Teruo Kuroda

Multidrug efflux transporters in the MATE family

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.

EfrAB, an ABC Multidrug Efflux Pump in Enterococcus faecalis

ABSTRACT A DNA fragment responsible for resistance to antimicrobial agents was cloned from the chromosomal DNA of Enterococcus faecalis ATCC 29212 by using drug-hypersensitive mutant Escherichia coli KAM32 as a host cell. Cells of E. coli KAM32 harboring a recombinant plasmid (pAEF82) carrying the DNA fragment became resistant to many structurally unrelated antimicrobial agents, such as norfloxacin, ciprofloxacin, doxycycline, acriflavine, 4′,6-diamidino-2-phenylindole, tetraphenylphosphonium chloride, daunorubicin, and doxorubicin. Since the sequence of the whole genome of E. faecalis is known, we sequenced several portions of the DNA insert in plasmid pAEF82 and identified two open reading frames within the insert. We designated the genes efrA and efrB. A search of the deduced amino acid sequences of EfrA and EfrB revealed that they are similar to each other and that they belong to the ATP-binding cassette (ABC) family of multidrug efflux transporters. Transformed E. coli KAM32 cells harboring efrAB showed energy-dependent efflux of acriflavine. The efflux activity was inhibited by reserpine, verapamil, and sodium-o-vanadate, known inhibitors of ABC efflux pumps.

A two-component multidrug efflux pump, EbrAB, in Bacillus subtilis.

Genes (ebrAB) responsible for ethidium resistance were cloned from chromosomal DNA of Bacillus subtilis ATCC 9372. The recombinant plasmid produced elevated resistance against ethidium bromide, acriflavine, pyronine Y, and safranin O not only in Escherichia coli but also in B. subtilis. It also caused an elevated energy-dependent efflux of ethidium in E. coli. EbrA and EbrB showed high sequence similarity with members of the small multidrug resistance (SMR) family of multidrug efflux pumps. Neither ebrA nor ebrB was sufficient for resistance, but introduction of the two genes carried on different plasmids conferred drug resistance. Thus, both EbrA and EbrB appear to be necessary for activity of the multidrug efflux pump. In known members of the SMR family, only one gene produces drug efflux. Thus, EbrAB is a novel SMR family multidrug efflux pump with two components.

An H+-Coupled Multidrug Efflux Pump, PmpM, a Member of the MATE Family of Transporters, from Pseudomonas aeruginosa

We cloned the gene PA1361 (we designated the gene pmpM), which seemed to encode a multidrug efflux pump belonging to the MATE family, of Pseudomonas aeruginosa by the PCR method using the drug-hypersensitive Escherichia coli KAM32 strain as a host. Cells of E. coli possessing the pmpM gene showed elevated resistance to several antimicrobial agents. We observed energy-dependent efflux of ethidium from cells possessing the pmpM gene. We found that PmpM is an H(+)-drug antiporter, and this finding is the first reported case of an H(+)-coupled efflux pump in the MATE family. Disruption and reintroduction of the pmpM gene in P. aeruginosa revealed that PmpM is functional and that benzalkonium chloride, fluoroquinolones, ethidium bromide, acriflavine, and tetraphenylphosphonium chloride are substrates for PmpM in this microorganism.

Molecular Cloning and Characterization of an ABC Multidrug Efflux Pump, VcaM, in Non-O1 Vibrio cholerae

ABSTRACT A gene responsible for multidrug resistance was cloned from the chromosomal DNA of non-O1 Vibrio cholerae NCTC 4716 by using as a host drug-hypersensitive Escherichia coli strain KAM32, which lacks major multidrug efflux pumps. E. coli cells transformed with the gene showed elevated levels of resistance to a number of structurally dissimilar drugs, such as tetracycline, norfloxacin, ciprofloxacin, doxorubicin, daunomycin, 4′,6-diamidino-2-phenylindole, and Hoechst 33342. We determined the nucleotide sequence and found one open reading frame. We designated the gene vcaM. The deduced product, VcaM, seems to be a polypeptide with 619 amino acid residues (69 kDa) that has a putative topology of six transmembrane segments in the N-terminal hydrophobic domain, followed by an ATP binding domain in the C-terminal hydrophilic region. The sequence of VcaM was shown to be similar to those of human multidrug resistance proteins P-glycoprotein MDR1 and lactococcal LmrA, which are driven by ATP. The efflux of Hoechst 33342 and doxorubicin from cells possessing VcaM was detected. The efflux activity was inhibited by reserpine and sodium o-vanadate, which are potent inhibitors of MDR1 and LmrA. Thus, we conclude that VcaM is a member of the family of multidrug efflux pumps of the ATP binding cassette type and the first experimentally proven example of a multidrug efflux pump of this family in gram-negative bacteria.

