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This study describes the complete genome sequence of Herbiconiux sp. strain L3-i23, acquired from an assembly of long reads and subsequently polished using short reads. The complete genome comprises a 3,139,863-bp chromosome with a GC content of 69.51% and a circular plasmid (39,507 bp).

This study investigated 116 clinical isolates of Campylobacter jejuni from Toyama, Japan, which were isolated from 2015 to 2019. Antimicrobial susceptibility testing and whole-genome sequencing were used for phenotypic and genotypic characterization to compare antimicrobial resistance (AMR) profiles and phylogenic linkage. The multilocus sequence typing approach identified 37 sequence types (STs) and 15 clonal complexes (CCs), including 7 novel STs, and the high frequency CCs were CC21 (27.7%), CC48 (10.9%), and CC354 (9.9%). Overall, 58.6% of the isolates were resistant to at least one of the antibiotics and 3.4% were resistant to three or more antibiotic classes. The AMR profiles and related resistant factors were as follows; fluoroquinolones (51.7%), mutation in QRDRs (GyrA T86I), tetracyclines (27.6%), acquisition of tet(O), ampicillin (5.2%), promoter mutation in blaOXA193, aminoglycosides (1.7%), acquisition of ant(6)-Ia and aph(3’)-III, chloramphenicol (0.9%), acquisition of cat. The resistance factors of fosfomycin (1 strain), sulfamethoxazole-trimethoprim (2 strain), and linezolid (1 strain) resistant isolates were unknown. The acquired resistance genes, tet(O), ant(6>)-Ia, aph(3’)-III, and cat, were located on pTet family plasmids. Furthermore, three pTet family plasmids formed larger plasmids that incorporated additional genes such as the Type IV secretion system.A comparison of pTet family plasmids in Japan has not been reported, and these results imply that the diversity of pTet family plasmids has increased. The prevalence of ST4526, belonging to CC21, in Japan has been reported, and it was also the major ST type (10.9%) in this study, suggesting that the ST4526 prevalence continues in Japan.

This study describes the draft genome sequence of Paenibacillus sp. strain L3-i20, obtained from an assembly of long reads and subsequently polished using short reads. The draft genome comprises a 5,308,756-bp chromosome with a GC content of 41.6% and no plasmids.

The emergence of multidrug-resistant Klebsiella pneumoniae is a worldwide problem. K. pneumoniae possesses numerous resistant genes in its genome. We isolated mutants resistant to various antimicrobials in vitro and investigated the importance of intrinsic genes in acquired resistance. The isolation frequency of the mutants was 10−7–10−9. Of the multidrug-resistant mutants, hyper-multidrug-resistant mutants (EB256-1, EB256-2, Nov1-8, Nov2-2, and OX128) were identified, and accelerated efflux activity of ethidium from the inside to the outside of the cells was observed in these mutants. Therefore, we hypothesized that the multidrug efflux pump, especially RND-type efflux pump, would be related to changes of the phenotype. We cloned all RND-type multidrug efflux pumps from the K. pneumoniae genome and characterized them. KexEF and KexC were powerful multidrug efflux pumps, in addition to AcrAB, KexD, OqxAB, and EefABC, which were reported previously. It was revealed that the expression of eefA was increased in EB256-1 and EB256-2: the expression of oqxA was increased in OX128; the expression of kexF was increased in Nov2-2. It was found that a region of 1,485 bp upstream of kexF, was deleted in the genome of Nov2-2. K. pneumoniae possesses more potent RND-multidrug efflux systems than E. coli. However, we revealed that most of them did not contribute to the drug resistance of our strain at basic levels of expression. On the other hand, it was also noted that the overexpression of these pumps could lead to multidrug resistance based on exposure to antimicrobial chemicals. We conclude that these pumps may have a role to maintain the intrinsic resistance of K. pneumoniae when they are overexpressed. The antimicrobial chemicals selected many resistant mutants at the same minimum inhibitory concentration (MIC) or a concentration slightly higher than the MIC. These results support the importance of using antibiotics at appropriate concentrations at clinical sites.

