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Pathogenic Leptospira species are the etiological agents of the widespread zoonotic disease leptospirosis. Most organisms, including Leptospira, require divalent cations for proper growth, but because of their high reactivity, these metals are toxic at high concentrations. Therefore, bacteria have acquired strategies to maintain metal homeostasis, such as metal import and efflux. By screening Leptospira biflexa transposon mutants for their ability to use Mn2+, we have identified a gene encoding a putative orphan ATP-binding cassette (ABC) ATPase of unknown function. Inactivation of this gene in both L. biflexa and L. interrogans strains led to mutants unable to grow in medium in which iron was replaced by Mn2+, suggesting an involvement of this ABC ATPase in divalent cation uptake. A mutation in this ATPase-coding gene increased susceptibility to Mn2+ toxicity. Recombinant ABC ATPase of the pathogen L. interrogans exhibited Mg2+-dependent ATPase activity involving a P-loop motif. The structure of this ATPase was solved from a crystal containing two monomers in the asymmetric unit. Each monomer adopted a canonical two-subdomain organization of the ABC ATPase fold with an α/β subdomain containing the Walker motifs and an α subdomain containing the ABC signature motif (LSSGE). The two monomers were arranged in a head-to-tail orientation, forming a V-shaped particle with all the conserved ABC motifs at the dimer interface, similar to functional ABC ATPases. These results provide the first structural and functional characterization of a leptospiral ABC ATPase.
Most eukaryotes and prokaryotes require divalent cations (mainly Fe2+, Zn2+, Mn2+, Ni2+, Cu2+, or Co2+) for proper growth and development, as these transition metals are necessary for numerous biological processes (1, 2). Iron, which is present at concentrations in the mM range in Escherichia coli (3), has traditionally been considered the most important transition metal in the physiology of bacteria, and several studies have led to a detailed understanding of how iron is acquired by bacterial cells (reviewed in reference 4). However, other divalent cations, such as Zn2+, Cu2+, and Mn2+, also play an important physiological role. Although Mn2+ is accumulated to a lesser extent than Fe2+ in cells (in the μM range in E. coli) (3), it is also involved in major enzymatic reactions and plays an important role in protection from reactive oxygen species (5). In fact, Fe2+ and Mn2+ are interchangeable in the metal binding site of many metalloenzymes (5, 6). However, the acquisition of Mn2+, as well as its role in the physiology of bacteria, remains poorly understood (7).
Although necessary for bacterial viability, divalent cations can be toxic when present at high concentrations. Therefore, bacteria have acquired strategies to maintain transition metal homeostasis, such as sensing fluctuations in metal cellular content or metal uptake, efflux, and sequestration (8). Metal homeostasis is of great importance for host-pathogen interactions (9). One defensive strategy of the host is to restrict the access of divalent cations to invading bacteria. Another recently discovered strategy is to harness the toxic properties of transition metals to eliminate invading pathogens. Among divalent cations, manganese has recently received attention. Indeed, mutation of the Mn2+ uptake system was shown to impair the virulence of several bacterial species, including Salmonella enterica serovar Typhimurium (10), Yersinia pestis (11), Bacillus anthracis (12), Streptococcus pneumoniae (13), Staphylococcus aureus (14), and Borrelia burgdorferi (15). Moreover, exporting excess Mn2+ has also proven necessary for infection by S. pneumoniae (16), Xanthomonas oryzae (17), and Neisseria meningitidis (18).
Leptospira spp. are motile, aerobic, chemoorganotrophic spirochetes. Pathogenic Leptospira spp. are the causative agents of leptospirosis, a zoonosis of global importance (19, 20). The lack of techniques for genetic manipulation of Leptospira spp. has greatly hindered investigation of their physiology, including metal homeostasis and metabolism. Indeed, targeted mutagenesis is extremely inefficient in pathogenic Leptospira (20). Our group has developed a system for random transposon mutagenesis using a mariner element, Himar1, in the saprophyte Leptospira biflexa (21) and the pathogen L. interrogans (22). This approach allowed the identification of specialized uptake systems for iron (21, 23).
