Efficient markerless genetic manipulation of Pasteurella multocida using lacZ and pheS m as selection markers

is a zoonotic conditional pathogen that infects multiple livestock species, causing substantial economic losses in the animal husbandry industry. An efficient markerless method for gene manipulation may facilitate the investigations of gene function and pathogenesis of . Herein, a temperature-sensitive shuttle vector was constructed using as a selection marker, and markerless , , and mutants of were subsequently constructed through blue-white colony screening. The screening efficiency of markerless deletion strains was improved by the system, and the method could be used for multiple gene deletions. However, the mutant was unavailable via this method. Therefore, we constructed a m screening system based on mutated phenylalanine tRNA synthetase as a counterselection marker to achieve deletion mutant. The transformed strain was sensitive to 20 mM -chloro-phenylalanine, demonstrating the feasibility of m as a counter-selective marker. The m system was used for markerless deletions of , , and as well as that could not be screened by the system. A comparison of screening efficiencies of the system showed that the m counterselection system was more efficient than the system and broadly applicable for mutant screening. The methods developed herein may provide valuable tools for genetic manipulation of .IMPORTANCE is a highly contagious zoonotic pathogen. An understanding of its underlying pathogenic mechanisms is of considerable importance and requires efficient species-specific genetic tools. Herein, we propose a screening system for mutants using or m screening markers. We evaluated the efficiencies of both systems, which were used to achieve markerless deletion of multiple genes. The results of this study support the use of or m as counterselection markers to improve counterselection efficiency in . This study provides an effective genetic tool for investigations of the virulence gene functions and pathogenic mechanisms of .