Studies On GlnA And GlnR Regulates TCA Cycle Enzymes Of Streptococcus Suis Serotype 2

Glutamine synthetase gene and its regulatory factors of Streptococcus suis serotype 2 (S. suis 2) are glnA and glnR. GlnR and glnA gene deletion mutantΔGlnA andΔGlnR were constructed to infect CD1 mice. The date showed thatΔGlnA andΔGlnR were attenuated andΔGlnA was significant decreased in virulence. The numbers of viableΔGlnA in various organs decreased significantly compared with wild strains. These results suggested that glutamine metabolism played an important role in the infection process of S. suis 2, particularly in the adhesion and colonization. The GlnR binding conserved sequences were found in the promoter region of the operon of aconitase, citrate synthase and isocitrate dehydrogenase, interstingly the product of isocitrate dehydrogenase is a-ketoglutarate and it can be generated into glutamine under the catalysis of glutamate dehydrogenase. We speculated that there was a close link between the glutamine metabolism and TCA cycle. In this study, S. suis 2 SC19 was the parent strain.1. The prokaryotic expression and purification of GlnR and GlnA and site-directed mutagenesis of GlnA in vitro.The glnA and glnR were amplified from SC19 genomic DNA respectively according to the reference sequence stated in the Genbank of NCBI (05ZYH33), and cloned into expression plasmid pET28a. The expression plasmids pET28a-GlnR and pET28a-GlnA were constructed. The key amino acid sites of GlnA were predicted and the amino acid sites of 54,67,133,308 were site-directed mutated by the overlap extension PCR. The expression plasmids were constructed. The expression plasmids were transformed into E. coli strain BL21. The proteins were obtained by inducing and expression. The enzyme activity of GlnA and mutated GlnA were tested after purification of proteins. The results showed that the sites of 54 and 67 amino acid mutations of GlnA were no enzyme activity.2. The study of TCA cycle enzymes were regulated by GlnA and GlnR.Real-time PCR was applied to reveal the differential transcription levels of aconitase, citrate synthase and isocitrate dehydrogenase amongΔGlnR,ΔGlnA and WT. The dates showed that these genes' transcription levels ofΔGlnR andΔGlnA were lower than that of WT. The promoter region of the operon of aconitase, citrate synthase and isocitrate dehydrogenase was amplified and the binding conserved region of GlnR was mutated. The results of electrophoretic mobility shift assay (EMSA) showed that GlnR could bind the conserved region of promoter region. However, the combination was not depend GlnA.3. Construction of the S. suis 2 aconitase mutant AAcnAThe upstream and downstream flanking regions of acnA and the erythromycin resistance gene were separately amplified respectively. The upstream, erythromycin resistance gene and downstream were cloned into pSET4s vector and obtained the resultant plasmids pSET4s-AcnA. The pSET4s-AcnA plasmid was transformed into SC19. Screened by resistance and temperature to find the colonies which is sensitive to spectinomycin not to erythromycin, then the colony was further confirmed by PCR and RT-PCR amplification, and DNA sequencing, respectively.4. Biological characteristics study of mutantΔAcnAA series biological characteristics ofΔAcnA and WT, including genetic stability, growth curve, morphology, hemolytic activity, infections to cells and median lethal dose to mice were tested. The results demonstrated thatΔAcnA could inhibits its post-exponential-phase growth and enhances the adherence and invasion to Hep-2 cell and not affet the virulence of S. suis 2. These results indicated that the affects to S. suis 2 virulence of GlnA and GlnR were not related to TCA cycle.