A Markerless Gene Deletion System in by Using the Copper-Inducible YoeB Toxin as a Counterselectable Marker.

is an important zoonotic pathogen causing severe infections in swine and humans. Induction of the YoeB toxin in resulted in cell death, leading to the speculation that YoeB can be a counterselectable marker. Herein, the counterselection potential of YoeB was assessed in . The gene was placed under the copper-induced promoter P. The P- construct was cloned into the shuttle vector pSET2 and introduced into to assess the effect of YoeB expression on growth. Reverse transcription quantitative PCR showed that copper induced expression. Growth curve analyses and spot dilution assays showed that YoeB expression inhibited growth both in liquid media and on agar plates, revealing that YoeB has the potential to be a counterselectable marker for . A SCIY cassette comprising the spectinomycin-resistance gene and copper-induced was constructed. Using the SCIY cassette and peptide-induced competence, a novel two-step markerless gene deletion method was established for . Moreover, using the Δ mutant generated by this method, we demonstrated that PmtA, a ferrous iron and cobalt efflux pump in , was negatively regulated by the PerR regulator.