The transport of metals through the bacterial cytoplasmic membrane is mediated by energy-coupled systems, such as ATP-binding cassette (ABC) and P-type transporters. Proton-coupled transporters, including Nramp (natural resistance associated with macrophage protein) and the resistance and nodulation (RND) and cation diffusion facilitator (CDF) proteins, can also mediate metal uptake (reviewed in reference 8). Analysis of Leptospira genomes indicated the presence of putative transporters of the Nramp and ABC families, but none of them has been characterized. Moreover, very little is known about divalent cation acquisition and homeostasis in Leptospira spp.
In this study, by screening L. biflexa transposon mutants for Mn2+-altered growth, we identified a gene encoding an orphan ABC ATPase of unknown function. We have investigated the functional and structural properties of this new ABC ATPase, encoded by LEPBIa2866 in L. biflexa and LIC12079 in L. interrogans.
L. biflexa serovar Patoc strain Patoc 1, L. interrogans serovar Manilae strain L495, and L. interrogans serovar Copenhageni strain Fiocruz L1-130 were grown aerobically at 30°C in Ellinghausen-McCullough-Johnson-Harris (EMJH) medium (24). When necessary, kanamycin and/or spectinomycin was added at 40 μg/ml. It should be noted that regular EMJH medium contains FeSO4 and ZnCl2 at concentrations of about 180 and 15 μM, respectively, and that MnCl2 is either absent from this medium or present at concentrations of less than 5 μM. When indicated, FeSO4 was omitted during the preparation of EMJH medium, and when necessary, MnCl2 was added at a final concentration of 100 μM.
Random insertion mutagenesis using the kanamycin-resistant transposon Himar1 was carried out in L. biflexa serovar Patoc strain Patoc 1 and L. interrogans serovar Manilae strain L495, as described previously (21, 22). To identify mutants of L. biflexa unable to grow in the presence of Mn2+, more than 2,000 random mutants were replica plated onto EMJH medium and iron-deficient EMJH medium containing 100 μM MnCl2. The insertion of a single transposon within each individual clone was confirmed by PCR and Southern blot hybridization, and the position of transposon insertion was determined by direct sequencing of genomic DNA. This allowed us to identify LEPBIa2866 (GI: 91178403) and LEPBIa2071 (GI: 167776217) mutants. LEPBIa2866- and LEPBIa2071-expressing plasmids used in mutant complementation were constructed by PCR amplifying these open reading frames (ORFs) with primer set 2751-2-5′ (5′-ACTCGCTGAAAAATGGGAATTCC-3′) and 2751-2-3′ (5′-TGCGGAGAACACATCGATATTGTC-3′) and primer set 1648-2-5′ (5′-ACAAAGTCCCTAGCCTCGATCCCC-3′) and 1648-2-3′ (5′-TTATGTTCTAACGACTAGTCATTCC-3′), respectively, and ligating the PCR products into the SmaI blunt-ended site of the spectinomycin-resistant L. biflexa-E. coli shuttle vector pGSBLe24 (25). Since the LEPBIa2866 ORF is cotranscribed with the upstream LEPBIa2865 ORF, complementation was performed with plasmid pGSBLe24 containing a transcriptional fusion between the L. interrogans hsp10 (groES) promoter and the LEPBIa2866-coding sequence, as described previously (26). Transposon insertion in the L. interrogans LEPBIa2866 ortholog (LIC12079, GI: 45657927) was identified in the L. interrogans serovar Manilae strain L495 mutant library (22).
L. biflexa cells were grown to exponential phase (about 108/ml) in EMJH medium and then incubated for 20 h in the absence or presence of 100 μM MnCl2. Cells were then diluted in complete EMJH medium without MnCl2 and plated on solid EMJH medium. After incubation for 1 week at 30°C, colonies were counted and percent survival (as a percentage of the total number of CFU) was calculated as the ratio of the number of CFU for cells incubated in the presence of MnCl2 to that for cells incubated in the absence of MnCl2.
MICs were determined by adding serial 2-fold dilutions of Mn2+ to L. interrogans cells (about 2 × 106/ml in EMJH medium). Bacteria were incubated at 30°C for 1 week before assessment by dark-field microscopy. The MIC was determined as the point at which growth was inhibited by 50%.
