Wakano Ogawa

Publications of Wakano Ogawa

• Functional study of the novel multidrug efflux pump KexD from Klebsiella pneumoniae.

  Authors: Wakano Ogawa, Motoyasu Onishi, Ruiting Ni, Tomofusa Tsuchiya, Teruo Kuroda

  Gene. 02/2012; 498(2):177-82.

  We cloned a gene, kexD, that provides a multidrug-resistant phenotype from multidrug-resistant Klebsiella pneumoniae MGH78578. The deduced amino acid sequence of KexD is similar to that of the innerWe cloned a gene, kexD, that provides a multidrug-resistant phenotype from multidrug-resistant Klebsiella pneumoniae MGH78578. The deduced amino acid sequence of KexD is similar to that of the inner membrane protein, RND-type multidrug efflux pump. Introduction of the kexD gene into Escherichia coli KAM32 resulted in a MIC that was higher for erythromycin, novobiocin, rhodamine 6G, tetraphenylphosphonium chloride, and ethidium bromide than that of the control. Intracellular ethidium bromide levels in E. coli cells carrying the kexD gene were lower than that in the control cells under energized conditions, suggesting that KexD is a component of an energy-dependent efflux pump. RND-type pumps typically consist of three components: an inner membrane protein, a periplasmic protein, and an outer membrane protein. We discovered that KexD functions with a periplasmic protein, AcrA, from E. coli and K. pneumoniae, but not with the periplasmic proteins KexA and KexG from K. pneumoniae. KexD was able to utilize either TolC of E. coli or KocC of K. pneumoniae as an outer membrane component. kexD mRNA was not detected in K. pneumoniae MGH78578 or ATCC10031. We isolated erythromycin-resistant mutants from K. pneumoniae ATCC10031, and some showed a multidrug-resistant phenotype similar to the drug resistance pattern of KexD. Two strains of multidrug-resistant mutants were investigated for kexD expression; kexD mRNA levels were increased in these strains. We conclude that changing kexD expression can contribute to the occurrence of multidrug-resistant K. pneumoniae.

• Quercetin diacylglycoside analogues showing dual inhibition of DNA gyrase and topoisomerase IV as novel antibacterial agents.

  Authors: Abugafar M L Hossion, Yoshito Zamami, Rafiya K Kandahary, Tomofusa Tsuchiya, Wakano Ogawa, Akimasa Iwado, Kenji Sasaki

  Journal of medicinal chemistry. 06/2011; 54(11):3686-703.

  A structure-guided molecular design approach was used to optimize quercetin diacylglycoside analogues that inhibit bacterial DNA gyrase and topoisomerase IV and show potent antibacterial activityA structure-guided molecular design approach was used to optimize quercetin diacylglycoside analogues that inhibit bacterial DNA gyrase and topoisomerase IV and show potent antibacterial activity against a wide spectrum of relevant pathogens responsible for hospital- and community-acquired infections. In this paper, such novel 3,7-diacylquercetin, quercetin 6''-acylgalactoside, and quercetin 2'',6''-diacylgalactoside analogues of lead compound 1 were prepared to assess their target specificities and preferences in bacteria. The significant enzymatic inhibition of both Escherichia coli DNA gyrase and Staphylococcus aureus topoIV suggest that these compounds are dual inhibitors. Most of the investigated compounds exhibited pronounced inhibition with MIC values ranging from 0.13 to 128 μg/mL toward the growth of multidrug-resistant Gram-positive methicillin-resistant S. aureus, methicillin sensitive S. aureus, vancomycin-resistant enterococci (VRE), vancomycin intermediate S. aureus, and Streptococcus pneumoniae bacterial strains. Structure-activity relationship studies revealed that the acyl moiety was absolutely essential for activity against Gram-positive organisms. The most active compound 5i was 512-fold more potent than vancomycin and 16-32-fold more potent than 1 against VRE strains. It also has realistic in situ intestinal absorption in rats and showed very low acute toxicity in mice. So far, this compound can be regarded as a leading antibacterial agent.

