Spontaneous and induced mutations to rifampicin, streptomycin and spectinomycin resistances in actinomycetes: mutagenic mechanisms and applications for strain improvement

Abstract

Chemical mutagenesis continues to be an important foundational methodology for the generation of highly productive actinomycete strains for the commercial production of antibiotics and other secondary metabolites. In the past, the determination of frequencies of chemically induced resistance to rifampicin (RifR), spectinomycin (SpcR) and streptomycin (StrR) have served as surrogate markers to monitor the efficiencies and robustness of mutagenic protocols. Recent studies indicate that high level RifR, SpcR and StrR phenotypes map to specific regions of the rpoB, rpsE and rpsL genes, respectively, in actinomycetes. Moreover, mutagenesis to RifR can occur spontaneously at many different sites in rpoB, and all six types of base-pair substitutions, as well as in-frame deletions and insertions, have been observed. The RifR/rpoB system provides a robust method to rank mutagenic protocols, to evaluate mutagen specificity and to study spontaneous mutagenesis mechanisms involved in the maintenance of high G+C content in Streptomyces species and other actinomycetes.

Investigation of auranofin and gold-containing analogues antibacterial activity against multidrug-resistant Neisseria gonorrhoeae

“…The bacteria were tested against both auranofin and rifampicin at 10× the MIC determined via agar dilution assay following the procedure that was previously described in 19 . Rifampicin was used as a positive control as it is known to be susceptible to spontaneous mutations 49,50 . Briefly, to prepare the media containing the drugs, GC agar base was autoclaved for 30 mins then human blood was added after cooling down to a final concentration of 5%.…”

Spatiotemporal Manipulation of the Mismatch Repair System of Pseudomonas putida Accelerates Phenotype Emergence

“…The occurrence of mutants developing resistance to either Rif or Str was investigated in bacterial populations after the treatments indicated above. Resistance to these antibiotics has been widely used for the investigation of spontaneous and induced mutagenesis processes in Gram-negative bacteria. − Rifampicin-resistant (Rif R ) and streptomycin-resistant (Str R ) phenotypes occur due to the appearance of mutations in the rpoB and rpsL genes, encoding the β-subunit of RNA polymerase and the 30S ribosomal protein S12, respectively. − Mutation frequencies were estimated by assessing the frequency of occurrence of Rif R or Str R cells on the total number of viable cells in the bacterial population for each tested experimental condition (Figure ). In all accelerated mutagenesis experiments, we observed a significantly higher number of Rif R and Str R mutants isolated in selective conditions in bacterial clones carrying a mutator device compared to their respective control strains (Figure ).…”

Identification of New Chromosomal Loci Involved in com Genes Expression and Natural Transformation in the Actinobacterial Model Organism Micrococcus luteus

“…For cell recovery and to allow segregation of mutations (single nucleotide polymorphisms, SNPs), samples of 0.05 mL were inoculated in 5 mL of fresh LB with kanamycin (LB Kan ) and incubated with aeration O/N at 30 °C. To assess the efficiency of mutagenesis, mutagen-induced rifampicin resistance was monitored [ 32 ]. Samples of EMS-treated cells were plated on full media supplemented with 1 µg/mL of rifampicin, and after growth, the frequency of resistant colonies was determined and compared with the frequency of spontaneous rifampicin-resistant colonies arising from cells not treated with the mutagen.…”