A LIC12079-expressing plasmid was constructed by amplifying the LIC12079 ORF (forward primer, 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTCGGAAAACCTGTATTTTCAGGGCAAAATCAATTCGTTATTATCCCTAG-3′; reverse primer, 5′-GGGGACCACTTTGTACAAGAAAGCTGGGTTTAATAGGTTCGTTTTTGTTTTGGGATCAC-3′) from L. interrogans serovar Copenhageni strain Fiocruz L1-130 and cloning it into the Gateway pDEST17 vector (Life Technologies). A single-mutation variant, LIC12079-K45A, was obtained by replacing the Lys 45 codon by an alanine codon using a QuikChange multisite-directed mutagenesis kit (Agilent Technologies) according to the manufacturer's recommendation and the primer 5′-AAGAAACGGAGCTGGCGCAAGTACTCTCGTAAATC-3′ (with the mutated codon indicated in bold). The codon substitution in the LIC12079-K45A variant was confirmed by DNA sequencing (Beckman Coulter Genomics).
E. coli strain BL21(DE3) ΔdnaK::kan (EN2) (27) was transformed with the pDEST17 expression vector carrying the LIC12079 or the LIC12079-K45A ORF and grown to an absorbance of about 0.7 at 600 nm at 30°C in Luria-Bertani (LB) medium supplemented with 30 μg/ml kanamycin and 100 μg/ml ampicillin. Protein expression was then induced with 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG) for 4 h. After centrifugation, cell pellets were resuspended in buffer A (50 mM NaH2PO4, pH 8.0, 300 mM NaCl, 10 mM imidazole, 10% glycerol) and lysed by sonication. The soluble fraction obtained after 60 min centrifugation at 40,000 × g at 4°C was loaded on nickel-nitrilotriacetic acid (Ni-NTA) resin (Qiagen) and washed with buffer A. His-tagged proteins were eluted with buffer B (50 mM NaH2PO4, pH 8.0, 300 mM NaCl, 250 mM imidazole, 10% glycerol). After elution, His-tagged proteins were dialyzed against buffer C (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM dithiothreitol [DTT]) and loaded onto a HiLoad 16/60 Superdex 200 (GE Healthcare) size exclusion chromatography column equilibrated with the same buffer. Elution was performed at a flow rate of 1.0 ml/min. Fractions containing the LIC12079 protein were pooled and concentrated. Protein concentrations were determined with Coomassie Plus protein assay reagent (Thermo Scientific).
The LIC12079 protein used in crystallization experiments was produced in the E. coli BLi5 strain [a BL21(DE3)-derived strain that carried a pDIA17 plasmid] (28). Cells were cultivated at 30°C in a microfermentor (Biopod Fogale Nanotech, France) with high-density medium (HDM) containing 100 μg/ml ampicillin and 30 μg/ml chloramphenicol until they reached an absorbance of 20 at 600 nm. Protein expression was induced with 1 mM IPTG for 15 h at 14°C. Cells were harvested and lysed by a French press in 50 mM Tris-HCl, pH 8.2, 100 mM NaCl, and 5 mM imidazole. Purification was performed by nickel immobilized-metal affinity chromatography using a HiTrap Chelating HP column (GE Healthcare), followed by size exclusion chromatographic step using a HiLoad 16/60 Superdex 75 column (GE Healthcare). Fractions containing the LIC12079 protein were pooled and concentrated to 5.2 mg/ml.
Preliminary crystallization screening was carried out by the sitting-drop vapor diffusion method with a Mosquito (TTP Labtech) automated crystallization system. A 400-nl (1:1) mixture of LIC12079 protein at 5.2 mg/ml and different crystallization solutions (672 commercially available solutions) equilibrated against 150 μl reservoir in Greiner plates was used. The protein crystallized only under conditions containing ammonium sulfate as a precipitant. After extensive screening and optimization, the best crystals were obtained by the hanging-drop method in Linbro plates at 18°C by mixing 1.5 μl of protein solution at 5.2 mg/ml with an equal volume of reservoir solution containing 1.2 M ammonium sulfate and 0.1 M sodium cacodylate, pH 6.5.
Crystals of LIC12079 were flash-cooled in liquid nitrogen using Paratone-paraffin (50%/50%) oil as the cryoprotectant. X-ray diffraction data were collected on beamline PROXIMA-1 at Synchrotron SOLEIL (St. Aubin, France). The diffraction images were integrated with the program XDS (29, 30), and crystallographic calculations were carried out with programs from the CCP4 program suite (31).
