Development of a counterselection system for efficient marker-free genetic manipulation in Avibacterium paragallinarum

ABSTRACT Avibacterium paragallinarum causes infectious coryza in chickens, a condition that is prevalent worldwide and causes considerable losses for the poultry sector. However, the scarcity of information concerning the virulence factors of the bacterium and the underlying pathogenic mechanisms is largely due to the lack of effective and practical tools for genetic manipulation. Herein, the point mutation A294G was introduced in the gene pheS of Av. paragallinarum to establish a counterselectable marker AppheS m. The host strain harboring the gene AppheS m exhibited sensitivity to para -chlorophenylalanine at a minimal inhibitory concentration of 20 mM, supporting the potential application of AppheS m as a counterselectable marker in Av. paragallinarum . The feasibility of this strategy was evaluated by generating an ApglgB insertion mutant harboring the gene AppheS m, followed by the seamless integration of the exogenous gene FtlpxE into the ApglgB locus via the replacement of the previously inserted gene AppheS m. This two-step recombination strategy successfully allowed the expression of the integrated gene FtlpxE , as assessed by the removal of 1-phosphate groups from lipid A. In addition, the effectiveness of the pheS m system in Av. paragallinarum was further evaluated by generating marker-free ApcpsB and ApneuB mutants, which exhibited defects in polysaccharide synthesis and sensitivity to the host complement system. To the best of our knowledge, this is the first study to demonstrate the potential application of AppheS m as a counterselectable marker in Av. Paragallinarum . Furthermore, the method developed herein is of tremendous importance as a tool for the genetic manipulation of this bacterium. IMPORTANCE Avibacterium paragallinarum is the etiological agent of infectious coryza, a significant respiratory ailment associated with growth inhibition and diminished egg production in poultry. Given the increasing incidence of antibiotic resistance in bacteria, there is an imperative need for the development of genetically engineered vaccines; however, the lack of effective tools for genetic manipulation in Av. paragallinarum has limited the scope of related research. Herein, we investigated the employment of the gene pheSm as a counterselectable marker for the screening of marker-free mutants of Av. paragallinarum . We report the successful and precise deletion of multiple genes in Av. paragallinarum and an instance of the effective seamless integration of the exogenous gene lpxE using this method. This study presents a valuable genetic tool for investigating the virulence genes and associated mechanisms of pathogenicity of this bacterium, as well as aiding the development of genetically engineered vaccines.