Crystal Structure of the Membrane Fusion Protein CusB from Escherichia coli

Gram-negative bacteria, such as Escherichia coli, frequently utilize tripartite efflux complexes belonging to the resistance-nodulation-division family to expel diverse toxic compounds from the cell. These systems contain a periplasmic membrane fusion protein (MFP) that is critical for substrate transport. We here present the x-ray structures of the CusB MFP from the copper/silver efflux system of E. coli. This is the first structure of any MFPs associated with heavy-metal efflux transporters. CusB bridges the inner-membrane efflux pump CusA and outer-membrane channel CusC to mediate resistance to Cu⁺ and Ag⁺ ions. Two distinct structures of the elongated molecules of CusB were found in the asymmetric unit of a single crystal, which suggests the flexible nature of this protein. Each protomer of CusB can be divided into four different domains, whereby the first three domains are mostly β-strands and the last domain adopts an entirely helical architecture. Unlike other known structures of MFPs, the α-helical domain of CusB is folded into a three-helix bundle. This three-helix bundle presumably interacts with the periplasmic domain of CusC. The N- and C-termini of CusB form the first β-strand domain, which is found to interact with the periplasmic domain of the CusA efflux pump. Atomic details of how this efflux protein binds Cu⁺ and Ag⁺ were revealed by the crystals of the CusB-Cu(I) and CusB-Ag(I) complexes. The structures indicate that CusB consists of multiple binding sites for these metal ions. These findings reveal novel structural features of an MFP in the resistance-nodulation-division efflux system and provide direct evidence that this protein specifically interacts with transported substrates.     

Screening and characterization of proteorhodopsin color-tuning mutations in Escherichia coli with endogenous retinal synthesis

Proteorhodopsin is photoactive 7-transmembrane protein, which uses all-trans retinal as a chromophore. Proteorhodopsin subfamilies are spectrally tuned in accordance with the depth of habitat of the host organisms, numerous species of marine picoplankton. We try to find residues critical for the spectral tuning through the use of random PCR mutagenesis and endogenous retinal biosynthesis. We obtained 16 isolates with changed color by screening in Escherichia coli with internal retinal biosynthesis system containing genes for beta-carotene biosynthesis and retinal synthase. Some isolates contained multiple substitutions, which could be separated to give 20 single mutations influencing the spectral properties. The color-changing residues are distributed through the protein except for the helix A, and about a half of the mutations is localized on the helices C and D, implying their importance for color tuning. In the pumping form of the pigment, absorption maxima in 8 mutants are red-shifted and in 12 mutants are blue-shifted compared to the wild-type. The results of flash-photolysis showed that most of the low pumping activity mutants possess slower rates of M decay and O decay. These results suggest that the color-tuning residues are not restricted to the retinal binding pocket, in accord with a recent evolutionary analysis.     

The Escherichia coli Cell Division Protein and Model Tat Substrate SufI (FtsP) Localizes to the Septal Ring and Has a Multicopper Oxidase-Like Structure

The Escherichia coli protein SufI (FtsP) has recently been proposed to be a component of the cell division apparatus. The SufI protein is also in widespread experimental use as a model substrate in studies of the Tat (twin arginine translocation) protein transport system. We have used SufI-GFP (green fluorescent protein) fusions to show that SufI localizes to the septal ring in the dividing cell. We have also determined the structure of SufI by X-ray crystallography to a resolution of 1.9 Å. SufI is structurally related to the multicopper oxidase superfamily but lacks metal cofactors. The structure of SufI suggests it serves a scaffolding rather than an enzymatic role in the septal ring and reveals regions of the protein likely to be involved in the protein-protein interactions required to assemble SufI at the septal ring.     

Heme/heme redox interaction and resolution of individual optical absorption spectra of the hemes in cytochrome bd from Escherichia coli

