ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, July 1998, p. 1778–1782 Copyright 1998, American Society for Microbiology. All Rights Reserved.
NorM, a Putative Multidrug Efflux Protein, of Vibrio parahaemolyticus and Its Homolog in Escherichia coli YUJI MORITA,1 KAZUYO KODAMA,1 SUMIKO SHIOTA,1 TOMOYUKI MINE,1 ATSUKO KATAOKA,1 TOHRU MIZUSHIMA,1 AND TOMOFUSA TSUCHIYA1,2,* Department of Microbiology, Faculty of Pharmaceutical Sciences,1 and Gene Research Center,2 Okayama University, Tsushima, Okayama 700-8530, Japan Received 17 February 1998/Returned for modification 27 March 1998/Accepted 6 May 1998 We found that cells of Vibrio parahaemolyticus possess an energy-dependent efflux system for norfloxacin. We
cloned a gene for a putative norfloxacin efflux protein from the chromosomal DNA of V. parahaemolyticus by
using an Escherichia coli mutant lacking the major multidrug efflux system AcrAB as the host and sequenced
the gene (norM). Cells of E. coli transformed with a plasmid carrying the norM gene showed elevated energy-
dependent efflux of norfloxacin. The transformants showed elevated resistance not only to norfloxacin and
ciprofloxacin but also to the structurally unrelated compounds ethidium, kanamycin, and streptomycin. These
results suggest that this is a multidrug efflux system. The hydropathy pattern of the deduced amino acid se-
quence of NorM suggested the presence of 12 transmembrane domains. The deduced primary structure of
NorM showed 57% identity and 88% similarity with that of a hypothetical E. coli membrane protein, YdhE. No
reported drug efflux protein in the sequence databases showed significant sequence similarity with NorM.
Thus, NorM seems to be a novel type of multidrug efflux protein. We cloned the ydhE gene from E. coli. Cells
of E. coli transformed with the cloned ydhE gene showed elevated resistance to norfloxacin, ciprofloxacin,
acriflavine, and tetraphenylphosphonium ion, but not to ethidium, when MICs were measured. Thus, it seems
that NorM and YdhE differ somehow in substrate specificity.
Drug efflux from cells is one of the major mechanisms of (18). Energy metabolism and energy coupling in membranes of drug resistance in bacteria. Many drug efflux systems are this microorganism are unique (11). Cells of V. parahaemolyti- known in many bacteria (4, 22, 31). Four major groups of cus show some natural resistance to some antimicrobial agents.
drug extrusion systems are known (4, 22, 31), i.e., the MF Thus, we were interested in drug efflux systems of V. parahae- (major facilitator) family, the SMR (small multidrug resis- molyticus. Cells of V. parahaemolyticus may possess unique tance) family, the RND (resistance nodulation cell division) drug efflux systems. During the course of our studies, we found family, and the ABC (ATP binding cassette) family. Mem- that cells of V. parahaemolyticus possess an energy-dependent brane transporters of the MF family possess 12 to 14 trans- efflux system for norfloxacin, a widely used new quinolone an- membrane domains. For example, Bcr (Escherichia coli) (1), timicrobial agent. Here we report the cloning and sequencing EmrB (E. coli) (14), EmrD (E. coli) (20), NorA (Staphylococ- of the gene for and characterization of a putative norfloxacin cus aureus) (34), QacA (S. aureus) (26), and Bmr (Bacillus efflux protein of V. parahaemolyticus and the cloning and char- subtilis) (21) are members of this family, and these systems acterization of the homolog gene found in E. coli.
mediate drug extrusion with different specificities. Transport- ers of the SMR family are rather small and usually possess four MATERIALS AND METHODS
transmembrane domains. Smr (or QacC) (S. aureus) (8, 13), QacE (Klebsiella aerogenes) (23), and EmrE (E. coli) (33) be- Bacteria and growth. V. parahaemolyticus AQ3334 (30) and E. coli TG1 [⌬(lac-
long to this family. An electrochemical potential of Hϩ across pro) supE thi hsd5/FЈtraD36 proAϩBϩ lacIq lacZ⌬M15], a derivative of K-12, and KAM2 and KAM3, derivatives of TG1, were used in this study. Cells of cell membranes seems to be the driving force for drug efflux V. parahaemolyticus AQ3334 were grown in LB medium (17) under aerobic by the MF and SMR family transporters. Transporters of the conditions at 37°C, and E. coli cells were grown in L medium (12) under aerobic RND family consist of several subunits (usually three), and an conditions at 37°C. Where indicated, drugs were added to the medium. Cell outer membrane protein(s) is involved in the drug transport.
growth was monitored turbidimetrically at 650 nm.
