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A point mutation in norA gene is responsible for quinolone resistance in Staphylococcus aureus
Vol.
172,
November
No.
3, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
Pages
15, 1990
Yoshihiro Ohshita, Keiichi
COMMUNICATIONS
1028-1034
Hiramatsu, and Takeshi Yokota
Department of Microbiology, Faculty of Medicine, Juntendo University, Hongo, Bunkyo-ku, Tokyo, Japan, 113
2-l-l
Received September 13, 1990 Summary!&io norA genes associated with hydrophilic guinolone resistance in Staphylococcus aureus were identified on the two reccmbinant plasmids pMR8736 and pSA209; the former was derived from a quinolone-resistant strain MR8736, and the latter was derived frcxn a fluoroquinolone-susceptible strain 209P. We compared functionsof these two genes, norA and norA respectively, by introducing them into -E. coli MC1061. Both genes expressed a novel protein of 52 kilodalton (kD) in size in MC1061. However, only norA could confer hydrophilic guinolone resistance to the host cell, which was accompanied by a significant decrease in the uptake of a hydrophilic quinolone, norfloxacin, by the cell. Subcloning and recombinant plasmid analyses localized the hydrophilic quinolone-resistance marker to the 0.5 kilobase &b-long &IHinfI DNA fragment of pMR8736. Nucleotide sequencing of this region and the corresponding region of pSA209 revealed that the hydrophilic quinolone resistance conferred by norA was caused by a single nucleotide substitution from A (adenosine) in norA to C (cytosine), which corresponded to a single amino acid substitution from Asp to Ala. Q1990Acadrmrc Press,Inc. Fluorcquinolone is a broad-spectrum antimicrobial agent useful for both Clinical use of this agent in gram-negative and gram-positive bacteria. recent years, however, has experienced frequent emergence of resistant populations of pathogenic bacteria such as -___ S. aureus (1,2) and Pseudomonasaerumechanisms for guinolone ginosa (3,4). Although at least three different resistance have been identified in -E. coli (5,6,7,8,9,10), little is known about the resistance mechanism in S. aureus. In view of the fact that methicillin-resistant Staphylococcus aureus (MMA), a major nosocomial pathogen throughout the world, has been reported to acquire fluorcquinolone resistance more frequently than its methicillin-susceptible counterpart (11,12), it is of great importance to elucidate mechanism of quinolone resistance in MRSA. Recently, a gene called & responsible for fluoroquinolone resistance has This gene has been been cloned from a resistant strain of MRSA (13). reported to carry homology with gyrase A and/or gyrase B of E. coli according
S. aureus, Staphylococcus aureus. E. coli, Escherichia ~ -MRSA, methicillin-resistant staphylococcus aureus. MIC, minimuminhibitory concentration. 0006-291X/90
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1028
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AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
to Southern hybridization experiments (13). However, a subsequent study has suggested that the gene functions by preventing cell uptake of hydrophilic guinolone antibiotics (14). In this paper, we report identification of a point mutation in the norA gene which is responsible for the expression of quinolone resistance, and also an identification of a novel protein of 52 kD in size which possibly is encoded by the norA gene and is involved in the transport of hydrophilic guinolone antibiotics across cell membrane.
DNA cloninq a& subcloninq. Cellular DNA extracted from a clinical MRSA and was ligated into the cloning strain MR8736 was digested with -dIII, vector pUCl8. The ligate was used to transform -E. coli MC1061. A transformant harboring reccmbinant plasmid pMR8736 was obtained by overnight selection on an L-agar plate containing 25 ug of ampicillin and 0.5 ug of norfloxacin per ml. The recombinant clone pSA209 was obtained by screening DNA library of an S. aureus strain 209P by colony hybridization using the radiolabeled 5.2 kb gsof pMR8736 as a probe. Subcloning of the cloned DNA fragments were performed by digestion with appropriate restriction enzymes followed by ligation into the corresponding restriction enzyme site of the cloning vector pUCl8. Construction of recombinant plasmids and deletion mutants. The recombinant plasmids between @I(?8736and pSA209 were constructed by crisscross ligation of DNA fragments cut out with Hind111 and HpaI from the inserted DNA of each plasmid, followed by recloning into the Hind111 site of pUC18. Deletion mutants of pMR8736 were constructed starting from pMR365, a subclone of pMR8736 carrying XbaI-XbaI DNA fragment, by using Kilo Sequence Deletion Kit (Takara Shuzo Co.,LM., Kyoto, Japan). MIC determination. MIC determination was performed by agar dilution method as described previously (15). Quinolone accumulation -in the a. inolone accumulation is the cell was determined as follows. A total of 10 P cells of bacteria in a log-phase of growth were collected and washed once with M9 medium. The pellet was resuspended in one ml of M9 medium containing various concentration of guinolone antibiotics. After incubation for 15 min at 37"C, cells were washed four times with M9 mediumand resuspended in 0.3 ml of distilled water. The cell suspension was boiled for 7 min, and cell debris was removed by centrifugation. The supernatant was tested for antimicrobial activity by blotting 30 ul portion of the supematant on a paper disk with 8 mmin diameter (Toy0 Roshi Co., Ltd., Tokyo, Japan) which was placed on an L-agar plate lawned with a quinolone-susceptible E. The -- coli NIHJC-2 cells. diameter of growth-inhibition zone around the disk was evaluated after 24 h incubation at 37'C. The amount of cell-associated guinolone was quantified by ccmparing the diameter of the inhibition zone with those produced by the disks blotted with various amounts of guinolone antibiotics. SDS-PAGE analysis of total bacterial cell lysate. Total cell lysate was prepared as follows. -CZiiFwere collected from one ml of overnight culture in L-broth by centrifugation. The pellet was resuspended in 22.5 ul of PE$&, added with 2.5 1.11of 2-mercaptoethnol and 25 ul of Tris-SDS Seprasol (Daiichi Pure Chemicals Co. Ltd., Tokyo, Japan), followed by heating in the boiling water for 5 min. After centrifugation at 12,000 q for 1 min at rocm temperature, ten ul of the supernatant was subjected to SDS-PAGE. The samples were electrophoresed in a 4120 gradient SDS-PAGEgel (J&iichi Pure Chemicals). The gel was stained with cocmassie brilliant blue R-250 (17). Nucleotide sequence determination. The nuclegtjide sequence was determined by the method of Maxamand Gillbert (18) using [ PI end-labeled single stranded IYWA fragments after purification through a Benzoylted-Naphthoylated DEAS cellulose column (19). 1029
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No.
