Contruction And Function Analysis Of The RovS Transcription Regulator Of Deleted Mutant Strain Of Streptococcus Agalactiae Isolated From Tilapia

Group B Streptococcus(GBS), also known as Streptococcus agalactiae, is a common pathogen, which can cause a clinical diseases of human being and animals. Recently,break-out of streptococcicosis of cultured tilapia fish, caused by Streptococcus agalactiae,severely affected healthy development of tilapia aquaculture and led to huge losses to tilapia culture all over the world. RovS protein is a member of the family of Rgg transcription regulation of S.agalactiae. RovS transcription regulation plays an important role in S.agalactiae, such as up- or down-regulating the expression of many genes, and cooperating with other transcriptional regulators to regulate the growth, multiplication and metabolism of bacteria. This research constructed rovS gene deletion mutant strain, and investigated the influence of rovS gene deletion on the biological characteristics between wild and mutant train, and the expression level of virulence-associated genes in bacteria.The results lay a theoretical foundation for further research on the pathogenesis mechanism of S. agalactiae isolated from tilapia.The two DNA fragments flanking the rovS gene were amplified by PCR from the genome of S.agalactiae. And the Erm gene was amplified from plasmid pAT18. Then a constructed recombinant vector, pSET4s-rF-Erm-rR, was electroporated into S.agalactiae.The ΔrovS deletion mutant strain has been screened through antibiotics which were Spectinomycin(Spc+, 100 μg·mL-1) and Erythromycin(Erm+, 50 μg·mL-1), and double temperatures(28 ℃, 37 ℃).The general biological characteristic of the wide strain and the mutant strain were compared under the same bacterial culture conditions. Firstly, S.agalactiae ZQ0910 and the ΔrovS mutant have no difference in the genetic stability and Gram stain. Secondly, it is found that the growth of the ΔrovS mutant strain was slower than S.agalactiae ZQ0910 by measuring OD600 values. Through observing shapes of wide type strain and the mutant strain in blood nutrient agar plates, the result shows that the ΔrovS strain deleted the hemolysis active. In addition, LD50 values wild-type strain and ΔrovS strain were respectively 3.16×106 cfu and 2.0×107 cfu. Thus the virulence or pathogenicity of ΔrovS strain is lower that of wild strain. Finally, the expressions of known and putative virulence genes of S.agalactiae ZQ0910 and the ΔrovS mutant strain were analyzed by quantitative real-time PCR. The results show that the expression of fbsA and sip increased in ΔrovSmutant compared to the wild-type. On the contrary, the transcription levels of the genes of gluC, gyrA, hlyA, pyrR and sodA decreased when rovS gene knocked out. However, the expression of ccpA, cpsC and scpB were not affected.