaemolyticus.

Siderophores are small-molecule iron chelators produced by many microorganisms that capture and uptake iron from the natural environment and host. Their biosynthesis in microorganisms is generally performed using non-ribosomal peptide synthetase (NRPS) or NRPS-independent siderophore (NIS) enzymes. Vibrio parahaemolyticus secretes its cognate siderophore vibrioferrin under iron-starvation conditions. Vibrioferrin is a dehydrated condensate composed of α-ketoglutarate, L-alanine, aminoethanol, and citrate, and pvsA (the gene encoding the ATP-grasp enzyme), pvsB (the gene encoding the NIS enzyme), pvsD (the gene encoding the NIS enzyme), and pvsE (the gene encoding decarboxylase) are engaged in its biosynthesis. Here, we elucidated the biosynthetic pathway of vibrioferrin through in vitro enzymatic reactions using recombinant PvsA, PvsB, PvsD, and PvsE proteins. We also found that PvsD condenses L-serine and citrate to generate O-citrylserine, and that PvsE decarboxylates O-citrylserine to form O-citrylaminoethanol. In addition, we showed that O-citrylaminoethanol is converted to alanyl-O-citrylaminoethanol by amidification with L-Ala by PvsA and that alanyl-O-citrylaminoethanol is then converted to vibrioferrin by amidification with α-ketoglutarate by PvsB.

Binding of AraC-Type Activator DesR to the Promoter Region of Vibrio vulnificus Ferrioxamine B Receptor Gene.

Vibrio vulnificus can utilize the xenosiderophore desferrioxamine B (DFOB) as an iron source under iron-restricted conditions. We previously identified in V. vulnificus that transcription of the desA gene encoding the outer membrane receptor for ferrioxamine B (FOXB) is activated by the AraC-type transcriptional regulator encoded by desR together with DFOB. In this study, we overexpressed and purified DesR as a glutathione S-transferase-fused protein and examined interaction between the promoter region of desA and DesR. Electrophoretic mobility shift assay (EMSA) revealed that DesR directly binds to the regulatory region of desA, and this binding was enhanced by the presence of DFOB in a concentration-dependent manner, while the presence of FOXB did not affect the potentiation of their binding. Moreover, EMSA identified that DNA fragments lacking a probable DesR binding sequence were unable to form complexes with DesR. Finally, deoxyribonuclease I footprinting assay demonstrated that the DNA binding sequence of DesR is located between -27 and -50 nucleotides upstream of the desA transcription start site. These results strongly indicate that DesR can directly activate the transcription of desA in cooperation with DFOB, which acts as a coactivator for DesR.

Iron-Utilization System in Vibrio vulnificus M2799.

Vibrio vulnificus is a Gram-negative pathogenic bacterium that causes serious infections in humans and requires iron for growth. A clinical isolate, V. vulnificus M2799, secretes a catecholate siderophore, vulnibactin, that captures ferric ions from the environment. In the ferric-utilization system in V. vulnificus M2799, an isochorismate synthase (ICS) and an outer membrane receptor, VuuA, are required under low-iron conditions, but alternative proteins FatB and VuuB can function as a periplasmic-binding protein and a ferric-chelate reductase, respectively. The vulnibactin-export system is assembled from TolCV1 and several RND proteins, including VV1_1681. In heme acquisition, HupA and HvtA serve as specific outer membrane receptors and HupB is a sole periplasmic-binding protein, unlike FatB in the ferric-vulnibactin utilization system. We propose that ferric-siderophore periplasmic-binding proteins and ferric-chelate reductases are potential targets for drug discovery in infectious diseases.

VuuB and IutB reduce ferric-vulnibactin in Vibrio vulnificus M2799.