Isolation of mutants. KAM3, which has a deletion in the chromosomal acrAB
AcrAB (E. coli) (16) and MexAB (Pseudomonas aeruginosa) genes, was obtained as follows. We tried to disrupt the acrAB genes, which code (24) are examples of members of this family. Energy coupling for the major multidrug efflux system of E. coli, by inserting the Mu phage. Cells in this family is not very clear. However, it is clear that at least of E. coli TG1 were infected with the Mud(Apr lac)I phage (5, 29). Cells were an electrochemical potential of Hϩ across cell membranes is diluted with L broth and spread onto an agar plate containing L broth and 40-␮g/ml ampicillin. After incubation at 37°C for 12 h, colonies were picked up involved in driving the drug efflux (16). Transporters of the and replica plated by using (i) an agar plate containing L broth and 40-␮g/ml ABC family utilize ATP as the energy source. LmrA (Lacto- ampicillin and (ii) an agar plate containing L broth, 40-␮g/ml ampicillin, and coccus lactis) (32) and MsrA (S. aureus) (25) are members of 50-␮g/ml methylene blue, an antimicrobial basic dye and substrate for the AcrAB system (16). We obtained two colonies which grew in the presence of methylene blue. One of them was designated KAM2. Thereafter, the Mu phage region was Vibrio parahaemolyticus, a slightly halophilic marine bacte- removed from the chromosome of KAM2 cells by heat induction at 42°C (5, 29), rium, is one of the major causes of food poisoning in Japan and colonies that were sensitive to both ampicillin and methylene blue were obtained. One of the colonies was designated KAM3. The KAM3 cells, as well as the KAM2 cells, were sensitive to many drugs that are known as sub- strates of the AcrAB system, although TG1 cells were resistant. The deletion * Corresponding author. Mailing address: Department of Microbi- in the acrAB region in KAM3 was confirmed by Southern blot analysis (data ology, Faculty of Pharmaceutical Sciences, Okayama University, Tsu- shima, Okayama 700-8530, Japan. Phone and Fax: 81-86-251-7957.
Assay of norfloxacin accumulation in cells. V. parahaemolyticus cells were
grown in the LB broth supplemented with 40 mM potassium lactate. The cells DRUG EFFLUX SYSTEM IN V. PARAHAEMOLYTICUS were harvested at the exponential phase of growth, washed with 0.2 M MOPS- Tris buffer (pH 7.0) containing 10 mM MgSO4, and suspended in the same buffer to 50 mg (wet weight)/ml. The assay mixture contained cells (10 mg [wet weight]/ ml) in the same buffer and 10 mM potassium lactate. After incubation at 25°C for 5 min, norfloxacin (100 ␮M, final concentration) was added to initiate the assay.
Samples (1 ml each) were taken at intervals, centrifuged at 7,000 ϫ g for 30 s at 4°C, and washed once with the same buffer. Where indicated, carbonyl cyanide m-chlorophenylhydrazone (CCCP) was added to the assay mixture at 100 ␮M.
The pellet was suspended in 1 ml of 100 mM glycine-HCl (pH 3.0). The suspen- sion was shaken vigorously for 1 h at room temperature and then centrifuged at 7,000 ϫ g for 5 min at room temperature. The supernatant was diluted twofold with the same buffer, and the fluorescence was measured with excitation at 277 nm and emission at 448 nm (10, 19) with a Hitachi F2000 fluorescence spectro- Assay of ethidium accumulation in cells. Cells were prepared and suspended
as described above for the norfloxacin accumulation assay. Potassium lactate (final concentration 20 mM) was added to the cell suspension (0.5 mg of protein/ ml), and the cell suspension was kept at 25°C for 5 min with gentle stirring.
