Developing a broadly applicable pyrF-based genetic manipulation system in Acinetobacter baumannii
Developing a broadly applicable pyrF-based genetic manipulation system in Acinetobacter baumannii
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在鲍曼不动杆菌中开发广泛适用的基于pyrF的基因操作系统
DOI：
10.1101/2020.11.05.370742
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发表时间：
2020-11
期刊：
影响因子：
--
通讯作者：
Rui Wang
中科院分区：
文献类型：
--
作者：
Run Xu;Can Gao;Shuqi Wu;Mengjiao Su;Chengfu Su;Xu Jia;Rui Wang
鲍曼不动杆菌具有多重耐药性，感染后死亡率高，是一种紧急致病菌。对鲍曼不动杆菌毒力和耐药基因进行深入的遗传分析是非常必要的。现有的鲍曼不动杆菌遗传操作方法主要依赖于使用抗生素作为选择标记，以及sacB/蔗糖作为反选择标记，这对于鲍曼不动杆菌的所有研究来说是不方便且不合适的。基于高度保守的pyrF基因及其保守的500bp侧翼序列，我们快速、轻松地生成了pyrF缺失突变体，作为3个模型菌株和11个临床菌株的尿嘧啶营养缺陷型宿主菌株。在这些pyrF缺失突变体中，构建用于以pyrF/5-FOA作为反选择进行基因编辑的pyrF携带载体既方便又省时。利用基于pyrF的基因操作系统，我们轻松高效地修饰了鲍曼不动杆菌AYE中I-F CRISPR-Cas系统的cas基因和CRISPR序列，并检测了这些突变体中的CRISPR干扰和适应。总之，基于pyrF的基因操作系统可广泛适用于大多数鲍曼不动杆菌菌株的高效无标记基因编辑。
Acinetobacter baumannii is an emergency pathogenic bacterium for its multidrug-resistance and high mortality rates after infection. In-depth genetic analysis of A. baumannii virulence and drug-resistant genes is highly desirable. Existing methods for genetic manipulation of A. baumannii mainly rely on the use of antibiotic as the selectable marker, and the sacB/sucrose as the counter-selectable marker, which is inconvenient and inappropriate for all research of A. baumannii. Based on the highly conserved pyrF gene and its conserved 500bp-flanking sequence, we quickly and easily generated the pyrF-deleted mutants as the uracil auxotrophic host strain in three model strains and 11 clinical strains. The pyrF-carried vectors constructed for gene editing with pyrF/5-FOA as the counter-selection were conveniently and time-saving in these pyrF-deleted mutants. Utilizing the pyrF-based genetic manipulation system, we easily and efficiently modified the cas gene and CRISPR sequence of I-F CRISPR-Cas system in A. baumannii AYE, and detected the CRISPR interference and adaptation in these mutants. In summary, the pyrF-based genetic manipulation system could be broadly applicable used for efficiently maker-less gene editing in most A. baumannii strains.
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