1098 CHEMOTHERAPY SEPT. 1992
VOL. 40 NO. 9 1099 A) B) Fig. 1. Construction the expression plasmids for the wild-type Escherichia coli GyrA (A) and GyrB (B) proteins.
Table 1. Quinolone-resistant mutations in the gyra gene Escherichia coli KL 16
VOL.40 NO.9 1101 キ ノ ロ ン薬 の ジ ャイ レ ー ス 阻 害 作 用 機 作 ー タ 省 略) 第1の タ イ プ の 変 異 で は 荷 電 と局 所 構 造 の 変 化 が 推 測 さ れ,第2の タ イ プ の 変 異 で は,荷 電 の 変 化 の み が起 きる と推 測 され る(デ ー タ省 略) 3.精 製GyrAお よびGyrB蛋 大 腸 菌KL16株(野 生 型),N-51株(gyrA変 株),N-24株(第1タ 31株(第2タ gyraお 白 の純 度 と比 活 性 イ プ 砂rB変 イ プgyrB変 よ びgyrB遺 異 株)よ よ びN- り ク ロ ー ン化 し た 伝 子 を 有 す る プ ラ ス ミ ドか ら, 材 料 と方 法 の 項 で述 べ た よ うに,発 築 しGyrAお 異 異 株)お よ びGyrB蛋 現 プ ラ ス ミ ドを構 白 を 大 量 生 産 し,精 製 し た 野 生 型 大 腸 菌KL 16株 お よ び キ ノ ロ ン耐 性gyrA変 異 株N-51のgyrA遺 (以 後AKL16お 伝 子 か ら生 産 さ れ たGyrA蛋 よ びAN51と キ ノ ロ ン耐 性gyrB変 記 載)お 異 株Nー24,N-31の 伝 子 か ら生 産 さ れ たGyrB蛋 よ びBN31と 白(以 記 載)はSDS-ポ 各gyrB遺 後BKL16,BN24お リ ア ク リル ア ミ ド ゲ ル 電 気 泳 動 に よ る チ ェ ッ クで は,90%以 た (Fig.2) し か し,DNAス 上 の純 度 で あ っ ーパ ー コ イ リング活 性 で 測 定 し た 各 蛋 白 の 比 活 性 はAKL16:2.0 106単 mg,an51:3.6 106単 で あ り,GyrB蛋 1/20 1/100程 Fig. 2. 白 の比 活 性 はGyrA蛋 位/mg 白 の比 活 性 の 度 で あ っ た この 結 果 はGyrB蛋 purified 4scherichia were 位/ 位/mg,BN31=3.6 104単 SDS-polyacrylamide gram 位/ 位/mg,BKL16:8,5 104単 mg,bn24:2.3 104単 coli. subjected gel GyrA and About 1-2ƒÊg to electrophoresis. 白 よ びKL16株, 白標 electrophoreto- GyrB proteins proteins each SDS-polyacrylamidegel KL 16; 2, GyrA N-51; 1, 3, GyrA GyrB KL 16; 4, GyrB N-24; 5, GyrB N-31.
1102 CHEMOTHERAPY SEPT. 1992 Table 3. Quinolone sensitivity wild-type and mutant Escherichia coli gyrases in the supercoiling reaction KL16: E. coli KL 16 having the wild-type gyra and gyrb genes. N-51: E. coli N-51 having the mutant gyra gene (Ser-83 to Leu) N-24: E. coil N-24 having the type 1 mutant gyrb gene (Asp-426 to Asn). N-31: E. coli N-31 having the type 2 mutant gyrb gene (Lys-447 to Glu). pbr 322 and phy 300 PLK: plasmids for E. coli and B. subtilis, respectively.
Table 4. Binding 3H-enoxacin with the gyrase-dna complexes a Kd values for the binding enoxacin were estimated by Scatchard plot analyses.