The structure was solved by the molecular replacement method with the program Phaser (32), as implemented in the CCP4 program package. The molecular replacement search model was the ABC ATPase SACOL2144 from S. aureus (Protein Data Bank [PDB] accession number 2IHY), with 35% sequence identity and 69% sequence similarity to LIC12079. The molecular replacement solution corresponded to two independent molecules in the crystallographic asymmetric unit, with a solvent content (Vs) of 52.4% and a Matthews coefficient (Vm) of 2.58 Å3 Da−1 (33). The structure was refined by alternate cycles of restrained maximum likelihood refinement with the program Refmac5 (34). Manual adjustments were made to the model with the Coot program (35). The crystal parameters, data collection statistics, and final refinement statistics are shown in Table 1. All structural figures were generated with the PyMOL program (36).
Purified LIC12079 and LIC12079-K45A were assayed for ATP hydrolysis by monitoring the production of inorganic phosphate at 37°C in 20 mM Tris-HCl, pH 8.0, and 1 mM DTT as described previously (37), using NaH2PO4 as a standard.
Atomic coordinates and structure factors have been deposited with the Protein Data Bank with entry code 4HZI.
The EMJH medium routinely used for the growth of Leptospira contains Fe2+, Mg2+, Ca2+, Zn2+, and, in some cases, low concentrations of Mn2+ (up to 5 μM). To test the ability of the L. biflexa wild-type (WT) strain to use divalent cations other than Fe2+, this metal was omitted from the EMJH medium during medium preparation, and consequently, the medium contained only trace amounts of iron. Various metals were then added to the iron-deficient medium in order to restore leptospiral growth. Mn2+ and Zn2+ permitted growth at a concentration of 100 μM, whereas the addition of Mg2+ (up to 500 μM) or Co2+ (up to 1 mM) failed to do so (data not shown). Thus, iron could be exchangeable with Mn2+ and Zn2+ in Leptospira.
In order to identify factors involved in manganese utilization in Leptospira, an L. biflexa transposon mutant library was screened for Mn2+-altered phenotypes. This allowed the identification of three mutants that were unable to grow in Fe2+-deficient EMJH medium supplemented with Mn2+. In two mutants, the transposon was inserted into the LEPBIa2071 ORF (see Fig. S1A in the supplemental material) that encodes a putative protein displaying similarities to predicted permeases of the major facilitator superfamily (MFS) of Cytophaga hutchinsonii (50% identity, 70% similarity), L. borgpetersenii (LBL_0798, LBJ_2309; 47% identity, 70% similarity), L. interrogans (LMANv1_2290003, LA1081, LIC12590; 47% identity, 69% similarity), and Nostoc punctiforme (43% identity, 62% similarity). MFS proteins transport small solutes, including sugars and inorganic ions, in response to chemiosmotic gradients (38). According to the phylogenic classification of the Transport Classification Database (TCDB; http://www.tcdb.org/), LEPBIa2071 could be related to bacterial siderophore exporters EntS (transporter classification number 2.A.1.38.1) and VabS (transporter classification number 2.A.1.38.2). Although most MFS proteins are predicted to contain 12 α-helical transmembrane-spanning segments (TMSs) (38), the L. biflexa and L. interrogans proteins contain only 10 predicted α-helical TMSs (data not shown).
The third mutant had a transposon insertion in LEPBIa2866, a gene encoding a protein that shares similarity with putative ABC transporter proteins of L. interrogans (LMANV1_3710001, LA1724, LIC12079; 40% identity, 61% similarity) and L. borgpetersenii (LBL_1280; 40% identity, 60% similarity) (see Fig. S1B in the supplemental material). This level of amino acid identity was consistent with the general genetic relatedness between saprophyte and pathogen strains. Similarities were also found with ORFs from other bacterial species, including Bacillus subtilis (38% identity, 56% similarity) and Streptomyces avermitilis (37% identity, 56% similarity). Since the L. interrogans orthologs exhibited more than 99% nucleotide sequence identity in L. interrogans serovars Lai (LA1724), Copenhageni (LIC12079), and Manilae (LMANV1_3710001), we further refer to LIC12079 throughout the text.