DcsB, one of the enzymes encoded in the D-cycloserine (D-CS) biosynthetic gene cluster, displays a high sequence homology to arginase, which contains two manganese ions in the active site. However, DcsB hydrolyzesNω-hydroxy-L-arginine, but not L-arginine, to supply hydroxyurea for the biosynthesis of D-CS. Here, the crystal structure of DcsB was determined at a resolution of 1.5 Å using anomalous scattering from the manganese ions. In the crystal structure, DscB generates an artificial dimer created by the open and closed forms. Gel-filtration analysis demonstrated that DcsB is a monomeric protein, unlike arginase, which forms a trimeric structure. The active center containing the binuclear manganese cluster differs between DcsB and arginase. In DcsB, one of the ligands of the MnAion is a cysteine, while the corresponding residue in arginase is a histidine. In addition, DcsB has no counterpart to the histidine residue that acts as a general acid/base during the catalytic reaction of arginase. The present study demonstrates that DcsB has a unique active site that differs from that of arginase.

Alpha‐1‐acid glycoprotein (AGP) is a major acute‐phase protein. Biosynthesis of AGP increases markedly during inflammation and infection, similar to nitric oxide (NO) biosynthesis. AGP variant A (AGP) contains a reduced cysteine (Cys149). Previously, we reported that S‐nitrosated AGP (SNO‐AGP) synthesized by reaction with a NO donor, possessed very strong broad‐spectrum antimicrobial activity (IC50 = 10−9‐10−6 M). In this study, using a cecal ligation and puncture animal model, we confirmed that AGP can be endogenously S‐nitrosated during infection. Furthermore, we examined the antibacterial property of SNO‐AGP against multidrug‐resistant Klebsiella pneumoniae and Pseudomonas aeruginosa to investigate the involvement of SNO‐AGP in the host defense system. Our results showed that SNO‐AGP could inhibit multidrug efflux pump, AcrAB‐TolC, a major contributor to bacterial multidrug resistance. In addition, SNO‐AGP decreased biofilm formation and ATP level in bacteria, indicating that SNO‐AGP can revert drug resistance. It was also noteworthy that SNO‐AGP showed synergistic effects with the existing antibiotics (oxacillin, imipenem, norfloxacin, erythromycin, and tetracycline). In conclusion, SNO‐AGP participated in the host defense system and has potential as a novel agent for single or combination antimicrobial therapy.

Through genome mining for fungal macrolide natural products, we discovered a characteristic family of putative macrolide biosynthetic gene clusters that contain a glycosylphosphatidylinositol-ethanolamine phosphate transferase (GPI-EPT) homologue. Through the heterologous expression of two clusters from Aspergillus kawachii and Colletotrichum incanum, new macrolides, including those with phosphoethanolamine or phosphocholine moieties, were formed. This study is the first demonstration of the tailoring steps catalyzed by GPI-EPT homologues in natural product biosynthesis, and it uncovers a new gene resource for phospholipid-resembling fungal macrolides.

DcsB, one of the enzymes encoded in the D-cycloserine (D-CS) biosynthetic gene cluster, displays a high sequence homology to arginase, which contains two manganese ions in the active site. However, DcsB hydrolyzes N

Tyrosinase (EC 1.14.18.1), which possesses two copper ions at the active center, catalyzes a rate-limiting reaction of melanogenesis, that is, the conversion of a phenol to the corresponding ortho-quinone. The enzyme from the genus Streptomyces is generated as a complex with a “caddie” protein that assists the transport of two copper ions into the active center. In this complex, the Tyr98 residue in the caddie protein was found to be accommodated in the pocket of the active center of tyrosinase, probably in a manner similar to that of l-tyrosine as a genuine substrate of tyrosinase. Under physiological conditions, the addition of the copper ion to the complex releases tyrosinase from the complex, in accordance with the aggregation of the caddie protein. The release of the copper-bound tyrosinase was found to be accelerated by adding reducing agents under aerobic conditions. Mass spectroscopic analysis indicated that the Tyr98 residue was converted to a reactive quinone, and resonance Raman spectroscopic a...

Drug-resistant bacteria including methicillin-resistant Staphylococcus aureus (MRSA), multidrug-resistant Pseudomonas aeruginosa, and vancomycin-resistant enterococci (VRE) have been spreading; however, the development of new antibacterial drugs has not progressed accordingly. Novel antibacterial drugs or their candidate seeds need to be developed for effective antibiotic therapy. Under these conditions, the search for novel compounds and novel targets is important. In Okayama University, as a part of the Drug Discovery for Intractable Infectious Diseases project, we are proceeding with the development of antibacterial drugs for the treatment of drug-resistant bacterial infections. We found that riccardin C (a natural product of liverwort) and 6,6'-dihydroxythiobinupharidine (from the crude drug Senkotsu) exhibited strong antibacterial activities, particularly against Gram-positive bacteria. We showed that riccardin C induced cell membrane leakage and that 6,6'-dihydroxythiobinupharidine inhibited DNA topoisomerase IV. Moreover, 6,6'-dihydroxythiobinupharidine exerted synergistic effects with already known anti-MRSA drugs as well as with vancomycin for VRE.

