GlnR negatively regulates glutamate-dependent acid resistance in Lactobacillus brevis

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 γ-aminobutyric acid (GABA) conversion from glutamate and was involved in glutamate-dependent acid resistance in Lactobacillus brevis. The glnR-deletion strain (ΔglnR) 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.