• Errata: Properties and Expression of a Multidrug Efflux Pump AcrAB-KocC from Klebsiella pneumoniae[Biol. Pharm. Bull. 31(4): 577-582 (2008)].

  Authors: Dai-Wei Li, Motoyasu Onishi, Takanori Kishino, Taira Matsuo, Wakano Ogawa, Teruo Kuroda, Tomofusa Tsuchiya

  Biological & pharmaceutical bulletin. 01/2011; 34(4):594.

• Design, synthesis, and biological evaluation of a novel series of quercetin diacylglucosides as potent anti-MRSA and anti-VRE agents.

  Authors: Abugafar M L Hossion, Nao Otsuka, Rafiya K Kandahary, Tomofusa Tsuchiya, Wakano Ogawa, Akimasa Iwado, Yoshito Zamami, Kenji Sasaki

  Bioorganic & medicinal chemistry letters. 09/2010; 20(17):5349-52.

  A series of novel quercetin diacylglucosides were designed and first synthesized by Steglich esterification on the basis of MRSA strains inhibiting natural compound A. The in vitro inhibition ofA series of novel quercetin diacylglucosides were designed and first synthesized by Steglich esterification on the basis of MRSA strains inhibiting natural compound A. The in vitro inhibition of different multi-drug resistant bacterial strains and Escherichia coli DNA gyrase B was investigated. In the series, compound 10h was up to 128-fold more potent against vancomycin-resistant enterococci and more effective than A, which represents a promising new candidate as a potent anti-MRSA and anti-VRE agent.

• Gene Cloning and Characteristics of the RND-Type Multidrug Efflux Pumps MuxABC-OpmB possessing two RND components in Pseudomonas aeruginosa.

  Authors: Takehiko Mima, Naoki Kohira, Yang Li, Hiroshi Sekiya, Wakano Ogawa, Teruo Kuroda, Tomofusa Tsuchiya

  Microbiology (Reading, England). 09/2009;

  muxA-muxB-muxC-opmB (formerly PA2528-PA2527-PA2526-opmB), encoding a putative Resistance Nodulation cell Division (RND)-type multidrug efflux pump system was cloned from Pseudomonas aeruginosa PAO1.muxA-muxB-muxC-opmB (formerly PA2528-PA2527-PA2526-opmB), encoding a putative Resistance Nodulation cell Division (RND)-type multidrug efflux pump system was cloned from Pseudomonas aeruginosa PAO1. The P. aeruginosa YM64, a drug hypersusceptible strain, introduced with muxABC-opmB showed elevated MICs of aztreonam, macrolides, novobiocin and tetracycline. Since muxB and muxC, both of which encode RND components, were essential for function, MuxABC-OpmB is thought to be a drug efflux pump with four components. One novobiocin resistant mutant, PMX725, isolated from P. aeruginosa PMX7 showed elevated resistance not only to novobiocin but also to aztreonam, macrolides, and tetracycline. The increased mRNA expression of muxABC-opmB was observed in the mutant PMX725 compared with the parental strain. Sequencing analysis revealed that a single nucleotide insertion occurred in the deduced promoter region for muxABC-opmB in PMX725. In this report, we characterized the last RND-type multidrug efflux pump predicted from genome sequence in P. aeruginosa.

• Gene Cloning and Characterization of EfmA, a Multidrug Efflux Pump, from Enterococcus faecium.

  Authors: Toshihiro Nishioka, Wakano Ogawa, Teruo Kuroda, Takashi Katsu, Tomofusa Tsuchiya

  Biological & pharmaceutical bulletin. 04/2009; 32(3):483-8.

