Common Distribution ofgad Operon inLactobacillus brevis and its GadA Contributes to Efficient GABA Synthesis toward Cytosolic Near-Neutral pH

Qinglong Wu¹Hein Min Tun²Yee-Song Law¹Ehsan Khafipour^2,3Nagendra P. Shah^1,4*

• ¹School of Biological Sciences, The University of Hong Kong, Hong Kong, Hong Kong
• ²Department of Animal Science, University of Manitoba, Winnipeg, MB, Canada
• ³Department of Medical Microbiology, University of Manitoba, Winnipeg, MB, Canada
• ⁴Victoria University, Melbourne, VIC, Australia

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 ofgad operon-encoded GAD system inLactobacillus brevis for its GABA production among varying species of LAB and bifidobacteria. Importantly, among four commonly distributed amino acid-dependent AR systems inLb. brevis, its GAD system was a major contributor to maintain cytosolic pH homeostasis by consuming protons via GABA synthesis. This highlights thatLb. 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 inLb. brevis. This is the first global report illustrating species-specific characteristic and mechanism of efficient GABA synthesis inLb. brevis.

Introduction

Many species of lactic acid bacteria (LAB) and bifidobacteria are of economic and health importance and have been extensively used for the production of yogurt, cheese, milk beverage, and fermented vegetables for thousands of years (Leroy and De Vuyst, 2004). These bacteria have been considered as potential probiotics for promoting human health (Hill et al., 2014). Interactions between ingested LAB or bifidobacteria and host are associated with immunomodulation (Bron et al., 2012;van Baarlen et al., 2013;Sivan et al., 2015), gut homeostasis maintenance (Gareau et al., 2010;Delzenne et al., 2011), and brain behavior (Tillisch et al., 2013;Möhle et al., 2016). Moreover, metabolites from these bacteria, such as lactate, have also been extensively evaluated for brain function and plasticity promotion (Herzog et al., 2013;Tang et al., 2014;Yang et al., 2014), bacteriocin for killing pathogens (Cotter et al., 2013;Martinez et al., 2013), exopolysaccharide as food texturing agent (Wu et al., 2014) and immune modulator (Fanning et al., 2012). In the past decades, γ-aminobutyric acid (GABA) has drawn a lot of attentions because GABA is the most abundant inhibitory neurotransmitter that maintains neuro functions of human central nervous system. Although GABA may not be able to cross human blood-brain barrier (Kuriyama and Sze, 1971;Boonstra et al., 2015), GABA approved as a food ingredient and its food carriers have shown anti-hypertensive and anti-depressant activities as two main functions to the host after oral administration (Diana et al., 2014;Wu and Shah, 2016). However, GABA content in natural animal and plant products is very low, thus solutions have been sought from microorganisms including LAB and bifidobacteria for their capability of producing GABA.

GABA synthesis via glutamate decarboxylation in bacteria has been associated with acid resistance (Su et al., 2011;De Biase and Pennacchietti, 2012;Teixeira et al., 2014). Glutamic acid decarboxylase (GAD) system encoded by thegad operon is responsible for glutamate decarboxylation and GABA secretion in bacteria and consists of two important elements—Glu/GABA antiporter GadC and GAD enzyme either GadA or GadB (Capitani et al., 2003). This system converts glutamate into GABA and while doing so consumes protons thus maintaining cytosolic pH homeostasis (Krulwich et al., 2011). Our recent review and another previous review summarized enormous reports presenting specific strains of LAB and bifidobacteria having varying capacities to produce GABA (Li and Cao, 2010;Wu and Shah, 2016). This suggests that the efficiency of GAD system in these bacteria depends on their activities of Glu/GABA antiporter and GAD enzyme. Thus, most studies have been carried out to characterize individual strains for their GABA production in varying conditions either controlled or natural fermentations (Li and Cao, 2010;Dhakal et al., 2012;Wu and Shah, 2015,2016). Among these reported strains, we found that isolates ofLactobacillus brevis appear to be efficient for GABA production (Wu and Shah, 2016). Importantly, these GABA-producingLb. brevis strains have different sources of origin such as human intestine, Korean kimchi and brewery, suggesting their varying genetic backgrounds but exhibiting similar GABA synthesis capacities (Wu and Shah, 2016).

