Molecular characterization of Rifr mutations in Pseudomonas aeruginosa and Pseudomonas putida |
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Authors: | Tatjana Jatsenko Andres Tover Radi Tegova Maia Kivisaar |
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Affiliation: | Department of Genetics, Institute of Molecular and Cell Biology, Tartu University and Estonian Biocentre, 23 Riia Street, 51010 Tartu, Estonia |
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Abstract: | The rpoB gene encoding for β subunit of RNA polymerase is a target of mutations leading to rifampicin resistant (Rifr) phenotype of bacteria. Here we have characterized rpoB/Rifr 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 Rifr 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 Rifr mutations characterized for P. aeruginosa grown at 37 °C and that characterized for P. putida grown at 30 °C were dissimilar but the differences almost disappeared when the mutants of both strain were isolated at the same temperature, at 30 °C. The strong Rifr phenotype of P. aeruginosa and P. putida was accompanied only with substitutions of these residues which belong to the putative Rif-binding pocket. Approximately 70% of P. aeruginosa mutants, which were isolated at 37 °C and expressed weak Rifr phenotype, contained base substitutions in the N-terminal cluster of the rpoB gene. The differences in the spectra of mutations at 30 °C and 37 °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 Rifr mutants from selective plates are critical when the rpoB/Rifr test system is employed for comparative studies of mutagenic processes in Pseudomonas species which are conventionally cultivated at different temperatures. |
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