3, 1990
BIOCHEMICAL
AND
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RESEARCH
COMMUNICATIONS
REXJL'ISANDDI!XXJSICN
Decreased uptake of hydrophilic quinolone antibiotics into the cell --harborinq pMR8736. Both of the recombinant plasmids @lR8736 and pSA209 had inserts of 5.2 kb in size, and shared an identical restriction map pattern except an additional EcoRI site in the latter plasmid (Fig. la). We concluded that these clones contained norA genes, because these clones had the same size and the same restriction enzyme pattern in terms of I$$, WuII, and HincII, with the previously reported DNA fragment carrying norA gene (13). Intrcduction of @JR8736 by transformation made MC1061 cells resistant to hydrophilic quinolone antibiotics such as norfloxacin and pipemidic acid (Table 1)(19,20). Susceptibility of the transformant to hydrophobic quinolone antibiotics was either unaltered or altered in a small degree (e.g. to tosufloxacin and ofloxacin; Table 1). Introduction of pSA209 into MC1061, on the other hand, did not cause any significant alteration of the host cell susceptibility to quinolone antibiotics (Table 1). The decreased norfloxacin susceptibility of the transformant harboring pMR8736 was accompanied by a decreased uptake of the drug by the cell (Table 2). This decrease of uptake was observed with norfloxacin, a hydrophilic quinolone, but was not observed with a hydrophobic quinolone, tosufloxacin (Table 2). This result was
the norA FIG. 1. (a) The restrict' ion map of 5.2kb Hind111 fragments containing gene. Abbreviation is as follows: B, WII; E, =I; H, &dIII; Hi, B&fI; Hp, &I; K, &I; P, EII; and X, XbaI. (b) Iocalization of the B gene on pMR8736. Restriction enzyme-generated subclones (pMR361-365) as well as exonucleaseIII-generated deletion mutants (pDP3640, pDP3622, pDP3619, pDs3625, (c)Iocalization of guinolone-resistance pDS3632, and pDS3601) were used. marker on p&lR8736. The chimeric plasmids were constructed fran pSA209 and @lR8736. E. coli MC1061 was transformed with each clone of (b) and (c), and MIC of norfloxacin (NFLX) was measured for each transfo-t. MIC value is presented to the left of each clone. 1030
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TABLE 1. Quinolone MIC
of
AND
susceptibility
of
transformed
MC1061
BIOPHYSICAL
with
none
NFL): PPA CPFX
0.025 3.13 0.025 0.2 0.1 0.2 0.1 6.25 0.025
ENX
OFLX LFLX
PFLX NA
TFLX
puc15
pSA209
0.025 3.13 0.025 0.2 0.1 0.2 0.1 6.25 0.05
pMR8736
0.05 6.25 0.05 0.39 0.2 0.2 0.1 6.25 0.05
COMMUNICATIONS
and its Index of increase ~MR8736~
QUlllOlGne
antlblotlcsa
MC1061
RESEARCH
transformants
MIC
with
16 16 16
0.39 50 0.39
1.56
8
0.39 0.39 0.2 6.25 0.05
4 2 2 1 1
coefficient
Partltlon
0.06gc 0.017" 0.115c 0.127' 0.391C O.llJ6C
0.32d 0.90d 1.14d 4.95d
0.069e
18.8 3.34c 0.347e
a Abbreviation is as follows: NFLX, norfloxacin; PPA, pipemidic acid; CPFX, ciprofloxacin; ENX, enoxacin; OELX, ofloxacin; LFLX, lcmefloxacin(NY198); pefloxacin; NA, nalidixic acid; TF'LX, tosufloxacin(T3262). pm, b Index was calculated by dividing MIC of MC1061(pMR8736) with MIC of MC1061. ' n-cctanol/50 mMsodium phosphate buffer(pH 7.0), cited from (20). d Chloroform/O.1 M phosphate buffer(pH 7.4). e n-cctanol/water, cited from (21).
consistent guinolone
with the antibiotics
report of others that confers resistance to hydrophilic by impairing their accumulation into the cell (14).
