Impact of gyrA and parC mutations on quinolone resistance, doubling time, and supercoiling degree of Escherichia coli
Impact of gyrA and parC mutations on quinolone resistance, doubling time, and supercoiling degree of Escherichia coli
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DOI：
10.1128/aac.43.4.868
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发表时间：
1999-04-01
影响因子：
4.9
通讯作者：
Heisig, P
中科院分区：
文献类型：
--
作者：
Bagel, S;Hüllen, V;Heisig, P
通过引入与喹诺酮类药物耐药相关的gyrA （S83L, D87G）和parC （S80I, E84K）突变，从喹诺酮类药物敏感的WT分离株中衍生出等基因突变体，这些突变体在喹诺酮类药物耐药、生长速度和全局超卷曲程度方面进行了表征。后者是通过使用一对携带超卷曲依赖启动子pgyrA和ptopA的报告质粒来确定的，它们分别与编码tem1 β -内酰胺酶的报告基因bla转录融合。携带两种质粒的突变体β -内酰胺酶特异性活性的商数（Qsc）被用来衡量其整体超旋度，这些Qsc数据与在含氯喹琼脂糖凝胶上分离质粒pBR322拓扑异构体的结果相当，表明抗性突变体的负超旋度相对于亲本有所降低。gyrA双突变（S83L + D87G）对喹诺酮类药物耐药的影响与单突变相似，parC突变（S80I）的表型表达依赖于至少一个gyrA突变的存在，高水平氟喹诺酮类药物耐药(环丙沙星MIG，>4 μ g/ml)需要gyrA双突变和一个parC突变（S80I或E84K）的组合，这些突变体表现出相当大的生长速率改变，全局超卷曲，或两者兼有。引入parC突变既不影响翻倍时间，也不影响超卷曲程度，而gyrA D87G突变的存在与DNA超卷曲程度的显著降低有关。
Isogenic mutants derived from quinolone-susceptible isolate WT by introducing gyrA (S83L, D87G) and parC (S80I, E84K) mutations associated with quinolone resistance were characterized with respect to quinolone resistance, growth rate, and degree of global supercoiling, The latter was determined by use of a pair of reporter plasmids carrying supercoiling-dependent promoters pgyrA and ptopA, respectively, transcriptionally fused to the reporter gene bla coding for TEM-1 beta-lactamase, The quotient (Qsc) of the beta-lactamase specific activity determined for a mutant carrying either plasmid was taken as a measure of the degree of global supercoiling, These Qsc data were comparable to results obtained from the separation of topoisomers of plasmid pBR322 on chloroquine-containing agarose gels and indicate a reduced degree of negative supercoiling in resistant mutants relative to the parent, WT. The S83L mutation in gyrA had the strongest influence on quinolone resistance while leaving other parameters nearly unaffected, The gyrA double mutation (S83L plus D87G) had an effect on quinolone resistance similar to that of a single mutation, Phenotypic expression of the parC mutation (S80I) was dependent on the presence of at least one gyrA mutation, Expression of high-level fluoroquinolone resistance (ciprofloxacin MIG, >4 mu g/ml) required a combination of the gyrA double mutation and one parC mutation (S80I or E84K), Such mutants showed considerable alterations of growth rate, global supercoiling, or both. Introduction of a parC mutation affected neither the doubling time nor the degree of supercoiling, while the presence of the gyrA D87G mutation was associated with a significant reduction in the degree of DNA supercoiling.