L. biflexa WT, LEPBIa2866 mutant, and LEPBIa2071 mutant cells were not able to grow in Fe2+-deficient EMJH medium (data not shown) (23). As shown in Fig. 1A, the addition of 100 μM MnCl2 to Fe2+-deficient EMJH medium did not allow the growth of either the LEPBIa2866 or the LEPBIa2071 mutant but could restore that of WT cells (Fig. 1A). Introduction of a recombinant plasmid harboring the LEPBIa2866 or LEPBIa2071 gene restored the ability of both mutants to use Mn2+ as a metal source (Fig. 1B). Notably, both mutants were able to grow in complete EMJH medium, indicating that their manganese-altered phenotype was not due to a general growth defect.
A search of our mutant library of the pathogen L. interrogans serovar Manilae (22) identified a mutant with a mutation in the gene LIC12079, an ortholog of LEPBIa2866. The effect of Mn2+ supplementation of Fe2+-deficient EMJH medium on the growth of the LIC12079 mutant was therefore investigated. L. interrogans WT cells, as well as the LIC12079 mutant cells, exhibited residual growth in Fe2+-deficient EMJH liquid medium, probably indicating an ability to acquire the trace amounts of Fe2+ present in the iron-deficient medium (23). The addition of Mn2+ to the Fe2+-deficient medium led to a slight increase in the doubling time of the WT cells, but it did not allow the growth of the LIC12079 mutant (Fig. 1C). These findings indicate that the ABC ATPase encoded by LEPBIa2866 or LIC12079 in Leptospira is involved in Mn2+ utilization and/or tolerance.
In order to determine whether the growth defect in Fe2+-deficient EMJH medium supplemented with Mn2+ observed upon inactivation of LEPBIa2866 or LIC12079 was partly due to an inability of these mutants to tolerate the presence of MnCl2, cell survival and growth were measured in complete EMJH medium in the presence of Mn2+. In EMJH medium (containing Fe2+), growth of L. biflexa WT and LEPBIa2866 mutant cells was not altered by the presence of 100 μM MnCl2 (Fig. 2A), indicating that the reduced growth of the LEPBIa2866 mutant observed when 100 μM MnCl2 was added to Fe2+-deficient medium could not be explained by a reduced tolerance toward this metal under these conditions. However, higher concentrations of MnCl2 impaired the growth of L. biflexa (data not shown). Survival of the WT L. biflexa cells was greater than that of LEPBIa2866 mutant cells after 20 h of incubation with 1 mM MnCl2 (Fig. 2B).
The growth of L. interrogans was more sensitive to the presence of MnCl2, since the addition of 100 μM MnCl2 to complete EMJH medium reduced its growth. This decrease in growth was even more pronounced in the LIC12079 mutant than in the wild type (Fig. 2C). In addition, cells of the L. interrogans WT strain showed significantly higher Mn2+ resistance (MIC, 1.25 mM) than LIC12079 mutant cells (MIC, 0.078 mM). These findings indicate that the LIC12079 ORF and its ortholog, LEPBIa2866, are involved in the reduction of Mn2+ toxicity in Leptospira spp.
The LIC12079 and its ortholog LEPBIa2866 ORFs are annotated as members of the large ABC transporter family. ABC ATPases couple the energy of ATP hydrolysis with the influx or efflux of a variety of substrates through cellular membranes but also with non-transport-related processes (39). A BLAST analysis of LIC12079 indicated that orthologs of these ORFs are present in the genomes of many Leptospira species (saprophyte, intermediate, and pathogen) and also in other bacteria, mostly Gram-positive species.
LIC12079 encodes a 30-kDa protein. Analysis of the primary sequence showed the presence of a single ABC module, but unlike ABC transporters of class 1, this module was not fused to any transmembrane domain (see Fig. S2 in the supplemental material). No gene encoding a protein with a predicted membrane or periplasmic localization was found in the close vicinity of LIC12079. The ORF immediately upstream (LIC12078) encodes a hypothetical protein containing a His/Asp (HD) domain found in members of a superfamily of metal-dependent phosphohydrolases (40). It is noteworthy that in L. biflexa, transcriptional analysis by reverse transcription-PCR revealed that the LIC12079 ortholog (LEPBIa2866) was cotranscribed with the LIC12078 ortholog (LEPBIa2865) (data not shown). Therefore, LEPBIa2866 and LIC12079 encode an orphan ABC ATPase with no predicted linked transmembrane partner.