Identification of the norA gene -on pMR8736. Localization of the norA --gene on pMR8736 was performed using norfloxacin-resistance phenotype as a marker. Various subclones and deletion mutants of pm8736 were constructed and introduced into Mc1061, and MIC of norfloxacin against each transformant was evaluated (Fig. lb). The m gene was localized within the 1.8 kb fragment demarcated by the left end of pDP3622and the right end of pDS3632 (Fig. lb). Identification of a protein as In order to -- a possible norA qene product. identify norA gene product, cell lysates were prepared from each transformant of MC1061 harboring pMR8736, pSA209, and pUCl8. A novel protein of 52 kD in size was identified in the transformants harboring m8736 and pSA209 but not in the transformant harboring pUCl8 (Fig. 2). Production of this protein TABLE 2. The accumulation of norfloxacin and tosufloxacin in MC1061 and its transformants NFLX (w/l
when
treated
MC1 061
MC1061 MCI061 MC1061
a b c d
(pUC18jb (pSA209jC (pMR8736jd
200 0.11 0.16 0.10 co.05
0
F
umulatlon
TFLX (uq/10
cells)
treated
with
concentrationa
drug Cells
ac
pg/ml f * -t
0.01 0.01 0.04
400 0.44 0.75 0.58 0.23
of
pg/ml 2 * f +
5
drug
when treated concentrationa
200
ug/ml
0.07 0.12 0.22 0.06
The values represent the means* SEM(n=4) of three experiments. MC1061harboring pUC18. MCI061 harboring pSA209. MC1061harboring pMR8736. 1031
ac
>I )l >I >I
.oo .oo .oo .oo
umulation
cells) with of 400
Ug/ml ,I ,l >I >I
.oo .oo -00 .oo
Vol.
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No.
3, 1990
correlated norA8736, deletion band with
well
with
which mutants the
BIOCHEMICAL
the
SDS-PAGE of membrane
fractions
shown). These data strongly protein of 52 kD in size is this protein was consistent localized
expression
was demonstrated pDP3640, pDP3622,
same migration
within
the
12
3
AND
rate
of
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
quinolone
resistance
conferred
by analysis pDS3632, and with
this
of
transformants
pDS3632
protein
(data
was also
by
carrying not
detectable
shown).
in the
of -___ S. aureus strains MR8736 and 209P (data not suggest, although not conclusive, that this encoded by the norA gene. The molecular weight of with the coding capacity of m gene which was
1.8 I& DNA fragment
of @IR8736.
kD
42-
3020-
02
A
14-
FIG. 2. SDS-PAGE analyses of cell lysates of MCI 061 transformed with various plasmids. Total cell lysates of transformants harboring pUCl8 (lane I), pSA209 (lane 2), and pMR8736 (lane 3) were analyzed. Molecular weight standards (Daiichi Pure Chemicals) were phosphorylase b, bovine serum albumin, aldolase, carbonic anhydrase, trypsin inhibitor, and lysozyme from the top in this order. The arrow indicates the 52 kD protein that is found only in the transformants harboring the norA gene. FIG. 3. (a) Nucleotide sequences of 0.6 kb FvuII-HinfI DNA fragments of pSA209 and pMH8736. Nucleotide sequences and predicted amino acid residues of pSA.209 are spelled out. Only the substituted nucleotide and amino acid in the pMR8736 are shown below the pSA209 sequences. The start of nucleotide numbering is C of =I1 site (GGCIG). The asterisks show a termination codon. An inverted repeated sequences of a probable transcription-termination signal are underlined. (b) Hydropathic profiles of the predicted 159 amino acid residues of pSA209 (left) and pMR8736 (right). The arrows indicate the position of the substituted amino acid. A span of consecutive residues of six was employed. 1032
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BIOCHEMICAL
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RESEARCH
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Identification of a point mutation --in the norA gene -which is responsible for guinolone resistance. To understand why pMR8736 but not pSA209 conferred we attempted further localization of the quinolone resistance to MCI061, resistance marker to a smaller region of @R8736 by constructing chimeric plasmids of pMR8736and pSA209, taking advantage of a unique HpaI restriction site shared by both plasmids (Fig. Ic). Quinolone-resistance marker was now localized within the 0.5 kb HpaI-HinfI fragment of pMR8736. The nucleotide sequences of this region of @lR8736 as well as the corresponding region of pSA209 are presented (Fig. 3a). Only one open reading frame which is long enough to encode a meaningful protein was observed in this region. The direction of transcription was frcm the HpaI restriction enzyme site to the HinfI restriction