Lactic acid bacteria often encounter a variety of multiple stresses in their natural and industrial fermentation environments. Glutamate decarboxylase (GAD) system is one of the most important acid resistance systems in lactic acid bacteria. In this study, we demonstrated that GlnR, a nitrogen regulator in Gram-positive bacteria, directly modulated -aminobutyric acid (GABA) conversion from glutamate and was involved in glutamate-dependent acid resistance in Lactobacillus brevis. The glnR-deletion strain (glnR) achieved a titer of 284.7 g/L GABA, a 9.8-fold higher than that of the wild-type strain. The cell survival of the glnR-deletion strain was significantly higher than that of the wild-type strain under the condition of acid challenge and was positively correlated with initial glutamate concentration and GABA production. Quantitative reverse transcription PCR assays demonstrated that GlnR inhibited the transcription of glutamate decarboxylase encoding gene (gadB), glutamate/GABA antiporter encoding gene (gadC), glutamine synthetase encoding gene (glnA) as well as the specific transcriptional regulator encoding gene (gadR) involved in gadCB operon regulation. Moreover, GABA production and glutamate-dependent acid resistance were absolutely abolished in the gadR-glnR-deletion strain. Electrophoretic mobility shift and DNase I footprinting assays revealed that GlnR directly bound to the 5'-untranslated regions of the gadR gene and gadCB operon, thus inhibited their transcription. These results revealed a novel regulatory mechanism of GlnR on glutamate-dependent acid resistance in Lactobacillus.

IMPORTANCE Free-living lactic acid bacteria often encounter acid stresses because of their organic acids producing features. Several acid resistant mechanisms, such as glutamate decarboxylase system, F₁F₀-ATPase proton pump and alkali production, are usually employed to relieve growth inhibition caused by acids. Glutamate decarboxylase system is vital for GAD-containing lactic acid bacteria to protect cells from DNA damage, enzyme inactivation and product yield loss in acidic habitats. In this study, we found that a MerR-type regulator GlnR was involved in glutamate-dependent acid resistance by directly regulating the transcription of the gadR gene and gadCB operon, resulting in an inhibition of GABA conversion from glutamate in L. brevis. This study represents a novel regulatory mechanism of GlnR to glutamate-dependent acid resistance and also provides a simple and novel strategy to engineer Lactobacillus strains to elevate their acid resistance as well as GABA conversion from glutamate.

乳酸菌在其自然和工业发酵环境中经常会遇到多种多重压力。谷氨酸脱羧酶（GAD）系统是乳酸菌中最重要的耐酸系统之一。在这项研究中，我们证明了革兰氏阳性细菌中的氮调节剂GlnR可直接调节谷氨酸的-氨基丁酸（GABA）转化，并参与了短乳杆菌中谷氨酸依赖的耐酸性。所述GLN R-缺失菌株（GLN R）来实现的284.7克/ L GABA，比野生型菌株的9.8倍高的效价。gln的细胞存活在酸攻击条件下，R-缺失菌株显着高于野生型菌株，并且与初始谷氨酸浓度和GABA产生正相关。定量逆转录PCR分析表明，GlnR抑制了谷氨酸脱羧酶编码基因（gad B），谷氨酸/ GABA反转运蛋白编码基因（gad C），谷氨酰胺合成酶编码基因（gln A）以及特异的转录调节因子编码基因（gad R）参与了gad CB操纵子调控。而且，gad R- gln中的GABA产生和谷氨酸依赖性的抗酸性被完全废除。R删除株。电泳迁移率变化和DNase I足迹测定表明，GlnR直接与gad R基因和gad CB操纵子的5'-非翻译区结合，从而抑制了它们的转录。这些结果揭示了GlnR对乳杆菌中谷氨酸依赖性酸抗性的新调节机制。

重要信息自由活动的乳酸菌由于会产生有机酸，因此经常会遇到酸胁迫。通常采用几种抗酸机理，例如谷氨酸脱羧酶系统，F ₁ F ₀ -ATPase质子泵和碱产生，来缓解由酸引起的生长抑制。谷氨酸脱羧酶系统对于含GAD的乳酸菌至关重要，可以保护细胞免受酸性环境中DNA的破坏，酶的失活和产品产量的损失。在这项研究中，我们发现，一个MERR型调节器GlnR是通过直接调节的转录涉及谷氨酸-依赖性耐酸性GAD受体基因和GADCB操纵子，导致短乳杆菌中谷氨酸的GABA转化受到抑制。这项研究代表了GlnR对谷氨酸依赖性酸抗性的新调节机制，并且还提供了一种工程改造乳杆菌菌株以提高其酸抗性以及从谷氨酸转化GABA的简单新颖的策略。