Currently, hundreds of species of LAB and bifidobacteria that have been systematically identified, and their genome sequences have been deposited in public databases including DDBJ/EMBL/GenBank databases. Due to the importance and applications of LAB and bifidobacteria in food industry and human health, it is crucial to gain insights into global diversity of GABA-producing bifidobacteria and LAB mainly includingLactobacillus andLactococcus. These bacteria are able to catabolize sugars to produce lactic acid efficiently (Leroy and De Vuyst, 2004), but they have also developed multiple acid resistance systems including F₀F₁-ATPase proton pump, sodium/proton antiporter, amino acid decarboxylation and deimination, alkali production and biofilm formation (Hutkins and Nannen, 1993;Zhao and Houry, 2010;Liu et al., 2015). Since multiple strategies as stated above in these bacteria are available to cope against acids, the linkage between bacterial acid resistance and GABA production capacity derived from GAD system has not been well established. Moreover, it is vital to unravel the mechanism of efficient GABA synthesis in these isolates especially inLb. brevis. In the present study,Lb. brevis NPS-QW-145 (hereafterLb. brevis 145) isolated previously from Korean kimchi was used as a model organism of high GABA-producing lactic acid bacterium (Wu and Shah, 2015). The chromosome of this organism has been completely sequenced in this study; genomic survey and biochemical toolkits have been carried out to illustrate efficient machinery of GABA production from this organism.

Materials and Methods

Bacterial Strains, Cultivation Conditions, and Genome Sequencing

GABA-producingLb. brevis 145 and a reference strain,Lb. plantarum WCFS1, were cultivated in Lactobacilli MRS medium (BD Company, Franklin Lakes, NJ). Unless otherwise stated,Lactobacillus strains used in this study were anaerobically cultivated in the above medium statically. For cloning and protein expression purposes,E. coli strains as indicated in Table1 were cultivated in Luria-Bertani (LB) medium with or without antibiotics supplemented to the medium. The model strain of high GABA-producingLb. brevis 145 was completely sequenced in this study (please refer to Supplementary Materials and Methods for details). The NCBI accession number for the chromosome ofLb. brevis NPS-QW-145 is CP015398.

Table 1. Bacterial strains, plasmids and primers used in this study.

Genomic Survey on the Distribution ofgad Operon and Genes Encoding Glutamate Decarboxylase in LAB and Bifidobacteria

Currently, there are more than a thousand of strains of LAB and bifidobacteria that have been sequenced and deposited in NCBI-GenBank database. Thus, genomic survey was carried out for the presence or absence ofgad operon and genes encoding glutamate decarboxylase in most of the sequenced strains (all assembly levels; 890 strains) ofBifidobacterium and LAB includingLactobacillus, Lactococcus, Leuconostoc, Pedicoccus, Oenococcus, Weissella, andStreptococcus thermophilus released in NCBI-GenBank genome database. Only the species having at least two strains sequenced were included in this genomic survey. In addition, the distribution of acid resistance systems including F₀F₁-ATPase system, amino acid/cation:proton antiporters, glutamic acid decarboxylase (GAD) system, tyrosine decarboxylase (TDC) system, agmatine deiminase (AgDI) system, arginine deiminase (ADI) system and urea system inLb. brevis was also surveyed in terms of presence or absence of above AR systems in the sequenced strains.

Extracellular pH (pH_ex) and Intracellular pH (pH_in) Measurements

Extracellular pH (pH_ex) was measured directly with the pH meter. The fluorescent probe–5(6)-carboxyfluorescein diacetate N-succinimidyl ester (cFDA-SE; Thermo Fisher Scientific) was used to label bacterial cells for intracellular pH (pH_in) measurement. There are several reports on the pH_in measurement of bacteria suspended in citrate-based buffer (Siegumfeldt et al., 2000;Teixeira et al., 2014); however, this buffer did not allow the discrimination between cells equilibrated to pH between 3.5 and 5.0 thus limiting the detection range of cFDA-SE probe (Hansen et al., 2016). Thus, phosphate-buffered saline (PBS) was used to re-suspend bacterial cells for pH_in measurement in the range of pH 3.5–7.0 in this study according to the previous studies (Breeuwer et al., 1996;Hansen et al., 2016). Briefly, the pH_ex of cultures was measured first, and bacterial cells were centrifuged, washed and re-suspended in PBS where its pH level was adjusted to the pH_ex of the cultures. The cell density was adjusted to an optical density (λ = 600) of 0.6–0.7, followed by the addition of both cFDA-SE and glucose to the final concentrations of 10 μM and 10 mM, respectively. The mixture was incubated in the dark at 37°C for 30 min. The stained cells were later harvested by centrifugation at 12,000 × g and 4°C for 5 min, and were re-suspended in the PBS buffer with the same pH_ex prior to the measurement.

Fluorescence intensities of the stained cells were measured in the Fluorescence Spectrophotometer F-7000 (Hitachi High-technologies, Shenzhen, China) at the excitation wavelengths of 488 nm (pH-sensitive) and 435 nm (pH-insensitive) by rapidly altering the monochromator between both wavelengths. The emission wavelength was started from 400 to 650 nm. Both excitation and emission slit width was 5 nm. For the calibration curve (pH 3.5–7.0) ofLb. brevis, the stained cells were suspended in PBS buffer having different pH values adjusted by hydrochloric acid (HCl). Valinomycin (0.2 mM in methanol; Sigma) and nigericin (0.2 mM in methanol; Sigma) were added to the final concentrations of 5 μM followed by incubation at 37°C for 10 min resulting in the equilibration of both potassium and proton ions across cell membrane. The equilibrated cells were later measure the same as described above.