Vibrio vulnificus, a pathogenic bacterium that causes serious infections in humans, requires iron for growth. Clinical isolate, V. vulnificus M2799, secretes a catecholate siderophore, namely, vulnibactin, to capture iron (III) from the environment. Growth experiments using a deletion mutant indicated that VuuB, a member of the FAD-containing siderophore-interacting protein family, plays a crucial role in Fe3+-vulnibactin reduction. IutB, a member of the ferric-siderophore reductase family, stands a substitute for VuuB in its absence. It remained unclear why V. vulnificus M2799 has two proteins with relevant functions. Here we biochemically characterized VuuB and IutB using purified recombinant proteins. Purified VuuB, a flavoprotein, catalyzed the reduction of Fe3+-nitrilotriacetic acid as its electron acceptor, in the presence of NADH as its electron donor and FAD as its cofactor. IutB catalyzed the reduction of Fe3+-nitrilotriacetic acid, in the presence of NADH, NADPH, or reduced glutathione as its electron donor. The optimal pH values and temperatures of VuuB and IutB were 7.0 and 37 °C, and 8.5 and 45 °C, respectively. On analyzing their ferric-chelate reductase activities, both VuuB and IutB were found to catalyze the reduction of Fe3+-aerobactin, Fe3+-vibriobactin, and Fe3+-vulnibactin. When the biologically relevant substrate, Fe3+-vulnibactin, was used, the levels of ferric-chelate reductase activities were similar between VuuB and IutB. Finally, the mRNA levels were quantified by qRT-PCR in M2799 cells cultivated under low-iron conditions. The number of vuuB mRNA was 8.5 times greater than that of iutB. The expression ratio correlated with the growth of their mutants in the presence of vulnibactin.

Identification of the heme acquisition system in Vibrio vulnificus M2799.

Vibrio vulnificus, the causative agent of serious, often fatal, infections in humans, requires iron for its pathogenesis. As such, it obtains iron via both vulnibactin and heme-mediated iron-uptake systems. In this study, we identified the heme acquisition system in V. vulnificus M2799. The nucleotide sequences of the genes encoding heme receptors HupA and HvtA and the ATP-binding cassette (ABC) transport system proteins HupB, HupC, and HupD were determined, and then used in the construction of deletion mutants developed from a Δics strain, which could not synthesize vulnibactin. Growth experiments using these mutants indicated that HupA and HvtA are major and minor heme receptors, respectively. The expressions of two proteins were analyzed by the quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR). Furthermore, complementation analyses confirmed that the HupBCD proteins are the only ABC transport system shared by both the HupA and HvtA receptors. This is the first genetic evidence that the HupBCD proteins are essential for heme acquisition by V. vulnificus. Further investigation showed that hupA, hvtA, and hupBCD are regulated by Fur. The qRT-PCR analysis of the heme receptor genes revealed that HupR, a LysR-family positive transcriptional activator, upregulates the expression of hupA, but not hvtA. In addition, ptrB was co-transcribed with hvtA, and PtrB had no influence on growth in low-iron CM9 medium supplemented with hemin, hemoglobin, or cytochrome C.

IutB participates in the ferric-vulnibactin utilization system in Vibrio vulnificus M2799.

Vibrio vulnificus, an opportunistic pathogen that causes a serious, often fatal, infection in humans, requires iron for its growth. This bacterium utilizes iron from the environment via the vulnibactin-mediated iron uptake system. The mechanisms of vulnibactin biosynthesis, vulnibactin export, and ferric-vulnibactin uptake systems have been reported, whereas the ferric-vulnibactin reduction mechanism in the cell remains unclear. The results of our previous study showed that VuuB, a member of the flavin adenine dinucleotide-containing siderophore-interacting protein family, is a ferric-vulnibactin reductase, but there are other reductases that can complement for the defective vuuB. The aim of this study was to identify these proteins that can complement the loss of function of VuuB. We constructed mutants of genes encoding putative reductases in V. vulnificus M2799, and analyzed their growth under low-iron conditions. Complementation analyses confirmed that IutB, which functions as a ferric-aerobactin reductase, participates in ferric-vulnibactin reduction in the absence of VuuB. This is the first genetic evidence that ferric-vulnibactin is reduced by a member of the ferric-siderophore reductase protein family. In the aerobactin-utilization system, IutB plays a major role in ferric-aerobactin reduction in V. vulnificus M2799, and VuuB and DesB can compensate for the defect of IutB. Furthermore, the expression of iutB and desB was found to be regulated by iron and a ferric uptake regulator.

The RND protein is involved in the vulnibactin export system in Vibrio vulnificus M2799.