  A DNA fragment responsible for resistance to antimicrobial agents was cloned from chromosomal DNA of Enterococcus faecium FN-1, a clinically isolated strain. Escherichia coli KAM32, aA DNA fragment responsible for resistance to antimicrobial agents was cloned from chromosomal DNA of Enterococcus faecium FN-1, a clinically isolated strain. Escherichia coli KAM32, a drug-hypersusceptible mutant, was used as a host for gene cloning. Cells of E. coli KAM32 harboring a recombinant plasmid (pTFM8) carrying the DNA fragment became resistant to fluoroquinolones, macrolides, ethidium bromide, 4',6-diamidino-2-phenylindole (DAPI) and tetraphenylphosphonium chloride (TPPCl). Three complete open reading frames (ORFs) were found in the DNA insert of pTFM8, and the deduced amino acid sequences of one of the ORFs showed high similarity to Mdt(A) from Lactococcus lactis. Mdt(A) is a multidrug efflux pump belonging to a major facilitator superfamily. We designated the ORF efmA. E. coli KAM32 cells harboring the efmA showed energy-dependent efflux of DAPI and TPP(+). We also observed norfloxacin/H(+) antiport due to EfmA. The mRNA expression of efmA was observed in E. faecium FN-1 grown without any exogenously added antimicrobial agents. Thus, we conclude that efmA is constitutively expressed under laboratory growth conditions and would contribute to intrinsic resistance against multiple antimicrobial agents in E. faecium FN-1.

• Synergistic Effect of Kaempferol Glycosides Purified from Laurus nobilis and Fluoroquinolones on Methicillin-Resistant Staphylococcus aureus.

  Authors: Mei-Hua Liu, Nao Otsuka, Kumiko Noyori, Sumiko Shiota, Wakano Ogawa, Teruo Kuroda, Tsutomu Hatano, Tomofusa Tsuchiya

  Biological & pharmaceutical bulletin. 04/2009; 32(3):489-92.

  In a previous study, we reported that two kaempferol glycosides isolated from Laurus nobilis L., kaempferol-3-O-alpha-L-(2'',4''-di-E-p-coumaroyl)-rhamnoside (C2) andIn a previous study, we reported that two kaempferol glycosides isolated from Laurus nobilis L., kaempferol-3-O-alpha-L-(2'',4''-di-E-p-coumaroyl)-rhamnoside (C2) and kaempferol-3-O-alpha-L-(2''-E-p-coumaroyl-4''-Z-p-coumaroyl)-rhamnoside (C3), showed strong antibacterial activities against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci. Thereafter we found that these compounds greatly reduced the minimum inhibitory concentrations (MICs) of some fluoroquinolones in MRSA. In other words, C2 and C3 greatly potentiated anti-MRSA activity of fluoroquinolones. The effect of C2 and C3 with fluoroquinolones was found to be synergistic. The potentiation activity was observed with hydrophilic fluoroquinolones, such as norfloxacin and ciprofloxacin, but not with hydrophobic quinolones. We also found that norfloxacin reduced MICs of C2 and C3. The effect was synergistic. Possible mechanism of the synergistic effect was discussed.

• Anti-methicillin Resistant Staphylococcus aureus (MRSA) Compounds Isolated from Laurus nobilis.

  Authors: Nao Otsuka, Mei-Hua Liu, Sumiko Shiota, Wakano Ogawa, Teruo Kuroda, Tsutomu Hatano, Tomofusa Tsuchiya

  Biological & pharmaceutical bulletin. 10/2008; 31(9):1794-7.

  We found that an extract from Laurus nobilis L. (Lauraceae) leaves showed antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA). We purified two flavonoids as theWe found that an extract from Laurus nobilis L. (Lauraceae) leaves showed antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA). We purified two flavonoids as the effective compounds and identified them as kaempferol 3-O-alpha-L-(2'',4''-di-E-p-coumaroyl)-rhamnoside (C2) and kaempferol 3-O-alpha-L-(2''-Z-p-coumaroyl-4''-E-p-coumaroyl)-rhamnoside (C3). Both compounds showed strong antibacterial activity not only against MRSA but also against vancomycin-resistant enterococci (VRE). There was low or no antibacterial activity of C2 and C3 for Streptococcus pneumoniae, Pseudomonas aeruginosa and Serratia marcescens.