Cytochrome bd is a terminal component of the respiratory chain of Escherichia coli catalyzing reduction of molecular oxygen to water. It contains three hemes, b₅₅₈, b₅₉₅, and d. The detailed spectroelectrochemical redox titration and numerical modeling of the data reveal significant redox interaction between the low-spin heme b₅₅₈ and high-spin heme b₅₉₅, whereas the interaction between heme d and either hemes b appears to be rather weak. However, the presence of heme d itself decreases much larger interaction between the two hemes b. Fitting the titration data with a model where redox interaction between the hemes is explicitly included makes it possible to extract individual absorption spectra of all hemes. The α- and β-band reduced-minus-oxidized difference spectra agree with the data published earlier ([22] J.G. Koland, M.J. Miller, R.B. Gennis, Potentiometric analysis of the purified cytochrome d terminal oxidase complex from Escherichia coli, Biochemistry 23 (1984) 1051-1056., and [23] R.M. Lorence, J.G. Koland, R.B. Gennis, Coulometric and spectroscopic analysis of the purified cytochrome d complex of Escherichia coli: evidence for the identification of “cytochrome a₁” as cytochrome b₅₉₅, Biochemistry 25 (1986) 2314-2321.). The Soret band spectra show λ_max = 429.5 nm, λ_min ≈ 413 nm (heme b₅₅₈), λ_max = 439 nm, λ_min ≈ 400 ± 1 nm (heme b₅₉₅), and λ_max = 430 nm, λ_min = 405 nm (heme d). The spectral contribution of heme d to the complex Soret band is much smaller than those of either hemes b; the Soret/α (ΔA₄₃₀:ΔA₆₂₉) ratio for heme d is 1.6.     

Membrane structure of CtrA3, a copper-transporting P-type-ATPase from Aquifex aeolicus

We have produced and characterized two new copper-transporting ATPases, CtrA2 and CtrA3 from Aquifex aeolicus, that belong to the family of heavy metal ion-transporting P_IB-type ATPases. CtrA2 has a CPC metal-binding sequence in TM6 and a CxxC metal-binding N-terminal domain, while CtrA3 has a CPH metal-binding motif in TM6 and a histidine-rich N-terminal metal-binding domain. We have cloned both copper pumps, expressed them in Escherichia coli and characterized them functionally. CtrA2 is activated by Ag⁺ and Cu⁺ and presumably transports reduced Cu⁺, while CtrA3 is activated by, and presumably transports, the oxidized copper ion. Both CtrA2 and CtrA3 are thermophilic proteins with an activity maximum at 75 °C. Electron cryomicroscopy of two-dimensional crystals of CtrA3 yielded a projection map at ∼7 Å resolution with density peaks, indicating eight membrane-spanning α-helices per monomer. A fit of the Ca-ATPase structure to the projection map indicates that the arrangement of the six central helices surrounding the ion-binding site in the membrane is conserved, and suggests the position of the two additional N-terminal transmembrane helices that are characteristic of the heavy metal, eight-helix P_1B-type ATPases.     

Crystal Structure of a Major Outer Membrane Protein from Thermus thermophilus HB27

The thermophilic eubacterium Thermus thermophilus belongs to one of the oldest branches of evolution and has a multilayered cell envelope that differs from that of modern Gram-negative bacteria. Its outer membrane contains integral outer membrane proteins (OMPs), of which only a few are characterized. TtoA, a new β-barrel OMP, was identified by searching the genome sequence of strain HB27 for the presence of a C-terminal signature sequence. The structure of TtoA was determined to a resolution of 2.8 Å, representing the first crystal structure of an OMP from a thermophilic bacterium. TtoA consists of an eight-stranded β-barrel with a large extracellular part to which a divalent cation is bound. A five-stranded extracellular β-sheet protrudes out of the membrane-embedded transmembrane barrel and is stabilized by a disulfide bridge. The edge of this β-sheet forms crystal contacts that could mimic interactions with other proteins. In modern Gram-negative bacteria, the C-terminal signature sequence of OMPs is required for binding to an Omp85 family protein as a prerequisite for its assembly. We present hints that a similar assembly pathway exists in T. thermophilus by an in vitro binding assay, where unfolded TtoA binds to the Thermus Omp85 family protein TtOmp85, while a mutant without the signature sequence does not.     