We investigated the role of the resistance-nodulation-cell division superfamily (RND) efflux system on intrinsic multidrug resistance in Serratia marcescens. We identified eight putative RND efflux system genes in the S. marcescens Db10 genome that included the previously characterized systems, sdeXY, sdeAB, and sdeCDE. Six out of the eight genes conferred multidrug resistance on KAM32, a drug hypersensitive strain of Escherichia coli. Five out of the eight genes conferred resistance to benzalkonium, suggesting the importance of RND efflux systems in biocide resistance in S. marcescens. The energy-dependent efflux activities of five of the pumps were examined using a rhodamine 6 G efflux assay. When expressed in the tolC-deficient strain of E. coli, KAM43, none of the genes conferred resistance on E. coli. When hasF, encoding the S. marcescens TolC ortholog, was expressed in KAM43, all of the genes conferred resistance on E. coli, suggesting that HasF is a major outer membrane protein that is used by all RND efflux systems in this organism. We constructed a sdeXY deletion mutant from a derivative strain of the clinically isolated multidrug-resistant S. marcescens strain and found that the sdeXY deletion mutant was sensitive to a broad spectrum of antimicrobial agents.

We previously showed that a naturally occurring macrocyclic bis(bibenzyl) derivative, riccardin C (RC), exhibits antibacterial activity towards methicillin-resistant Staphylococcus aureus (MRSA), with a potency comparable to that of the clinically used drug vancomycin. Here, we synthesized a series of RC derivatives to explore the structure-activity relationships (SAR). The SAR results clearly indicated that the number and positions of the phenolic hydroxyl groups are primary determinants of the anti-MRSA activity. Pharmacological characterization of the macrocyclic bis(bibenzyl) derivatives, together with fragment compounds and their dimers, indicated that the macrocycles and the fragment compounds elicit anti-MRSA activity with different mechanism(s) of action. The macrocyclic bis(bibenzyl)s are bactericidal, while the fragment compounds are bacteriostatic, showing only weak bactericidal activity. Treatment with a macrocyclic bis(bibenzyl) derivative significantly changed the intracellular Na+ and K+ concentrations of Staphylococcus aureus, and transmission electron microscopy revealed that treated cells developed intracellular lamellar mesosomal-like structures. These results indicated that the macrocyclic compound directly damages the gram-positive bacterial membrane, resulting in increased permeability.

A V. cholerae MATE transporter was crystallized using the lipidic cubic phase (LCP) method. X-ray diffraction data sets were collected from single crystals obtained in a sandwich plate and a sitting-drop plate to resolutions of 2.5 and 2.2 Å, respectively., Multidrug and toxic compound extrusion (MATE) transporters, one of the multidrug exporter families, efflux xenobiotics towards the extracellular side of the membrane. Since MATE transporters expressed in bacterial pathogens contribute to multidrug resistance, they are important therapeutic targets. Here, a MATE-transporter homologue from Vibrio cholerae, VcmN, was overexpressed in Escherichia coli, purified and crystallized in lipidic cubic phase (LCP). X-ray diffraction data were collected to 2.5 Å resolution from a single crystal obtained in a sandwich plate. The crystal belonged to space group P212121, with unit-cell parameters a = 52.3, b = 93.7, c = 100.2 Å. As a result of further LCP crystallization trials, crystals of larger size were obtained using sitting-drop plates. X-ray diffraction data were collected to 2.2 Å resolution from a single crystal obtained in a sitting-drop plate. The crystal belonged to space group P212121, with unit-cell parameters a = 61.9, b = 91.8, c = 100.9 Å. The present work provides valuable insights into the atomic resolution structure determination of membrane transporters.