The primary sequences of LIC12079 and LEPBIa2866 were aligned with the primary sequence of the glucose transporter of Sulfolobus solfataricus (GlcV), a well-characterized ABC transporter. The sequence of LIC12079 exhibited all typical ABC ATPase motifs (41) (see Fig. S2 in the supplemental material), including the Walker A (GRNGAGKS) and Walker B (FLIMDE) motifs of a P-loop nucleoside triphosphatase (NTPase) (42), a putative Q-loop sequence (QQENSIQR) located between the Walker motifs, and a putative H loop (THR) located downstream from the Walker B motif. An LSSGE sequence was found upstream of the Walker B motif. The LSSGE sequence corresponds to the ABC transporter family LSGGQ signature (43), a motif unique to ABC ATPases that, together with the Walker motifs, interacts with ATP.
The ability of recombinant LIC12079 protein to hydrolyze ATP was tested. To avoid contamination by two other major E. coli ATPases, the DnaK and GroEL/GroES chaperones, recombinant LIC12079 was produced in a BL21(DE3) ΔdnaK strain (27), and the purification protocol contained a size exclusion chromatography step to separate the 30-kDa LIC12079 from the 870-kDa GroEL/GroES complex. The purified wild-type LIC12079 exhibited ATPase activity (Fig. 3A). Replacing the lysine residue of the Walker A motif (Lys45 in LIC12079), known to be involved in ATP hydrolysis, by alanine resulted in significantly decreased ATPase activity (Fig. 3A).
As expected for a P-loop NTPase, the ability of LIC12079 to hydrolyze ATP depended on the presence of Mg2+ (Fig. 3B). Despite a putative role of this ABC ATPase in Mn2+ uptake or utilization, this cation did not support LIC12079 ATPase activity in a manner comparable to that of Mg2+. ATPase hydrolysis by LIC12079 was also increased in the presence of sodium or potassium salts (data not shown). These findings demonstrate that the recombinant LIC12079 protein was purified in a native conformation exhibiting ATPase activity.
In order to gain insight into the structural organization of the ABC ATPase LIC12079, its crystal structure was determined. This protein crystallized in the absence of nucleotide with two independent molecules in the asymmetric unit. The final model consisted of residues 4 to 262 for both polypeptide chains, with no visible density from residues Gln90 to Gln95 in both chains or from residues Phe109 to Arg116 in chain B. Both polypeptides adopted identical folding and could be closely superimposed, with an overall root mean square deviation of 0.58 Å in alpha-carbon positions. A bound sulfate ion was present in each monomer.
Each ABC ATPase monomer adopted a two-subdomain organization of the canonical ABC ATPase fold (Fig. 4). The ABC α/β subdomain of LIC12079 (residues 4 to 86 and 164 to 228) displayed a central α helix packed between two β sheets, a three-stranded antiparallel sheet, and a five-stranded parallel sheet followed by an additional antiparallel β strand. In this domain, the Walker A motif folded into the canonical loop-helix structure and the Walker B motif was located in the parallel β sheet. The ABC α subdomain (residues 87 to 163) contained four α helices. The ABC signature motif (LSSGE in LIC12079) was contained in a central helix. The highly exposed Q loop (Gln89-Arg96) connecting the two subdomains was disordered, displaying weak discontinuous electron density in both chains in the crystal structure. This disorder could be due to the absence of nucleotide in the active site. An additional α/β structure (residues 229 to 262) was present at the C terminus of the α/β ABC subdomain.
Close structural similarity with the ABC ATPase domain of GlcV from S. solfataricus in its ADP-bound form was observed (44) (see Fig. S3 in the supplemental material). In LIC12079, the Walker A motif and the strictly conserved residues Asp170 and Glu171 (the putative catalytic base) at the C terminus of the Walker B motif were present with a conserved spatial orientation. Other structural motifs, the D loop (Ser174-Asp177) and the H loop (Thr203-His204-Arg205), could be closely superimposed with the corresponding residues in the GlcV ATPase structure. The ABC signature motif (LSSGE in LIC12079), partially exposed to solvent at the beginning of the central α helix in the ABC α subdomain, also adopted a similar conformation in the superimposed structures.
