Teruo Kuroda

Publications of Teruo Kuroda

• 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.

• 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.

• Patch clamp analysis of the respiratory chain in Bacillus subtilis.

  Authors: Koji Nakamura, Satoshi Ikeda, Taira Matsuo, Aiko Hirata, Masaya Takehara, Tetsuo Hiyama, Fujio Kawamura, Iwao Kusaka, Tomofusa Tsuchiya, Teruo Kuroda, Isamu Yabe

  Biochimica et biophysica acta. 01/2011; 1808(4):1103-7.

  Bacillus subtilis is a representative Gram-positive bacterium. In aerobic conditions, this bacterium can generate an electrochemical potential across the membrane with aerobic respiration. Here, weBacillus subtilis is a representative Gram-positive bacterium. In aerobic conditions, this bacterium can generate an electrochemical potential across the membrane with aerobic respiration. Here, we developed the patch clamp method to analyze the respiratory chain in B. subtilis. First, we prepared giant protoplasts (GPs) from B. subtilis cells. Electron micrographs and fluorescent micrographs revealed that GPs of B. subtilis had a vacuole-like structure and that the intravacuolar area was completely separated from the cytoplasmic area. Acidification of the interior of the isolated and purified vacuole-like structure, due to H(+) translocation after the addition of NADH, revealed that they consisted of everted cytoplasmic membranes. We called these giant provacuoles (GVs) and again applied the patch clamp technique. When NADH was added as an electron donor for the respiratory system, a significant NADH-induced current was observed. Inhibition of KCN and 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO) demonstrated that this current is certainly due to aerobic respiration in B. subtilis. This is the first step for more detailed analyses of respiratory chain in B. subtilis, especially H(+) translocation mechanism.

• The protein encoded by NCgl1221 in Corynebacterium glutamicum functions as a mechanosensitive channel.

  Authors: Ken-ichi Hashimoto, Koji Nakamura, Teruo Kuroda, Isamu Yabe, Tsuyoshi Nakamatsu, Hisashi Kawasaki

  Bioscience, biotechnology, and biochemistry. 01/2010; 74(12):2546-9.

  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 forThe 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.

• 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.

• Identification and characterization of the NA+/H+ antiporter NHAS3 from the thylakoid membrane of synechocystis SP. PCC 6803.

  Authors: Kenta Tsunekawa, Toshiaki Shijuku, Mitsuo Hayashimoto, Yoichi Kojima, Kiyoshi Onai, Megumi Morishita, Masahiro Ishiura, Teruo Kuroda, Tatsunosuke Nakamura, Hiroshi Kobayashi, Mayuko Sato, Kiminori Toyooka, Ken Matsuoka, Tatsuo Omata, Nobuyuki Uozumi

  The Journal of biological chemistry. 05/2009;

  Na+/H+ antiporters influence proton or sodium motive force across the membrane. Synechocystis sp. PCC 6803 has six genes encoding Na+/H+ antiporters, nhaS1-5 and sll0556. In this study, the functionNa+/H+ antiporters influence proton or sodium motive force across the membrane. Synechocystis sp. PCC 6803 has six genes encoding Na+/H+ antiporters, nhaS1-5 and sll0556. In this study, the function of NhaS3 was examined. NhaS3 was essential for growth of Synechocystis, and loss of nhaS3 was not complemented by expression of the E. coli Na+/H+ antiporter NhaA. Membrane fractionation followed by immunoblotting as well as immunogold labeling revealed that NhaS3 was localized in the thylakoid membrane of Synechocystis. NhaS3 was shown to be functional over a pH range from pH 6.5 to 9.0 when expressed in E. coli. A reduction in the copy number of nhaS3 in the Synechocystis genome rendered the cells more sensitive to high Na+ concentrations. NhaS3 had no K+/H+ exchange activity itself, but enhanced K+ uptake from the medium when expressed in an E. coli potassium uptake mutant. Expression of nhaS3 increased after shifting from low CO2 to high CO2 conditions. Expression of nhaS3 was also found to be controlled by the circadian rhythm. Gene expression peaked at the beginning of subjective night. This coincided with the time of the lowest rate of CO2 consumption due to the ceasing of O2-evolving photosynthesis. This is the first report of a Na+/H+ antiporter localized in thylakoid membrane. Our results suggested a role of NhaS3 in the maintenance of ion homeostasis of H+, Na+ and K+, in supporting the conversion of photosynthetic products and in the supply of energy in the dark.