Macrocyclic bis(bibenzyl)-type phenolic natural products, found exclusively in bryophytes, exhibit potent antibacterial activity towards methicillin-resistant Staphylococcus aureus (anti-MRSA activity). Here, in order to identify the minimum essential structure for cell membrane leakage-mediated anti-MRSA activity of these compounds, we synthesized acyclic fragment structures and evaluated their anti-MRSA activity. The activities of all of the acyclic fragments tested exhibited similar characteristics to those of the macrocycles, i.e., anti-MRSA bactericidal activity, an enhancing effect on influx and efflux of ethidium bromide (EtBr: fluorescent DNA-binder) in Staphylococcus aureus cells, and bactericidal activity towards a Staphylococcus aureus strain resistant to 2-phenoxyphenol (4). The latter result suggests that they have a different mechanism of action from 4, which is a FabI inhibitor previously proposed to be the minimum active fragment of riccardin-type macrocycles. Thus, cyclic structure is not a necessary condition for cell membrane leakage-mediated anti-MRSA activity of macrocyclic bis(bibenzyl)s.

In an attempt to discover inhibitory compounds against pore-forming toxins, some of the major toxins produced by bacteria, we herein examined the effects of four kinds of indolo[3,2-b]quinoline derivatives on hemolysis induced by the aerolysin-like hemolysin (ALH) of Aeromonas sobria and also by the alpha-hemolysin of Staphylococcus aureus. The results showed that hemolysis induced by ALH was significantly reduced by every derivative, while that induced by alpha-hemolysis was significantly reduced by three out of the four derivatives. However, the degrees of reduction induced by these derivatives were not uniform. Each derivative exhibited its own activity to inhibit the respective hemolysin. Compounds 1 and 2, which possessed the amino group bonding the naphthalene moiety at the C-11 position of indolo[3,2-b]quinoline, had strong inhibitory effects on the activity of ALH. Compound 4 which consisted of benzofuran and quinoline had strong inhibitory effects on the activity of alpha-hemolysin. These results indicated that the amino group bonding the naphthalene moiety of compounds 1 and 2 assisted in their ability to inhibit ALH activity, while the oxygen atom at the 10 position of compound 4 strengthened its interaction with alpha-hemolysin. These compounds also suppressed the hemolytic activity of the supernatant of A. sobria or A. hydrophila, suggesting that these compounds were effective at the site of infection of these bacteria.

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Multidrug and toxic compound extrusion (MATE) transporters efflux toxic compounds, using a Na(+) or H(+) gradient across the membrane. Although the structures of MATE transporters have been reported, the cation-coupled substrate transport mechanism remains controversial. Here we report crystal structures of VcmN, a Vibrio cholerae MATE transporter driven by the H(+) gradient. High-resolution structures in two distinct conformations associated with different pHs revealed that the rearrangement of the hydrogen-bonding network around the conserved Asp35 induces the bending of transmembrane helix 1, as in the case of the H(+)-coupled Pyrococcus furiosus MATE transporter. We also determined the crystal structure of the D35N mutant, which captured a unique conformation of TM1 facilitated by an altered hydrogen-bonding network. Based on the present results, we propose a common step in the transport cycle shared among prokaryotic H(+)-coupled MATE transporters.

Methicillin-resistant Staphylococcus aureus (MRSA) is a major problem in clinical settings, and because it is resistant to most antimicrobial agents, MRSA infections are difficult to treat. We previously reported that synthetic macrocyclic bis(bibenzyl) derivatives, which were originally discovered in liverworts, had anti-MRSA activity. However, the action mechanism responsible was unclear. In the present study, we elucidated the action mechanism of macrocyclic bis(bibenzyl) RC-112 and its partial structure, IDPO-9 (2-phenoxyphenol). Survival experiments demonstrated that RC-112 had a bactericidal effect on MRSA, whereas IDPO-9 had bacteriostatic effects. IDPO-9-resistant mutants exhibited cross-resistance to triclosan, but not to RC-112. The mutation was identified in the fabI, enoyl-acyl carrier protein reductase gene, a target of triclosan. We have not yet isolated the RC-112-resistant mutant. On the other hand, the addition of RC-112, unlike IDPO-9, caused the inflow of ethidium and propidium into S. aureus cells. RC-112-dependent ethidium outflow was observed in ethidium-loaded S. aureus cells. Transmission electron microscopy also revealed that S. aureus cells treated with RC-112 had intracellular lamellar mesosomal-like structures. Intracellular Na+ and K+ concentrations were significantly changed by the RC-112 treatment. These results indicated that RC-112 increased membrane permeability to ethidium, propidium, Na+, and K+, and also that the action mechanism of IDPO-9 was different from those of the other compounds.