Vibrio vulnificus, an opportunistic marine bacterium that causes a serious, often fatal, infection in humans, requires iron for its pathogenesis. This bacterium exports vulnibactin for iron acquisition from the environment. The mechanisms of vulnibactin biosynthesis and ferric-vulnibactin uptake systems have recently been reported, while the vulnibactin export system has not been reported. Mutant growth under low-iron concentration conditions and a bioassay of the culture supernatant indicate that the VV1_0612 protein plays a crucial role in the vulnibactin secretion as a component of the resistance-nodulation-division (RND)-type efflux system in V. vulnificus M2799. To identify which RND protein(s) together with VV1_0612 TolC constituted the RND efflux system for vulnibactin secretion, deletion mutants of 11 RND protein-encoding genes were constructed. The growth inhibition of a multiple mutant (Δ11) of the RND protein-encoding genes was observed 6 h after the beginning of the culture. Furthermore, ΔVV1_1681 exhibited a growth curve that was similar to that of Δ11, while the multiple mutant except ΔVV1_1681 showed the same growth as the wild-type strain. These results indicate that the VV1_1681 protein is involved in the vulnibactin export system of V. vulnificus M2799. This is the first genetic evidence that vulnibactin is secreted through the RND-type efflux systems in V. vulnificus.

Identification and characterization of Aeromonas hydrophila genes encoding the outer membrane receptor of ferrioxamine B and an AraC-type transcriptional regulator.

We found that, under iron-limiting conditions, Aeromonas hydrophila ATCC 7966(T) could utilize the xenosiderophore desferrioxamine B (DFOB) for growth by inducing the expression of its own outer membrane receptor. Two consecutive genes, desR and desA, were selected as candidates involved in DFOB utilization. The presence of the ferric-uptake regulator boxes in their promoters suggested that these genes are under iron-dependent regulation. Mutation of desA, a gene that encodes the outer membrane receptor of ferrioxamine B, disrupted the growth of the amonabactin-deficient mutant in the presence of DFOB. ß-Galactosidase reporter assays and reverse transcriptase-quantitative PCR demonstrated that desR, a gene that encodes an AraC-like regulator homolog is required for the induction of desA transcription in the presence of DFOB and under iron-limiting conditions. The functions of desA and desR were analyzed using complementation experiments. Our data provided evidence that DesA is powered primarily by the TonB2 system.

Identification and characterization of a cluster of genes involved in biosynthesis and transport of acinetoferrin, a siderophore produced by Acinetobacter haemolyticus ATCC 17906T.

Acinetobacter haemolyticus ATCC 17906(T) is known to produce the siderophore acinetoferrin under iron-limiting conditions. Here, we show that an operon consisting of eight consecutive genes, named acbABCD and actBCAD, participates in the biosynthesis and transport of acinetoferrin, respectively. Transcription of the operon was found to be iron-regulated by a putative Fur box located in the promoter region of the first gene, acbA. Homology searches suggest that acbABCD and actA encode enzyme proteins involved in acinetoferrin biosynthesis and an outer-membrane receptor for ferric acinetoferrin, respectively. Mutants defective in acbA and actA were unable to produce acinetoferrin or to express the ferric acinetoferrin receptor under iron-limiting conditions. These abilities were rescued by complementation of the mutants with native acbA and actA genes. Secondary structure analysis predicted that the products of actC and actD may be inner-membrane proteins with 12 membrane-spanning helices that belong to the major facilitator superfamily proteins. ActC showed homology to Sinorhizobium meliloti RhtX, which has been characterized as an inner-membrane importer for ferric rhizobactin 1021 structurally similar to acinetoferrin. Compared to the parental ATCC 17906(T) strain, the actD mutant displayed about a 35 % reduction in secretion of acinetoferrin, which was restored by complementation with actD, suggesting that ActD acts as an exporter of the siderophore. Finally, the actB product was significantly similar to hypothetical proteins in certain bacteria, in which genes encoding ActBCA homologues are arranged in the same order as in A. haemolyticus ATCC 17906(T). However, the function of ActB remains to be clarified.

Role of periplasmic binding proteins, FatB and VatD, in the vulnibactin utilization system of Vibrio vulnificus M2799.

Vibrio vulnificus, an opportunistic marine bacterium that causes a serious, often fatal, infection in humans, requires iron for its pathogenesis. This bacterium uses iron from the environment via the vulnibactin-mediated-iron-uptake system. In this study, we constructed the deletion mutants of the genes encoding the proteins involved in the vulnibactin-mediated-iron-uptake system, isochorismate synthase (ICS), vulnibactin utilization protein (VuuB), periplasmic ferric-vulnibactin binding protein (FatB), and ferric-vulnibactin receptor protein (VuuA). The Δics and ΔvuuA mutants were unable to grow under low-iron concentration conditions compared with the isogenic wild-type, indicating that the involvement of ICS in the vulnibactin biosynthesis pathway and uptake of ferric-vulnibactin through the VuuA receptor protein are essential for V. vulnificus M2799 growth under low-iron concentration conditions. Similar growth impairment was also observed in ΔfatB, with growth recovery of this mutant observed 6 h after the beginning of the culture. These results indicate that there must be other periplasmic ferric-vulnibactin binding proteins in V. vulnificus M2799 that complement the defective fatB gene. Complementary growth studies confirmed that VatD protein, which functions as a periplasmic ferric-aerobactin binding protein, was found to participate in the ferric-vulnibactin uptake system in the absence of FatB. Furthermore, the expression of ics, vuuB, fatB, vuuA, and vatD genes was found to be regulated by iron and the ferric uptake regulator.

