nfxB as a novel target for analysis of mutation spectra in Pseudomonas aeruginosa

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 (Cip(r)). In this work, we evaluated the use of nfxB/Cip(r) 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/Cip(r) 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/Cip(r) 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.

Figure 1. Distribution of mutations in nfxB.

The position of all mutations detected in nfxB among 240 Cip^r clones derived independently from different genetic backgrounds or treatment with mutagens is plotted. (A) The number of base substitutions (blue bars) and 1-bp deletions and insertions (red bars) detected at each nucleotide position of the open reading frame is shown. (B) >1-bp deletions (red bars) and duplications (blue bars) are displayed over the open reading frame (black bar) and promoter region (grey bar) of nfxB.

Figure 2. 3D-structural model of NfxB.

(A) Ribbon representation of the homology-modeled NfxB dimer based on the TetR-like transcriptional repressor LfrR. Helices α1–α3 are shown in blue, α4 in light blue, α5–α7b in green, and α8 and α9 are in yellow. N-terminal helices α1 to α3 and C-terminal helices α8 and α9 corresponded to LfrR helices involved in DNA-binding and dimerization, respectively. (B) Front (above) and top-down (below) visualization of electrostatic potential distribution in the NfxB dimer as determined using APBS. Positive and negative potentials are indicated in blue and red, respectively. Note that a large positive charged surface is located on the N-terminal loop between helices α1 and α2 and helices α2 and α3, which could form the putative DNA-binding channel in the NfxB dimer.

Figure 3. Amino acid substitutions in NfxB.

Amino acid changes in NfxB caused by missense mutations are indicated with the one letter nomenclature. Amino acid substitutions for proline are shown in red, and changes to residues that differ in polarity or charge from the original one are depicted in blue. The secondary structure obtained from the NfxB homology model is shown (Nomenclature H: Helix; T: Turn; -: Coil). The nine α-helices are numbered from α1 to α9.

Figure 4. Spontaneous and mutagen-induced mutations in nfxB.

The nfxB mutations detected in the wild-type (WT) and mutator strains (mutS, mutT, mutY and mutM), and the WT strain after exposure to the mutagens 2-aminopurine (2AP), cisplatin (CPT) and hydrogen peroxide (HP) are summarized. The percentage of each mutation type relative to the total number of mutations detected for each strain or treatment is plotted using a color code.

Figure 5. Identification of nfxB mutants using a luminescence-based reporter.

(A) Colonies of the mutT strain selected at 1.0 µg/mL Cip after 48 h of incubation. Examples of colonies with basal (non-mutated) and increased (nfxB mutants) luminescence are indicated (horizontal and vertical arrows, respectively). (B–C) Colonies of the mutT strain selected at a subinhibitory Cip concentration (0.4 µg/mL) after 24 h (B) and 56 h (C) of incubation. Detection of a subpopulation of cells with increased luminescence arising from colonies with basal luminescence indicates the emergence of nfxB mutants (examples are indicated by arrows).