Two new flavonoids, bakuisoflavone (1) and bakuflavanone (2), together with 15 known compounds, were isolated from the fruits of Psoralea corylifolia, and their structures were characterized by spectroscopic data. The effects of the isolated compounds on methicillin-resistant Staphylococcus aureus were also examined. We found that two compounds, isobavachalcone (10) and bakuchiol (12), showed noticeable antibacterial effects on the MRSA strains examined. Quantitation of the major constituents, including anti-MRSA constituents, was then performed. The results showed individual contents of 1.26%–16.49% (w/w) among the examined compounds in the ethyl acetate extract from P. corylifolia fruits.

Background Multidrug-resistant bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin resistant enterococci (VRE), cause serious infections at clinical sites, for which the development of new drugs is necessary. We screened candidates for new antibiotics and investigated its action mechanism. Methods An antimicrobial compound was isolated from an extract of Nuphar japonicum . Its chemical structure was determined by NMR, MS, and optical rotation. We measured its minimum inhibitory concentration (MIC) using the microdilution method. The effects of the compound on DNA gyrase and DNA topoisomerase IV were investigated with DNA supercoiling, decatenation, and cleavage assay. Results We isolated and identified 6,6′-dihydroxythiobinupharidine as the antimicrobial compound. The MIC of this compound was 1–4 μg/mL against various MRSA and VRE strains. We also demonstrated that this compound inhibited DNA topoisomerase IV (IC 50 was 10–15 μM), but not DNA gyrase in S. aureus , both of which are known to be the targets of quinolone antibiotics and necessary for DNA replication. However, this compound only exhibited slight cross-resistance to norfloxacin-resistant S. aureus , which indicated that DTBN might inhibit other targets besides topoisomerase IV. These results suggest that 6,6′-dihydroxythiobinupharidine may be a potent candidate or seed for novel antibacterial agents. Conclusions DTBN from N. japonicum showed anti-MRSA and anti-VRE activities. DTBN might be involved in the inhibition of DNA topoisomerase IV. General significance DTBN might be useful as a seed compound. The information on the inhibition mechanism of DTBN will be useful for the modification of DTBN towards developing novel anti-MRSA and anti-VRE drug.

Three new ellagitannin oligomers, isorugosins H (1), I (2), and J (3), together with 11 known hydrolyzable tannins were isolated from an aqueous acetone extract of the fresh leaves of Liquidambar formosana. Their chemical structures were elucidated based on spectroscopic data and chemical conversion into known hydrolyzable tannins. The bridging mode of the valoneoyl groups between their sugar moieties has been identified only in this plant species. Additionally, the effects of the isorugosins isolated from this species on drug-resistant bacteria were evaluated and showed that isorugosin A (4) exhibited the most potent antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA). The isorugosins also had a suppressing effect on pigment formation in Pseudomonas aeruginosa. The isorugosin-protein complexes were analyzed using size-exclusion chromatography and polyacrylamide gel electrophoresis to clarify the relationship of their antibacterial properties with their protein interaction potency as hydrolyzable tannins. The results suggested that the antibacterial properties of hydrolyzable tannins are not simply a result of their binding activity to proteins, but are due to other factors such as the accessibility of polyphenolic acyl groups to bacterial membranes.

Licorice, which is the underground part of Glycyrrhiza species, has been used widely in Asian and Western countries as a traditional medicine and as a food additive. Our continuous investigation on the constituents of roots and stolons of Glycyrrhiza uralensis led to the isolation of two new phenolics, in addition to 14 known compounds. Structural studies including spectroscopic and simple chemical derivatizations revealed that both of the new compounds had 2-aryl-3-methylbenzofuran structures. An examination of the effectiveness of licorice phenolics obtained in this study on vancomycin-resistant strains Enterococcus faecium FN-1 and Enterococcus faecalis NCTC12201 revealed that licoricidin showed the most potent antibacterial effects against both of E. faecalis and E. faecium with a minimum inhibitory concentration (MIC) of 1.9 × 10−5 M. 8-(γ,γ-Dimethylallyl)-wighteone, isoangustone A, 3'-(γ,γ-dimethylallyl)-kievitone, glyasperin C, and one of the new 3-methyl-2-phenylbenzofuran named neoglycybenzofuran also showed potent anti-vancomycin-resistant Enterococci effects (MIC 1.9 × 10−5–4.5 × 10−5 M for E. faecium and E. faecalis). The HPLC condition for simultaneous detection of the phenolics in the extract was investigated to assess the quality control of the natural antibacterial resource, and quantitative estimation of several major phenolics in the extract with the established HPLC condition was also performed. The results showed individual contents of 0.08%–0.57% w/w of EtOAc extract for the major phenolics in the materials examined.

