GlnR Negatively Regulates Glutamate-Dependent Acid Resistance in Lactobacillus brevis

Abstract

Lactic acid bacteria often encounter a variety of multiple stresses in their natural and industrial fermentation environments. The 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 mutant) achieved a titer of 284.7 g/liter GABA, which is 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 the glutamate decarboxylase-encoding gene (gadB), glutamate/GABA antiporter-encoding gene (gadC), glutamine synthetase-encoding gene (glnA), and 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 inhibiting 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 acid-producing features. Several acid resistance mechanisms, such as the glutamate decarboxylase system, F1Fo-ATPase proton pump, and alkali production, are usually employed to relieve growth inhibition caused by acids. The 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 mechanism for GlnR's regulation of 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.

A Feedback Regulatory Loop Containing McdR and WhiB2 Controls Cell Division and DNA Repair in Mycobacteria

“…Most MerR proteins respond to metal ions, antibiotics, or drug-like compounds and activate the transcription of detoxification-related genes to eliminate the toxicity of substances ( 15 , 16 ). However, some MerR proteins, such as HonC and GlnR ( 17 , 18 ), inhibit the expression of their target genes, indicating an alternative regulatory mechanism of MerR proteins. Rv1830 is a MerR family protein that has been characterized as an essential regulator in M. tuberculosis ( 9 , 10 , 19 ), but its regulatory role has not been characterized.…”

Molecular Analysis of Glutamate Decarboxylases in Enterococcus avium

“…The GAD system plays important roles in GABA biosynthesis and acid tolerance (De Biase et al, 1999;Cotter et al, 2005;De Biase and Pennacchietti, 2012;Feehily et al, 2014;Gong et al, 2020). Recently, most studies have focused on the distribution and biophysiological function of the GAD system in food-grade lactic acid bacteria (e.g., L. brevis, L. plantarum, L. reuteri, Lc.…”

“…The proton-consuming decarboxylation of glutamate to GABA, which is then exported out of the cell, increases bacterial tolerance to acid stress while maintaining cell viability in acidic environments (Small and Waterman, 1998;Shin et al, 2014;Krumbeck et al, 2016;Yunes et al, 2016;Lyu et al, 2018;Bajic et al, 2019;Gong et al, 2019). Therefore, the GAD system plays an important role in resistance to acid stress in many types of bacteria (De Biase et al, 1999;Cotter et al, 2005;De Biase and Pennacchietti, 2012;Feehily et al, 2014;Gong et al, 2020).…”

Lactobacilli and human dental caries: more than mechanical retention

“…Various factors have been shown to play a role in regulation of acid-tolerance response, including GadR, GlnR and two-component signal transduction systems [60][61][62]. GadR in L. brevis functions as a positive regulator of GAD and glutamate/GABA antiporter, thus the GABA conversion from glutamate, and is expressed at a much higher level in the strains isolated from acidic habitats [61].…”