Attempts to determine the structure of LIC12079 in complex with AMP, ADP, or ATP by cocrystallization or soaking were unsuccessful, possibly due to the presence of a bound sulfate ion (from the crystallization solution) at the nucleotide binding site (Fig. 5A). The Walker A motif of LIC12079 could be closely superimposed with the corresponding motif in the ADP-bound structure of GlcV from S. solfataricus (Fig. 5B). Indeed, residues Gly42, Gly44, Lys45, and Ser46 in the Walker A motif, which could make hydrogen bonds with the β phosphate in the ATP-binding site, were strictly conserved in spatial orientation with the corresponding residues in GlcV (see also Fig. S2 and S3 in the supplemental material). The equivalent residues in LIC12079 interacted with a sulfate ion located at the equivalent position of the ADP β-phosphate group in the GlcV ADP-bound form (Fig. 5B). By homology with other ABC transporters (45), the adenine moiety of the nucleotide could be stabilized by interactions with the side chain of Tyr14 in LIC12079 (data not shown).
The two LIC12079 monomers were arranged in a head-to-tail orientation, forming a V-shaped particle (Fig. 6A). The interface between the two molecules included two C-terminal β strands and the two conserved D and H loops (Fig. 6B). All the other ABC conserved motifs were also located near the interface between the two monomers, with the Walker A and B motifs of each monomer facing the C loop of the other. This arrangement is similar to that of other ABC ATPase dimers (46). Interactions between the two monomers were hydrophobic and electrostatic, with a buried surface area of 2,960 Å2, as calculated by the European Bioinformatics Institute PISA web-based server (47). Analysis by dynamic light scattering and size exclusion chromatography indicated a dimeric self-association of the protein in solution (data not shown).
Manganese is an important divalent cation metal that has a dual effect on cell growth; it can be a nutrient as well as a toxic element. In the present study, we identified and characterized a novel ABC ATPase in Leptospira whose inactivation led to the decreased growth of the fast-growing saprophyte L. biflexa and the slow-growing pathogen L. interrogans when Fe2+ was replaced by Mn2+ and led to reduced survival in the presence of toxic concentrations of Mn2+. The observed phenotypes differed slightly between the two leptospiral species. In L. biflexa, Mn2+ could restore the growth of wild-type cells in the absence of Fe2+ but not that of LEPBIa2866 ABC ATPase mutant cells. Even though Mn2+ was toxic for this strain when used at higher concentrations, 100 μM MnCl2 was not lethal (Fig. 2), and the inability of L. biflexa LEPBIa2866 mutant cells to grow in Fe2+-deficient medium in the presence of Mn2+ could therefore be explained by an inability to take up Mn2+. When iron was not added to the medium, L. interrogans cells still exhibited residual growth. Addition of 100 μM MnCl2, a toxic concentration for L. interrogans (Fig. 2), greatly reduced the growth of the ABC ATPase mutant, perhaps reflecting a lower tolerance of these cells toward Mn2+. Under these conditions, the sensitivity of L. interrogans to Mn2+ precluded the discrimination between Mn2+ uptake and sensitivity.
The difference in phenotypes between L. biflexa and L. interrogans mutants could be explained if the ABC ATPase encoded by LEPBIa2866 or LIC12079 is involved in both the uptake and the efflux of Mn2+, implying interaction of a single ABC ATPase with different transmembrane components involved in the uptake and the export of Mn2+. Examples of a single ABC ATPase component serving several transporter systems have recently been reported in the literature (48–50), although to our knowledge not with both import and export systems. Alternatively, the LEPBIa2866 or LIC12079 ATPase could serve in a non-transport-related process that allows utilization of Mn2+. Further experiments will be necessary to define the precise role of this ATPase in Mn2+ transport, tolerance, or utilization.