Characterization of a gene encoding the outer membrane receptor for ferric enterobactin in Aeromonas hydrophila ATCC 7966(T).

Aeromonas hydrophila ATCC 7966(T) produces a catecholate siderophore amonabactin in response to iron starvation. In this study, we determined that this strain utilizes exogenously supplied enterobactin (Ent) for growth under iron-limiting conditions. A homology search of the A. hydrophila ATCC 7966(T) genomic sequence revealed the existence of a candidate gene encoding a protein homologous to Vibrio parahaemolyticus IrgA that functions as the outer membrane receptor for ferric Ent. SDS-PAGE showed induction of IrgA under iron-limiting conditions. The growth of the double mutant of irgA and entA (one of the amonabactin biosynthetic genes) was restored when it was complemented with irgA in the presence of Ent. Moreover, a growth assay of three isogenic tonB mutants indicated that the tonB2 system exclusively provides energy for IrgA to transport ferric Ent. Finally, reverse transcriptase-quantitative PCR revealed that the transcription of irgA and the TonB2 system cluster genes is iron-regulated, consistently with the presence of a predicted Fur box in the promoter region.

Characterization of Vibrio parahaemolyticus genes encoding the systems for utilization of enterobactin as a xenosiderophore.

We determined the ability of Vibrio parahaemolyticus to utilize enterobactin (Ent) as a xenosiderophore. Homology searches of the V. parahaemolyticus genomic sequence revealed the presence of genes that are homologous to the V. cholerae ferric Ent utilization genes, which consist of the iron-repressible outer-membrane protein genes irgA and vctA, and the ATP-binding cassette transport system operon vctPDGC. Moreover, the irgB and vctR genes, which encode transcriptional regulators, were also found immediately upstream of irgA and vctA, respectively. Growth assays of V. parahaemolyticus indicated that both irgA and vctA mutants grew well in the presence of Ent under iron-limiting conditions, whereas both the irgA/vctA double mutant and the vctPDGC mutant barely grew under the same conditions. In addition, growth assays of three isogenic tonB mutants demonstrated that the TonB2 system, and to a lesser extent the TonB1 system, can provide energy for both IrgA and VctA to transport ferric Ent. SDS-PAGE analysis showed that expression of both IrgA and VctA was enhanced by the presence of Ent. Complementation of the irgB and vctR mutants with their respective genes resulted in the increased expression of IrgA and VctA, respectively. Finally, reverse transcriptase-quantitative PCR revealed that transcription of the Ent utilization system genes is iron-regulated, and that transcription of irgA and vctA under iron-limiting conditions is further activated by proteins encoded by irgB and vctR, respectively, together with Ent.

NK1.1(+) cells regulate neutrophil migration in mice with Acinetobacter baumannii pneumonia.

Acinetobacter baumannii is a major cause of both community-associated and nosocomial infections worldwide. These infections are difficult to treat because the bacterium rapidly develops resistance to multiple antibiotics. However, little is known about the nature of the innate cellular response to A. baumannii infection. In the present study, we identified the cells infiltrating the lungs of mice with Acinetobacter pneumonia and analyzed their response to infection. Normal mice eradicated the A. baumannii infection within 3 days of inoculation. Neutrophils were rapidly recruited to the lungs, followed by macrophages and NK1.1(+) cells. Neutrophil-depleted mice showed acute and severe symptoms, and all of the mice died within 3 days of inoculation. The majority of macrophage-depleted mice responded in a similar manner to the control mice. These results indicate that neutrophils are essential for the elimination of A. baumannii. Half of NK1.1(+) cell-depleted mice died within 1 day of inoculation and the number of infiltrating neutrophils was lower than that in control mice up until 3 days post-inoculation. Moreover, the expression levels of keratinocyte chemoattractant protein (KC) decreased in NK1.1(+) cell-depleted mice. These results indicate that NK1.1(+) cells recruit neutrophils during the early phase of Acinetobacter infection by increasing KC expression.

