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

Abstract

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 YoeBVp can be a counterselectable marker. Herein, the counterselection potential of YoeBVp was assessed in S. suis. The yoeBVp gene was placed under the copper-induced promoter PcopA. The PcopA-yoeBVp construct was cloned into the S. suis-E. coli shuttle vector pSET2 and introduced into S. suis to assess the effect of YoeBVp expression on S. suis growth. Reverse transcription quantitative PCR showed that copper induced yoeBVp expression. Growth curve analyses and spot dilution assays showed that YoeBVp expression inhibited S. suis growth both in liquid media and on agar plates, revealing that YoeBVp has the potential to be a counterselectable marker for S. suis. A SCIY cassette comprising the spectinomycin-resistance gene and copper-induced yoeBVp 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.

Highly Effective Markerless Genetic Manipulation of Streptococcus suis Using a Mutated PheS-Based Counterselectable Marker

“…This small difference between the two MICs makes counter-selection escape easy to happen, thus limiting the counter-selection efficiency. For the yeoB system, induction of the toxin only partially inhibits the growth of yeoB -containing strain, it is not surprising that the final culture needs to be enriched for several generations in presence of the inducer, but still, the efficiency could not reach 100% ( Zheng et al, 2021 ). All in all, the P 1503 - mPheS developed in this study is the most efficient CSM for S. suis up to now.…”

“…Only very recently, counterselectable markers (CSMs) were inspected for their use in S. suis genetic manipulation. The well-known Bacillus subtilis levansucrase SacB and the Vibrio parahaemolyticus toxin YoeB have been tested for their potential for counter-selection in S. suis ( Zhang et al, 2019 ; Zheng et al, 2021 ). Though the target markerless mutations have been successfully selected by these CSMs, the notorious counter-selection escape of the SacB-based system and the incomplete growth inhibition of the YoeB-based system strongly limited the efficiency of these systems and urged us to explore a more effective CSM alternative for S. suis .…”

Streptococcus suis subtilisin-like serine proteases SspA-1 and SspA-2 interplay with complement C3a and C5a to facilitate bacterial immune evasion and infection

“…The sspA-1 gene deletion strain Δ sspA-1 was constructed via a two-step procedure as described previously [ 32 ]. In the first step, upstream and downstream arms of sspA-1 , SCIY cassette were amplified using primers sspA-1 -P1/P2, sspA-1 -P5/P6, and sspA-1 -P3/P4, respectively.…”