Molecular characterization of Rif(r) mutations in Pseudomonas aeruginosa and Pseudomonas putida

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 24h 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 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 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.