A new Na+/H+ antiporter, NhaD, of Vibrio parahaemolyticus

A gene encoding an Na+/H+ antiporter was cloned from chromosomal DNA of Vibrio parahaemolyticus, a slightly halophilic bacterium, and expressed in Escherichia coli cells. The gene enabled mutant E. coli cells, which were unable to grow in the presence of 10 mM LiCl (or 0.2 M NaCl) because of the lack of major Na+(Li+)/H+ antiporters, to grow under such conditions. We detected Na+/H+ antiport activity due to the gene in membrane vesicles prepared from E. coli cells that harbored the plasmid carrying the gene. Li+ was also a substrate for this antiporter. Activity of this antiporter was pH-dependent with highest activity at pH 8.5 to 9 and no activity at 7.0 to 7.5. Restriction mapping and a Southern blot analysis revealed that the cloned gene was different from the nhaA and the nhaB of V. parahaemolyticus. We designated the gene nhaD. The gene was sequenced, and the amino acid sequence of the NhaD protein was deduced. The NhaD is a unique Na+/H+ antiporter with respect to the primary structure compared with known Na+/H+ antiporters.

Functional Cloning and Characterization of a Multidrug Efflux Pump, MexHI-OpmD, from a Pseudomonas aeruginosa Mutant

ABSTRACT We isolated mutant YM644, which showed elevated resistance to norfloxacin, ethidium bromide, acriflavine, and rhodamine 6G, from Pseudomonas aeruginosa YM64, a strain that lacks four major multidrug efflux pumps. The genes responsible for the resistance were mexHI-opmD. Elevated ethidium extrusion was observed with cells of YM644 and YM64 harboring a plasmid carrying the genes. Disruption of the genes in the chromosomal DNA of YM644 made the cells sensitive to the drugs.

Characterization of a Tobacco TPK-type K+ Channel as a Novel Tonoplast K+ Channel Using Yeast Tonoplasts

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 ∼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 Ca2+, but a cytosolic concentration of 45 μm Ca2+ 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.

CLONING AND SEQUENCING OF A NOVEL NA+/H+ ANTIPORTER GENE FROM PSEUDOMONAS AERUGINOSA

We cloned a gene for Na+/H+ antiporter from chromosomal DNA of Pseudomonas aeruginosa. Introduction of the gene into host Escherichia coli mutant cells lacking all of the major Na+/H+ antiporters enabled the cells to grow in the presence of 0.2 M NaCl, although the original host cells could not. Membrane vesicles prepared from cells of the transformant possessing the cloned gene showed Na+/H+ antiport activity. As a result of DNA sequencing, we found one open reading frame (nhaP). The deduced amino acid sequence suggests that the Na+/H+ antiporter (NhaP) of P. aeruginosa consists of 424 amino acid residues with molecular mass of 45486 Da, and hydropathy analysis suggested the presence of 12 putative transmembrane domains. We found no bacterial Na+/H+ antiporter which showed significant sequence similarity with the NhaP in the protein sequence database. The NhaP showed partial sequence similarity with animal Na+/H+ exchangers. Thus, the NhaP of P. aeruginosa is unique among bacterial antiporters.

Gene Cloning and Characterization of VcrM, a Na+-Coupled Multidrug Efflux Pump, from Vibrio cholerae Non-O1

We cloned a DNA fragment responsible for drug resistance from chromosome of Vibrio cholerae non‐O1. Nucleotide sequence analysis of this fragment revealed the presence of a single open reading frame encoding a protein consisting of 445 amino acid residues. We designated the gene as vcrM. Hydropathy analysis of the deduced amino acid sequence of VcrM suggests the presence of 12 trans‐membrane segments. A dendrogram showed that VcrM is a member of the DinF‐subfamily within the MATE family of multidrug efflux pumps. Expression of the cloned vcrM gene in drug‐hypersensitive Escherichia coli KAM32 cells made them resistant to acriflavine, 4′, 6‐diamidino‐2‐phenylindole, Hoechst 33342, rhodamine 6G, tetraphenylphosphonium chloride (TPPCl) and ethidium bromide. Efflux of acriflavine due to VcrM was dependent on Na+ or Li+. Moreover, Na+ efflux was observed with VcrM when TPPCl was added to Na+‐loaded cells. Therefore, we conclude that VcrM is a Na+/drug antiporter‐type multidrug efflux pump.