Common Distribution of gad Operon in Lactobacillus brevis and its GadA Contributes to Efficient GABA Synthesis toward Cytosolic Near-Neutral pH

Abstract

Many strains of lactic acid bacteria (LAB) and bifidobacteria have exhibited strain-specific capacity to produce γ-aminobutyric acid (GABA) via their glutamic acid decarboxylase (GAD) system, which is one of amino acid-dependent acid resistance (AR) systems in bacteria. However, the linkage between bacterial AR and GABA production capacity has not been well established. Meanwhile, limited evidence has been provided to the global diversity of GABA-producing LAB and bifidobacteria, and their mechanisms of efficient GABA synthesis. In this study, genomic survey identified common distribution of gad operon-encoded GAD system in Lactobacillus brevis for its GABA production among varying species of LAB and bifidobacteria. Importantly, among four commonly distributed amino acid-dependent AR systems in Lb. brevis, its GAD system was a major contributor to maintain cytosolic pH homeostasis by consuming protons via GABA synthesis. This highlights that Lb. brevis applies GAD system as the main strategy against extracellular and intracellular acidification demonstrating its high capacity of GABA production. In addition, the abundant GadA retained its activity toward near-neutral pH (pH 5.5-6.5) of cytosolic acidity thus contributing to efficient GABA synthesis in Lb. brevis. This is the first global report illustrating species-specific characteristic and mechanism of efficient GABA synthesis in Lb. brevis.

Keywords: Lactobacillus brevis; acid resistance; genomic survey; glutamic acid decarboxylase; γ-aminobutyric acid (GABA).

Figure 1

Common distribution and arrangement of gad operon and genes encoding glutamic acid decarboxylase in the genomes of Lb. brevis. (A) General GABA production in bacteria from GAD pathway and putrescine degradation pathways (Puu pathway and ADC pathway). (B) Phylogeny of amino acids sequences of four components in gad operon demonstrating two isoforms of glutamate decarboxylases and the highly-conserved four genetic components in Lb. brevis. (C) Common distribution of gad operon in all the sequenced strains of Lb. brevis. Denotations: GABA-AT, GABA aminotransferase; SSADH, succinic semialdehyde dehydrogenase; gadA, glutamate decarboxylase isoform A; gadB, glutamate decarboxylase isoform B, gadR, transcriptional regulator; gadC, Glu/GABA antiporter. The phylogenetic tree was generated from MEGA (version 6.0) after MUSCLE alignment of amino acids sequences of each component in gad operon. The length of each component (locus tag indicated) in gad operon is indicated in the braces. The chromosome of a model strain Lb. brevis NPS-QW-145 was completely sequenced in this study and its NCBI accession no. is CP015398. All gene loci and genome data were collected from NCBI genome database (genome assembly and annotation report) on 10 January 2016.

Figure 2

GAD system in Lb. brevis improves cell viability by maintaining intracellular pH homeostasis. (A) Carbohydrate metabolism and amino acid-dependent acid resistance (AR) systems in the model strains Lb. brevis 145. (B) Effect of amino acid-dependent ARs' substrates on survival rate of Lb. brevis cells (12-h cultures; acid-adapted cells) during acid resistance assay (37°C and 2-h incubation) carried out in Lactobacilli MRS medium (pH 2.5). (C) Effect of amino acid-dependent ARs' substrates on intracellular pH (pH_in) of Lb. brevis cells (3-h cultures; non-acid-adapted cells) upon acid challenge tested at 37°C (extracellular pH–pH_ex decreased from pH 6.5 to pH 3.5. Glutamate, arginine and agmatine were dissolved in PBS buffer (pH 3.5) and tyrosine was dissolved in 0.1 M hydrochloric acid (HCl; after addition of tyrosine, pH_in of the cell was out of detection range but was still calculated from the equation of standard curve (pH_in = −0.1141 × ${\text{RFU}}_{488/435}^2$ + 1.4035 × RFU_488/435 + 2.6307; R² = 0.9849; pH range: 3.5–7.0) of pH and RFU_488/435 (RFU, relative fluorescence units). Cells were suspended in phosphate-buffered saline but not citrate-based buffer for pH_in measurements ranging from pH 3.5 to pH 7.0. Denotations: GAD, glutamate decarboxylase; TDC, tyrosine decarboxylase; PTC, putrescine carbamoyltransferase; OTC, ornithine carbamoyltransferase; ADI, arginine deiminase; AgDI, agmatine deiminase; CK, carbamate kinase; TR, transcriptional regulator; A/O, arginine/ornithine antiporter; Ag/P, agmatine/putrescine antiporter; Glu/GABA, glutamate/GABA antiporter. Experiments were performed in triplicates and data is presented as mean ± standard derivation (SD). ^*p < 0.05; n.s., not significant.

Figure 3

GAD system is a major contributor for acid resistance of Lb. brevis. (A) Growth curve of Lb. brevis in lactobacilli MRS medium supplemented with extra glutamate, arginine, agmatine and tyrosine. (B) Lactic acid and acetic acid production. (C) Intracellular pH (pH_in; cFDA-SE as the probe) and extracellular pH (pH_ex) of Lb. brevis cells incubated in lactobacilli MRS medium with (ARS) or without (as control) amino acid-dependent ARs' substrates. (D) Relative gene expression of key genes of different amino acid-dependent AR systems in Lb. brevis at 18 h normalized to that at 6 h of cultivation. (E–H) Changes in the concentrations of the end products from each amino acid-dependent AR system. The experiment was carried out in triplicates and data was presented as mean ± standard derivation (SD).

Figure 4

GadA supports GABA synthesis in Lb. brevis toward a weak pH range of cytosolic acidity. (A) Temperature effect on Gads tested under constant pH 4.8. (B) Acidity effect on Gads tested under constant 37°C. (C) Enzyme kinetics tested under their optimal pH and temperature. (D) Activities of wild-type Lb. brevis GadA and its mutants. (E) Phylogeny of representative Gads from LAB and Bifidobacterium. The well-characterized Lb. plantarum GadB was used as a reference Gad. The phylogenetic tree was generated from MEGA (version 6.0) after MUSCLE alignment of amino acids sequences of Gads. GraphPad Prism version 6.0 was used to generate kinetic curves of three Gads. The enzyme assay was carried out in duplicates and data is presented as mean ± standard derivation (SD). ^*p < 0.05.