Assembly of the MexAB-OprM multidrug pump of Pseudomonas aeruginosa: component interactions defined by the study of pump mutant suppressors

In an effort to identify key domains of the Pseudomonas aeruginosa MexAB-OprM drug efflux system involved in component interactions, extragenic suppressors of various inactivating mutations in individual pump constituents were isolated and studied. The multidrug hypersusceptibility of P. aeruginosa expressing MexB with a mutation in a region of the protein implicated in oligomerization (G220S) was suppressed by mutations in the alpha/beta domain of MexA. MexB(G220S) showed a reduced ability to bind MexA in vivo while representative MexA suppressors (V66M and V259F) restored the MexA-MexB interaction. Interestingly, these suppressors also restored resistance in P. aeruginosa expressing OprM proteins with mutations at the proximal (periplasmic) tip of OprM that is predicted to interact with MexB, suggesting that these suppressors generally overcame defects in MexA-MexB and MexB-OprM interaction. The multidrug hypersusceptibility arising from a mutation in the helical hairpin of MexA implicated in OprM interaction (V129M) was suppressed by mutations (T198I and F439I) in the periplasmic alpha-helical barrel of OprM. Again, the MexA mutation compromised an in vivo interaction with OprM that was restored by the T198I and F439I substitutions in OprM, consistent with the hairpin domain mediating MexA binding to this region of OprM. Interestingly, these OprM suppressor mutations restored multidrug resistance in P. aeruginosa expressing MexB(G220S). Finally, the oprM(T198I) suppressor mutation enhanced the yields of all three constituents of a MexA-MexB-OprM(T198I) pump as detected in whole-cell extracts. These data highlight the importance of MexA and interactions with this adapter in promoting MexAB-OprM pump assembly and in stabilizing the pump complex.     

Role of the membrane fusion protein in the assembly of resistance-nodulation-cell division multidrug efflux pump in Pseudomonas aeruginosa

The tripartite xenobiotic-antibiotic transporter of Pseudomonas aeruginosa consists of the inner membrane transporter (e.g., MexB, MexY), the periplasmic membrane-fusion-protein (e.g., MexA, MexX), and the outer membrane channel protein (e.g., OprM). These subunits were assumed to assemble into a transporter unit during export of the substrates. However, subunit interaction and their specificity in native form remained to be elucidated. To address these important questions, we analyzed the role of the individual subunits for the assembly of MexAB-OprM by pull-down assay tagging only one of the subunits. We found stable MexA-MexB-OprM complex without chemical cross-linking that withstand all purification procedures. Results of bi-partite interactions analysis showed tight association between MexA and OprM in the absence of MexB, whereas the expression systems lacking MexA failed to co-purify MexB or OprM. None of the heterologous subunit combinations such as MexA+MexY(his)+OprM and MexX+MexB(his)+OprM showed interaction. These results implied that the membrane fusion protein is central to the tripartite xenobiotic transporter assembly.     

Differential impact of MexB mutations on substrate selectivity of the MexAB-OprM multidrug efflux pump of Pseudomonas aeruginosa

The integral inner membrane resistance-nodulation-division (RND) components of three-component RND-membrane fusion protein-outer membrane factor multidrug efflux systems define the substrate selectivity of these efflux systems. To gain a better understanding of what regions of these proteins are important for substrate recognition, a plasmid-borne mexB gene encoding the RND component of the MexAB-OprM multidrug efflux system of Pseudomonas aeruginosa was mutagenized in vitro by using hydroxylamine and mutations compromising the MexB contribution to antibiotic resistance identified in a DeltamexB strain. Of 100 mutants that expressed wild-type levels of MexB and showed increased susceptibility to one or more of carbenicillin, chloramphenicol, nalidixic acid, and novobiocin, the mexB genes of a representative 46 were sequenced, and 19 unique single mutations were identified. While the majority of mutations occurred within the large periplasmic loops between transmembrane segment 1 (TMS-1) and TMS-2 and between TMS-7 and TMS-8 of MexB, mutations were seen in the TMSs and in other periplasmic as well as cytoplasmic loops. By threading the MexB amino acid sequence through the crystal structure of the homologous RND transporter from Escherichia coli, AcrB, a three-dimensional model of a MexB trimer was obtained and the mutations were mapped to it. Unexpectedly, most mutations mapped to regions of MexB predicted to be involved in trimerization or interaction with MexA rather than to regions expected to contribute to substrate recognition. Intragenic second-site suppressor mutations that restored the activity of the G220S mutant version of MexB, which was compromised for resistance to all tested MexAB-OprM antimicrobial substrates, were recovered and mapped to the apparently distal portion of MexB that is implicated in OprM interaction. As the G220S mutation likely impacted trimerization, it appears that either proper assembly of the MexB trimer is necessary for OprM interaction or OprM association with an unstable MexB trimer might stabilize it, thereby restoring activity.     

