Zheng Chengkun, Wei Man, Qiu Jun, Li Jinquan
Joint International Research Laboratory of Agriculture and Agri-Product Safety, The Ministry of Education of China, Yangzhou University, Yangzhou 225009, China.
Jiangsu Key Laboratory of Zoonosis, Yangzhou University, Yangzhou 225009, China.
Microorganisms. 2021 May 19;9(5):1095. doi: 10.3390/microorganisms9051095.
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.
是一种重要的人畜共患病原体，可在猪和人类中引起严重感染。诱导产生YoeB毒素会导致细胞死亡，这引发了YoeB可能是一种可反向选择标记的推测。在此，评估了YoeB在中的反向选择潜力。将基因置于铜诱导型启动子P的控制下。将P-构建体克隆到穿梭载体pSET2中，并导入以评估YoeB表达对生长的影响。逆转录定量PCR表明铜诱导了表达。生长曲线分析和斑点稀释试验表明，YoeB的表达在液体培养基和琼脂平板上均抑制了的生长，这表明YoeB有潜力成为的可反向选择标记。构建了一个包含壮观霉素抗性基因和铜诱导的SCIY盒。利用SCIY盒和肽诱导的感受态，为建立了一种新型的两步无标记基因缺失方法。此外，利用通过该方法产生的Δ突变体，我们证明了中的亚铁和钴外排泵PmtA受PerR调节因子的负调控。