Ethidium bromide was added at 10 ␮M to the cell suspension to initiate the assay. CCCP was added at 100 ␮M where indicated. The fluorescence of the assay mixture was measured with excitation and emission wavelengths of 500 and Gene cloning and sequencing. The gene responsible for norfloxacin efflux was
FIG. 1. Accumulation of norfloxacin in cells of V. parahaemolyticus. V. para- cloned from V. parahaemolyticus cells as follows. Chromosomal DNA was pre- haemolyticus cells were grown in LB medium supplemented with 40 mM potas- pared from cells of V. parahaemolyticus by the method of Berns and Thomas (2).
sium lactate. Norfloxacin was added to the cell suspensions at a final concentra- The DNA was partially digested with Sau3AI, and fragments of 4 to 10 kbp were tion of 100 ␮M. After 15 min, CCCP was added to the suspensions at a final separated by sucrose density gradient centrifugation. The DNA fragments were concentration of 100 ␮M. Portions were removed at the times shown, and the ligated into pBR322 (which had been digested with BamHI and dephosphory- concentration of norfloxacin extracted from the cells was determined with a lated with bacterial alkaline phosphatase) by using T4 DNA ligase. Competent cells of E. coli KAM3 were transformed with the ligated hybrid plasmids and were spread on agar plates containing L broth, 0.05-␮g/ml norfloxacin, 60-␮g/ml ampicillin, and 1.5% agar. The plates were incubated at 37°C for 24 h, and the RESULTS AND DISCUSSION
clones formed were picked up. Plasmids contained in the transformants were isolated, reintroduced into KAM3 cells, and spread on the plates again. The Norfloxacin efflux in cells of V. parahaemolyticus. We mea-
plates were incubated at 37°C for 24 h. Plasmids contained in the retransfor- sured norfloxacin accumulation and tested the effect of an Hϩ mants were prepared. One of the resulting hybrid plasmids that carried a gene for the norfloxacin efflux system was designated pMVP3.
conductor, CCCP, on the accumulation in cells of V. parahae- The DNA insert in pMVP3 was digested with several restriction endonucleases molyticus. A certain level of norfloxacin accumulation in the and subcloned into pBR322. The resulting hybrid plasmids were introduced into cells was observed, and the accumulation level increased after KAM3 cells, and the transformants were tested for sensitivity or resistance to addition of CCCP (Fig. 1), suggesting the existence of a nor- floxacin efflux system in V. parahaemolyticus that is driven by an The nucleotide sequence was determined by the dideoxy chain termination electrochemical potential of Hϩ. It is not clear whether Hϩ is method (27) with a DNA sequencer (ALF Express, Pharmacia Biotech).
The ydhE gene of E. coli was cloned as follows. Chromosomal DNA was directly or indirectly coupled to the drug efflux.
prepared from cells of E. coli KAM3 by the method of Berns and Thomas (2).
Cloning of the gene for a putative norfloxacin efflux protein.
The DNA was digested with SspI and SphI, which should cut out the whole ydhE We cloned a fragment of the chromosomal DNA of V. para- gene (EMBL nucleotide sequence database accession no. AE000261), judging haemolyticus, which enabled norfloxacin-hypersensitive E. coli from the DNA sequence of the E. coli genome. The SspI-SphI fragments were KAM3 cells to grow in the presence of norfloxacin. We ob- ligated into pBR322 (which had been digested with EcoRV and SphI and de- phosphorylated with bacterial alkaline phosphatase) by using T4 DNA ligase.
tained about 40 candidate hybrid plasmids. However, it seemed Competent cells of E. coli KAM3, which are very sensitive to norfloxacin, were that the DNA inserts in all of the candidate plasmids contained transformed with the ligated hybrid plasmids and spread on plates containing L the same DNA portion, judging from restriction maps of those broth, 0.05-␮g/ml norfloxacin, 60-␮g/ml ampicillin, and 1.5% agar. The plates plasmids. We tested whether the plasmids carry a gene(s) re- were incubated at 37°C for 24 h, and the clones formed were picked up. Plasmids sponsible for norfloxacin efflux or not by measuring norfloxa- contained in the transformants were checked for whether they contained the cin accumulation and the effect of CCCP in E. coli cells trans- expected SspI-SphI fragment. One of the resulting hybrid plasmids that carried the ydhE gene was designated pMEC2.
formed with the plasmids. A considerable level of norfloxacin Sequence data were analyzed with GENETYX sequence analysis software accumulation was observed with the host cells (E. coli KAM3).
