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

Abstract

UNLABELLED: 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 geneofto establish a counterselectable markerm. The host strain harboring the genem exhibited sensitivity to-chlorophenylalanine at a minimal inhibitory concentration of 20 mM, supporting the potential application ofm as a counterselectable marker in. The feasibility of this strategy was evaluated by generating aninsertion mutant harboring the genem, followed by the seamless integration of the exogenous geneinto thelocus via the replacement of the previously inserted genem. This two-step recombination strategy successfully allowed the expression of the integrated gene, as assessed by the removal of 1-phosphate groups from lipid A. In addition, the effectiveness of them system inwas further evaluated by generating marker-freeandmutants, 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 ofm as a counterselectable marker in. Furthermore, the method developed herein is of tremendous importance as a tool for the genetic manipulation of this bacterium. IMPORTANCE: 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 inhas 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. We report the successful and precise deletion of multiple genes inand 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.