Summary Multidrug and toxic compound extrusion (MATE) transporters efflux toxic compounds using a Na+ or H+ gradient across the membrane. Although the structures of MATE transporters have been reported, the cation-coupled substrate transport mechanism remains controversial. Here we report crystal structures of VcmN, a Vibrio cholerae MATE transporter driven by the H+ gradient. High-resolution structures in two distinct conformations associated with different pHs revealed that the rearrangement of the hydrogen-bonding network around the conserved Asp35 induces the bending of transmembrane helix 1, as in the case of the H+-coupled Pyrococcus furiosus MATE transporter. We also determined the crystal structure of the D35N mutant, which captured a unique conformation of TM1 facilitated by an altered hydrogen-bonding network. Based on the present results, we propose a common step in the transport cycle shared among prokaryotic H+-coupled MATE transporters.

We synthesized a series of macrocyclic bis(bibenzyl) derivatives, including riccardin-, isoplagiochin- and marchantin-class structures, and evaluated their antibacterial activity towards methicillin-resistant Staphylococcus aureus (anti-MRSA activity). The structure–activity relationships and the results of molecular dynamics simulations indicated that bis(bibenzyl)s with potent anti-MRSA activity commonly have a 4-hydroxyl group at the D-benzene ring and a 2-hydroxyl group at the C-benzene ring in the hydrophilic part of the molecule, and an unsubstituted phenoxyphenyl group in the hydrophobic part of the molecule containing the A–B-benzene rings. Pharmacological characterization of the bis(bibenzyl) derivatives and 2-phenoxyphenol fragment 25, previously proposed as the minimum structure of riccardin C 1 for anti-MRSA activity, indicated that they have different action mechanisms: the bis(bibenzyl)s are bactericidal, while 25 is bacteriostatic, showing only weak bactericidal activity.

Resistance nodulation cell division (RND)-type efflux transporters play the main role in intrinsic resistance to various antimicrobial agents in many gram-negative bacteria. Here, we estimated 12 RND-type efflux transporter genes in Vibrio parahaemolyticus. Because VmeAB has already been characterized, we cloned the other 11 RND-type efflux transporter genes and characterized them in Escherichia coli KAM33 cells, a drug hypersusceptible strain. KAM33 expressing either VmeCD, VmeEF, or VmeYZ showed increased minimum inhibitory concentrations (MICs) for several antimicrobial agents. Additional four RND-type transporters were functional as efflux pumps only when co-expressed with VpoC, an outer membrane component in V. parahaemolyticus. Furthermore, VmeCD, VmeEF, and VmeYZ co-expressed with VpoC exhibited a broader substrate specificity and conferred higher resistance than that with TolC of E. coli. Deletion mutants of these transporter genes were constructed in V. parahaemolyticus. TM32 (ΔvmeAB and ΔvmeCD) had significantly decreased MICs for many antimicrobial agents and the number of viable cells after exposure to deoxycholate were markedly reduced. Strains in which 12 operons were all disrupted had very low MICs and much lower fluid accumulation in rabbit ileal loops. These results indicate that resistance nodulation cell division-type efflux transporters contribute not only to intrinsic resistance but also to exerting the virulence of V. parahaemolyticus.

Multidrug and toxic compound extrusion (MATE) family transporters are conserved in the three primary domains of life (Archaea, Bacteria and Eukarya), and export xenobiotics using an electrochemical gradient of H+ or Na+ across the membrane1, 2. MATE transporters confer multidrug resistance to bacterial pathogens3, 4, 5, 6 and cancer cells7, thus causing critical reductions in the therapeutic efficacies of antibiotics and anti-cancer drugs, respectively. Therefore, the development of MATE inhibitors has long been awaited in the field of clinical medicine8, 9. Here we present the crystal structures of the H+-driven MATE transporter from Pyrococcus furiosus in two distinct apo-form conformations, and in complexes with a derivative of the antibacterial drug norfloxacin and three in vitro selected thioether-macrocyclic peptides, at 2.1?3.0?A resolutions. The structures, combined with functional analyses, show that the protonation of Asp?41 on the amino (N)-terminal lobe induces the bending of TM1, which in turn collapses the N-lobe cavity, thereby extruding the substrate drug to the extracellular space. Moreover, the macrocyclic peptides bind the central cleft in distinct manners, which correlate with their inhibitory activities. The strongest inhibitory peptide that occupies the N-lobe cavity may pave the way towards the development of efficient inhibitors against MATE transporters.