(Software Development Co.). The SwissProt and GenBank databases were Addition of CCCP increased the accumulation level to some extent, supporting the presence of some norfloxacin efflux Southern blot analysis. Chromosomal DNAs were prepare from cells of
system(s) in E. coli KAM3 (6). Cells harboring the plasmids E. coli, V. parahaemolyticus, V. alginolyticus, P. aeruginosa, and S. aureus as described above. The DNAs were digested with several restriction enzymes and showed very low levels of norfloxacin accumulation compared subjected to agarose gel electrophoresis. DNA fragments were blotted onto a with those of the host cells. The norfloxacin accumulation in Hybond-N (Amersham Co.) nylon membrane by the capillary blotting method as cells harboring one of the plasmids, pMVP3, is shown in Fig. 2.
suggested by the manufacturer. The probes used were three DNA fragments Addition of CCCP to the assay mixture greatly increased the derived from the acrRAB genes of E. coli, which are shown below. Southern blot accumulation level (Fig. 2). The norfloxacin accumulation level analysis was performed with the enhanced-chemiluminescence detection system after the addition of CCCP was very similar in the host cells (Amersham Co.) as suggested by the manufacturer.
Drugs susceptibility test. The MICs of drugs were determined in Mueller-
and the transformed cells. Thus, we conclude that the elevat- Hinton broth (Difco) containing various drugs at various concentrations. Cells in ed norfloxacin efflux is due to the gene(s) carried on plasmid the test medium were incubated at 37°C for 24 h, and growth was judged Inhibition of the drug efflux in KAM3/pMVP3 cells by Other. Protein contents were determined by the method of Lowry et al. (15).
CCCP indicates that an electrochemical potential of Hϩ is the The chemicals and enzymes used in this study were from commercial sources.
Nucleotide sequence accession number. The nucleotide sequence data re-
driving force for the drug extrusion. Some drug/Hϩ antiport- ported in this paper have been deposited in the DDBJ, EMBL, and GenBank ers, such as the tetracycline/Hϩ antiporter (9), are known in nucleotide sequence databases under accession no. AB010463.
microbial cells. It is very likely that the norfloxacin extrusion FIG. 3. Restriction maps of pMVP3 and its deletion derivatives. DNA re- gions derived from V. parahaemolyticus chromosomal DNA and carried by each plasmid are shown. Growth of E. coli KAM3 cells harboring each plasmid on an agar plate containing L broth, 0.05-␮g/ml norfloxacin, and 60-␮g/ml ampicillin is shown on the right. Plus signs indicate that cells grew, and minus signs indicate FIG. 2. Accumulation of norfloxacin in host and transformant cells. Cells of that cells did not grow. The arrow shows the position and direction of the norM E. coli KAM3 and KAM3/pMVP3 were grown in L medium supplemented with 40 mM potassium lactate. Norfloxacin was added to the cell suspensions at a final concentration of 100 ␮M. After 15 min, CCCP was added to the suspensions at a final concentration of 100 ␮M. Portions were removed at the times shown, and the concentration of norfloxacin extracted from the cells was determined with a fluorescence spectrophotometer. Symbols: E, KAM3; F, KAM3/pMVP3.
