Suis Response Regulator CovR Target Gene Identification And Regulation Of Phosphorylation System Stk / Stp Prokaryotic Protein Expression

Streptococcus suis （S. suis） is an important pathogenic bacterium that infects piglets and causes many serious disease such as arthritis, meningitis and sepsis. It can be transmitted to human beings through wounds and other routes. It not only poses a threat to the swine industry, but also affects the public health and safety. In recent years, studies have shown that two-component signal transduction system plays an important role in regulating the expression of pathogen virulence genes and forming of bacterial pathogenicity.A TCSTS response regulator CovR was found to regulate nearly8.9%of the S. suis2genes directly or indirectly in our previous studies on the pathogenic mechanism of the bacterium. These genes include some of the known or potential S. suis2pathogenicity related genes. The findings suggested that covR is a global repressor in virulence regulation of S. suis2. To better understanding the role of CovR in pathogenic process, our research has been focused on the virulence mechanism of CovR.The chromatin immunoprecipitation experiment was performed to screen the target genes directly regulated by CovR. S. suis205ZYH33strain was fixed with1%formaldehyde so as to cross-link DNA and protein together. The genomic DNA was disrupted into fragments of100-500bp, and then immunoprecipitated with anti-CovR monoclonal antibody. After that, DNA fragments were extracted, purified, and ultimately detected by the general PCR assay. Finally, we successfully screened out2target genes ssu05₁668and ssu05₁732.It was found that CovR was regulated by eukaryotic-like serine/threonine protein kinase e in S. agalactiae and S. pneumoniae in recent years. We speculate that the Stk/Stp phosphorylation system may also exist in S. suis, which may modulate CovR by phosphorylation or dephosphorylation. The present study has identified two homologous enzymes of stk and stp by of analying the genome of S. suis2Chinese high virulent strain05ZYH33. The ssu05₀428gene encoding a serine/threonine protein kinase （Stk） and ssu05₀427gene encoding its corresponding phosphatase （Stp）. The target DNA fragments were successfully amplified using the genomic template of Chinese strains05ZYH33. Subsequently, target genes were inserted into pMD18-T vector, and then subcloned into prokaryotic expression vector pET28a to generate recombinant expression plasmids: pET28a-stk, pET28a-stp and pET28a-N-stk. The Stp and N-Stk proteins were purified by His affinity chromatography.In conclusion, this study identified the target genes of CovR and prokaryotic expressed and purified the recombinant N-Stk protein and Stp protein successfully. It lays the foundation for further investigatation the role of CovR phosphorylation in S. suis2pathogenic process and systematically illustrating the CovR signaling pathway.