1104 CHEMOTHERAPY SEPT, 1992 1) Gellert M, Mizuuchi K, O'Dea M H, Ito T, Tomizawa J: Nalidixic acid resistance: a second. genetic character involved in DNA gyrase activity, Proc. Natl. Acad. Sci. USA 74: 4772 `4776, 1977 2) Sugino A, Peebles C L, Kreuzer K N, Cozzarelli N R: Mechanism action nalidixic acid: purification Escherichia coil nala gene product and its relationship to DNA gyrase and a novel nicking-closing activity. Proc. Natl. Acad. Sci. USA 74: 4772 `4776, 1977 3) Wolfson J S, Hooper D C, Swartz M N. Mechanism action and resistance to quinolone antimicrobial agents, In Quinolone antimicrobial agents (Wolfson J S, Hooper DC eds.), p 5 `34, American Society for Microbiology, Washington, D.C. 1989 4) Shen L L, Pernet A G. Mechanism inhibition DNA gyrase by analogues nalidixic acid: the target the drugs is DNA. Proc. Natl. Acad. Sci. USA 82:307 `311, 1985 5) Shen L L, Kohlbrenner W E, Weigl D, Baranowski J: Mechanism quinolone inhibition DNA gyrase. J. Biol. Chem. 264: 2973 `2978, 1989 6) Shen L L, Mitscher L A, Sharma P N O'Donnell T J, Chu D W T, Cooper C S, Rosen T, Pernet A G: Mechanism inhibition DNA gyrase by quinolone antibacterials: a cooperative drug- DNA binding model. Biochemistry 28: 3886 ` 3894, @ 1989 7) Yoshida H, Kojima T, Yamagishi J, Nakamura S: Quinolone-resistant mutations the gyra gene Escherichia coli. Mol. Gen. Genet. 211: 1 `7, 1988 8) Yamagishi J, Yoshida H, Yamayoshi M, Nakamura S: Nalidixic acid-resistant mutations the gyrb gene Escherichia coli. Mol. Gen. Genet. 204: 367 `373, 1986 9) Yoshida H, Bogaki M, Nakamura M, Nakamura S: Quinolone resistance-determining region in the DNA gyrase gyra gene Escherichia coli. Antimicrob. Agents Chemother. 34: 1271 `1272, 1990 10) Yoshida H, Bogaki M, Nakamura M, Yamanaka L M, Nakamura S: Quinolone resistancedetermining region in the DNA gyrase gyrb gene Escherichia coli. Antimicrob. Agents Chemother. 35: 1647 `1650, 1991 11) Wilkie N M, Clements J B, Boll W, Mantei N, Lonsdale D, Weissmann C: Hybrid plasmids containing an active thymidine kinase gene Herpes simplex virus I. Nucleic Acids Res. 7: 859 12) Holmes D S, Quigley M: A rapid boiling method for the preparation bacterial plasmids. Anal. Biochem. 114: 193 `197, 1981 13) Messing J: New M 13 vectors for cloning. Methods in Enzymol. 101: 20 `78, 1983
VOL. 40 NO. 9 1105 14) Miller J H: Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor N Y, 1972 16) Yamada M, Furutani Y, Notake M, Yamagishi J, Yamayoshi M, Fukui T, Nomura H, Komiya M, Kuwashima J, Nakano K, Sohmura Y, Nakamura S: Efficient production human tumor necrosis factor in Escherichia coli. J. Biotechnol. 3: 141 `153, 1985 17) Gellert M, Fisher L M, O'Dea M H: DNA gyrase: purification and catalytic properties a fragment gyrase B protein. Proc. Natl. Acad. Sci. USA. 76: 6289 `6293, 1979 18) Sato K, Inoue Y, Fujii T, Aoyama H, Inoue M, Mitsuhashi S: Purification and properties DNA gyrase from a fluoroquinolone-resistant strain Escherichia coli, Antimicrob. Agents Chemother, 30: 777 `780, 1986 19) Horowitz D S, Wang J C: Mapping the active site tyrosine Escherichia coli. DNA gyrase. J. Biol. Chem. 262: 5339 `5344, 1987 20) Adachi T, Mizuuchi M, Robinson E A, Appella E, O'Dea, Gellert M. Mizuuchi K: DNA sequence the E. coli gyrb gene: application a new sequencing strategy. Nucleic Acids Res, 15: 771 ` 784, 1987 21) Brown P O, Peebles C L, Cozzarelli N R: A topoisomerase from Escherichia coli related to DNA gyrase. Proc. Natl. Acad. Sci, USA. 76: 6110 `6114, 1979 Mechanism action quinolones on Escherichia coli DNA gyrase Hiroaki Yoshida Bioscience Research Laboratories, Dainippon Pharmaceutical Co., Ltd., Enoki 33-94, Suita, Osaka 564, Japan Seven point mutations were detected in 10 quinolone-resistant gyra mutants Escherichia coli KL 16, i.e., Ser-83 to Leu (4 strains), Ser-83 to Trp, Asp-87 to Asn, Gly-81 to Cys, Ala-84 to Pro, Ala -67 to Ser and Gln-106 to His. Among 13 quinolone-resistant gyrb mutants E. coli KL 16, 2 point mutations were detected, Asp-426 to Asn (9 strains) and Lys-447 to Glu (4 strains). The former mutation (type 1) confers resistance to all the quinolones tested, while the latter mutation (type 2) results in resistance to acidic quinolones and hypersusceptibility to amphoteric quinolones. Almost all the gyra and gyrb mutations are believed to occur on protein surfaces, based on cpmputer analysis. Mutant DNA gyrases reconstituted from wild-type GyrA (or GyrB) and mutant GyrB (or GyrA) proteins were resistant to or hypersensitive to quinolones as expected from the MICs for the corresponding mutants. 3H-enoxacin was bound to gyrase-dna complexes but not to gyrase alone or to DNA alone. The amount enoxacin bound to the GyrA mutant gyrase-dna complex was the same as that bound to the wild-type gyrase-dna complex, but the binding affinity to the former was one-tenth the binding to the latter. The amount enoxacin bound to the type 1 GyrB mutant gyrase-dna complex was one-seventh that bound to the wild-type complex. Enoxacin was bound to the type 2 GyrB mutant gyrase-dna complex in the same amount as to the wild-type complex with five-times greater affinity for the former than the latter. These data suggest that quinoloneresistance DNA gyrase can be explained by the decreased amount quinolone binding or decreased quinolone binding affinity to gyrase-dna complexes.