Acid Resistance and Challenge Assays

Acid resistance assay was carried out based on previously method described (Seo et al., 2015). Briefly, cells ofLb. brevis 145 grown for 12 h (early stationary phase) in Lactobacilli MRS broth were inoculated into Lactobacilli MRS broth (pH 2.5) supplemented with or without 1 g/L of MSG, arginine, agmatine sulfate or tyrosine. The initial cell density after inoculation for acid challenge was 1.4 × 10⁸ CFU/mL. Cells were incubated at 37°C for 2 h statically. Plate count method was then applied to assess the survival rates ofLb. brevis cells under above conditions. For acid challenge assay, the 3-hLb. brevis cultures (lag phase, not acid-adapted cells) were centrifuged, washed and suspended in PBS buffer (pH 7.0), followed by cFDA-SE staining as described above. The stained cells were then placed in fluorescence spectrophotometer and their fluorescence emission intensities were measured every 5 min at 37°C. Acid challenge was achieved at the point of 5 min by adding hydrochloric acid that changed pH_ex from 7.0 to 3.5. After another 5 min, the stock solution (100 mM) of individual substrate (glutamate, arginine, agmatine, and tyrosine) was added to the final concentration of 10 mM in the solution. The changes in pH_in were recorded accordingly as per the above description.

Growth ofLb. brevis 145 in Lactobacilli MRS Broth Containing Arginine, Glutamate, Agmatine, and Tyrosine

Cells ofLb. brevis 145 grown for 12 h (early stationary phase) in Lactobacilli MRS broth were inoculated into Lactobacilli MRS broth (pH 6.5) supplemented with or without the mixture of MSG, arginine, agmatine sulfate salt and tyrosine (1 g/L each; Sigma). However, tyrosine was partially dissolved in MRS broth (pH 6.5), and the insoluble tyrosine (solid form in the broth) was removed by membrane filtering (0.45 μm). The initial cell density after inoculation for acid challenge was 1.0 × 10⁸ CFU/mL and the cells were incubated at 37°C for 36 h statically. Samples were collected at the time point of 0, 3, 6, 9, 12, 18, 24, and 36 h. Subsequently, measurements of acids, amino acids, amines and gene expression were carried out as discussed below. Plate count method was used to assess the cell viability, and pH_in and pH_ex measurements were carried out as stated above.

Measurements of Acids Production

Acids in the culture broth were analyzed by HPLC (Model Shimadzu LC-2010A, Shimadzu Corporation, Kyoto, Japan) equipped with Aminex HPX-87H column (300 × 7.8 mm; Bio-Rad). Briefly, cell-free supernatants (diluted if necessary) after removal of bacterial cells and passing through Acrodisc^® syringe filter with 0.20 μm Supor^® membrane (Pall, Ann Arbor, MI, USA) were injecting into the HPLC system. Acids were eluted with 5 mM sulfuric acid with a flow of 0.60 mL/min. The temperature of the column was maintained at 50°C and the absorbance of the detector was set to 210 nm for detection.

Measurements of Amino Acids and Amines

Amino acids including glutamic acid, GABA, arginine, ornithine and tyrosine were separated and quantified as per the method we have previously developed (Wu and Shah, 2016). Briefly, cell-free supernatant was collected after centrifugation of fresh cultures. The derivatization was achieved by adding 200 μL acetonitrile, 200 μL NaHCO₃ (1 M, pH 9.8 adjusted with NaOH), 300 μL distilled water and 100 μL dansyl chloride solution (40 g/L; dissolved in acetonitrile) to 100 μL supernatant (diluted if necessary), then the mixture was kept in an oven (40°C) for 60 min. After derivatization, 100 μL of 20% (v/v) acetic acid was added to stop the reaction and centrifuged at 10,000 × g and 20°C for 2 min. The supernatant was passed through a 0.22 μm membrane filter before HPLC analysis.

Amines including agmatine, putrescine, and tyramine in the supernatants were analyzed as previously described (Dugo et al., 2006). Briefly, 1.6 mL of 10 g/L dansyl chloride solution (dissolved in acetone) was added to 1.5 mL of the supernatant (diluted if necessary). The pH of the mixture was adjusted to pH 8.2–8.3 with 40 g/L of NaCO₃ solution, then the mixture was heated in a water-bath (40°C) for 60 min in dark. After derivatization, acetone was removed under a stream of nitrogen and then the volume was made up to 5 mL with acetonitrile and centrifuged at 1000 × g and 20°C for 2 min. The supernatant was passed through a 0.22 μm membrane filter before HPLC analysis.

