A pyrF-Based Efficient Genetic Manipulation Platform in Acinetobacter baumannii To Explore the Vital DNA Components of Adaptive Immunity for I-F CRISPR-Cas

Acinetobacter baumannii is an important pathogenic bacterium with multidrug resistance which causes infections with high mortality rates. In-depth genetic analysis of A. baumannii virulence and drug-resistant genes is highly desirable. In this study, we utilized the conserved pyrF-flanking fragment to rapidly generate uracil auxotrophy hosts with pyrF deleted in model and clinical A. baumannii strains and then introduced the pyrF gene as the selectable and counterselectable marker to establish a series of gene manipulation vectors. For gene deletion with the suicide pyrF-based plasmid, the second-crossover colonies screened with the pyrF/5-fluoroorotic acid (5-FOA) system were obtained more quickly and efficiently than those screened with the sacB/sucrose system. By using the replicative plasmid, the recognized protospacer-adjacent motif (PAM) bias for type I-F CRISPR was experimentally revealed in A. baumannii AYE. Interestingly, interference recognized only the PAM-CC sequence, whereas adaptation priming tolerates 4 PAM sequences. Furthermore, we also performed a rapid and extensive modification of the I-F CRISPR-Cas elements and revealed that the role of double-nucleotide sequence mutants at the end of the repeat could be critical during both CRISPR interference and priming; we also found strong biases for A and demonstrated that adaptation could tolerate certain sequence and size variations of the leader in A. baumannii. In conclusion, this pyrF-based genetic manipulation system was readily applicable and efficient for exploring the genetic characteristics of A. baumannii. IMPORTANCE In this study, we developed the widely applicable and efficient pyrF-based selection and counterselection system in A. baumannii for gene manipulation. In most cases, this pyrF/5-FOA genetic manipulation system was very effective and enabled us to obtain marker-free mutants in a very short period of time. Utilizing this system and the separate mechanism of interference and/or primed adaptation, our experiments revealed some recognition mechanism differences for the key DNA elements of PAM, leader, and repeat in the priming adaptation process of the I-F CRISPR-Cas systems of A. baumannii, which provided some new and original insights for the study of the molecular mechanisms of these processes and laid a foundation for further studies.