Study About The Effection Of CodY Protein On Nitrogen Metabolism In Streptococcus Thermophilus

Streptococcus thermophilus has an important role in the formation of the yogurt texture andflavor as a key part of the yoghurt culture. Strains need plenty of carbon, nitrogen supply when theyare growing, so using of carbon and nitrogen sources of srrains is to become an important mean toimprove the growth of strains. For less regulation of nitrogen metabolism in Streptococcusthermophilus that have been reported, majority of data is inferred from Lactococcus lactis. CodY isa global transcription factor in Gram-positive bacteria. Over200genes can be regulated inexpression levels. These genes generally are suppressed when the logarithmic growth period, whenthe lack of nutrients into the stable and then be induced to help the cells adapt to the period ofnutrient deprivation. According to published Streptococcus thermophilus genomic information（NC₀06449and NC₀08532） inferred, Streptococcus thermophilus should contain a similarregulatory proteins CodY as fermentation agent in yogurt. Therefore, CodY protein of overexpression was built in this study as Streptococcus thermophilus mutant for analysising themechanism of the protein in streptococcus thermophilic. Trying to find out the effecttion of CodYprotein on nitrogen metabolism of streptococcus thermophilic.According to the analysis of endogenous plasmids of thermophilic streptococcus KLDS3.0503in this study and confirm it can be used as a host cell, and CodY genes was amplified out bydesigning the corresponding primers. Constructing over expression of CodY plasmid andself-joined plasmid,and both were successfully electrical transformed into no plasmid strainsKLDS3.0503. Using expression vector pNZCodY that have been built and was induced by Nisin,the protein got efficient expression in Lactococcus lactis NZ9000. His-CodY was successfullyexpressed in L. lactis NZ9000and purified by affinity chromatography using a HisTrap FFcolumn.Getting the change trend of the OD values by SDS-PAGE protein electrophoresis.Findout genes for prtM, amid, ilvA, ilvC, ilvD1, gdhA, codY,bcaT,argF, pepA,pepS, pepN andpepC according to the complete genome sequence in the NCBI in Streptococcus thermophilus ontheir nitrogen metabolism,though RT-Real Time PCR, comparing thirteen genes associated withnitrogen metabolism and instructed that the CodY protein is able to control the expression of thesegenes.This study got following conclusions:1. Identified the Streptococcus thermophilus KLDS3.0503through the16SrRNA gene sequence determination, and confirmed which may be used as host cells by plasmid analysis.2. CodY gene fragment was successfully cloned from Streptococcus thermophilusKLDS3.0503.3. Excessive expression vector and build self-serialized body got copy in Streptococcusthermophilus successfully, and both the growth trend OD value monitoring, derived the codY genecan regulate the growth trend of the original strain.4. Determine the the CodY protein expression differences significantly by SDS-PAGE proteinelectrophoresis, and the existing CodY protein was purified and identified the two coincide.5. By quantitative PCR method, derived that relative expression levels of prtM、amiD、ilvA、ilvC、ilvD1、gdhA、bcaT、argF、pepS、pepC have significantly reduced only pepA, pepN expressionlevels increased slightly, codY gene is overexpression. It explained that Cody protein can regulatethirteen genes in nitrogen metabolism in Streptococcus thermophilus.