Dansyl amino acids and dansyl amines were separated and detected using a previously developed gradient elution mode (Wu et al., 2015) by HPLC (Model Shimadzu LC-2010A, Shimadzu) equipped with a Kromasil 5 μ 100A C18 column (250 × 4.6 mm; Phenomenex). All the standards of amino acids and amines were derivatized and analyzed as the same in the above procedures. The absorbance for dansyl-amino acids and dansyl-amines were recorded at 275 nm and 254 nm, respectively.

Total RNA Extraction and Real-Time Quantitative PCR Assay

Total RNA extraction using hot SDS/phenol method, DNase I treatment, cDNA synthesis, qPCR assay were carried out as per previously described (Wu et al., 2015). The expression of target genes (gadA, gadB, aguB, arcB, andtyrDC inLb. brevis 145;gadB inLb. plantarum WCFS1) listed in Table1 was quantified by real-time qPCR assay. Reference genes includingtuf inLb. brevis andldhD inLb. plantarum listed in Table1 were used to normalize the expression of target genes in both strains. The efficacy of qPCR amplification using each pair of primers was in the range of 90–110%. Comparative critical threshold method (2^−ΔΔCt) was used to calculate the relative gene expression. The qPCR assay was performed in duplicates for each sample and independent experiments were carried out in triplicates.

Glutamate Decarboxylation Assay

For the procedure of cloning, hetero-expression and purification of GadA and GadB fromLb. brevis 145 and GadB fromLb. plantarum WCFS1, please refer to Supplementary Materials and Methods for details. The effects of pH (3.0–6.6) and temperature (30–90°C) on the purified GADs includingLb. plantarum GadB,Lb. brevis GadB, andLb. brevis GadA and its mutants (Lb. brevis GadA Δ5 andLb. brevis GadA +GSHM) were carried out. Low concentrations of PLP such as 20 μM is sufficient to activate the activities of bothLb. plantarum GadB andLb. brevis GadB (Fan et al., 2012;Yu et al., 2012;Shin et al., 2014), thus the reaction mixture consisted of 5 μL of 1 M monosodium glutamate (MSG; Sigma), 5 μL of 2 mM pyridoxal 5′-phosphate (PLP; Sigma), 500 μL of McIlvaine (citrate-phosphate) buffer, and 10 μg of GAD. Previous studies have indicated the PLP- and sulfate ion-dependent activation ofLb. brevis GadA and its homologousLb. zymae GadB (Ueno et al., 1997;Park et al., 2014); thus, forLb. brevis GadA and its mutants, the reaction mixture contained 5 μL of 1 M MSG, 5 μL of 20 mM PLP, 500 μL of sulfate buffer (0.8 M sodium sulfate and 50 mM sodium acetate; different pH), and 10 μg of GAD. The kinetics of these enzymes was determined under their optimal pH and temperature, and the concentrations of substrate (glutamate) ranged from 0.9 to 36 mM.

Statistical Analysis

All presented data in the bar charts and tables correspond to means ± standard deviation. Significant difference was carried out by using IBM SPSS Statistics 20.0 version.

Results

Genomic Survey Identifies Common Distribution ofgad operon inLb. brevis

Currently, there have been two major pathways reported for GABA production either through the degradation of putrescine (Puu and ADC pathways) or via the decarboxylation of glutamate (GAD pathway) in bacteria (Figure1A). However, GABA production from putrescine degradation is not common in LAB and bifidobacteria due to the absence of Puu and ADC pathways. Interestingly, genes (gadA andgadB) encoding glutamate decarboxylases (GadA and GadB) and an intactgad operon includinggadR (regulator),gadA andgadC (Glu/GABA antiporter) have been identified in all of the sequenced strains ofLb. brevis having varying origins excluding the one of strain WK12 with incomplete (contig levels) genome sequence (Figure1C). The location ofgadB is far away fromgad operon inLb. brevis suggesting there may be two different gene regulations forgadA andgadB in this species. However, arrangement ofgadA andgadC close to each other in the genome could ensure the timely co-regulation of transcription and translation ofgadA andgadC for GABA production (De Biase and Pennacchietti, 2012). Moreover, phylogenetic analysis of four gene components (gadR, gadC, gadA, andgadB) indicated the presence of two isoforms of glutamate decarboxylases, GadA and GadB, inLb. brevis; all of the four components from varyingLb. brevis strains are highly conserved in this species (Figure1B). This implies that co-existence ofgadA andgadB inLb. brevis contributes to its GABA synthesizing capacity.

Figure 1. Common distribution and arrangement ofgad operon and genes encoding glutamic acid decarboxylase in the genomes ofLb. 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 ingad operon demonstrating two isoforms of glutamate decarboxylases and the highly-conserved four genetic components inLb. brevis.(C) Common distribution ofgad operon in all the sequenced strains ofLb. 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 ingad operon. The length of each component (locus tag indicated) ingad operon is indicated in the braces. The chromosome of a model strainLb. 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.

