Blaschke Ulrike, Suwono Beneditta, Zafari Sachli, Ebersberger Ingo, Skiebe Evelyn, Jeffries Cy M, Svergun Dmitri I, Wilharm Gottfried
Robert Koch-Institute, Project Group P2, Wernigerode, Germany.
Applied Bioinformatics Group, Institute of Cell Biology and Neuroscience, Goethe University, Frankfurt/Main, Germany.
Protein Expr Purif. 2018 Nov;151:78-85. doi: 10.1016/j.pep.2018.06.009. Epub 2018 Jun 22.
Acinetobacter baumannii appears as an often multidrug-resistant nosocomial pathogen in hospitals worldwide. Its remarkable persistence in the hospital environment is probably due to intrinsic and acquired resistance to disinfectants and antibiotics, tolerance to desiccation stress, capability to form biofilms, and is possibly facilitated by surface-associated motility. Our attempts to elucidate surface-associated motility in A. baumannii revealed a mutant inactivated in a putative DNA-(adenine N6)-methyltransferase, designated A1S_0222 in strain ATCC 17978. We recombinantly produced A1S_0222 as a glutathione S-transferase (GST) fusion protein and purified it to near homogeneity through a combination of GST affinity chromatography, cation exchange chromatography and PD-10 desalting column. Furthermore we demonstrate A1S_0222-dependent adenine methylation at a GAATTC site. We propose the name AamA (Acinetobacteradenine methyltransferase A) in addition to the formal names M.AbaBGORF222P/M.Aba17978ORF8565P. Small angle X-ray scattering (SAXS) revealed that the protein is monomeric and has an extended and likely two-domain shape in solution.
鲍曼不动杆菌在全球医院中常表现为一种多重耐药的医院病原体。它在医院环境中具有显著的持久性，这可能归因于其对消毒剂和抗生素的固有及获得性耐药性、对干燥应激的耐受性、形成生物膜的能力，并且可能借助表面相关运动性得以促进。我们试图阐明鲍曼不动杆菌表面相关运动性的研究揭示了一株在假定的DNA - （腺嘌呤N6） - 甲基转移酶中失活的突变体，在菌株ATCC 17978中该酶被命名为A1S_0222。我们通过谷胱甘肽S - 转移酶（GST）融合蛋白重组表达A1S_0222，并通过GST亲和层析、阳离子交换层析和PD - 10脱盐柱的组合将其纯化至近乎均一。此外，我们证明了A1S_0222在GAATTC位点依赖的腺嘌呤甲基化。除了正式名称M.AbaBGORF222P/M.Aba17978ORF8565P之外，我们提议将其命名为AamA（不动杆菌腺嘌呤甲基转移酶A）。小角X射线散射（SAXS）表明该蛋白是单体，并且在溶液中具有延伸的、可能为双结构域的形状。