enzyme site. The region contain&i a part of a structural gene coding for the carboxyl terminal 159 amino acid residues. Prediction of the amino acid sequence of this open reading frame revealed a remarkably high composition of hydrophobic amino acids (Fig. 3b). Nucleotide sequence of the corresponding region of pMR8736was identical with that of pSA209 except for a single nucleotide change from A (of pSA209) to C (of pMH8736) which caused amino acid substitution from Asp (of pSA209) to Ala (of pMR8736) at the 27th residue from the carboxyl terminus of the protein. Neither of the nucleotide and peptide sequences shared homolcgy with previously reported sequences including those of gyrase A and gyrase B genes of S. aureus (19). All clinical S. aureus strains so far tested were found to carry a norA -___ gene irrespective of their susceptibility towards quinolone antibiotics (data not shown). In view of this observation, the norA gene, not like a transposonassociated drug-resistance gene, seemsto code for a protein which is involved in scme essential physiological activity in the cell. As a point mutation of m gene, designated norA8736, was shown to cause quinolone resistance by preventing cell accumulation of guinolone antibiotics, we consider that such a physiological function of the norA gene product is the transport of hydrophilic quinolone molecules across cell membrane. Consistent with this view is the highly hydrophobic nature of the deduced amino acid sequences of the norA protein which is reminiscent of membrane-spanning regions of several transport proteins. We are not unaware of the fact that the identification of a protein of 52 kD in size as the norA gene product is inconclusive, since we cannot rule out the possibility that the protein is an intrinsic protein of -L E coli MC1061 whose expression was induced by the norA gene product. We also do not know whether the norA gene functions by enhancing efflux or impairing import of the guinolone antibiotics across cell membrane. Existence of such an efflux system of fluorcquinolone antibiotics in _L E coli has recently been suggested (5,8). The study designed to solve these questions are now under way. We thank K. ubukata of Teikyo University for a precious discussion. This work was partly supported by grants from the Japanese Ministry of Education. 1033
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BIOPHYSICAL
RESEARCH
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1. Chapman,S.T.,Speller,D.C.E.,and Reeves,D.S.(1985)Iancet,2,39. 2. Humphreys,H.,and Mulvihill,E.(1985)Lancet,2,383. 3. Daikos,G.L.,Lolans,V.T.,and Jackson,G.G.(1988)Antimicrob.Agents
Chemother.
32,785-787. 4.
5. 6. 7. 8. 9.
Masecar,B.L.,Celesk,R.A.,and Robillard,N.J.(1990)Antimicrob.Agents Chemother.34,281-286. Cohen,S.P.,Hooper,D.C.,Wolfson,J.S.,Souza,K.S.,McMurry,L.M.,and Ievy,S.B. (1988)Antimicrob.Agents Chemother.32,1187-1191. Cullen,M.E.,Wyke,A.W.,Kurcda,R.,and Fisher,L.M.(1989)Antimicrob. Agents Chemother.33,886-894. Hirai,K.,Aoyama,H.,Suzue,A.,Irikura,T.,Iyobe,S.,and Mitsuhashi,S.(1986) Antimicrob.Agents Chemother.30,248-253. Hooper,D.C.,Wolfson,J.S.,Souza,K.S.,Ng,E.Y.,McHugh,G.L.,and Swartz,M.N. (1989)Antimicrob.Agents Chemother.33,283-290. Yamagishi,J.,Yoshida,H.,Yamayoshi,M.,and Nakamura,S.(1986)Mol.Gen.Genet. 204,367-373.
10.
Yoshida,H.,Kojima,T.,Yamagishi,J.,and
Nakamura,S.(1988)Mol.Gen.Genet.
211,1-7.
Schaefler,S.(1989)J.Clinic.Microb.27,335-336. 12. Shalit,I.,Berger,S.A.,Gorea,A.,and Frimerman,H.(1989)Antimicrob.Agents Chemother.33,593-594. 13. lJbukata,K.,Itoh-Yamashita,N.,and Konno,M.(1989)Antimicrob.Agents 11.
Chemother.
33,1535-1539.
Ubukata,K.,and Konno,M. (1990) The 3rd Yoshida,H.,Bogaki,M.,Nakamura,S., International Symposium on New Quinolones(manuscript No.160), VamOUVer, Canada. 15. Utsui,Y.,and Yokota,T.(1985)Antimicrob.Agents Chemother.28,397-403. 16. Koshland,D.,and Botstein,D.(1980)Ce11,20,749-760. 17. Wilson,C.M.(1983)Metha& in Enzymolcgy(Hirs,C.H.W.,and Timasheff,S.N., eds.),Vo1.91,Part 1,236-247,Academic Press, New York. 18. Maxam,A.M.,and Gilbert,W.(1977)Proc.Natl.Acad.Sci.USA.74,560 19. Kiger,J.A.,and .Sinsheimer,R.L.(1969)J.Mol.Bio1.40,467. Tamai,I.,Okezaki,E.,Nagata,O.,and Kato,H.(l988) 20. Tsuji,A.,Sato,H.,Kume,Y., Antimicrob.Agents Chemother.,32,190-194. 21. Rosen,T.,Chu,D.T.,Lico,I.M.,Femandes,P.B.,Marsh,K.,Shen,L.,Cepa,V.G., and Permet,A.G.(1988)5. Med. Chem.,31,1598-1611. 22. Hopewell,R.,Oram,M.,Briesewitz,R.,and Fisher,L.M.(1990)J.Bacteriol.l72 14.
3481-3484.