Global Diversity of GABA-Producing LAB and Bifidobacteria

In order to gain a global view on the distribution ofgad operon and genes encoding glutamate decarboxylases in LAB and bifidobacteria, a genomic survey was carried out on the sequenced LAB and bifidobacteria species, of which at least two strains have been sequenced and deposited in GenBank database (accessed on 10 January 2016). As shown in Table2, many strains carrygadA orgadB, butgadC is only present in the genomes of limited strains indicating their strain-specific characteristic of GABA production. Amonggad operon-positive strains,Lb. brevis is the only one species that has close to 100% probability to carry the intactgad operon in their chromosomes. All the sequenced strains ofB. dentium possessedgadBC operon in their chromosomes. However, there is very little information on GABA production fromB. dentium with onlygadB gene, whereas there are several reports on high GABA production fromLb. brevis with twogad genes (Li and Cao, 2010;Dhakal et al., 2012;Wu and Shah, 2016). Thus, the present study focused on onlyLb. brevis for its efficient GABA synthesis. Moreover, it was found that other common reported GABA-producing species such asLb. reuteri andLc. lactis showed strain-specific GABA biosynthesis at the genetic level.

Table 2. Distribution ofgad operon and genes encoding glutamate decarboxylases in the sequenced lactic acid bacteria and bifidobacteria.

Although, there are several reports on GABA-producingLb. plantarum without sequencing their genomes (Li and Cao, 2010;Wu and Shah, 2016), in this study we found thatgadC is absent in all the sequenced strains ofLb. plantarum (Table2). Hence, a fermentation study was carried out using the sequencedLb. plantarum WCFS1 as a model organism. Although, there was an incomplete pathway for GABA metabolism in this organism based on KEGG pathway (Figures S1A,B), its viable counts,gadB transcripts level, acid profiles and GABA production did not significantly change after glutamate supplementation to the medium (Figures S1C–H). AlthoughgadB transcript was increased in the stationary phase suggesting an enhanced GABA synthesis,gadC in LAB is required and specific for exchanging extracellular glutamate with intracellular GABA (Figure S1D). Our cultivation experiment onLb. plantarum WCFS1 complied with previous gene knock-out studies in other bacteria with intactgad operon that GadC is an important element for GABA production (Cotter et al., 2001;Lu et al., 2013).

Diverse and Constant Acid Resistance Systems inLb. brevis

A genomic survey on the distribution of amino acid-dependent AR systems in all the sequenced strains ofLb. brevis was carried out. Except for universal amino acid-dependent AR systems including F₀F₁-ATPase system and amino acid/cation:proton antiporter, extra amino acid-dependent AR systems including GAD system, tyrosine decarboxylase (TDC) system, agmatine deiminase (AgDI) system and arginine deiminase (ADI) system are commonly present in all sequenced strains ofLb. brevis (Figure2A and Table3).

Figure 2. GAD system inLb. brevis improves cell viability by maintaining intracellular pH homeostasis. (A) Carbohydrate metabolism and amino acid-dependent acid resistance (AR) systems in the model strainsLb. brevis 145.(B) Effect of amino acid-dependent ARs' substrates on survival rate ofLb. 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) ofLb. 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.

Table 3. Acid resistance systems in the sequencedLactobacillus brevis based on NCBI annotated protein database.

Activation of GAD System inLb. brevis Upon Challenge with Exogenous Acid

Although, the contribution of AR systems for acid resistance in other LAB such asLb. reuteri has been characterized (Teixeira et al., 2014), individual contribution of above AR systems to resist exogenous acids inLb. brevis has not been well demonstrated. In this study, it was found that both glutamate and arginine were more effective in increasing the survivability ofLb. brevis significantly (p < 0.05) when exposed to acidic condition (pH 2.5; HCl-adjusted) for 2 h than that by agmatine and tyrosine (Figure2B). Moreover, both glutamate and arginine were also able to increase the intracellular pH ofLb. brevis significantly (p < 0.05) than agmatine and tyrosine after challenging cells with hydrochloric acid (Figure2C). This suggests that glutamate and arginine increased cell viability ofLb. brevis by increasing intracellular pH of the cell thus maintaining its metabolic activity. However, the increased level of intracellular pH ofLb. brevis cells by arginine and glutamate differed greatly. This may be due to the stages of cells selected for acid resistance experiment (12-h culture; acid-adapted cells) and acid challenge assay (3-h cultures; non-acid-adapted cells). The stages of cells may affect the contents of key cytosolic enzymes including GadA, GadB, ADI, and OTC from the four amino acid-dependent AR systems (Figure3D). In addition, the increase in the intracellular pH by glutamate may be explained by GABA synthesis that consumed cytosolic protons inLb. brevis upon acid challenge. Since the function of GadC is activated under acidic conditions (Ma et al., 2012), GAD system inLb. brevis may be activated by acid challenge.

