The Regulatory Function Of SloR In Streptococcus Suis Serotype 2

The transcription factor SloR of Streptococcus suis serotype 2（S.suis 2） is one of the bacterial DtxR family of transcription factors, this family was explored firstly from the research of the uptake mechanism of iron ions in Gram-positive bacteria with high GC content. The investigation result shows these proteins have similar functions with the Fur family, participating in the metabolism of metal ions, antioxidant responses of the cell and all that. But this family has different gene sequences and protein structures from the Fur family. Investigations have shown that the microorganisms with the Fur dependent regulation mechanism of iron ions use the DtxR protein family to regulate the transport of manganese ions. Some transcription factors of the DtxR family associate with the bacterial virulence, such as the DtxR protein is the transcription factor of diphtheria toxin which is the virulence factor of Corynebacterium diphtheria（C.diphtheria）. while others affect many bacterial life activities and the expression of the virulence with metal ions.According to the function of the DtxR family reported, the functional characterization of sloR gene in S.suis 2 was carried out with the parent stain of S.suis 2 SC-19, the main content involves the following aspects.1. Construction of the S.suis 2 sloR gene mutant △sloRWith the template of SC-19 strain and referring to the 05ZYH33 genome sequence of S.suis 2, the upstream and downstream flanking regions of the sloR gene （SSU05₂087） were separately amplified, and cloned into the thermosensitive suicide vector of pSET4s. At the same time, the erythromycin resistance gene was cloned into the vector between the flanking regions and the recombinant plasmid pSET4s-sloR was obtained. Then the recombinant plasmid was transformed into SC-19. The colony was screened by resistance and temperature with the insensitivity to erythromycin and the sensitivity to spectinomycin, then the mutant △sloR was obtained through PCR tests and other verifications.2. Biological characteristics research of the mutant △sloR of sloR geneA series biological characteristics of mutant △sloR and parent strain SC-19 were carried out, including the genetic stability of the mutant, measured difference in growth between the two strains under different culture conditions, mutant hemolytic activity change, the determination of the median lethal dose in mice and the uptake difference on different metal ions of two strains. The result shows that there is no difference in hemolytic activity analysis and no difference under the culture condition of TSB between two strains. While △sloR has a higher manganese ion concentration than the parent strain according to the ICP-MS analysis under CDM culture condition. The result of median lethal dose in mice displays the difference between mutant and parent strain insignificant. These description indicates that SloR does not directly regulate the expression of the virulence of S.suis 2. Not only SloR regulates the uptake of manganese ion, it affects many metabolic activity of the cell.3. The prokaryotic expression and purification of SloR and the investigation of SloR regulation mechanismThe sloR gene was amplified with the template of SC-19 strain and referring to the 05ZYH33 genome sequence of S.suis 2 and cloned into the expression vector pET-30a（+）. The prokaryotic expression plasmid pET30a-sloR was constructed and then transformed into E.coli expression strain BL21. After induced expression, the target protein was obtained.According to the reported reference, some target genes were found and regulated by SloR in Streptococcus mutans（S.mutans）, the homologous genes were found in S.suis 2. These genes may be related to the target genes regulated by SloR in S.suis 2. The promoters of these genes were amplified and carried out in "electrophoresis gel mobility shift analysis（EMSA） with SloR. The results showed that SloR did not combine with the promoter DNA if there was no manganese ions; after the proper dose manganese ions were added into the reaction, SloR still did not combine with the promoter DNA.For further study of the SloR regulatory mechanism in S.suis 2, the DAP-seq（DNA-Affinity-Purification-Sequencing） method was used to studying transcriptional mechanism. The following was the general process, mixed the purified tag-labeled transcription factor with the genomic fragments and interacted, then the sample flew through the column and only the specific DNA fragments combined with the proteins were captured, then the DNA fragments were enriched and sequenced. Proper method was selected to analyse the obtained data and further verification was needed for the resluts. The principle of DAP-seq is similar to the classic chromatin immunoprecipitation, and the advantage is that the operation in vitro can greatly improve the enrichment recovery efficiency of specific DNA fragments. Several times have been attempped, and the approach needs further validation and improvement.