Abstract
nfxB encodes a negative regulator of the mexCD-oprJ genes for drug efflux in the opportunistic pathogen Pseudomonas aeruginosa. Inactivating mutations in this transcriptional regulator constitute one of the main mechanisms of resistance to ciprofloxacin (Cipr). In this work, we evaluated the use of nfxB/Cipr as a new test system to study mutation spectra in P. aeruginosa. The analysis of 240 mutations in nfxB occurring spontaneously in the wild-type and mutator backgrounds or induced by mutagens showed that nfxB/Cipr offers several advantages compared with other mutation detection systems. Identification of nfxB mutations was easy since the entire open reading frame and its promoter region were sequenced from the chromosome using a single primer. Mutations detected in nfxB included all transitions and transversions, 1-bp deletions and insertions, >1-bp deletions and duplications. The broad mutation spectrum observed in nfxB relies on the selection of loss-of-function changes, as we confirmed by generating a structural model of the NfxB repressor and evaluating the significance of each detected mutation. The mutation spectra characterized in the mutS, mutT, mutY and mutM mutator backgrounds or induced by the mutagenic agents 2-aminopurine, cisplatin and hydrogen peroxide were in agreement with their predicted mutational specificities. Additionally, this system allowed the analysis of sequence context effects since point mutations occurred at 85 different sites distributed over the entire nfxB. Significant hotspots and preferred sequence contexts were observed for spontaneous and mutagen-induced mutation spectra. Finally, we demonstrated the utility of a luminescence-based reporter for identification of nfxB mutants previous to sequencing analysis. Thus, the nfxB/Cipr system in combination with the luminescent reporter may be a valuable tool for studying mutational processes in Pseudomonas spp. wherein the genes encoding the NfxB repressor and the associated efflux pump are conserved.
Highlights
Mutation is an essential cellular process that contributes to the genetic variability and acts as the driving force of evolution
Sequence analysis of nfxB in ciprofloxacin resistant clones In order to evaluate the use of nfxB as a new target to analyze mutation spectra, we screened a large collection (240) of ciprofloxacin resistant (Cipr) clones derived independently from different genetic backgrounds or treatment with mutagens (2-aminopurine, cisplatin or hydrogen peroxide)
Several mutation detection systems have been developed for bacteria, the majority are well suited for use in E. coli
Summary
Mutation is an essential cellular process that contributes to the genetic variability and acts as the driving force of evolution. Genetic analysis of mutator and antimutator genes has been used to define the molecular mechanisms of spontaneous mutagenesis in E. coli [2] These processes are evolutionarily well conserved, it has been suggested that there are considerable differences in the mechanisms leading to mutation among bacteria [1]. Members of this genus are capable of thriving in highly diverse ecological niches due to their versatile metabolic capacity and broad potential for adaptation to fluctuating environmental conditions This unique feature of Pseudomonas spp. implies a remarkable degree of genomic diversity and genetic adaptability to customize its genome to fit the requirements for survival in diverse niches [4].
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have