Centro de Investigaciones en Química Biológica de Córdoba (CIQUIBIC), CONICET, Departamento de Química Biológica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Ciudad Universitaria, Córdoba, Argentina.
PLoS One. 2013 Jun 7;8(6):e66236. doi: 10.1371/journal.pone.0066236. Print 2013.
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.
nfxB 基因编码一种负调控因子，可调节铜绿假单胞菌中药物外排的 mexCD-oprJ 基因。该转录调控因子的失活突变是铜绿假单胞菌对环丙沙星（Cip(r)）耐药的主要机制之一。在这项工作中，我们评估了 nfxB/Cip(r) 作为一种新的测试系统，用于研究铜绿假单胞菌中的突变谱。分析了野生型和突变体背景下自发产生的或诱变剂诱导的 240 个 nfxB 突变，结果表明 nfxB/Cip(r) 与其他突变检测系统相比具有几个优势。由于可以使用单个引物从染色体上测序整个开放阅读框及其启动子区域，因此很容易鉴定 nfxB 突变。在 nfxB 中检测到的突变包括所有的转换和颠换、1bp 缺失和插入、>1bp 缺失和重复。在 nfxB 中观察到的广泛突变谱依赖于功能丧失性变化的选择，这一点我们通过生成 NfxB 阻遏物的结构模型并评估每个检测到的突变的意义来证实。在 mutS、mutT、mutY 和 mutM 突变体背景下或诱变剂 2-氨基嘌呤、顺铂和过氧化氢诱导下的突变谱的特征与其预测的突变特异性一致。此外，该系统允许分析序列上下文效应，因为点突变发生在整个 nfxB 上分布的 85 个不同位点。在自发和诱变诱导的突变谱中观察到显著的热点和首选序列上下文。最后，我们展示了基于发光的报告基因在 nfxB 突变体鉴定之前进行测序分析的应用。因此，nfxB/Cip(r) 系统与发光报告基因相结合可能是研究铜绿假单胞菌中突变过程的有价值的工具，其中编码 NfxB 阻遏物和相关外排泵的基因是保守的。