• Properties and expression of a multidrug efflux pump AcrAB-KocC from Klebsiella pneumoniae.

  Authors: Dai-Wei Li, Motoyasu Onishi, Takanori Kishino, Taira Matsuo, Wakano Ogawa, Teruo Kuroda, Tomofusa Tsuchiya

  Biological & pharmaceutical bulletin. 05/2008; 31(4):577-82.

  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 strainWe 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.

• Functional gene cloning and characterization of the SsmE multidrug efflux pump from Serratia marcescens.

  Authors: Yusuke Minato, Fereshteh Shahcheraghi, Wakano Ogawa, Teruo Kuroda, Tomofusa Tsuchiya

  Biological & pharmaceutical bulletin. 04/2008; 31(3):516-9.

  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 acidWe 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.

• SmdAB, a heterodimeric ABC-Type multidrug efflux pump, in Serratia marcescens.

  Authors: Taira Matsuo, Jing Chen, Yusuke Minato, Wakano Ogawa, Tohru Mizushima, Teruo Kuroda, Tomofusa Tsuchiya

  Journal of bacteriology. 02/2008; 190(2):648-54.

  We cloned genes, designated smdAB, that encode a multidrug efflux pump from the chromosomal DNA of clinically isolated Serratia marcescens NUSM8906. For cells of the drug-hypersensitive strainWe cloned genes, designated smdAB, that encode a multidrug efflux pump from the chromosomal DNA of clinically isolated Serratia marcescens NUSM8906. For cells of the drug-hypersensitive strain Escherichia coli KAM32 harboring a recombinant plasmid carrying smdAB, structurally unrelated antimicrobial agents such as norfloxacin, tetracycline, 4',6-diamidino-2-phenylindole (DAPI), and Hoechst 33342 showed elevated MICs. The deduced amino acid sequences of both SmdA and SmdB exhibited similarities to the sequences of ATP-binding cassette (ABC)-type multidrug efflux pumps. The efflux of DAPI and Hoechst 33342 from E. coli cells expressing SmdAB was observed, and the efflux activities were inhibited by sodium o-vanadate, which is a well-known ATPase inhibitor. The introduction of smdA or smdB alone into E. coli KAM32 did not elevate the MIC of DAPI; thus, both SmdA and SmdB were required for function. These results indicate that SmdAB is probably a heterodimeric multidrug efflux pump of the ABC family in S. marcescens.

• VmeAB, an RND-type multidrug efflux transporter in Vibrio parahaemolyticus.

  Authors: Taira Matsuo, Katsuhiko Hayashi, Yuji Morita, Motohiro Koterasawa, Wakano Ogawa, Tohru Mizushima, Tomofusa Tsuchiya, Teruo Kuroda

  Microbiology (Reading, England). 01/2008; 153(Pt 12):4129-37.

  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.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.

• Gene cloning and characterization of KdeA, a multidrug efflux pump from Klebsiella pneumoniae.

  Authors: Yu Ping, Wakano Ogawa, Teruo Kuroda, Tomofusa Tsuchiya

  Biological & pharmaceutical bulletin. 11/2007; 30(10):1962-4.

  We cloned a gene related to multidrug resistance from a drug-resistant Klebsiella pneumoniae strain MGH78578. We designated the gene kdeA, which encodes a protein possessing 12 hydrophobic regions.We cloned a gene related to multidrug resistance from a drug-resistant Klebsiella pneumoniae strain MGH78578. We designated the gene kdeA, which encodes a protein possessing 12 hydrophobic regions. The deduced amino acid sequence of KdeA is similar to that of MdfA, a well-characterized multidrug efflux pump from Escherichia coli. Introduction of the kdeA gene into cells of the drug-hypersusceptible E. coli strain KAM32 resulted in elevated minimum inhibitory concentrations of chloramphenicol, norfloxacin, acriflavine, and ethidium bromide. We observed elevated energy-dependent ethidium efflux activity with cells carrying kdeA compared with control cells. We also observed expression of kdeA in cells of K. pneumoniae under normal growth conditions.