1034
172,
November
No.
3, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
Pages
15, 1990
Yoshihiro Ohshita, Keiichi
COMMUNICATIONS
1028-1034
Hiramatsu, and Takeshi Yokota
Department of Microbiology, Faculty of Medicine, Juntendo University, Hongo, Bunkyo-ku, Tokyo, Japan, 113
2-l-l
Received September 13, 1990 Summary!&io norA genes associated with hydrophilic guinolone resistance in Staphylococcus aureus were identified on the two reccmbinant plasmids pMR8736 and pSA209; the former was derived from a quinolone-resistant strain MR8736, and the latter was derived frcxn a fluoroquinolone-susceptible strain 209P. We compared functionsof these two genes, norA and norA respectively, by introducing them into -E. coli MC1061. Both genes expressed a novel protein of 52 kilodalton (kD) in size in MC1061. However, only norA could confer hydrophilic guinolone resistance to the host cell, which was accompanied by a significant decrease in the uptake of a hydrophilic quinolone, norfloxacin, by the cell. Subcloning and recombinant plasmid analyses localized the hydrophilic quinolone-resistance marker to the 0.5 kilobase &b-long &IHinfI DNA fragment of pMR8736. Nucleotide sequencing of this region and the corresponding region of pSA209 revealed that the hydrophilic quinolone resistance conferred by norA was caused by a single nucleotide substitution from A (adenosine) in norA to C (cytosine), which corresponded to a single amino acid substitution from Asp to Ala. Q1990Acadrmrc Press,Inc. Fluorcquinolone is a broad-spectrum antimicrobial agent useful for both Clinical use of this agent in gram-negative and gram-positive bacteria. recent years, however, has experienced frequent emergence of resistant populations of pathogenic bacteria such as -___ S. aureus (1,2) and Pseudomonasaerumechanisms for guinolone ginosa (3,4). Although at least three different resistance have been identified in -E. coli (5,6,7,8,9,10), little is known about the resistance mechanism in S. aureus. In view of the fact that methicillin-resistant Staphylococcus aureus (MMA), a major nosocomial pathogen throughout the world, has been reported to acquire fluorcquinolone resistance more frequently than its methicillin-susceptible counterpart (11,12), it is of great importance to elucidate mechanism of quinolone resistance in MRSA. Recently, a gene called & responsible for fluoroquinolone resistance has This gene has been been cloned from a resistant strain of MRSA (13). reported to carry homology with gyrase A and/or gyrase B of E. coli according
S. aureus, Staphylococcus aureus. E. coli, Escherichia ~ -MRSA, methicillin-resistant staphylococcus aureus. MIC, minimuminhibitory concentration. 0006-291X/90
Copyright All rights
$1.50
0 1990 by Academic Press, Inc. of reproduction in arty form reserved.
1028
coli.
Vol.
172, No. 3, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
to Southern hybridization experiments (13). However, a subsequent study has suggested that the gene functions by preventing cell uptake of hydrophilic guinolone antibiotics (14). In this paper, we report identification of a point mutation in the norA gene which is responsible for the expression of quinolone resistance, and also an identification of a novel protein of 52 kD in size which possibly is encoded by the norA gene and is involved in the transport of hydrophilic guinolone antibiotics across cell membrane.
DNA cloninq a& subcloninq. Cellular DNA extracted from a clinical MRSA and was ligated into the cloning strain MR8736 was digested with -dIII, vector pUCl8. The ligate was used to transform -E. coli MC1061. A transformant harboring reccmbinant plasmid pMR8736 was obtained by overnight selection on an L-agar plate containing 25 ug of ampicillin and 0.5 ug of norfloxacin per ml. The recombinant clone pSA209 was obtained by screening DNA library of an S. aureus strain 209P by colony hybridization using the radiolabeled 5.2 kb gsof pMR8736 as a probe. Subcloning of the cloned DNA fragments were performed by digestion with appropriate restriction enzymes followed by ligation into the corresponding restriction enzyme site of the cloning vector pUCl8. Construction of recombinant plasmids and deletion mutants. The recombinant plasmids between @I(?8736and pSA209 were constructed by crisscross ligation of DNA fragments cut out with Hind111 and HpaI from the inserted DNA of each plasmid, followed by recloning into the Hind111 site of pUC18. Deletion mutants of pMR8736 were constructed starting from pMR365, a subclone of pMR8736 carrying XbaI-XbaI DNA fragment, by using Kilo Sequence Deletion Kit (Takara Shuzo Co.,LM., Kyoto, Japan). MIC determination. MIC determination was performed by agar dilution method as described previously (15). Quinolone accumulation -in the a. inolone accumulation is the cell was determined as follows. A total of 10 P cells of bacteria in a log-phase of growth were collected and washed once with M9 medium. The pellet was resuspended in one ml of M9 medium containing various concentration of guinolone antibiotics. After incubation for 15 min at 37"C, cells were washed four times with M9 mediumand resuspended in 0.3 ml of distilled water. The cell suspension was boiled for 7 min, and cell debris was removed by centrifugation. The supernatant was tested for antimicrobial activity by blotting 30 ul portion of the supematant on a paper disk with 8 mmin diameter (Toy0 Roshi Co., Ltd., Tokyo, Japan) which was placed on an L-agar plate lawned with a quinolone-susceptible E. The -- coli NIHJC-2 cells. diameter of growth-inhibition zone around the disk was evaluated after 24 h incubation at 37'C. The amount of cell-associated guinolone was quantified by ccmparing the diameter of the inhibition zone with those produced by the disks blotted with various amounts of guinolone antibiotics. SDS-PAGE analysis of total bacterial cell lysate. Total cell lysate was prepared as follows. -CZiiFwere collected from one ml of overnight culture in L-broth by centrifugation. The pellet was resuspended in 22.5 ul of PE$&, added with 2.5 1.11of 2-mercaptoethnol and 25 ul of Tris-SDS Seprasol (Daiichi Pure Chemicals Co. Ltd., Tokyo, Japan), followed by heating in the boiling water for 5 min. After centrifugation at 12,000 q for 1 min at rocm temperature, ten ul of the supernatant was subjected to SDS-PAGE. The samples were electrophoresed in a 4120 gradient SDS-PAGEgel (J&iichi Pure Chemicals). The gel was stained with cocmassie brilliant blue R-250 (17). Nucleotide sequence determination. The nuclegtjide sequence was determined by the method of Maxamand Gillbert (18) using [ PI end-labeled single stranded IYWA fragments after purification through a Benzoylted-Naphthoylated DEAS cellulose column (19). 1029
Vol.
