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

Chengkun Zheng,Man Wei,Jun Qiu,Jinquan Li

doi:10.3390/microorganisms9051095

Chengkun Zheng, Man Wei + Show 2 more

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https://doi.org/10.3390/microorganisms9051095

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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.

Highlights

Streptococcus suis is a Gram-positive, facultative anaerobe that threatens the swine industry and public health worldwide [1]
We identified a chromosomal type II toxin-antitoxin system, YefM-YoeB, in Vibrio parahaemolyticus; induction of YoeBVp in Escherichia coli resulted in cell death [31]
Using the ∆perR mutant generated by this method, we demonstrated that pmtA, a gene encoding a ferrous iron and cobalt efflux pump in S. suis [32] was negatively regulated by the PerR regulator

Summary

Introduction

Streptococcus suis is a Gram-positive, facultative anaerobe that threatens the swine industry and public health worldwide [1]. It is responsible for various swine diseases, including meningitis, septicemia, pneumonia, endocarditis, and arthritis [2]. S. suis is considered one of the most important bacterial pathogens that lead to significant economic losses to the swine industry [3]. Two great outbreaks of human S. suis infections occurred in China in 1998 and 2005, resulting in 25 cases with 14 deaths and 215 cases with 39 deaths, respectively [7,8]. S. suis still frequently caused sporadic human cases worldwide [9,10,11,12,13,14]

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