High-throughput Sorting of an Anticalin Library via EspP-mediated Functional Display on the Escherichia coli Cell Surface

We demonstrate that small engineered single-chain binding proteins based on the lipocalin scaffold, so-called Anticalins, can be functionally displayed on the Gram-negative bacterial cell envelope. To this end, the β-domains of five different bacterial autotransporters (the IgA protease from Neisseria gonorrhoeae, the esterase EstA from Pseudomonas aeruginosa, the YpjA autotransporter from E. coli K12, the AIDA-I adhesin from enteropathogenic E. coli O127:H27 strain 2787 and the protease EspP from enterohemorrhagic E. coli O157:H7 strain EDL933) were compared with respect to display level, functional variance, and bacterial cell viability. Use of the EspP autotransporter led to a system with high genetic stability for the display of fully functional Anticalins in high density on the cell surface of E. coli as shown by quantitative flow cytofluorimetry. This system was applied to engineer an immunostimulatory Anticalin that binds and blocks the extracellular region of human CTLA-4 to achieve a slower dissociation rate. A combinatorial library of the original Anticalin was generated by error-prone PCR, subjected to E. coli cell surface display, and applied to repeated cycles of cell sorting after incubation with the fluorescently labelled target protein under competition with the unlabelled extracellular domain of CTLA-4. The resulting Anticalin variants, which were expressed and purified as soluble proteins, showed more than eightfold decelerated target dissociation, as revealed by real time surface plasmon resonance analysis. Hence, the EspP autotransporter-mediated E. coli surface display in combination with high-throughput fluorescence-activated cell sorting (FACS) provides an efficient strategy to select for Anticalins, and possibly other small protein scaffolds, with improved binding properties, which is particularly useful for in vitro affinity maturation but may also serve for the selection of novel target specificity from naive libraries.     

Selecting Optimum Eukaryotic Integral Membrane Proteins for Structure Determination by Rapid Expression and Solubilization Screening

A medium-throughput approach is used to rapidly identify membrane proteins from a eukaryotic organism that are most amenable to expression in amounts and quality adequate to support structure determination. The goal was to expand knowledge of new membrane protein structures based on proteome-wide coverage. In the first phase, membrane proteins from the budding yeast Saccharomyces cerevisiae were selected for homologous expression in S. cerevisiae, a system that can be adapted to expression of membrane proteins from other eukaryotes. We performed medium-scale expression and solubilization tests on 351 rationally selected membrane proteins from S. cerevisiae. These targets are inclusive of all annotated and unannotated membrane protein families within the organism's membrane proteome. Two hundred seventy-two targets were expressed, and of these, 234 solubilized in the detergent n-dodecyl-β-d-maltopyranoside. Furthermore, we report the identity of a subset of targets that were purified to homogeneity to facilitate structure determinations. The extensibility of this approach is demonstrated with the expression of 10 human integral membrane proteins from the solute carrier superfamily. This discovery-oriented pipeline provides an efficient way to select proteins from particular membrane protein classes, families, or organisms that may be more suited to structure analysis than others.     

Involvement of the AtoS-AtoC signal transduction system in poly-(R)-3-hydroxybutyrate biosynthesis in Escherichia coli

The AtoS–AtoC signal transduction system in E. coli, which induces the atoDAEB operon for the growth of E. coli in short-chain fatty acids, can positively modulate the levels of poly-(R)-3-hydroxybutyrate (cPHB) biosynthesis, a biopolymer with many physiological roles in E. coli. Increased amounts of cPHB were synthesized in E. coli upon exposure of the cells to acetoacetate, the inducer of the AtoS–AtoC two-component system. While E. coli that overproduce both components of the signal transduction system synthesize higher quantities of cPHB (1.5–4.5 fold), those that overproduce either AtoS or AtoC alone do not display such a phenotype. Lack of enhanced cPHB production was also observed in cells overexpressing AtoS and phosphorylation-impaired AtoC mutants. The results were not affected by the nature of the carbon source used, i.e., glucose, acetate or acetoacetate. An E. coli strain with a deletion in the atoS–atoC locus (ΔatoSC) synthesized lower amounts of cPHB compared to wild-type cells. When the ΔatoSC strain was transformed with a plasmid carrying a 6.4-kb fragment encoding the AtoS–AtoC system, cPHB biosynthesis was restored to the level of the atoSC⁺ cells. Introduction of a multicopy plasmid carrying a functional atoDAEB operon, but not one with a promoterless operon, resulted in increased cPHB synthesis only in atoSC⁺ cells in the presence of acetoacetate. These results indicate that the presence of both a functional AtoS–AtoC two-component signal transduction system and a functional atoDAEB operon is critical for the enhanced cPHB biosynthesis in E. coli.     

pKILLIN: a versatile positive-selection cloning vector based on the toxicity of Killin in Escherichia coli

