A Markerless Gene Deletion System in Streptococcus suis by Using the Copper-Inducible Vibrio parahaemolyticus YoeB Toxin as a Counterselectable Marker

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