Study On The Mechanism Of Global Transcription Regulator Fnr Involved In The Degradation Of Nitrite By Lactobacillus Plantarum WU14

During the pickling process of pickles and kimchi,nitrite pollution is easy to occur,and strong carcinogenic N-nitroso compounds such as nitrosamines are generated.Long-term intake may cause mutagenic and carcinogenic risks.Lactobacillus plantarum is widely present in various pickles and pickles.Its main metabolite lactic acid can replace NaNO2 as a food preservative,and it can also produce nitrite reductase(Nir)degradation sauce NaNO2 in pickles and pickles.Nitrite reductase is an oxidoreductase that can reduce nitrite to NO and NH3,reduce the content of NaNO2,and reduce the harm to the human body.The expression of this enzyme is regulated by a variety of transcription factors such as GlnR.The transcription factors Fumarate and Nitrate Reduction Protein(Fnr)are upstream of the bacterial transcriptional regulatory network,but the mechanism of direct or indirect regulation of Nir is unknown.Studying the regulatory mechanism of Fnr on Nir degradation of nitrite is of great significance for speeding up the search for safe and effective nitrite biodegradation methods and ensuring national food safety.In this study,the relative expression levels of fnr and nir of L.plantarum WU14 with high nitrite reductase Nir production under NaNO2 stress were analyzed by real-time fluorescence quantitative PCR(qRT-PCR),and gel resistance Hysteresis experiment(EMS A)verified the interaction of WU14 Fnr protein with glnR and nir genes in vitro.The main findings are as follows:1.The fnr and glnR genes were connected to the pGEX-6p-1 expression vector,and heterologous expression was successfully achieved in E.coli Transetta.The purified Fnr and GlnR proteins were obtained by GST tags;2.The interaction between the regulatory proteins Fnr/GlnR and PglnR/Pnir promoter was verified in vitro using EMSA experiments.The results show that GlnR can specifically bind to the Pnir promoter,while Fnr can specifically bind to PglnR and Pnir promoter Under nitrite stress,Fnr of L.plantarum WU14 can directly regulate the transcriptional expression of Nir,or indirectly regulate the transcriptional expression of Nir through GlnR;3.The relative expression levels of fnr and nir transcription levels of L.plantarum WU14 strain under aerobic/anaerobic and NaNO2 stress conditions were analyzed by qRTPCR.The results showed that the expression levels of fnr and nir under anaerobic conditions were slightly higher than those under aerobic conditions when cultured for 6-12 h,and the addition of NaNO2 during aerobic culture could increase the expression levels of the two genes.This study aims to reveal the regulatory mechanism of nitrite degradation by L.plantarum WU14.The experimental results show that the expression of nitrite reductase Nir is regulated by Fnr protein.