Figure 3. GAD system is a major contributor for acid resistance ofLb. brevis. (A) Growth curve ofLb. 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) ofLb. 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 inLb. 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).

GAD System inLb. brevis Is a Major Contributor for Resisting Endogenous Acid

To understand the association between GAD system for endogenous acid (lactic acid) resistance and GABA production capacity ofLb. brevis, a 24-h course of cultivation experiment onLb. brevis 145 in Lactobacilli MRS broth supplemented with four amino acid-dependent AR's substrates (glutamate, arginine, agmatine and tyrosine) was carried out. Firstly, it was observed that the AR's substrates were able to increase the intracellular pH ofLb. brevis cells by about 0.5 compared to that inLb. brevis without the AR's substrates supplementation (Figures3A–C). In addition, results indicate thatLb. brevis maintained its intracellular pH level in the near-neutral range (pH 5.5–6.5) or close to neutral range (pH 6.5–7.0) when extracellular pH decreased spontaneously by secreting protons during its lactic acid production (Figures3B,C). Moreover, gene transcription ofgadB andaguB increased by at least 2 fold whereasgadA mRNA was highly up-regulated by about 55 fold at the point of 18 h (stationary phase) when compared to that at 6 h (logarithmic phase) (Figure3D). In combination with the higher rate of GABA production at 18 h than that at 6 h (Figure3G), it appears that GAD system was more active in stationary phase than that in lag and logarithmic phases. In general, changes in the end metabolites of four amino acid-dependent AR systems also demonstrated that utilization of the AR's substrates varied with phases. Based on the metabolic data in Figures3E–H and gene expression data in Figure3D, TDC and ADI pathways were active in early cultivation (Figures3E,F) but were down-regulated in late cultivation (Figure3D), while GAD and AgDI pathways were more active in stationary phase (Figures3G,H). Although a gene encoding lysine decarboxylase was found inLb. brevis 145, the lysine content little changed suggesting an inactive state of this AR system (Figure S2).

The acid resistance and challenge assays may not be able to indicate actual contributions of AgDI and TDC pathways for resisting acids (Figures2B,C); however, changes in the content of metabolites during different cultivation stages evidenced that all the four amino acid-dependent AR systems contributed to intracellular pH homeostasis (Figure3C). Since GABA production fromLb. brevis was increased in the stationary and late stages, its GAD system played an important role in bacterial acid resistance because its lactic acid accumulated largely during late cultivation (Figure3B).

GadA Supports GABA Synthesis inLb. brevis toward a Weak pH Range

Two Gads, GadA and GadB, fromLb. brevis and a reference type I GadB fromLb. plantarum were hetero-expressed and purified for enzyme assay (Figure S3). Two mutants ofLb. brevis GadA (wild-type; WT),Lb. brevis GadA +GSHM (extra GSHM to N-terminus) andLb. brevis GadA Δ5 (5 amino acids deleted from N-terminus), were constructed in this study. As shown in Figure4A,Lb. brevis GadA (WT) exhibited a narrow activity spectrum toward the change of temperature, but still retained about 50% of the highest activity at 37°C, whereasLb. brevis GadB andLb. plantarum GadB were heat-stable enzymes. More interestingly,Lb. brevis GadB andLb. plantarum GadB retained similar catalytic spectrum in the range of pH 3.0–5.5 whileLb. brevis GadA (WT) exhibited activity toward a weak acidity range (pH 5.5–6.6) (Figure4B). Thein vitro enzyme kinetic assay (Figure4C and Table4) demonstrated that catalytic efficiency ofLb. brevis GadA (WT) was largely lower than that of two GadB enzymes. However, these three Gads had a very similar K_m value suggesting a similar level of substrate saturation (Table4). Moreover,Lb. brevis GadA (WT) exhibited a significant (p < 0.05) activity than its two mutants with modifications to its N-terminus (Figure4D). Previous gel filtration studies have indicated the tetramer state ofLb. brevis GadA (Hiraga et al., 2008), the monomer state ofLb. brevis GadB (Yu et al., 2012;Shi et al., 2014) whileLb. plantarum GadB function as dimers (Shin et al., 2014). This suggests that N-terminus ofLb. brevis GadA may be critical to the tetramer formation via incorporation to its N-terminus. The phylogenetic analysis of representative Gads from LAB andBifidobacterium highlighted thatLb. brevis GadA was a type III group of Gads which differed largely fromLb. brevis GadB (sub-group 2) andLb. plantarum GadB (sub-group 1) which were classified in type I Gads (Figure4E). Overall,Lb. brevis, as a cell factory, exhibited GABA biosynthesizing capacity in a board range of acidity, especially in near-neutral pH range (pH 5.5–6.5).