• Molecular cloning and characterization of a multidrug efflux pump, SmfY, from Serratia marcescens.

  Authors: Fereshteh Shahcheraghi, Yusuke Minato, Jing Chen, Tohru Mizushima, Wakano Ogawa, Teruo Kuroda, Tomofusa Tsuchiya

  Biological & pharmaceutical bulletin. 05/2007; 30(4):798-800.

  We cloned a gene smfY for multidrug efflux pump from chromosomal DNA of Serratia marcescens using drug-hypersensitive Escherichia coli KAM32 as the host, and characterized the pump. E. coliWe cloned a gene smfY for multidrug efflux pump from chromosomal DNA of Serratia marcescens using drug-hypersensitive Escherichia coli KAM32 as the host, and characterized the pump. E. coli KAM32/pESM42 carrying the smfY showed significantly increased MICs of various drugs including DAPI, norfloxacin, benzalkonium chloride, acriflavine and ethidium bromide, compared with the control. We also detected energy-dependent ethidium and acriflavine efflux due to the SmfY. Sequence analysis revealed that the SmfY was a multidrug efflux pump of the MF (Major Facilitator) superfamily transporters. This is the first report of a multidrug efflux pump belonging to the MF superfamily in S. marcescens.

• Molecular cloning and characterization of all RND-type efflux transporters in Vibrio cholerae non-O1.

  Authors: M Mushfequr Rahman, Taira Matsuo, Wakano Ogawa, Motohiro Koterasawa, Teruo Kuroda, Tomofusa Tsuchiya

  Microbiology and immunology. 02/2007; 51(11):1061-70.

  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 sixResistance 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.

• KmrA multidrug efflux pump from Klebsiella pneumoniae.

  Authors: Wakano Ogawa, Motohiro Koterasawa, Teruo Kuroda, Tomofusa Tsuchiya

  Biological & pharmaceutical bulletin. 04/2006; 29(3):550-3.

  We cloned a gene responsible for multidrug resistance from the chromosomal DNA of Klebsiella pneumoniae MGH78578 that showed multidrug resistance. We designated the gene kmrA. The deduced amino acidWe cloned a gene responsible for multidrug resistance from the chromosomal DNA of Klebsiella pneumoniae MGH78578 that showed multidrug resistance. We designated the gene kmrA. The deduced amino acid sequence of KmrA was similar to that of SmvA that is responsible for methyl viologen-resistance in Salmonella enterica sv. Typhi and Typhimurium. Introduction of the cloned kmrA gene into drug-hypersensitive Escherichia coli KAM32 cells made them resistant to acriflavine, 4',6-diamidino-2-phenylindole (DAPI), Hoechst 33342, tetraphenylphosphonium chloride (TPPCl), methyl viologen and ethidium bromide. We observed elevated energy-dependent efflux of ethidium in E. coli cells carrying the kmrA gene compared with control cells. We also cloned the smvA gene from S. enterica sv. Typhimurium LT2 and investigated the resistance pattern for several drugs. The pattern was similar between KmrA from K. pneumoniae and SmvA from S. enterica.

• Multidrug resistance in Klebsiella pneumoniae MGH78578 and cloning of genes responsible for the resistance.

  Authors: Wakano Ogawa, Dai-Wei Li, Ping Yu, Anowara Begum, Tohru Mizushima, Teruo Kuroda, Tomofusa Tsuchiya

  Biological & pharmaceutical bulletin. 09/2005; 28(8):1505-8.