• 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.

• Multidrug efflux transporters in the MATE family.

  Authors: Teruo Kuroda, Tomofusa Tsuchiya

  Biochimica et biophysica acta. 01/2009;

  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 haveThe 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.

• 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.

• Characterization of a tobacco TPK-type K+ channel as a novel tonoplast K+ channel using yeast tonoplasts.

  Authors: Shin Hamamoto, Junichiro Marui, Ken Matsuoka, Kyohei Higashi, Kazuei Igarashi, Tsuyoshi Nakagawa, Teruo Kuroda, Yasuo Mori, Yoshiyuki Murata, Yoichi Nakanishi, Masayoshi Maeshima, Isamu Yabe, Nobuyuki Uozumi

  The Journal of biological chemistry. 02/2008; 283(4):1911-20.

  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 NicotianaThe 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.

• 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.

• Antimicrobial activity of oleanolic acid from Salvia officinalis and related compounds on vancomycin-resistant enterococci (VRE).

  Authors: Kumiko Horiuchi, Sumiko Shiota, Tsutomu Hatano, Takashi Yoshida, Teruo Kuroda, Tomofusa Tsuchiya

  Biological & pharmaceutical bulletin. 07/2007; 30(6):1147-9.

  An extract from Salvia officinalis (Sage) leaves showed antimicrobial activity against vancomycin-resistant enterococci (VRE). We isolated the effective compound and identified it as oleanolic acid,An extract from Salvia officinalis (Sage) leaves showed antimicrobial activity against vancomycin-resistant enterococci (VRE). We isolated the effective compound and identified it as oleanolic acid, a triterpenoid. We also tested antimicrobial activity of similar triterpenoids, ursolic acid, uvaol, betulinic acid and betulin. We found that ursolic acid also showed antimicrobial activity against VRE. The minimum inhibitory concentrations (MICs) of oleanolic acid and ursolic acid were 8 and 4 microg/ml, respectively. These two compounds also showed antimicrobial activity against Streptococcus pneumoniae and methicillin-resistant Staphylococcus aureus (MRSA). These compounds showed bactericidal activity against VRE at least for 48 h when added at concentrations that were two-times higher than their MICs.

• 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.

• Potentiation of antimicrobial activity of aminoglycosides by carnosol from Salvia officinalis.

  Authors: Kumiko Horiuchi, Sumiko Shiota, Teruo Kuroda, Tsutomu Hatano, Takashi Yoshida, Tomofusa Tsuchiya

  Biological & pharmaceutical bulletin. 03/2007; 30(2):287-90.

  We found that a crude extract from Salvia officinalis (sage) reduced the minimum inhibitory concentrations (MICs) of aminoglycosides in vancomycin-resistant enterococci (VRE). We isolated theWe found that a crude extract from Salvia officinalis (sage) reduced the minimum inhibitory concentrations (MICs) of aminoglycosides in vancomycin-resistant enterococci (VRE). We isolated the effective compound from the extract and identified it as carnosol, one of diterpenoids. Carnosol showed a weak antimicrobial activity, and greatly reduced the MICs of various aminoglycosides (potentiated the antimicrobial activity of aminoglycosides) and some other types of antimicrobial agents in VRE. Carnosic acid, a related compound, showed the similar activity. The effect of carnosol and carnosic acid with gentamicin was synergistic.

• 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.