Figure 4. GadA supports GABA synthesis inLb. 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-typeLb. brevis GadA and its mutants.(E) Phylogeny of representative Gads from LAB andBifidobacterium. The well-characterizedLb. 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.

Table 4. Kinetic parameters ofLb. brevis GadA,Lb. brevis GadB andLb. plantarum GadB under optimal conditions.

Discussion

GABA synthesis can also be derived from putrescine degradation process via either Puu pathway or arginine decarboxylase (ADC) pathway as shown in Figure1A in some bacterial species such asEscherichia coli (Kurihara et al., 2008,2010); however, based on our KEGG survey on arginine and proline metabolism map of complete sequenced bacterial strains, these two pathways are not commonly present in LAB and bifidobacteria (data not shown). Thus, GABA production from LAB and bifidobacteria is mainly derived from GAD pathway if it is present in them. Due to the important biofunctionalities of GABA and GABA-producing LAB and bifidobacteria, several studies have characterized GABA production from individual strains (Li and Cao, 2010;Dhakal et al., 2012;Diana et al., 2014;Wu and Shah, 2016). The diversity of GABA-producing LAB and bifidobacteria has been poorly understood either at genetic level or from experimental insights. Our genomic survey conducted on most of the sequenced strains of LAB and bifidobacteria released in GenBank has clearly indicated that the genetics ofLb. brevis differed with other species of LAB and bifidobacteria in terms of GABA production: (1) an intactgad operon, and (2) two genes (gadA &gadB) encoding glutamate decarboxylases in their genomes (Figure1C and Table2). Thus, GABA production fromLb. brevis appears to be species-specific based on our current genetic observation (Table2) and previously reported GABA yields from various strains ofLb. brevis (Li and Cao, 2010;Dhakal et al., 2012;Wu and Shah, 2016). This differs with the strain-specificity of GABA biosynthesis in other species such asLactococcus lactis andLb. reuteri (Table2). To our knowledge, this is the first global insights into genetic diversity of GABA producer among above food-grade bacteria.

It is clear that GAD system is one of AR systems in bacteria to cope against acidic environments thus allowing them to maintain metabolic potential and cell viability (De Biase and Pennacchietti, 2012). Multiple AR systems including universal and amino acids-based AR systems function in the bacteria, but the contribution of GAD system to bacterial acid resistance has not been clearly demonstrated so far. SinceLactobacillus genus includingLb. brevis is a high lactic acid producer (Leroy and De Vuyst, 2004), excessive intracellular protons from endogenous or exogenous acids can be neutralized or eliminated by their AR systems. Among the four amino acid-dependent AR systems that were commonly identified inLb. brevis, our results indicate the major contribution of GAD system for acid resistance and challenge in this organism upon either short-term or long-term acidic exposure, and either extracellular or intracellular acidification (Figures2,3). For instance, high amount of lactic acid was produced and accumulated in the medium during stationary phase ofLb. brevis cultivation, this organism utilized the highly active GAD system that decarboxylated glutamate into GABA greatly at this stage contributing to the increase in cytosolic pH (Figure3). This highlights thatLb. brevis, a lactic acid producer, applies GAD system as the main strategy for its acid resistance thus reflecting its high capacity of GABA production.

Considering high GABA production fromLb. brevis and its GAD system, it is crucial to understand the mechanism of efficient GABA synthesis for this phenomenon. Several studies have reported individualLb. brevis GadB tested either in acetate buffer or citrate-phosphate buffer exhibiting its activity spectrum toward acidic conditions (pH 3.5–5.5) but not near-neutral pH (pH 5.5–6.5;Yu et al., 2012;Seo et al., 2013;Lin et al., 2014), but the activity of only one GadA fromLb. brevis IFO 12005 has been reported twice focusing on its activation of enzymatic activity duringin vitro conditions (Ueno et al., 1997;Hiraga et al., 2008). However, these studies did not provide systematic and comparative insights into this decarboxylation machinery inLb. brevis. In general,Lb. brevis GadA retained its activity from pH 4.0 to pH 6.6 in the sulfate buffer (Figure4B). This showed similar activity spectrum, particularly in near-neutral pH range (pH 5.5–6.5), with another reportedLb. brevis IFO 12005 GadA tested in pyridine-HCl buffer after sulfate activation and enzyme purification via gel filtration (Ueno et al., 1997;Hiraga et al., 2008). The characteristic ofLb. brevis GadA differs greatly withLb. brevis GadB since the former requires enzyme activation by high concentrations of sulfate (Hiraga et al., 2008). As forLb. brevis GadB, our study and previous reports showed its active activity in a more acidic range (pH 3.0–5.5; Figure4B;Yu et al., 2012;Seo et al., 2013). Taken together, GadA and GadB inLb. brevis cells may help them synthesize GABA toward a broad range of intracellular pH (pH 3.0–6.6).