Members of a series of macrocyclic bis(bibenzyl) riccardin-class derivatives were found to exhibit antibacterial activity towards methicillin-resistant Staphylococcus aureus (anti-MRSA activity). Structure–activity relationship (SAR) studies were conducted, focusing on the number and position of the hydroxyl groups. The minimum essential structure for anti-MRSA activity was also investigated.

In an attempt to discover inhibitory compounds against pore-forming toxins, some of the major toxins produced by bacteria, we herein examined the effects of four kinds of indolo[3,2-b]quinoline derivatives on hemolysis induced by the aerolysin-like hemolysin (ALH) of Aeromonas sobria and also by the alpha-hemolysin of Staphylococcus aureus. The results showed that hemolysis induced by ALH was significantly reduced by every derivative, while that induced by alpha-hemolysis was significantly reduced by three out of the four derivatives. However, the degrees of reduction induced by these derivatives were not uniform. Each derivative exhibited its own activity to inhibit the respective hemolysin. Compounds 1 and 2, which possessed the amino group bonding the naphthalene moiety at the C-11 position of indolo[3,2-b]quinoline, had strong inhibitory effects on the activity of ALH. Compound 4 which consisted of benzofuran and quinoline had strong inhibitory effects on the activity of alpha-hemolysin. These results indicated that the amino group bonding the naphthalene moiety of compounds 1 and 2 assisted in their ability to inhibit ALH activity, while the oxygen atom at the 10 position of compound 4 strengthened its interaction with alpha-hemolysin. These compounds also suppressed the hemolytic activity of the supernatant of A. sobria or A. hydrophila, suggesting that these compounds were effective at the site of infection of these bacteria.

The function of the NCgl1221-encoded protein of Corynebacterium glutamicum was analyzed using Bacillus subtilis as host because a method for preparing the giant provacuole required for electrophysiological studies has been established. Expression of NCgl1221 in a strain deficient in mscL and ykuT, both of which encode mechanosensitive channels, resulted in an 8.9-fold higher cell survival rate upon osmotic downshock than the control. Electrophysiological investigation showed that the giant provacuole prepared from this strain, expressing NCgl1221, exhibited significantly higher pressure-dependent conductance than the control. These findings show that the NCgl1221-encoded protein functions as a mechanosensitive channel.

The MATE (Multidrug And Toxic Compound Extrusion) family is the most recently categorized one among five multidrug efflux transporter families. As far as we know, about twenty MATE transporters have been characterized so far. According to the information in sequence databases, huge numbers of MATE transporters seem to be present in various microorganisms. In this review, we would like to summarize the properties of the MATE-family transporters.

We cloned a gene responsible for multidrug resistance from chromosomal DNA of Serratia marcescens, and determined the nucleotide sequence. We designated the gene as ssmE. The deduced amino acid sequence of SsmE showed high similarity with the small multidrug resistance (SMR)-type multidrug efflux pumps. Cells of Escherichia coli KAM32, a drug hyper-susceptible mutant, transformed with a plasmid pESM437 carrying the ssmE gene showed elevated minimum inhibitory concentrations of structurally unrelated antimicrobial agents. E. coli KAM32/pESM437 showed elevated energy dependent efflux of ethidium. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis revealed that ssmE was expressed in cells of S. marcescens.

We previously reported that we had cloned genes responsible for multidrug resistance from the chromosomal DNA of Klebsiella pneumoniae MGH78578 using a drug-hypersusceptible Escherichia coli strain as a host. One of the recombinant plasmids pETV6 conferred resistance to host cells against a wide range of antimicrobial agents, dyes and detergents. It was revealed that this plasmid carried the acrBKp gene and a part of the acrAKp gene coding for a multidrug efflux pump belonging to the RND family. We cloned the whole acrAKpBKp operon of K. pneumoniae and characterized the pump. The AcrAB pump utilized TolC as an outer membrane component in cells of E. coli. Elevated energy-dependent efflux of ethidium was observed with cells possessing AcrAKp BKp-TolC. We cloned a gene coding for an ortholog of TolC from chromosomal DNA of K. pneumoniae, and designated it kocC. It seems that the AcrAKpBKp-KocC complex functions as a potent multidrug efflux pump in K. pneumoniae. We observed a higher level of expression of acrAKp in K. pneumoniae MGH78578, a multidrug resistant strain, compared with ATCC10031, a drug susceptible strain.