172,
No.
3, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
REXJL'ISANDDI!XXJSICN
Decreased uptake of hydrophilic quinolone antibiotics into the cell --harborinq pMR8736. Both of the recombinant plasmids @lR8736 and pSA209 had inserts of 5.2 kb in size, and shared an identical restriction map pattern except an additional EcoRI site in the latter plasmid (Fig. la). We concluded that these clones contained norA genes, because these clones had the same size and the same restriction enzyme pattern in terms of I$$, WuII, and HincII, with the previously reported DNA fragment carrying norA gene (13). Intrcduction of @JR8736 by transformation made MC1061 cells resistant to hydrophilic quinolone antibiotics such as norfloxacin and pipemidic acid (Table 1)(19,20). Susceptibility of the transformant to hydrophobic quinolone antibiotics was either unaltered or altered in a small degree (e.g. to tosufloxacin and ofloxacin; Table 1). Introduction of pSA209 into MC1061, on the other hand, did not cause any significant alteration of the host cell susceptibility to quinolone antibiotics (Table 1). The decreased norfloxacin susceptibility of the transformant harboring pMR8736 was accompanied by a decreased uptake of the drug by the cell (Table 2). This decrease of uptake was observed with norfloxacin, a hydrophilic quinolone, but was not observed with a hydrophobic quinolone, tosufloxacin (Table 2). This result was
the norA FIG. 1. (a) The restrict' ion map of 5.2kb Hind111 fragments containing gene. Abbreviation is as follows: B, WII; E, =I; H, &dIII; Hi, B&fI; Hp, &I; K, &I; P, EII; and X, XbaI. (b) Iocalization of the B gene on pMR8736. Restriction enzyme-generated subclones (pMR361-365) as well as exonucleaseIII-generated deletion mutants (pDP3640, pDP3622, pDP3619, pDs3625, (c)Iocalization of guinolone-resistance pDS3632, and pDS3601) were used. marker on p&lR8736. The chimeric plasmids were constructed fran pSA209 and @lR8736. E. coli MC1061 was transformed with each clone of (b) and (c), and MIC of norfloxacin (NFLX) was measured for each transfo-t. MIC value is presented to the left of each clone. 1030
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BIOCHEMICAL
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TABLE 1. Quinolone MIC
of
AND
susceptibility
of
transformed
MC1061
BIOPHYSICAL
with
none
NFL): PPA CPFX
0.025 3.13 0.025 0.2 0.1 0.2 0.1 6.25 0.025
ENX
OFLX LFLX
PFLX NA
TFLX
puc15
pSA209
0.025 3.13 0.025 0.2 0.1 0.2 0.1 6.25 0.05
pMR8736
0.05 6.25 0.05 0.39 0.2 0.2 0.1 6.25 0.05
COMMUNICATIONS
and its Index of increase ~MR8736~
QUlllOlGne
antlblotlcsa
MC1061
RESEARCH
transformants
MIC
with
16 16 16
0.39 50 0.39
1.56
8
0.39 0.39 0.2 6.25 0.05
4 2 2 1 1
coefficient
Partltlon
0.06gc 0.017" 0.115c 0.127' 0.391C O.llJ6C
0.32d 0.90d 1.14d 4.95d
0.069e
18.8 3.34c 0.347e
a Abbreviation is as follows: NFLX, norfloxacin; PPA, pipemidic acid; CPFX, ciprofloxacin; ENX, enoxacin; OELX, ofloxacin; LFLX, lcmefloxacin(NY198); pefloxacin; NA, nalidixic acid; TF'LX, tosufloxacin(T3262). pm, b Index was calculated by dividing MIC of MC1061(pMR8736) with MIC of MC1061. ' n-cctanol/50 mMsodium phosphate buffer(pH 7.0), cited from (20). d Chloroform/O.1 M phosphate buffer(pH 7.4). e n-cctanol/water, cited from (21).
consistent guinolone
with the antibiotics
report of others that confers resistance to hydrophilic by impairing their accumulation into the cell (14).