Assembly of the MexAB-OprM multidrug efflux system of Pseudomonas aeruginosa: identification and characterization of mutations in mexA compromising MexA multimerization and interaction with MexB

The membrane fusion protein (MFP) component, MexA, of the MexAB-OprM multidrug efflux system of P. aeruginosa is proposed to link the inner (MexB) and outer (OprM) membrane components of this pump as a probable oligomer. A cross-linking approach confirmed the in vivo interaction of MexA and MexB, while a LexA-based assay for assessing protein-protein interaction similarly confirmed MexA multimerization. Mutations compromising the MexA contribution to antibiotic resistance but yielding wild-type levels of MexA were recovered and shown to map to two distinct regions within the N- and C-terminal halves of the protein. Most of the N-terminal mutations occurred at residues that are highly conserved in the MFP family (P68, G72, L91, A108, L110, and V129), consistent with these playing roles in a common feature of these proteins (e.g., oligomerization). In contrast, the majority of the C-terminal mutations occurred at residues poorly conserved in the MFP family (V264, N270, H279, V286, and G297), with many mapping to a region of MexA that corresponds to a region in the related MFP of Escherichia coli, AcrA, that is implicated in binding to its RND component, AcrB (C. A. Elkins and H. Nikaido, J. Bacteriol. 185:5349-5356, 2003). Given the noted specificity of MFP-RND interaction in this family of pumps, residues unique to MexA may well be important for and define the MexA interaction with its RND component, MexB. Still, all but one of the MexA mutations studied compromised MexA-MexB association, suggesting that native structure and/or proper assembly of the protein may be necessary for this.     

Antibiotics potentiating potential of catharanthine against superbug Pseudomonas aeruginosa

Multidrug resistance (MDR) put an alarming situation like preantibiotic era which compels us to invigorate the basic science of anti-infective chemotherapy. Hence, the drug resistant genes/proteins were explored as promising drug targets. Keeping this thing in mind, proteome of Pseudomonas aeruginosa PA01 was explored, which resulted in the identification of tripartite protein complexes (MexA, MexB, and OprM) as promising drug target for the screening of natural and synthetic inhibitors. The purpose of present investigation was to explore the drug resistance reversal potential mechanism of catharanthine isolated from the leaves of Catharanthus roseous. Hence, the test compound catharanthine was in silico screened using docking studies against the above receptors, which showed signi?cant binding affinity with these receptors. In order to validate the in silico findings, in vitro evaluation of the test compound was also carried out. In combination, catharanthine reduced the minimum inhibitory concentration MIC of tetracycline (TET) and streptomycin up to 16 and 8 folds, respectively. Further, in time kill assay, catharanthine in combination with TET reduced the cell viability in concentration dependent manner and was also able to reduce the mutation prevention concentration of TET. It was also deduced that drug resistance reversal potential of catharanthine was due to inhibition of the efflux pumps.     

Mutations affecting DNA-binding activity of the MexR repressor of mexR-mexA-mexB-oprM operon expression

We have isolated 25 MexR mutants that retained their dimerizing ability but were unable to bind mexOP DNA. Surprisingly, 20 mutations were located in the hydrophobic core region at alpha4, W1, alpha2, alpha3, and beta2, and only 3 were in positively charged residues. These results verified that DNA binding is mediated by distinct regions of MexR and showed the importance of the hydrophobic core region of the DNA-binding domain.     

Linkage of the efflux-pump expression level with substrate extrusion rate in the MexAB-OprM efflux pump of Pseudomonas aeruginosa

The amount of the subunit proteins of the MexAB-OprM efflux pump in Pseudomonas aeruginosa was quantified by the immunoblotting method. A single cell of the wild-type strain contained about 2500, 1000, and 1200 copies of MexA, MexB, and OprM, respectively, and their stoichiometry therefore was 2:1:1. The mexR mutant produced an eightfold higher level of these proteins than did wild-type cells. Assuming that MexB and OprM exist as a trimer in a pump assembly, the total number of MexAB-OprM per wild-type cell was calculated to be about 400 assemblies. The substrate efflux rate of MexAB-OprM was calculated from the fluorescent intensity of ethidium in intact cells that a single cell extruded ethidium at a maximum of about 3 x 10(-19) mol s(-1) and, therefore, the turnover rate of a single pump unit was predicted to be about 500 s(-1).     

