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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\n                      <jats:italic toggle=\"yes\">pheS<\/jats:italic>\n                      of\n                      <jats:italic toggle=\"yes\">Av. paragallinarum<\/jats:italic>\n                      to establish a counterselectable marker\n                      <jats:italic toggle=\"yes\">AppheS<\/jats:italic>\n                      m. The host strain harboring the gene\n                      <jats:italic toggle=\"yes\">AppheS<\/jats:italic>\n                      m exhibited sensitivity to\n                      <jats:italic toggle=\"yes\">para<\/jats:italic>\n                      -chlorophenylalanine at a minimal inhibitory concentration of 20 mM, supporting the potential application of\n                      <jats:italic toggle=\"yes\">AppheS<\/jats:italic>\n                      m as a counterselectable marker in\n                      <jats:italic toggle=\"yes\">Av. paragallinarum<\/jats:italic>\n                      . The feasibility of this strategy was evaluated by generating an\n                      <jats:italic toggle=\"yes\">ApglgB<\/jats:italic>\n                      insertion mutant harboring the gene\n                      <jats:italic toggle=\"yes\">AppheS<\/jats:italic>\n                      m, followed by the seamless integration of the exogenous gene\n                      <jats:italic toggle=\"yes\">FtlpxE<\/jats:italic>\n                      into the\n                      <jats:italic toggle=\"yes\">ApglgB<\/jats:italic>\n                      locus via the replacement of the previously inserted gene\n                      <jats:italic toggle=\"yes\">AppheS<\/jats:italic>\n                      m. This two-step recombination strategy successfully allowed the expression of the integrated gene\n                      <jats:italic toggle=\"yes\">FtlpxE<\/jats:italic>\n                      , as assessed by the removal of 1-phosphate groups from lipid A. In addition, the effectiveness of the\n                      <jats:italic toggle=\"yes\">pheS<\/jats:italic>\n                      m system in\n                      <jats:italic toggle=\"yes\">Av. paragallinarum<\/jats:italic>\n                      was further evaluated by generating marker-free\n                      <jats:italic toggle=\"yes\">ApcpsB<\/jats:italic>\n                      and\n                      <jats:italic toggle=\"yes\">ApneuB<\/jats:italic>\n                      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\n                      <jats:italic toggle=\"yes\">AppheS<\/jats:italic>\n                      m as a counterselectable marker in\n                      <jats:italic toggle=\"yes\">Av. Paragallinarum<\/jats:italic>\n                      . Furthermore, the method developed herein is of tremendous importance as a tool for the genetic manipulation of this bacterium.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>IMPORTANCE<\/jats:title>\n                    <jats:p>\n                      <jats:italic toggle=\"yes\">Avibacterium paragallinarum<\/jats:italic>\n                      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\n                      <jats:italic toggle=\"yes\">Av. paragallinarum<\/jats:italic>\n                      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\n                      <jats:italic toggle=\"yes\">Av. paragallinarum<\/jats:italic>\n                      . We report the successful and precise deletion of multiple genes in\n                      <jats:italic toggle=\"yes\">Av. paragallinarum<\/jats:italic>\n                      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.\n                    <\/jats:p>\n                  <\/jats:sec>","DOI":"10.1128\/aem.02398-25","type":"journal-article","created":{"date-parts":[[2026,3,30]],"date-time":"2026-03-30T12:48:41Z","timestamp":1774874921000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":1,"title":["Development of a counterselection system for efficient marker-free genetic manipulation in\n                    <i>Avibacterium paragallinarum<\/i>"],"prefix":"10.1128","volume":"92","author":[{"given":"Juan","family":"Sun","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/05v9jqt67","id-type":"ROR","asserted-by":"publisher"}],"name":"College of Veterinary Medicine, South China Agricultural 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