The invention of DNA cloning over 40 years ago marked the advent of molecular biology. The technique has now become a routine practice in any modern biomedical laboratory. Although positive-selection of recombinants in DNA cloning seems to be superior to blue/white selection based on the disruption of the lacZ gene, it is rarely practiced due to its high background, lack of multiple cloning sites, and inability to express the genes of interest or purify the protein products. Here we report the creation of a new positive-selection cloning vector dubbed pKILLIN, which overcomes all of the above pitfalls. The essence behind its high cloning efficiency is the extreme toxicity and small size of the toxic domain of killin, a recently discovered p53 target gene. Insertion inactivation of killin within the multiple cloning site via either blunt- or sticky-end ligation not only serves as a highly efficient cloning trap, but also may allow any cloned genes to be expressed as His-tagged fusion proteins for subsequent purification. Thus, pKILLIN is a versatile positive-selection vector ideal for cloning PCR products, making DNA libraries, as well as routine cloning and bacterial expression of genes.     

Entamoeba histolytica: Effect on virulence, growth and gene expression in response to monoxenic culture with Escherichia coli 055

Monoxenic cultivation of pathogenic Entamoeba histolytica trophozoites with Escherichia coli serotype 055 which binds strongly to the Gal/GalNAc amoebic lectin, markedly improved the growth of E. histolytica and produced a significant decrease in cysteine proteinase activity and a lower cytopathic activity on monolayer cells after 3 months of monoxenic culture. However, after long term monoxenic culture (12 months) the proteolytic and cytopathic activities were recovered and the amoebic growth reached the maximum yield. Employing the GeneFishing^R technology and DNA macroarrays we detected differentially gene expression related to the amoebic interaction with bacteria. A number of differentially expressed genes encoding metabolic enzymes, ribosomal proteins, virulence factors and proteins related with cytoskeletal and vesicle trafficking were found. These results suggest that E. coli 055 has a nutritional role that strongly supports the amoebic growth, and is also able to modulate some biological activities related with amoebic virulence.     

A tRNA-dependent cysteine biosynthesis enzyme recognizes the selenocysteine-specific tRNA in Escherichia coli

The essential methanogen enzyme Sep-tRNA:Cys-tRNA synthase (SepCysS) converts O-phosphoseryl-tRNA^Cys (Sep-tRNA^Cys) into Cys-tRNA^Cys in the presence of a sulfur donor. Likewise, Sep-tRNA:Sec-tRNA synthase converts O-phosphoseryl-tRNA^Sec (Sep-tRNA^Sec) to selenocysteinyl-tRNA^Sec (Sec-tRNA^Sec) using a selenium donor. While the Sep moiety of the aminoacyl-tRNA substrates is the same in both reactions, tRNA^Cys and tRNA^Sec differ greatly in sequence and structure. In an Escherichia coli genetic approach that tests for formate dehydrogenase activity in the absence of selenium donor we show that Sep-tRNA^Sec is a substrate for SepCysS. Since Sec and Cys are the only active site amino acids known to sustain FDH activity, we conclude that SepCysS converts Sep-tRNA^Sec to Cys-tRNA^Sec, and that Sep is crucial for SepCysS recognition.     

Kinetics of charge translocation in the passive downhill uptake mode of the Na/H antiporter NhaA of Escherichia coli

The Na⁺/H⁺ antiporter NhaA is the main Na⁺ extrusion system in E. coli. Using direct current measurements combined with a solid supported membrane (SSM), we obtained electrical data of the function of NhaA purified and reconstituted in liposomes. These measurements demonstrate NhaA's electrogenicity, its specificity for Li⁺ and Na⁺ and its pronounced pH dependence in the range pH 6.5-8.5. The mutant G338S, in contrast, presents a pH independent profile, as reported previously. A complete right-side-out orientation of the NhaA antiporter within the proteoliposomal membrane was determined using a NhaA-specific antibody based ELISA assay. This allowed for the first time the investigation of NhaA in the passive downhill uptake mode corresponding to the transport of Na⁺ from the periplasmic to the cytoplasmic side of the membrane. In this mode, the transporter has kinetic properties differing significantly from those of the previously investigated efflux mode. The apparent K_m values were 11 mM for Na⁺ and 7.3 mM for Li⁺ at basic pH and 180 mM for Na⁺ and 50 mM for Li⁺ at neutral pH. The data demonstrate that in the passive downhill uptake mode pH regulation of the carrier affects both apparent K_m as well as turnover (V_max).     

