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Studies on GlnA and GlnR Regulates TCA Cycle Enzymes of Streptococcus Suis Serotype 2

Author: ChangHaiTao
Tutor: ChenHuanChun；BeiWeiCheng
School: Huazhong Agricultural University
Course: Preventive Veterinary Medicine
Keywords: Streptococcus suis type 2 GlnA and GlnR TCA cycle Aconitase
CLC: S852.611
Type: Master's thesis
Year: 2011
Downloads: 28
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Abstract

Streptococcus suis type 2 (Streptococcus suis Serotype 2, S. suis 2) gene encoding glutamine synthetase and its transcriptional regulator of glnA and glnR. CD1 mice were infected by constructing the glnA and glnR, the gene deletion the strains ΔGlnA and ΔGlnR found mutant ΔGlnR and ΔGlnA virulence has declined and the ΔGlnA virulence decreased very significantly in the number of viable cells of various tissues and organs with the wild-type strain phase ratio was significantly reduced. These results indicate that the glutamine synthetase Streptococcus suis type 2 infection process, in particular, plays an important role in the adhesion and colonization process. Through bioinformatics analysis, aconitase, citrate synthase and isocitrate dehydrogenase operon promoter region found that a the GlnR combination of conservative sequence isocitrate product of α-ketoglutarate dehydrogenase generated glutamic acid but also in the role of glutamate dehydrogenase and glutamine metabolism. Therefore, we hypothesized that certain glutamine metabolism and the citric acid cycle (TCA cycle). Pigs, Streptococcus type 2 SC19 for the parent strain, carry out the following tasks: 1. GlnR GlnA original nuclear expression, purified and GlnA of in vitro fixed-point mutation reference NCBI's Genbank pig Hammer type 2 05ZYH33 genetic group sequence, to SC19's genetic group DNA as a template, PCR amplification the glnA and glnR of genes amplified genes connected to the prokaryotic expression vector pET28a constructed expression plasmid pET28a-glnR was pET28a-glnA. The predicted the possible key amino acids GlnA sites and mediated by overlap extension PCR directed mutagenesis GlnA the 54,67,133 and 308 amino acids in a site-directed mutagenesis, the same prokaryotic expression vector was transformed into E. coli strain BL21 induced expression, to give the target protein. The purified and detecting the target protein the GlnA and amino acid sites the mutation GlnA enzyme activity, enzyme activity is lost after the 67 and 133 amino acid mutations is GlnA Key Enzymes loci. The the GlnR and GlnA of regulation of the TCA cycle enzymes using real-time fluorescence quantitative PCR detection ΔGlnR the the AGlnA mutant and WT wild strain TCA cycle aconitase, citrate synthase and isocitrate dehydrogenase gene expression situation. The results showed: aconitase, citrate synthase and isocitrate dehydrogenase ΔGlnR, AGlnA expression than WT. PCR amplification of the promoter of the operon, a site-directed mutagenesis and DNA binding GlnR found GlnR with the conserved regions of the promoter binding by electrophoretic mobility shift assay, and the binding does not depend GlnA interaction. Streptococcus suis type 2 aconitase the Construction of the mutant gene AAcnA the PCR amplification of the the aconitase gene upstream fragment, downstream fragment and erythromycin resistance gene upstream fragment, erythromycin resistance The genes downstream fragment order to be connected to the the Thermosensitive the suicide plasmid pSET4s on, get the recombinant plasmid pSET4s-AcnA. Transformation of the recombinant plasmid pSET4s-AcnA electric SC19 competent cells and spectinomycin sensitive to erythromycin sensitive colonies screened through resistance and temperature to confirm mutant ΔAcnA then PCR, RT-PCR and sequencing successfully constructed. The 4 mutant ΔAcnA Biological Characteristics of the mutants of ΔAcnA and WT series of biological characteristics (genetic stability, growth curves, morphology, hemolytic activity, cells infected experimental mice and the median lethal dose). The results show that ΔAcnA late log phase growth rate has slowed down, increased adhesion and invasive capacity of Hep-2 cells, and other features compared with the wild-type strain was no significant difference, indicating that the the aconitase pig chain aureus virulence of type 2 has no effect. These the results indicate GlnA and GlnR of Streptococcus suis type 2 virulence is not to play a role in regulation of the TCA cycle.

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