Developing a broadly applicable pyrF-based genetic manipulation system in Acinetobacter baumannii

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.