The role of the invariant glutamate 95 in the catalytic site of Complex I from Escherichia coli

Replacement of glutamate 95 for glutamine in the NADH- and FMN-binding NuoF subunit of E. coli Complex I decreased NADH oxidation activity 2.5-4.8 times depending on the used electron acceptor. The apparent K_m for NADH was 5.2 and 10.4 μM for the mutant and wild type, respectively. Analysis of the inhibitory effect of NAD⁺ on activity showed that the E95Q mutation caused a 2.4-fold decrease of K_i^NAD+ in comparison to the wild type enzyme. ADP-ribose, which differs from NAD⁺ by the absence of the positively charged nicotinamide moiety, is also a competitive inhibitor of NADH binding. The mutation caused a 7.5-fold decrease of K_i^ADP-ribose relative to wild type enzyme. Based on these findings we propose that the negative charge of Glu95 accelerates turnover of Complex I by electrostatic interaction with the negatively charged phosphate groups of the substrate nucleotide during operation, which facilitates release of the product NAD⁺. The E95Q mutation was also found to cause a positive shift of the midpoint redox potential of the FMN, from − 350 mV to − 310 mV, which suggests that the negative charge of Glu95 is also involved in decreasing the midpoint potential of the primary electron acceptor of Complex I.     

Tracing Ancestors and Relatives of Escherichia coli B, and the Derivation of B Strains REL606 and BL21(DE3)

Antecedents of Escherichia coli B have been traced through publications, inferences, and personal communication to a strain from the Institut Pasteur in Paris used by d'Herelle in his studies of bacteriophages as early as 1918 (a strain not in the current collection). This strain appears to have passed from d'Herelle to Bordet in 1920, and from Bordet to at least three other laboratories by 1925. The strain that Gratia received from Bordet was apparently passed to Bronfenbrenner by 1924 and from him to Luria around 1941. Delbrück and Luria published the first paper calling this strain B in 1942. Its choice as the common host for phages T1-T7 by the phage group that developed around Delbrück, Luria, and Hershey in the 1940s led to widespread use of B along with E. coli K-12, chosen about the same time for biochemical and genetic studies by Tatum and Lederberg. Not all currently available strains related to B are descended from the B of Delbrück and Luria; at least three strains with somewhat different characteristics were derived independently by Hershey directly from the Bronfenbrenner strain, and a strain that appears to have passed from Bordet to Wollman is in the current Collection of the Institut Pasteur. The succession of manipulations and strains that led from the B of Delbrück and Luria to REL606 and BL21(DE3) is given, established in part through evidence from their recently determined complete genome sequences.     

Identification of Interacting Regions within the Coiled Coil of the Escherichia coli Structural Maintenance of Chromosomes Protein MukB

MukB, a divergent structural maintenance of chromosomes (SMC) protein, is important for chromosome segregation and condensation in Escherichia coli and other γ-proteobacteria. MukB and canonical SMC proteins share a common five-domain structure in which globular N- and C-terminal regions combine to form an ABC-like ATPase domain. This ATPase domain is connected to a central, globular dimerization domain, commonly called the “hinge” domain, by a long antiparallel coiled coil. Although the ATPase and hinge domains of SMC proteins have been the subject of extensive investigation, little is known about the coiled coil, in spite of its clear importance for SMC function. This limited knowledge is primarily due to a lack of structural information. We report here the first experimental constraints on the relative alignment of the N- and C-terminal halves of the MukB coiled coil, obtained by a combination of limited proteolysis and site-directed cross-linking approaches. Using these experimental constraints, phylogenetic data, and coiled-coil prediction algorithms, we propose a pairing scheme for the discontinuous segments in the coiled coil. This structural model will not only facilitate the study of the physiological role of this unusually long and flexible antiparallel coiled coil but also help to delineate the boundaries between MukB domains.     

