Ferrero L, Cameron B, Manse B, Lagneaux D, Crouzet J, Famechon A, Blanche F
Département des Biotechnologies, Rhône-Poulenc Rorer S.A., Centre de Recherche de Vitry-Alfortville, Vitry-sur-Seine, France.
Mol Microbiol. 1994 Aug;13(4):641-53. doi: 10.1111/j.1365-2958.1994.tb00458.x.
A 4.6 kb Staphylococcus aureus DNA fragment containing DNA gyrase-like genes (grlA and grlB) was cloned and sequenced. The proteins GrlA and GrlB exhibit more than 30% identity with E. coli DNA topoisomerase IV subunits and with the gyrase subunits from S. aureus and Escherichia coli. The combined E. coli cell extracts of GrlA and GrlB overproducing strains catalysed ATP-dependent relaxation and decatenation specific to DNA topoisomerase IV. The temperature-sensitive phenotype of Salmonella typhimurium parC and parE mutants was complemented by the S. aureus grlA and grlB genes, when the two genes were co-expressed. These results show that GrlA and GrlB are the subunits of S. aureus DNA topoisomerase IV. The GyrA subunit of DNA gyrase has been previously defined as a primary target of quinolones based on genetic and biochemical experiments essentially carried out in E. coli. Single-point mutations occurring in the 'quinolone resistance-determining region' (QRDR) of GyrA were found in bacteria exhibiting quinolone resistance, the most common mutation being a substitution of Ser-83 on the E. coli GyrA sequence. We analysed eight S. aureus fluoroquinolone-resistant clinical isolates and observed that mutations in the QRDR of GyrA are not present in the low-quinolone-resistant isolates. In contrast, Ser-80 of GrlA, which corresponds to Ser-83 of E. coli GyrA, is substituted to Phe or Tyr in both high- and low-quinolone-resistant isolates. We propose that DNA topoisomerase IV is a primary target of fluoroquinolones in S. aureus.
克隆并测序了一个含有DNA促旋酶样基因（grlA和grlB）的4.6 kb金黄色葡萄球菌DNA片段。GrlA和GrlB蛋白与大肠杆菌DNA拓扑异构酶IV亚基以及金黄色葡萄球菌和大肠杆菌的促旋酶亚基具有30%以上的同源性。GrlA和GrlB过量表达菌株的大肠杆菌细胞提取物组合催化了DNA拓扑异构酶IV特有的ATP依赖性松弛和解连环反应。当金黄色葡萄球菌的grlA和grlB基因共表达时，鼠伤寒沙门氏菌parC和parE突变体的温度敏感表型得到了互补。这些结果表明，GrlA和GrlB是金黄色葡萄球菌DNA拓扑异构酶IV的亚基。基于主要在大肠杆菌中进行的遗传和生化实验，DNA促旋酶的GyrA亚基先前已被定义为喹诺酮类药物的主要靶点。在表现出喹诺酮耐药性的细菌中发现了GyrA“喹诺酮耐药决定区”（QRDR）中发生的单点突变，最常见的突变是大肠杆菌GyrA序列上Ser-83的替换。我们分析了8株耐氟喹诺酮类金黄色葡萄球菌临床分离株，发现低喹诺酮耐药分离株中不存在GyrA的QRDR突变。相反，在高喹诺酮耐药和低喹诺酮耐药分离株中，与大肠杆菌GyrA的Ser-83对应的GrlA的Ser-80都被替换为Phe或Tyr。我们提出DNA拓扑异构酶IV是金黄色葡萄球菌中氟喹诺酮类药物的主要靶点。