The tonoplast K(+) membrane transport system plays a crucial role in maintaining K(+) homeostasis in plant cells. Here, we isolated cDNAs encoding a two-pore K(+) channel (NtTPK1) from Nicotiana tabacum cv. SR1 and cultured BY-2 tobacco cells. Two of the four variants of NtTPK1 contained VHG and GHG instead of the GYG signature sequence in the second pore region. All four products were functional when expressed in the Escherichia coli cell membrane, and NtTPK1 was targeted to the tonoplast in tobacco cells. Two of the three promoter sequences isolated from N. tabacum cv. SR1 were active, and expression from these was increased approximately 2-fold by salt stress or high osmotic shock. To determine the properties of NtTPK1, we enlarged mutant yeast cells with inactivated endogenous tonoplast channels and prepared tonoplasts suitable for patch clamp recording allowing the NtTPK1-related channel conductance to be distinguished from the small endogenous currents. NtTPK1 exhibited strong selectivity for K(+) over Na(+). NtTPK1 activity was sensitive to spermidine and spermine, which were shown to be present in tobacco cells. NtTPK1 was active in the absence of Ca(2+), but a cytosolic concentration of 45 microM Ca(2+) resulted in a 2-fold increase in the amplitude of the K(+) current. Acidification of the cytosol to pH 5.5 also markedly increased NtTPK1-mediated K(+) currents. These results show that NtTPK1 is a novel tonoplast K(+) channel belonging to a different group from the previously characterized vacuolar channels SV, FV, and VK.

Genes vmeA and vmeB, encoding a multidrug efflux transporter in the halophilic bacterium Vibrio parahaemolyticus, have been cloned using a drug-hypersusceptible Escherichia coli strain as the host. Cells of E. coli KAM33 (DeltaacrAB DeltaydhE) carrying the vmeAB region from V. parahaemolyticus conferred much higher MICs for a variety of antimicrobial agents than did control cells. Cells possessing VmeAB under energized conditions maintained very low intracellular concentrations of ethidium. This was as expected for an energy-dependent efflux system, and supports the notion--based on sequence homology--that VmeAB belongs to the resistance nodulation cell division (RND) family of multidrug efflux transporters. It is likely that VmeAB forms functional complexes with the outer-membrane protein TolC in E. coli, because introduction of vmeAB into cells of E. coli KAM43, which lacks the tolC gene, failed to elevate the MICs for any of the antimicrobial agents tested. Therefore, a V. parahaemolyticus homologue of tolC was also cloned, designated vpoC, and was introduced together with vmeAB into cells of E. coli KAM43. The MICs of all agents tested were raised and were comparable to the values observed in E. coli KAM33 harbouring a plasmid carrying vmeAB. Finally, a vmeAB-deficient mutant of V. parahaemolyticus was constructed (designated TM3). TM3 showed slightly higher susceptibility than the parental V. parahaemolyticus to some antimicrobial agents. Survival rate of the TM3 when exposed to deoxycholate decreased compared with that of the parent.

Resistance Nodulation cell Division (RND) efflux transporters are thought to be involved in mediating multidrug resistance in Gram-negative bacteria, including Vibrio cholerae non-O1. There are six operons for putative RND-type efflux transporters present in the chromosome of V. cholerae O1 including two operons, vexAB and vexCD, which had already been identified. All of the six operons were cloned from V. cholerae non-O1, NCTC4716 by the PCR method, introduced, and expressed in cells of drug hypersusceptible Escherichia coli KAM33 (DeltaacrAB, DeltaydhE). Only vexEF conferred elevated minimum inhibitory concentrations (MICs) of some antimicrobial agents in the E. coli cells. However, VexEF did not confer increased MIC of any drug tested in tolC-deficient E. coli KAM43 cells. On the other hand, when E. coli KAM43 was transformed with vexAB, vexCD or vexEF together with tolC(Vc) of V. cholerae NCTC4716, we observed elevated MICs of various antimicrobial agents. Among them, E. coli KAM43 expressing both VexEF and TolC(Vc) showed much higher MICs and much broader substrate specificity than the other two. We also observed ethidium efflux activity via VexEF-TolC(Vc), and the activity required Na(+). Thus, VexEF-TolC (Vc) is either a Na(+)-activated or a Na(+)-coupled transporter. To our knowledge, this is the first report on the requirement of Na(+) for an RND-type efflux transporter.