A -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 -flanking fragment to rapidly generate uracil auxotrophy hosts with deleted in model and clinical A. baumannii strains and then introduced the gene as the selectable and counterselectable marker to establish a series of gene manipulation vectors. For gene deletion with the suicide -based plasmid, the second-crossover colonies screened with the /5-fluoroorotic acid (5-FOA) system were obtained more quickly and efficiently than those screened with the /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 -based genetic manipulation system was readily applicable and efficient for exploring the genetic characteristics of A. baumannii. In this study, we developed the widely applicable and efficient -based selection and counterselection system in A. baumannii for gene manipulation. In most cases, this /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.