Understanding the Differences between Genome Sequences of Escherichia coli B Strains REL606 and BL21(DE3) and Comparison of the E. coli B and K-12 Genomes

Each difference between the genome sequences of Escherichia coli B strains REL606 and BL21(DE3) can be interpreted in light of known laboratory manipulations plus a gene conversion between ribosomal RNA operons. Two treatments with 1-methyl-3-nitro-1-nitrosoguanidine in the REL606 lineage produced at least 93 single-base-pair mutations (∼ 90% GC-to-AT transitions) and 3 single-base-pair GC deletions. Two UV treatments in the BL21(DE3) lineage produced only 4 single-base-pair mutations but 16 large deletions. P1 transductions from K-12 into the two B lineages produced 317 single-base-pair differences and 9 insertions or deletions, reflecting differences between B DNA in BL21(DE3) and integrated restriction fragments of K-12 DNA inherited by REL606. Two sites showed selective enrichment of spontaneous mutations. No unselected spontaneous single-base-pair mutations were evident. The genome sequences revealed that a progenitor of REL606 had been misidentified, explaining initially perplexing differences. Limited sequencing of other B strains defined characteristic properties of B and allowed assembly of the inferred genome of the ancestral B of Delbrück and Luria. Comparison of the B and K-12 genomes shows that more than half of the 3793 proteins of their basic genomes are predicted to be identical, although ∼ 310 appear to be functional in either B or K-12 but not in both. The ancestral basic genome appears to have had ∼ 4039 coding sequences occupying ∼ 4.0 Mbp. Repeated horizontal transfer from diverged Escherichia coli genomes and homologous recombination may explain the observed variable distribution of single-base-pair differences. Fifteen sites are occupied by phage-related elements, but only six by comparable elements at the same site. More than 50 sites are occupied by IS elements in both B and K, 16 in common, and likely founding IS elements are identified. A signature of widespread cryptic phage P4-type mobile elements was identified. Complex deletions (dense clusters of small deletions and substitutions) apparently removed nonessential genes from ∼ 30 sites in the basic genomes.     

Production of fully assembled and active Aquifex aeolicus F1FO ATP synthase in Escherichia coli

Background

F₁F_O ATP synthases catalyze the synthesis of ATP from ADP and inorganic phosphate driven by ion motive forces across the membrane. A number of ATP synthases have been characterized to date. The one from the hyperthermophilic bacterium Aquifex aeolicus presents unique features, i.e. a putative heterodimeric stalk. To complement previous work on the native form of this enzyme, we produced it heterologously in Escherichia coli.

Methods

We designed an artificial operon combining the nine genes of A. aeolicus ATP synthase, which are split into four clusters in the A. aeolicus genome. We expressed the genes and purified the enzyme complex by affinity and size-exclusion chromatography. We characterized the complex by native gel electrophoresis, Western blot, and mass spectrometry. We studied its activity by enzymatic assays and we visualized its structure by single-particle electron microscopy.

Results

We show that the heterologously produced complex has the same enzymatic activity and the same structure as the native ATP synthase complex extracted from A. aeolicus cells. We used our expression system to confirm that A. aeolicus ATP synthase possesses a heterodimeric peripheral stalk unique among non-photosynthetic bacterial F₁F_O ATP synthases.

Conclusions

Our system now allows performing previously impossible structural and functional studies on A. aeolicus F₁F_O ATP synthase.

General significance

More broadly, our work provides a valuable platform to characterize many other membrane protein complexes with complicated stoichiometry, i.e. other respiratory complexes, the nuclear pore complex, or transporter systems.     

Crystal Structure of the Multidrug Exporter MexB from Pseudomonas aeruginosa

We report here the crystal structure of the Pseudomonas aeruginosa multidrug exporter MexB, an intensively studied member of the resistance-nodulation-cell division family of secondary active transporters, at 3.0 Å. MexB forms an asymmetric homotrimer where each subunit adopts a different conformation representing three snapshots of the transport cycle similar to the recently determined structures of its close homologue AcrB from Escherichia coli, so far the sole structurally characterized member of the superfamily. As for AcrB, the conformations of two subunits can be clearly assigned to either the binding step or the extrusion step in the transport process. Unexpectedly, a remarkable conformational shift in the third subunit is observed in MexB, which has potential implications for the assembly of the tripartite MexAB-OprM drug efflux system. Furthermore, an n-dodecyl-d-maltoside molecule was found bound to the internal multidrug-binding cavity, which might indicate that MexB binds and transports detergent molecules as substrates. As the only missing piece of the puzzle in the MexAB-OprM system, the X-ray structure of MexB completes the molecular picture of the major pump mediating intrinsic and acquired multidrug resistance in P. aeruginosa.