Differential behaviors of Staphylococcus aureus and Escherichia coli type II DNA topoisomerases
Differential behaviors of Staphylococcus aureus and Escherichia coli type II DNA topoisomerases
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DOI：
10.1128/aac.40.12.2714
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发表时间：
1996-12-01
影响因子：
4.9
通讯作者：
Crouzet, J
中科院分区：
文献类型：
--
作者：
Blanche, F;Cameron, B;Crouzet, J
将编码DNA促旋酶亚基的金黄色葡萄球菌gyrA和gyrB基因克隆到大肠杆菌中，并在T7启动子-T7 RNA聚合酶系统的控制下共表达，得到可溶性促旋酶，并纯化至均一。纯化的促旋酶与从S.金黄色葡萄球菌中不含可检测量的拓扑异构酶。coli宿主的拓扑异构酶TV亚基GrlA和GrlB。aureus在E.大肠杆菌中分离纯化得到的拓扑异构酶IV具有ATP依赖的脱链和DNA松弛活性。这种酶比促旋酶对典型的氟喹诺酮类抗菌剂如环丙沙星或司帕沙星的抑制更敏感，这为遗传研究提供了强有力的支持，该研究表明拓扑异构酶IV是S.金黄色。用氧氟沙星获得的结果表明，这种氟喹诺酮也可以主要靶向促旋酶。与S.金黄色葡萄球菌促旋酶与完全抑制DNA超螺旋的浓度的环丙沙星或司帕沙星一起孵育。这表明，这些药物不稳定开放的DNA-促旋酶复合物，至少在标准的体外孵育条件下，但更可能主要干扰DNA断裂步骤，与E. coli促旋酶、S. aureus旋转酶催化DNA超螺旋和S.金黄色葡萄球菌拓扑异构酶IV催化的脱连环反应被谷氨酸钾或天冬氨酸钾显著刺激（700和350 mM谷氨酸分别抑制500和50倍），而拓扑异构酶IV依赖性DNA松弛被350 mM谷氨酸抑制3倍，二羧酸氨基酸对II型拓扑异构酶活性的影响的相关性进行了讨论，关于细胞内的渗透组成S.金黄色。
Staphylococcus aureus gyrA and gyrB genes encoding DNA gyrase subunits were cloned and coexpressed in Escherichia coli under the control of the T7 promoter-T7 RNA polymerase system, leading to soluble gyrase which was purified to homogeneity. Purified gyrase was catalytically indistinguishable from the gyrase purified from S. aureus and did not contain detectable amounts of topoisomerases from the E. coli host, Topoisomerase TV subunits GrlA and GrlB from S. aureus were also expressed in E. coli and were separately purified to apparent homogeneity, Topoisomerase IV, which was reconstituted by mixing equimolar amounts of GrlA and GrlB, had both ATP-dependent decatenation and DNA relaxation activities in vitro. This enzyme was more sensitive than gyrase to inhibition by typical fluoroquinolone antimicrobial agents such as ciprofloxacin or sparfloxacin, adding strong support to genetic studies which indicate that topoisomerase IV is the primary target of fluoroquinolones in S. aureus. The results obtained with ofloxacin suggest that this fluoroquinolone could also primarily target gyrase. No cleavable complex could be detected with S. aureus gyrase upon incubation with ciprofloxacin or sparfloxacin at concentrations which fully inhibit DNA supercoiling. This suggests that these drugs do not stabilize the open DNA-gyrase complex, at least under standard in vitro incubation conditions, but are more likely to interfere primarily with the DNA breakage step, contrary to what has been reported with E. coli gyrase, Both S. aureus gyrase catalyzed DNA supercoiling and S. aureus topoisomerase IV-catalyzed decatenation were dramatically stimulated by potassium glutamate or aspartate (500- and 50-fold by 700 and 350 mM glutamate, respectively), whereas topoisomerase IV-dependent DNA relaxation was inhibited 3-fold by 350 mM glutamate, The relevance of the effect of dicarboxylic amino acids on the activities of type II topoisomerases is discussed with regard to the intracellular osmolite composition of S. aureus.