Generation of markerless and multiple-gene knockout in Glaesserella parasuis based on natural transformation and Flp recombinase

Glaesserella parasuis is an important bacterial pathogen that affects the swine industry worldwide. Research on the pathogenic mechanism and genetically engineered vaccine remains undeveloped because an effective markerless and multiple-gene knockout system is unavailable for G. parasuis yet. To establish a markerless knockout, deleted allelic genes with kanamycin resistance (Kan R ) cassettes were introduced into the genome of G. parasuis by using natural transformation with suicide plasmids. Then, the Kan R cassette was excised with a thermosensitive plasmid pGF conferring a constitutive Flp expression. To realize the markerless and multiple-gene knockout, plasmid pGAF was constructed by placing the Flp gene under the control of an arabinose-inducible promoter. Firstly, pGAF was introduced into G. parasuis by electroporation, and the marked mutants were produced following natural transformation. Finally, the Kan R cassette was excised from the genome by the inducible expression of Flp upon arabinose action. Based on the natural transformation and the inducible expression of Flp, the markerless single-gene knockout mutants of Δ hsdR , Δ neuA2 , Δ espP2 , Δ apd , and Δ nanH were constructed. In addition, a five-gene knockout mutant of Δ hsdR Δ neuA2 Δ espP2 Δ apd Δ nanH was generated by successive natural transformation with five suicide plasmids. Taken together, a markerless and multiple-gene deletion system was established for G. parasuis in the present study for the first time. This system is simple, efficient, and easy to manipulate for G. parasuis ; thus, our technique will substantially aid the understanding of the etiology, pathogenesis, and genetic engineering of G. parasuis and other bacteria that can be naturally transformed in laboratory conditions. Key points • Flp recombinase excised the Kan R gene flanked by FRT sites in Glaesserella parasuis. • The regulatory expression of Flp enabled a multiple-gene knockout for G. parasuis. • The technique will promote the understanding of Glässer’s disease pathogens. Graphical abstract