  Klebsiella pneumoniae MGH78578, a clinical isolate, showed high level of resistance to many antimicrobial agents. We cloned genes responsible for drug resistance from chromosomal DNA of K. pneumoniaeKlebsiella pneumoniae MGH78578, a clinical isolate, showed high level of resistance to many antimicrobial agents. We cloned genes responsible for drug resistance from chromosomal DNA of K. pneumoniae MGH78578 by shotgun method using Escherichia coli KAM32, a drug hypersensitive strain, as host. We obtained 43 hybrid plasmids that made host cells resistant to several antimicrobial agents. We classified them into 17 groups based on growth properties in the presence of each one of 9 antimicrobial agents and on restriction patterns of each hybrid plasmid. Analysis of the cloned genes must be very useful for investigation of major parts of multidrug resistance systems including multidrug efflux pumps in K. pneumoniae MGH78578 in which genome sequence is available.

• Gene cloning and characterization of four MATE family multidrug efflux pumps from Vibrio cholerae non-O1.

  Authors: Anowara Begum, M Mushfequr Rahman, Wakano Ogawa, Tohru Mizushima, Teruo Kuroda, Tomofusa Tsuchiya

  Microbiology and immunology. 02/2005; 49(11):949-57.

  There are six putative genes for multidrug and toxic compound extrusion (MATE) family multidrug efflux pumps in the chromosome of Vibrio cholerae. We have so far analyzed two MATE family pumps in V.There are six putative genes for multidrug and toxic compound extrusion (MATE) family multidrug efflux pumps in the chromosome of Vibrio cholerae. We have so far analyzed two MATE family pumps in V. cholerae non-O1 NCTC4716. Here we cloned four remaining genes for putative MATE family efflux pumps by the PCR method from this microorganism and designated them as vcmB, vcmD, vcmH and vcmN. Each one of the four genes was introduced and expressed in the drug hypersusceptible host Escherichia coli KAM32 cells. We observed elevated MICs of multiple antimicrobial agents, such as fluoroquinolones, aminoglycosides, ethidium bromide and Hoechst 33342 in the transformants. Energydependent efflux of substrate was observed with the transformed cells. We found that efflux activities of VcmB, VcmD and VcmH were Na+-dependent, but that of VcmN was Na+-independent. Thus, all six of the MATE family multidrug efflux pumps of V. cholerae non-O1 have been characterized. We also found that all six genes were expressed in cells of V. cholerae non-O1.

• Purification, reconstitution, and characterization of Na(+)/serine symporter, SstT, of Escherichia coli.

  Authors: Young-Mog Kim, Wakano Ogawa, Eiji Tamai, Teruo Kuroda, Tohru Mizushima, Tomofusa Tsuchiya

  Journal of biochemistry. 08/2002; 132(1):71-6.

  A gene encoding Na(+)/serine symporter (SstT) of Escherichia coli has been cloned and sequenced in our laboratory [Ogawa et al. (1998) J. Bacteriol. 180, 6749-6752]. In an attempt to overproduce theA gene encoding Na(+)/serine symporter (SstT) of Escherichia coli has been cloned and sequenced in our laboratory [Ogawa et al. (1998) J. Bacteriol. 180, 6749-6752]. In an attempt to overproduce the protein and purify it, we first constructed a plasmid pTSTH in which the modified sstT gene (sstT gene with 8 successive codons for His at the 3'-terminus) is located downstream from the trc promoter. Upon induction by IPTG, the His-tagged SstT protein was overproduced (about 15% of total membrane proteins), and showed activity as high as the wild type SstT. The His-tagged SstT was solubilized with octylglucoside and purified to homogeneity using a nickel nitrilotriacetic acid (Ni(2+)-NTA) affinity resin. The N-terminal sequence (20 amino acid residues) of the purified protein showed that the sequence was identical to that deduced from the DNA sequence of the sstT gene and that the initiation methionine was excised. The purified His-tagged SstT was reconstituted into liposomes by the detergent dilution method. Reconstituted proteoliposomes mediated the transport of serine driven by an artificially imposed electrochemical Na(+) gradient. The K(m) and the V(max) values for serine transport with the proteoliposomes were 0.82 microM and 0.37 nmol/min/mg protein, respectively. Serine transport was inhibited by L-threonine, but not by other amino acids. The purified protein was stable for at least 6 months at -80 degrees C.