The mexR repressor of the mexAB-oprM multidrug efflux operon in Pseudomonas aeruginosa: characterization of mutations compromising activity

Mutations in mexR yield a multidrug resistance phenotype in nalB mutants of Pseudomonas aeruginosa as a result of derepression of the mexAB-oprM multidrug efflux operon. MexR produced by several nalB strains carried single amino acid changes that compromised MexR stability or its ability to dimerize. Changes at residues L95 and R21, however, produced a stable MexR protein capable of dimerization and, thus, likely compromised DNA binding.     

Assignment of the outer-membrane-subunit-selective domain of the membrane fusion protein in the tripartite xenobiotic efflux pump of Pseudomonas aeruginosa

Early in vivo experiments revealed that the MexA-MexB dipartite pump unit of Pseudomonas aeruginosa conferred drug resistance to the cells, which expressed OprM, but not to the OprN-bearing cells. While the MexE-MexF unit interplayed with either the outer membrane subunits. Taking advantage of this subunit selectivity, we selected the MexA mutant that gained the ability to interplay with OprN. Four mutants have been isolated and all showed an amino acid substitution (Q116R) in the coiled-coil domain of MexA. The hybrid protein bearing the coiled-coil domain of MexA and the remainder domains from MexE retained the ability to interplay with OprM, but lost the functional interplay with OprN. These results established that the coiled-coil domain of the membrane fusion protein is responsible for selecting the compatible outer membrane subunit.     

Structure and heme binding properties of Escherichia coli O157:H7 ChuX

For many pathogenic microorganisms, iron acquisition from host heme sources stimulates growth, multiplication, ultimately enabling successful survival and colonization. In gram‐negative Escherichia coli O157:H7, Shigella dysenteriae and Yersinia enterocolitica the genes encoded within the heme utilization operon enable the effective uptake and utilization of heme as an iron source. While the complement of proteins responsible for heme internalization has been determined in these organisms, the fate of heme once it has reached the cytoplasm has only recently begun to be resolved. Here we report the first crystal structure of ChuX, a member of the conserved heme utilization operon from pathogenic E. coli O157:H7 determined at 2.05 Å resolution. ChuX forms a dimer which remarkably given low sequence homology, displays a very similar fold to the monomer structure of ChuS and HemS, two other heme utilization proteins. Absorption spectral analysis of heme reconstituted ChuX demonstrates that ChuX binds heme in a 1:1 manner implying that each ChuX homodimer has the potential to coordinate two heme molecules in contrast to ChuS and HemS where only one heme molecule is bound. Resonance Raman spectroscopy indicates that the heme of ferric ChuX is composed of a mixture of coordination states: 5‐coordinate and high‐spin, 6‐coordinate and low‐spin, and 6‐coordinate and high‐spin. In contrast, the reduced ferrous form displays mainly a 5‐coordinate and high‐spin state with a minor contribution from a 6‐coordinate and low‐spin state. The ν_Fe‐CO and ν_C‐O frequencies of ChuX‐bound CO fall on the correlation line expected for histidine‐coordinated hemoproteins indicating that the fifth axial ligand of the ferrous heme is the imidazole ring of a histidine residue. Based on sequence and structural comparisons, we designed a number of site‐directed mutations in ChuX to probe the heme binding sites and dimer interface. Spectral analysis of ChuX and mutants suggests involvement of H65 and H98 in heme coordination as mutations of both residues were required to abolish the formation of the hexacoordination state of heme‐bound ChuX.     

Structural mechanism of ATP‐dependent DNA binding and DNA end bridging by eukaryotic Rad50

The Mre11–Rad50–Nbs1 (MRN) complex is a central factor in the repair of DNA double‐strand breaks (DSBs). The ATP‐dependent mechanisms of how MRN detects and endonucleolytically processes DNA ends for the repair by microhomology‐mediated end‐joining or further resection in homologous recombination are still unclear. Here, we report the crystal structures of the ATPγS‐bound dimer of the Rad50^NBD (nucleotide‐binding domain) from the thermophilic eukaryote Chaetomium thermophilum (Ct) in complex with either DNA or CtMre11^RBD (Rad50‐binding domain) along with small‐angle X‐ray scattering and cross‐linking studies. The structure and DNA binding motifs were validated by DNA binding experiments in vitro and mutational analyses in Saccharomyces cerevisiae in vivo. Our analyses provide a structural framework for the architecture of the eukaryotic Mre11–Rad50 complex. They show that a Rad50 dimer binds approximately 18 base pairs of DNA along the dimer interface in an ATP‐dependent fashion or bridges two DNA ends with a preference for 3′ overhangs. Finally, our results may provide a general framework for the interaction of ABC ATPase domains of the Rad50/SMC/RecN protein family with DNA.     

