Department of Biomedical and Molecular Sciences, Queen's University, Kingston, ON K7L 3N6, Canada.
Microbiology (Reading). 2013 Oct;159(Pt 10):2058-2073. doi: 10.1099/mic.0.069286-0. Epub 2013 Aug 7.
The mexCD-oprJ multidrug efflux operon of Pseudomonas aeruginosa is regulated by the NfxB repressor. Two forms of NfxB have been reported [Shiba et al. (1995). J Bacteriol 177, 5872) although mutagenesis studies here confirm that the larger protein (199 amino acids, 22.4 kDa) is the functional repressor. NfxB binds upstream of the mexCD-oprJ transcription initiation site to a region containing two inverted repeats, both of which are required for binding. Two-hybrid assays confirmed that NfxB is a multimer, with the C-terminal two-thirds of the repressor required for multimerization. Random mutagenesis identified several mutations within the C-terminal region of NfxB required for multimerization, all of which mapped to a three-helix subdomain of the C-terminal region in a structural model of the repressor, which may thus represent the multimerization domain. These mutations compromised NfxB binding to its target DNA in electromobility shift assays, and their introduction into the chromosome of P. aeruginosa enhanced mexCD-oprJ expression and promoted multidrug resistance, consistent with the functional NfxB repressor being a multimer. Site-directed and spontaneous nfxB mutants showing increased mexCD-oprJ expression and multidrug resistance were also recovered, with mutations mapping to the three-helix subdomain again impacting multimerization and DNA binding. Mutations mapping to the N-terminal helix-turn-helix motif implicated in DNA binding did not impact multimerization although they did render the repressor insoluble and unsuitable for mobility shift assays. Size exclusion column chromatography demonstrated that wild-type NfxB forms tetramers in solution, although a mutant form of the repressor carrying a G192D substitution near the C terminus of the protein and compromised for DNA binding and repressor activity forms dimers. These results suggest that NfxB operates as a tetramer (dimer of dimers) and that the C terminus of the protein serves as a tetramerization domain.
铜绿假单胞菌的 mexCD-oprJ 多药外排操纵子受 NfxB 阻遏物调控。已有报道称存在两种形式的 NfxB [Shiba 等人，（1995）。J Bacteriol 177，5872]，尽管此处的诱变研究证实较大的蛋白（199 个氨基酸，22.4 kDa）是功能性阻遏物。NfxB 与 mexCD-oprJ 转录起始位点上游结合，结合于包含两个反向重复序列的区域，这两个重复序列均为结合所必需。双杂交测定法证实 NfxB 是一种多聚体，该阻遏物的 C 末端三分之二对于多聚化是必需的。随机诱变鉴定出 NfxB 中对于多聚化必需的 C 末端区域的几个突变，所有这些突变都位于阻遏物 C 末端区域的三个螺旋亚结构域内，在阻遏物的结构模型中，该亚结构域可能代表多聚化结构域。这些突变使 NfxB 在电泳迁移率变动测定中与靶 DNA 的结合受到损害，并且将这些突变引入铜绿假单胞菌的染色体中增强了 mexCD-oprJ 的表达并促进了多药耐药性，这与功能性 NfxB 阻遏物是多聚体一致。还恢复了显示 mexCD-oprJ 表达增加和多药耐药性增强的 NfxB 点突变和自发突变，这些突变再次定位于三个螺旋亚结构域，影响多聚化和 DNA 结合。定位于涉及 DNA 结合的 N 端螺旋-转角-螺旋基序的突变不影响多聚化，尽管它们确实使阻遏物不可溶且不适合于电泳迁移率变动测定。尺寸排阻柱层析法表明，野生型 NfxB 在溶液中形成四聚体，尽管一种携带靠近蛋白 C 末端的 G192D 取代的突变形式的阻遏物不能与 DNA 结合并且阻遏物活性受到损害，该突变体形成二聚体。这些结果表明，NfxB 作为四聚体（二聚体的二聚体）发挥作用，并且蛋白的 C 末端作为四聚化结构域。
