GlnR-mediated Regulation For Nitrogen Metabolism In Lactobacillus Brevis

γ-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in themammalian brain and has has several well-known physiological functions. Four strainswere isolated from traditional pickles.They were anerobic, gram-positive and rod-shaped.According to the partial16S rDNA of the strains, three of them were Lactobacillus brevisand the other one is Lactobacillus plantarum. As an important strain of lactobacillus,Lactobacillus brevis has been widely used in food and medicine industries.GlnR is one of the global transcriptional regulators for nitrogen metabolism that canbe found in most Gram-positive bacteria. The GlnR belongs to the OmpR wingedhelix-turn-helix family as well as MerR family. GlnR activates or represses expression ofvarious genes involved in nitrogen metabolism in response to changes in nitrogenavailability. We focused on the global regulator GlnR-mediated nitrogen metabolismregulation in the highest γ-aminobutyric acid-producing strain Lactobacillus brevis BS6, aswell as the relationship between GlnR and the production of GABA.glnRA、 amtB-glnK and glnPQ operons play a crucial role in central nitrogenmetabolism. With the genomic DNA as template, the full-length sequence of Lactobacillusbrevis BS6glnR gene, and the promoter sequence containing GlnR box of glnRA,amtB-glnK and glnPQ operons were obtained by PCR. By adopting the method of bacterialone hybrid, the results proved the combination between GlnR and the promoter of the threeoperons in vivo, which indicated GlnR played a significant role in nitrogen regulation of L.brevis. The relative expression of glnR and genes regulated by GlnR under differentnitrogen source concentrations were analyzed by real time quantitative PCR. Our resultsshowed that GlnR may inhibit the expression of the three operons under nitrogen excessconditions.GAD catalyzes the decarboxylation of L-glutamate to GABA.The GAD activity andGABA production of4Lactobacillus brevis strains were determine by Berthelotcolorimetric and pre-staining paper chromatography.The results indicated that GABAproduction was positively correlated with GAD activity.Upstream sequences of gadCBoperon were obtained by Tail-PCR.In addition,another gltX gene identified belonging togadCB operon was comfirmed by RT-PCR. The full-length sequence of gadCB operon was5663bp. Degenerate PCR results showed that GabT coding gene was absent inLactobacillus brevis BS6, which also demonstrateed the importance of GAD in thesynthesis of GABA.To explore the regulation of GlnR on GABA production, the glnRknockout vector pUCnR was constructed, which can bu used for establishing a△glnRmutant.This may contribute to elucidating the relation between GlnR and GABAproduction at the molecular level.