We checked whether the cloned DNA fragment is really from V. parahaemolyticus, and whether a similar gene is pres- ent in other bacteria, by Southern blot analysis. A DNA frag- ment derived from pMVP36 was used as a probe. We detected system in KAM3/pMVP3 is a drug/ion antiporter. However, it a dense hybridized band with chromosomal DNA of V. para- is not clear whether this system utilizes Hϩ as the counterion haemolyticus (data not shown). The position of the band was for the antiport. Many ion-coupled systems in membranes of exactly the same as that of the control (pMVP36). Thus, we vibrios utilize Naϩ instead of Hϩ (28). Although cells of believe that the DNA insert in pMVP36 is from the chromo- V. parahaemolyticus possess a primary respiratory Naϩ pump somal DNA of V. parahaemolyticus. We also detected a less (30), Naϩ-coupled membrane processes, such as Naϩ/solute dense band with chromosomal DNA of V. alginolyticus. Thus, symport, are sensitive to an Hϩ conductor (unpublished re- it seems that cells of V. alginolyticus possess a similar gene.
sults). Therefore, we cannot distinguish between an Hϩ-cou- Chromosomal DNAs from other bacteria tested, P. aeruginosa pled system and an Naϩ-coupled system from the effects of an Hϩ conductor. We tested the effect of Naϩ on the activity of norfloxacin extrusion in cells of both V. parahaemolyticus and E. coli transformed with pMVP3. However, no clear effect was TABLE 1. Susceptibilities of study strains to different compounds observed (data not shown). We also tried to detect norfloxacin/ Hϩ antiport by the quinacrine fluorescence quenching method with everted membrane vesicles prepared from cells of KAM3/ pMVP3. However, we were unable to detect Hϩ efflux due to norfloxacin influx (data not shown).
Several plasmids carrying different portions of pMVP3 were constructed, and the ability to confer norfloxacin resistance on E. coli KAM3 was tested (Fig. 3). Among the plasmids that con- ferred norfloxacin resistance, plasmid pMVP36 possessed the shortest DNA insert derived from V. parahaemolyticus DNA We tested the susceptibility of cells of KAM3/pMVP36 to many drugs (Table 1). pMVP36 made KAM3 cells resistant to rather hydrophilic new quinolones, such as norfloxacin and ciprofloxacin, but not to hydrophobic quinolones, such as spa- floxacin and nalidixic acid. Cells harboring pMVP36 were also resistant to structurally different antimicrobial agents, such as ethidium and streptomycin (and kanamycin). Thus, it seems that pMVP36 carries a gene for multidrug resistance. Our re- sults are consistent with the idea that the gene encodes a pro- tein mediating the extrusion of norfloxacin and ethidium from cells. In fact, we observed elevated ethidium efflux in cells of KAM3/pMVP36 compared with cells of KAM3 (data not shown).
DRUG EFFLUX SYSTEM IN V. PARAHAEMOLYTICUS FIG. 4. Amino acid sequence alignment. The deduced amino acid sequence of V. parahaemolyticus NorM (NorM VPARA) and the amino acid sequences of E. coli YdhE (YdhE ECOLI) and H. influenzae YdhE (YdhE HAEIN) are aligned. The numbers on both sides refer to the position of the nearest residue on each line.
Asterisks and dots indicate residues in YdhE of E. coli that are identical and similar to those in NorM, respectively.
and S. aureus, showed no hybridized band under our experi- residues (Fig. 4) with a molecular mass of 49,422 Da and is very rich in hydrophobic residues, indicating that the protein is a Gene and protein sequences. We determined the sequence
membrane protein. A hydropathy analysis done by the method of 2,243 nucleotides of the DNA insert in pMVP36. We found of Eisenberg et al. (7) revealed that NorM possesses 12 hydro- only one open reading frame, which was preceded by a Shine- phobic regions which may be transmembrane domains (data Dalgarno sequence in this region, which is likely to be the gene not shown). Therefore, it is likely that NorM is a member of (which we designated norM) encoding the norfloxacin efflux protein (NorM). Several promoterlike sequences were present Homolog of NorM. We searched for amino acid sequence
upstream of norM. The amino acid sequence deduced from the homology between NorM and the reported sequences in a pro- norM gene revealed that NorM consists of 456 amino acid tein sequence database (SwissProt). No sequence homology or similarity was observed between NorM and the reported iya. 1994. Properties of the Naϩ/Hϩ antiporter in Vibrio parahaemolyticus.
members of the MF family. The first extrusion system for J. Biochem. 115:1162–1165.
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