Identification of the norA gene -on pMR8736. Localization of the norA --gene on pMR8736 was performed using norfloxacin-resistance phenotype as a marker. Various subclones and deletion mutants of pm8736 were constructed and introduced into Mc1061, and MIC of norfloxacin against each transformant was evaluated (Fig. lb). The m gene was localized within the 1.8 kb fragment demarcated by the left end of pDP3622and the right end of pDS3632 (Fig. lb). Identification of a protein as In order to -- a possible norA qene product. identify norA gene product, cell lysates were prepared from each transformant of MC1061 harboring pMR8736, pSA209, and pUCl8. A novel protein of 52 kD in size was identified in the transformants harboring m8736 and pSA209 but not in the transformant harboring pUCl8 (Fig. 2). Production of this protein TABLE 2. The accumulation of norfloxacin and tosufloxacin in MC1061 and its transformants NFLX (w/l
when
treated
MC1 061
MC1061 MCI061 MC1061
a b c d
(pUC18jb (pSA209jC (pMR8736jd
200 0.11 0.16 0.10 co.05
0
F
umulatlon
TFLX (uq/10
cells)
treated
with
concentrationa
drug Cells
ac
pg/ml f * -t
0.01 0.01 0.04
400 0.44 0.75 0.58 0.23
of
pg/ml 2 * f +
5
drug
when treated concentrationa
200
ug/ml
0.07 0.12 0.22 0.06
The values represent the means* SEM(n=4) of three experiments. MC1061harboring pUC18. MCI061 harboring pSA209. MC1061harboring pMR8736. 1031
ac
>I )l >I >I
.oo .oo .oo .oo
umulation
cells) with of 400
Ug/ml ,I ,l >I >I
.oo .oo -00 .oo
Vol.
172,
No.
3, 1990
correlated norA8736, deletion band with
well
with
which mutants the
BIOCHEMICAL
the
SDS-PAGE of membrane
fractions
shown). These data strongly protein of 52 kD in size is this protein was consistent localized
expression
was demonstrated pDP3640, pDP3622,
same migration
within
the
12
3
AND
rate
of
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
quinolone
resistance
conferred
by analysis pDS3632, and with
this
of
transformants
pDS3632
protein
(data
was also
by
carrying not
detectable
shown).
in the
of -___ S. aureus strains MR8736 and 209P (data not suggest, although not conclusive, that this encoded by the norA gene. The molecular weight of with the coding capacity of m gene which was
1.8 I& DNA fragment
of @IR8736.
kD
42-
3020-
02
A
14-
FIG. 2. SDS-PAGE analyses of cell lysates of MCI 061 transformed with various plasmids. Total cell lysates of transformants harboring pUCl8 (lane I), pSA209 (lane 2), and pMR8736 (lane 3) were analyzed. Molecular weight standards (Daiichi Pure Chemicals) were phosphorylase b, bovine serum albumin, aldolase, carbonic anhydrase, trypsin inhibitor, and lysozyme from the top in this order. The arrow indicates the 52 kD protein that is found only in the transformants harboring the norA gene. FIG. 3. (a) Nucleotide sequences of 0.6 kb FvuII-HinfI DNA fragments of pSA209 and pMH8736. Nucleotide sequences and predicted amino acid residues of pSA.209 are spelled out. Only the substituted nucleotide and amino acid in the pMR8736 are shown below the pSA209 sequences. The start of nucleotide numbering is C of =I1 site (GGCIG). The asterisks show a termination codon. An inverted repeated sequences of a probable transcription-termination signal are underlined. (b) Hydropathic profiles of the predicted 159 amino acid residues of pSA209 (left) and pMR8736 (right). The arrows indicate the position of the substituted amino acid. A span of consecutive residues of six was employed. 1032
Vol.
172,
No.
3, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Identification of a point mutation --in the norA gene -which is responsible for guinolone resistance. To understand why pMR8736 but not pSA209 conferred we attempted further localization of the quinolone resistance to MCI061, resistance marker to a smaller region of @R8736 by constructing chimeric plasmids of pMR8736and pSA209, taking advantage of a unique HpaI restriction site shared by both plasmids (Fig. Ic). Quinolone-resistance marker was now localized within the 0.5 kb HpaI-HinfI fragment of pMR8736. The nucleotide sequences of this region of @lR8736 as well as the corresponding region of pSA209 are presented (Fig. 3a). Only one open reading frame which is long enough to encode a meaningful protein was observed in this region. The direction of transcription was frcm the HpaI restriction enzyme site to the HinfI restriction enzyme site. The region contain&i a part of a structural gene coding for the carboxyl terminal 159 amino acid residues. Prediction of the amino acid sequence of this open reading frame revealed a remarkably high composition of hydrophobic amino acids (Fig. 3b). Nucleotide sequence of the corresponding region of pMR8736was identical with that of pSA209 except for a single nucleotide change from A (of pSA209) to C (of pMH8736) which caused amino acid substitution from Asp (of pSA209) to Ala (of pMR8736) at the 27th residue from the carboxyl terminus of the protein. Neither of the nucleotide and peptide sequences shared homolcgy with previously reported sequences including those of gyrase A and gyrase B genes of S. aureus (19). All clinical S. aureus strains so far tested were found to carry a norA -___ gene irrespective of their susceptibility towards quinolone antibiotics (data not shown). In view of this observation, the norA gene, not like a transposonassociated drug-resistance gene, seemsto code for a protein which is involved in scme essential physiological activity in the cell. As a point mutation of m gene, designated norA8736, was shown to cause quinolone resistance by preventing cell accumulation of guinolone antibiotics, we consider that such a physiological function of the norA gene product is the transport of hydrophilic quinolone molecules across cell membrane. Consistent with this view is the highly hydrophobic nature of the deduced amino acid sequences of the norA protein which is reminiscent of membrane-spanning regions of several transport proteins. We are not unaware of the fact that the identification of a protein of 52 kD in size as the norA gene product is inconclusive, since we cannot rule out the possibility that the protein is an intrinsic protein of -L E coli MC1061 whose expression was induced by the norA gene product. We also do not know whether the norA gene functions by enhancing efflux or impairing import of the guinolone antibiotics across cell membrane. Existence of such an efflux system of fluorcquinolone antibiotics in _L E coli has recently been suggested (5,8). The study designed to solve these questions are now under way. We thank K. ubukata of Teikyo University for a precious discussion. This work was partly supported by grants from the Japanese Ministry of Education. 1033
Vol.
172,
No.
3, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
1. Chapman,S.T.,Speller,D.C.E.,and Reeves,D.S.(1985)Iancet,2,39. 2. Humphreys,H.,and Mulvihill,E.(1985)Lancet,2,383. 3. Daikos,G.L.,Lolans,V.T.,and Jackson,G.G.(1988)Antimicrob.Agents
Chemother.
32,785-787. 4.
5. 6. 7. 8. 9.
Masecar,B.L.,Celesk,R.A.,and Robillard,N.J.(1990)Antimicrob.Agents Chemother.34,281-286. Cohen,S.P.,Hooper,D.C.,Wolfson,J.S.,Souza,K.S.,McMurry,L.M.,and Ievy,S.B. (1988)Antimicrob.Agents Chemother.32,1187-1191. Cullen,M.E.,Wyke,A.W.,Kurcda,R.,and Fisher,L.M.(1989)Antimicrob. Agents Chemother.33,886-894. Hirai,K.,Aoyama,H.,Suzue,A.,Irikura,T.,Iyobe,S.,and Mitsuhashi,S.(1986) Antimicrob.Agents Chemother.30,248-253. Hooper,D.C.,Wolfson,J.S.,Souza,K.S.,Ng,E.Y.,McHugh,G.L.,and Swartz,M.N. (1989)Antimicrob.Agents Chemother.33,283-290. Yamagishi,J.,Yoshida,H.,Yamayoshi,M.,and Nakamura,S.(1986)Mol.Gen.Genet. 204,367-373.
10.
Yoshida,H.,Kojima,T.,Yamagishi,J.,and
Nakamura,S.(1988)Mol.Gen.Genet.
211,1-7.
Schaefler,S.(1989)J.Clinic.Microb.27,335-336. 12. Shalit,I.,Berger,S.A.,Gorea,A.,and Frimerman,H.(1989)Antimicrob.Agents Chemother.33,593-594. 13. lJbukata,K.,Itoh-Yamashita,N.,and Konno,M.(1989)Antimicrob.Agents 11.
Chemother.
33,1535-1539.
Ubukata,K.,and Konno,M. (1990) The 3rd Yoshida,H.,Bogaki,M.,Nakamura,S., International Symposium on New Quinolones(manuscript No.160), VamOUVer, Canada. 15. Utsui,Y.,and Yokota,T.(1985)Antimicrob.Agents Chemother.28,397-403. 16. Koshland,D.,and Botstein,D.(1980)Ce11,20,749-760. 17. Wilson,C.M.(1983)Metha& in Enzymolcgy(Hirs,C.H.W.,and Timasheff,S.N., eds.),Vo1.91,Part 1,236-247,Academic Press, New York. 18. Maxam,A.M.,and Gilbert,W.(1977)Proc.Natl.Acad.Sci.USA.74,560 19. Kiger,J.A.,and .Sinsheimer,R.L.(1969)J.Mol.Bio1.40,467. Tamai,I.,Okezaki,E.,Nagata,O.,and Kato,H.(l988) 20. Tsuji,A.,Sato,H.,Kume,Y., Antimicrob.Agents Chemother.,32,190-194. 21. Rosen,T.,Chu,D.T.,Lico,I.M.,Femandes,P.B.,Marsh,K.,Shen,L.,Cepa,V.G., and Permet,A.G.(1988)5. Med. Chem.,31,1598-1611. 22. Hopewell,R.,Oram,M.,Briesewitz,R.,and Fisher,L.M.(1990)J.Bacteriol.l72 14.
3481-3484.
1034