Structure of reconstituted bacterial membrane efflux pump by cryo-electron tomography

Complexes of OprM and MexA, two proteins of the MexA-MexB-OprM multidrug efflux pump from Pseudomonasaeruginosa, an opportunistic Gram-negative bacterium, were reconstituted into proteoliposomes by detergent removal. Stacks of protein layers with a constant height of 21 nm, separated by lipid bilayers, were obtained at stoichiometry of 1:1 (w/w). Using cryo-electron microscopy and tomography, we showed that these protein layers were composed of MexA-OprM complexes self-assembled into regular arrays. Image processing of extracted sub-tomograms depicted the architecture of the bipartite complex sandwiched between two lipid bilayers, representing an environment close to that of the native whole pump (i.e. anchored between outer and inner membranes of P. aeruginosa). The MexA-OprM complex appeared as a cylindrical structure in which we were able to identify the OprM molecule and the MexA moiety. MexA molecules have a cylindrical shape prolonging the periplasmic helices of OprM, and widening near the lipid bilayer. The flared part is likely composed of two MexA domains adjacent to the lipid bilayer, although their precise organization was not reachable mainly due to their flexibility. Moreover, the intermembrane distance of 21 nm indicated that the height of the bipartite complex is larger than that of the tripartite AcrA-AcrB-TolC built-up model in which TolC and AcrB are docked into contact. We proposed a model of MexA-OprM taking into account features of previous models based on AcrA-AcrB-TolC and our structural results providing clues to a possible mechanism of tripartite system assembly.     

Development of germ‐line‐specific CRISPR‐Cas9 systems to improve the production of heritable gene modifications in Arabidopsis

The Streptococcus‐derived CRISPR/Cas9 system is being widely used to perform targeted gene modifications in plants. This customized endonuclease system has two components, the single‐guide RNA (sgRNA) for target DNA recognition and the CRISPR‐associated protein 9 (Cas9) for DNA cleavage. Ubiquitously expressed CRISPR/Cas9 systems (UC) generate targeted gene modifications with high efficiency but only those produced in reproductive cells are transmitted to the next generation. We report the design and characterization of a germ‐line‐specific Cas9 system (GSC) for Arabidopsis gene modification in male gametocytes, constructed using a SPOROCYTELESS (SPL) genomic expression cassette. Four loci in two endogenous genes were targeted by both systems for comparative analysis. Mutations generated by the GSC system were rare in T1 plants but were abundant (30%) in the T2 generation. The vast majority (70%) of the T2 mutant population generated using the UC system were chimeras while the newly developed GSC system produced only 29% chimeras, with 70% of the T2 mutants being heterozygous. Analysis of two loci in the T2 population showed that the abundance of heritable gene mutations was 37% higher in the GSC system compared to the UC system and the level of polymorphism of the mutations was also dramatically increased with the GSC system. Two additional systems based on germ‐line‐specific promoters (pDD45‐GT and pLAT52‐GT) were also tested, and one of them was capable of generating heritable homozygous T1 mutant plants. Our results suggest that future application of the described GSC system will facilitate the screening for targeted gene modifications, especially lethal mutations in the T2 population.     

Selection and structural analysis of de novo proteins from an α3β3 genetic library

The construction of novel functional proteins has been a key area of protein engineering. However, there are few reports of functional proteins constructed from artificial scaffolds. Here, we have constructed a genetic library encoding α3β3 de novo proteins to generate novel scaffolds in smaller size using a binary combination of simplified hydrophobic and hydrophilic amino acid sets. To screen for folded de novo proteins, we used a GFP‐based screening system and successfully obtained the proteins from the colonies emitting the very bright fluorescence as a similar intensity of GFP. Proteins isolated from the very bright colonies (vTAJ) and bright colonies (wTAJ) were analyzed by circular dichroism (CD), 8‐anilino‐1‐naphthalenesulfonate (ANS) binding assay, and analytical size‐exclusion chromatography (SEC). CD studies revealed that vTAJ and wTAJ proteins had both α‐helix and β‐sheet structures with thermal stabilities. Moreover, the selected proteins demonstrated a variety of association states existing as monomer, dimer, and oligomer formation. The SEC and ANS binding assays revealed that vTAJ proteins tend to be a characteristic of the folded protein, but not in a molten‐globule state. A vTAJ protein, vTAJ13, which has a packed globular structure and exists as a monomer, was further analyzed by nuclear magnetic resonance. NOE connectivities between backbone signals of vTAJ13 suggested that the protein contains three α‐helices and three β‐strands as intended by its design. Thus, it would appear that artificially generated α3β3 de novo proteins isolated from very bright colonies using the GFP fusion system exhibit excellent properties similar to folded proteins and would be available as artificial scaffolds to generate functional proteins with catalytic and ligand binding properties.