Identification and characterization of an outer membrane receptor gene in Acinetobacter baumannii required for utilization of desferricoprogen, rhodotorulic acid, and desferrioxamine B as xenosiderophores.

In this study, we found that Acinetobacter baumannii utilized exogenously supplied desferricoprogen, rhodotorulic acid, and desferrioxamine B for growth under iron-limiting conditions. The ferric uptake regulator (Fur) titration assay method was then successfully applied to select iron-regulated genes in A. baumannii genomic libraries. Part of the nucleotide sequence homologous to Escherichia coli, fhuE, obtained from one of the positive clones allowed us to clone the entire gene, which was named fhuE. The fhuE gene had an amino acid sequence consistent with the N-terminal amino acid sequence of the 76-kDa iron-repressible outer membrane proteins in A. baumannii. Reverse transcription-polymerase chain reaction analysis demonstrated that fhuE mRNA is transcribed under iron-limiting conditions, consistent with the presence of a sequence homologous to the consensus Fur box in the promoter region. Disruption of fhuE resulted in the loss of expression of the 76-kDa protein. In addition, the double disruptant of fhuE and basD, which encodes one of the biosynthetic genes for the cognate siderophore acinetobactin, was unable to grow in the presence of desferricoprogen, rhodotorulic acid or desferrioxamine B. However, growth of the double disruptant was restored by complementation with fhuE, demonstrating that A. baumannii FhuE functions as the receptor common to coprogen, ferric rhodotorulic acid and ferrioxamine B.

Identification of genes, desR and desA, required for utilization of desferrioxamine B as a xenosiderophore in Vibrio furnissii.

We found that Vibrio (V.) furnissii ATCC35016 can gain iron through a xenosiderophore desferrioxamine B (DFOB) for its growth under iron-limiting conditions, concurrent with the expression of the 79-kDa iron-repressible outer membrane protein (IROMP) in response to the presence of DFOB. Based on the sequence of the ferrioxamine B (an iron-bound form of DFOB) receptor gene in V. vulnificus, two V. furnissii genes, termed desA and desR, encoding the 79-kDa IROMP and AraC-type transcriptional regulator, respectively, were identified and cloned. Nucleotide sequences located in the promoter regions of both desR and desA predicted the presence of consensus ferric uptake regulation (Fur)-binding sequences. The transcription of both genes was negatively regulated by exogenous iron levels. Deletion of the desA gene abolished the ability of V. furnissii to utilize DFOB, and neither desA mRNA nor DesA was detected in the deletion mutant of desR regardless of the presence of DFOB. The functions of DesA and DesR as the ferrioxamine B receptor and transcriptional activator for desA, respectively, were confirmed by complementation of desA and desR deletion mutants.

Crystallization and preliminary X-ray crystallographic analysis of BxlA, an intracellular beta-D-xylosidase from Streptomyces thermoviolaceus OPC-520.

BxlA from Streptomyces thermoviolaceus OPC-520, together with the extracellular BxlE and the integral membrane proteins BxlF and BxlG, constitutes a xylanolytic system that participates in the intracellular transport of xylan-degradation products and the production of xylose. To elucidate the mechanism of the hydrolytic degradation of xylooligosaccharides to xylose at the atomic level, X-ray structural analysis of BxlA was attempted. The recombinant BxlA protein (molecular weight 82 kDa) was crystallized by the hanging-drop vapour-diffusion method at 289 K. The crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 142.2, b = 129.5, c = 101.4 A, beta = 119.8 degrees , and contained two molecules per asymmetric unit (V(M) = 2.47 A(3) Da(-1)). Diffraction data were collected to a resolution to 2.50 A and provided a data set with an overall R(merge) of 8.3%.

Chitinase inhibitor allosamidin promotes chitinase production of Streptomyces generally.

