Molecular characterization of Rifr mutations in Pseudomonas aeruginosa and Pseudomonas putida
Molecular characterization of Rifr mutations in Pseudomonas aeruginosa and Pseudomonas putida
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DOI：
10.1016/j.mrfmmm.2009.10.015
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发表时间：
2010-01-05
影响因子：
2.3
通讯作者：
Kivisaar, Maia
中科院分区：
文献类型：
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作者：
Jatsenko, Tatjana;Tover, Andres;Kivisaar, Maia
编码 RNA 聚合酶 β 亚基的 rpoB 基因是导致细菌利福平耐药 (Rif(r)) 表型的突变目标。在这里，我们对铜绿假单胞菌和恶臭假单胞菌中的 rpoB/Rif(r) 系统进行了表征，作为研究突变过程的测试系统。我们发现，除了在铺板 24 小时后在 Rif 选择性平板上出现清晰可见的大菌落外，小菌落还在这些平板上生长了 48 小时。突变体菌落在选择性平板上的时间依赖性出现是由突变体的不同水平的 Rif 抗性引起的。对 rpoB 基因的 Rif(r) 簇进行了测序并分析了 360 个铜绿假单胞菌突变体和 167 个恶臭假单胞菌突变体。以在 37°C 生长的铜绿假单胞菌为特征的 Rif(r) 突变谱与以在 30°C 生长的恶臭假单胞菌为特征的 Rif(r) 突变谱并不相似，但当两种菌株的突变体在相同温度（30°C）下分离时，差异几乎消失。铜绿假单胞菌和恶臭假单胞菌的强 Rif(r) 表型仅伴随着属于推定的 Rif 结合袋的这些残基的取代。大约 70% 的铜绿假单胞菌突变体（在 37 摄氏度下分离并表达弱 Rif(r) 表型）在 rpoB 基因的 N 末端簇中含有碱基取代。 30 摄氏度和 37 摄氏度下突变谱的差异可以通过几种突变体在利福平存在下的温度敏感生长来解释。因此，我们的结果表明，当采用 rpoB/Rif(r) 测试系统对通常在不同温度下培养的假单胞菌属物种的诱变过程进行比较研究时，细菌生长的温度和从选择性平板中分离 Rif(r) 突变体的时间至关重要。 (C) 2009 Elsevier B.V. 保留所有权利。
The rpoB gene encoding for beta subunit of RNA polymerase is a target of mutations leading to rifampicin resistant (Rif(r)) phenotype of bacteria. Here we have characterized rpoB/Rif(r) system in Pseudomonas aeruginosa and Pseudomonas putida as a test system for studying mutational processes. We found that in addition to the appearance of large colonies which were clearly visible on Rif selective plates already after 24 h of plating, small colonies grew up on these plates for 48 h. The time-dependent appearance of the mutant colonies onto selective plates was caused by different levels of Rif resistance of the mutants. The Rif(r) clusters of the rpoB gene were sequenced and analyzed for 360 mutants of P. aeruginosa and for 167 mutants of P. putida. The spectrum of Rif(r) mutations characterized for P. aeruginosa grown at 37 degrees C and that characterized for P. putida grown at 30 degrees C were dissimilar but the differences almost disappeared when the mutants of both strain were isolated at the same temperature, at 30 degrees C. The strong Rif(r) phenotype of P. aeruginosa and A putida was accompanied only with substitutions of these residues which belong to the putative Rif-binding pocket. Approximately 70% of A aeruginosa mutants, which were isolated at 37 degrees C and expressed weak Rif(r) phenotype, contained base substitutions in the N-terminal cluster of the rpoB gene. The differences in the spectra of mutations at 30 degrees C and 37 degrees C can be explained by temperature-sensitive growth of several mutants in the presence of rifampicin. Thus, our results imply that both the temperature for the growth of bacteria and the time for isolation of Rif(r) mutants from selective plates are critical when the rpoB/Rif(r) test system is employed for comparative studies of mutagenic processes in Pseudomonas species which are conventionally cultivated at different temperatures. (C) 2009 Elsevier B.V. All rights reserved.