The Leptospira ATPase described here possesses all the canonical motifs of ABC ATPases (39). In this study, we have experimentally demonstrated that LIC12079 displays ATPase activity and exhibits the typical ABC ATPase structural architecture. A BLAST analysis showed that it shares close sequence homology with mostly uncharacterized ABC ATPases that are predicted to energize the transport of solutes, including iron, molybdenum, sulfate, and amino acids. LIC12079 has been classified as a member of the YLU ABC family in the phylogenic and functional classification of ABC systems (http://www.pasteur.fr/abcisse). This family includes orphan ABC ATPases with no defined function or substrate, and no member of this family has been characterized so far. Our study therefore provides the first characterization of a YLU family member and establishes experimental evidence that members of this ABC ATPase family might be involved in divalent cation utilization and tolerance.
The transmembrane component that could mediate the transport energized by the LEPBIa2866 or LIC12079 ATPase is still unknown, and no gene encoding a predicted transmembrane protein could be identified in the close vicinity of these ORFs in L. biflexa or L. interrogans. It is noteworthy that comparing the chromosomal loci of LEPBIa2866 or LIC12079 orthologs in all the genomes available in the databases showed that this organization is conserved in saprophyte, intermediate, and pathogen Leptospira species. Similar to the findings for Leptospira, the S. aureus LIC12079 homolog, SACOL2144, was not found in the vicinity of genes encoding membrane proteins. Interestingly, another transposon insertion identified during the screening of the L. biflexa mutant library was in ORF LEPBIa2071, encoding a putative permease of the MFS transporter family. MFS permeases use the proton motive force rather than ATP hydrolysis as an energy source for solute transport (38). Recently, a functional interplay between an MFS permease and an ABC ATPase in macrolide efflux has been described (51). It would be interesting to investigate whether a possible functional relationship exists between LEPBIa2866 ATPase and the permease encoded by LEPBIa2071 in divalent cation transport and to study the specificity of the transport system energized by the ABC ATPase LEPBIa2866 or LIC12079, which might not be restricted to Mn2+.
It is interesting to note that the ORFs immediately upstream of the ABC ATPase genes LEPBIa2866 (in L. biflexa) and LIC12079 (in L. interrogans) encode putative phosphatases from the HD family (LEPBIa2865 and LIC12078, respectively). Similarly, the closest structurally related ortholog of LIC12079, the S. aureus SACOL2144, is adjacent to an ORF encoding a haloacid dehalogenase (HAD)-like phosphatase (SACOL2143). Since all these phosphatases usually require divalent cations for activity (40), one can speculate that the LEPBIa2866 and LIC12079 ATPases are involved in either the import or incorporation of divalent cations used by the upstream HD phosphatase.
Our study has led to the determination of the crystal structure of an orphan ABC ATPase dimer. ABC ATPases are subject to nucleotide-dependent conformational changes (52). In the ATP-bound form of the dimer, two ABC monomers form a closed interface allowing ATP to interact with the P loop of one ABC ATPase monomer and the ABC signature motif of the second monomer. ATP hydrolysis leads to a conformation with a wider interface between the two monomers. The crystal structure determined in this study showed the two LIC12079 monomers arranged as a dimer with a wide groove at the interface, consistent with the typical conformation of an ADP-bound or nucleotide-free ABC ATPase dimer.
To date, less than 0.03% of all structures available in the Protein Data Bank are of leptospiral origin. In this study, we report the first structure of an ABC ATPase from Leptospira. In addition, by presenting a functional characterization of an orphan ABC ATPase of the YLU family in Leptospira spp., this study contributes to a better understanding of the physiology of these bacteria.
P.R. was supported by CAPES (Brasil) and a Fiocruz-Pasteur grant. H.L. was supported by a grant from the Région Ile-de-France. G.L.M. was a recipient of an NHMRC Peter Doherty Fellowship. Aspects of the work were supported by the Australian Research Council and the National Health and Medical Research Council, the Institut Pasteur, and the French Ministry of Research (ANR-08-MIE-018).
We acknowledge SOLEIL for provision of synchrotron radiation facilities, and we thank the staff of beamline PROXIMA-1 for assistance. We are grateful to O. Chesneau, E. Dassa, A. Eshghi, G. Karimova, A. Lambert, and A. Ullmann for valuable comments on the manuscript.
Published ahead of print 11 October 2013
Supplemental material for this article may be found at http://dx.doi.org/10.1128/JB.00915-13.