Allosamidin is a family 18 chitinase inhibitor produced by Streptomyces. In its producing strain, Streptomyces sp. AJ9463, allosamidin promotes production of the family 18 chitinase originated from chi65 in a chitin medium through the two-component regulatory system encoded by chi65R and chi65S, which were present at the 5'-upstream region of chi65. In this study, we showed generality of the allosamidin's effect. Allosamidin enhanced production of the family 18 chitinases originated from chi65h of Streptomyces halstedii MF425, another allosamidin producer, chiC of Streptomyces coelicolor A3(2) and chiIII of Streptomyces griseus. All the three chitinase genes had high homology to chi65 and two genes homologous to chi65S and chi65R were present at their 5'-upstream regions. When allosamidin's effect was tested with six Streptomyces strains randomly isolated from soil, allosamidin enhanced chitinase production of all strains. All six strains possessed a set of three genes homologous to chi65, chi65S and chi65R. Analysis of 16S rDNA indicated that allosamidin-sensitive strains are distributed widely in Streptomyces. These observations suggested that allosamidin can affect the common regulatory system for production of a chitinase with a two-component regulatory system in Streptomyces.

Transcriptional regulation of the ferric aerobactin receptor gene by a GntR-like repressor IutR in Vibrio furnissii.

We found that Vibrio furnissii can utilize aerobactin (AERO) as a xenosiderophore. A homology search of its genome revealed that this bacterium possesses genes encoding an AERO-mediated iron acquisition system similar to that of V. vulnificus. The system consists of the ABC transporter gene vatCDB, the GntR-type transcriptional repressor gene iutR, and the outer membrane receptor gene iutA. The functions of the vatCDB operon and iutA in V. furnissii were confirmed by the inability of the corresponding deletion mutants to utilize AERO. Reverse transcription-quantitative PCR revealed that iutA transcription under iron-limiting conditions was extensively activated by the addition of AERO to the growth medium; therefore, we focused on elucidating this phenomenon. Electrophoretic mobility shift and DNase I footprinting assays revealed that glutathione S-transferase-fused IutR (GST-IutR) bound directly to a specific palindromic sequence in the iutA promoter region. However, GST-IutR did not bind to this sequence when either AERO or ferric AERO was present in the assay mixture. These in vitro findings suggest that, under iron-limiting conditions, iutA transcription in V. furnissii is artfully regulated both by IutR, acting as a direct repressor of iutA, and by AERO, acting as an effector for IutR, leading to the derepression of iutA transcription.

Crystallization and preliminary X-ray crystallographic analysis of BxlE, a xylobiose transporter from Streptomyces thermoviolaceus OPC-520.

Together with the integral membrane proteins BxlF and BxlG, BxlE isolated from Streptomyces thermoviolaceus OPC-520 forms an ATP-binding cassette (ABC) transport system that mediates the uptake of xylan. To clarify the structural basis of sugar binding by BxlE at the atomic level, recombinant BxlE was crystallized using the hanging-drop vapour-diffusion method at 290 K. The crystals belonged to the monoclinic space group P2(1), with unit-cell parameters a = 44.63, b = 63.27, c = 66.40 A, beta = 103.05 degrees, and contained one 48 kDa molecule per asymmetric unit (V(M) = 1.96 A3 Da(-1)). Diffraction data collected to a resolution of 1.65 A using a rotating-anode X-ray source gave a data set with an overall R(merge) of 2.6% and a completeness of 91.3%. A data set from a platinum derivative is being used for phasing by the SAD method.

Crystal structure of the solute-binding protein BxlE from Streptomyces thermoviolaceus OPC-520 complexed with xylobiose.

BxlE from Streptomyces thermoviolaceus OPC-520 is a xylo-oligosaccharide (mainly xylobiose)-binding protein that serves as the initial receptor for the bacterial ABC-type xylo-oligosaccharide transport system. To determine the ligand-binding mechanism of BxlE, X-ray structures of ligand-free (open form) and ligand (xylobiose)-bound (closed form) BxlE were determined at 1.85 Å resolution. BxlE consists of two globular domains that are linked by two ß-strands, with the cleft at the interface of the two domains creating the ligand-binding pocket. In the ligand-free open form, this pocket consists of a U-shaped and negatively charged groove located between the two domains. In the xylobiose-bound closed form of BxlE, both the N and C domains move to fold the ligand without conformational changes in either domain. Xylobiose is buried in the groove and wrapped by the N-domain mainly via hydrogen bond interactions and by the C-domain primarily via non-polar interactions with Trp side chains. In addition to the concave shape matching the binding of xylobiose, an inter-domain salt bridge between Asp-47 and Lys-294 limits the space in the ligand-binding site. This domain-stabilized mechanism of ligand binding to BxlE is a unique feature that is not observed with other solute-binding proteins.