Construction And Biological Characteristics Of Streptococcus Suis DeoR Gene Deletion Strain

Streptococcus suis is an important human-animal disease that has caused huge economic losses to the global pig industry and poses a great threat to human life.Streptococcus suis can regulate the expression of relevant genes according to the variable internal environment of the host during the process of host infection,thus enhancing its ability to colonize the host.Transcriptional regulators are an important mechanism by which bacteria respond to dynamic environments.DeoR（Deoxyribonucleoside operon Repressor）protein family is a regulatory factor,which exists in many bacteria.According to the regulation function of DeoR on other bacteria,it is predicted that DeoR may have certain regulation effect on glucose metabolism and pathogenicity of Streptococcus suis.However,there are few reports on its regulation in S.suis,so it is of great significance to study its regulation in S.suis.The details of the study are as follows:1.Isolation,identification,virulence gene and drug resistance analysis of Streptococcus suisIn this study,pericardial effusion from clinically infected pigs was collected and inoculated in 5%rabbit blood plate.Suspected colonies were purified and cultured,examined by Gram staining,identified by PCR,identified by 16S r RNA sequencing,drug sensitivity test,serotype identification and virulence gene identification.The results showed that streptococcus suis type 2 strain was successfully isolated and named SS21-1.The isolates had six virulence genes including gapdh+,orf2+,fbps+,mrp+,sly+,epf+.Drug sensitivity test showed that the strain was sensitive to cefuroxime,ceftriaxone,cefotaxime,ofloxacin,norfloxacin,tetracycline,ampicillin,penicillin,gentamicin;Resistant to lincomycin,kanamycin,erythromycin.16S r RNA sequence analysis showed that the strain had high homology with strains P1/7,05ZYH33 and 98HAH33 in Gen Bank and belonged to the same evolutionary branch groupⅠ.2.Analysis of biological information of DeoR gene of Streptococcus suis type 2Due to the widespread and highly conserved deoR gene in SS,related biomolecular informatics websites were used to conduct protein sequence homology retrieval,functional domain retrieval,signal peptide prediction and transmembrane region prediction for the transcriptional regulatory factor deoR.DeoR gene encoded 247 amino acids with a molecular weight of 27.9 k Da.Small DNA-binding domains with helix-turn-helix（HTH）motifs at the n-terminal.DeoR protein may be in cytoplasm without transmembrane region and does not encode signal peptide.The isoelectric point（PL）of DeoR family transcription proteins was 6.24.3.Construction and biological characterization of Streptococcus suis deoR gene deletion strainIn this study,SS21-1△deoR gene deletion strain ss21-1 was successfully constructed by homologous recombination technique.The biological characteristics and pathogenicity of the deletion strain were compared with that of the parent strain.The results showed that there were no significant differences in colony morphology and hemolysis characteristics between the missing strain and the wild strain.The growth rate of deletion plant was higher than that of wild plant in logarithmic phase,but lower than that of wild plant in stable phase.Wild seedlings of sugar and lack the utilization rate of the experiment,two strains can grow in the glucose,sucrose,raffinose,in glucose and raffinose as the only under the condition of glycogen,lack of the growth rate is reduced,no significant differences between OD₆₀₀ of two strains,under the condition of the sucrose as the sole glycogen,the lack of a significant reduction in the growth rate and OD₆₀₀;The wild strains were moderately sensitive to doxycycline,while the deletion type was sensitive to doxycycline.The pathogenicity of the missing strain was higher than that of the wild strain.The anti-phagocytic ability,whole blood survival rate and colonization ability of the mutant were better than those of the wild type.Therefore,deoR gene can not only regulate glucose metabolism,but also enhance the survival ability of bacteria in blood and tissues and organs,thus enhancing the ability of Streptococcus suis type 2 to infect mice.In addition,the deletion of deoR gene made it more difficult for RAW264.7 cells to clear Streptococcus suis type 2,which further confirmed that deoR gene could regulate streptococcus suis type 2 related virulence genes,thus regulating its pathogenicity.In summary,this experiment firstly isolated and identified a Streptococcus suis type2 strain SS21-1,and through the analysis of biological information of deoR gene and the construction of its deletion strain,it was further clarified that DeoR has a certain degree of regulatory effect on sugar metabolism and pathogenicity of Streptococcus suis.The above findings laid a theoretical foundation for the in-depth exploration of the pathogenic mechanism of Streptococcus suis type 2.