As shown in Figure3C, intracellular pH ofLb. brevis cells during a standard 24-h course of cultivation in MRS medium was in the range of pH 5.8–6.5. This indicates that glutamate decarboxylation may be very slow due to limited activities of both Gads in this near-neutral pH range (pH 5.5–6.5). Moreover, pH_in ofLb. brevis cells decreased in the late incubation (24–36 h) thus may increase GABA synthesis by the enhanced activities of both GadA and GadB. This may be able to explain why resting cells could be used for GABA production (Zhang et al., 2012). Moreover, transmembrane potential, which contributes to amino acid-dependent acid resistance in bacteria, could be increased by glutamate decarboxylation (Teixeira et al., 2014). The transmembrane ΔpH in the ARS group was less than that in the control after the incubation of 9 h (Figure3C) indicating that transmembrane potential in ARS group may be higher than that in the control; this effect may be attributed by glutamate decarboxylation inLb. brevis.

The tetramer formation ofLb. brevis GadAin vitro needs high content of sulfate ions which is not similar to that in thein vivo conditions (Hiraga et al., 2008). This may be due to the fact thatin vitro catalytic activity does not fully representin vivo situation of the enzyme, though in general it concurs to thatin vivo in bacteria (Davidi et al., 2016). However, we observed that glutamate was almost decarboxylated into GABA within 24 h (Figure3G), even at the supplementation level of 10 g/L of monosodium glutamate (Wu and Shah, 2015), and GadA showed similar spectrum of enzyme activity either in sulfate buffer (Figure4D) or in pyridine-HCl buffer (Hiraga et al., 2008). This may suggest that the pH value rather than the composition of the buffer affects the activity of GadA greatly after the activation process for GadA.

The near-neutral intracellular pH ofLb. brevis cells limited activities of cytosolic Gads though GadA and GadB were highly regulated (Figures3C,D,4B). Thus, approaches to improve their activities in near-neutral pH (pH 5.5–6.5) would be of economic and biological importance. Two studies that applied mutation strategy toLb. brevis GadB demonstrated that these mutants extended their activities toward near-neutral acidity (Yu et al., 2012;Shi et al., 2014). However, we observed the extended activity of GadA in near-neutral pH range (pH 5.5–6.5) supporting GABA synthesis inLb. brevis offering a novel insight into the diversity of Gads from LAB and bifidobacteria (Figure4B). Although detailed regulatory mechanism for GABA synthesis inLb. brevis is not clear, thegadA mRNA transcript became more abundant thangadB mRNA transcript inLb. brevis at the stationary phase; this may contribute to its high GABA production by this organism during late cultivation (Figures3D,G). The classification of Gads from LAB and bifidobacteria also indicated thatLb. brevis GadA and GadB were unique Gads differing with other Gads (Figure4E). In general, we concluded thatLb. brevis GadA, a novel type III Gad that may play a more important role thanLb. brevis GadB, exhibited its activity toward weak pH range (pH 5.5–7.0) of bacterial cytosolic acidity. This supports the efficient GABA synthesis inLb. brevis as a microbial cell factory.

Apart from the contribution of GAD system to acid resistance during late cultivation ofLb. brevis, our results showed that its ADI system was active in early cultivation, specifically in lag and log phases (Figures2,3D,F). This implies that ADI pathway plays a key role in early acid resistance by alkali production.

In conclusion, GAD system was commonly distributed inLb. brevis among various species of LAB and bifidobacteria, and was the major contributor for acid resistance thus exhibiting a high capacity of GABA production of the cell. GadA in GAD system played a key role in GABA synthesis via its extended activity toward a near-neutral pH range (pH 5.5–6.5) of cytosolic acidity ofLb. brevis cells. Further understanding on tetramer formation and structural insights ofLb. brevis GadA would be necessary to unravel the decarboxylation machinery in a weak acidic condition.

Author Contributions

QW and NS designed the study; HT and EK performed the draft genome sequencing ofLactobacillus brevis NPS-QW-145; QW performed the experiments; QW and YL analyzed the data; QW and NS wrote the manuscript.

Funding

This work is partially supported by Seed Funding Programme for Basic Research (project code: 201511159156), University Research Committee, The University of Hong Kong.

Conflict of Interest Statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Acknowledgments

The authors thank the approval from Prof. Hongzhe Sun for instrumental usage, and Dr. Xinming Yang and Ms. Ya Yang for their technical assistance in fluorescence spectrophotometry and gel-filtration chromatography at the Department of Chemistry of The University of Hong Kong.

Supplementary Material

The Supplementary Material for this article can be found online at:http://journal.frontiersin.org/article/10.3389/fmicb.2017.00206/full#supplementary-material

References

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

Citation: Wu Q, Tun HM, Law Y-S, Khafipour E and Shah NP (2017) Common Distribution ofgad Operon inLactobacillus brevis and its GadA Contributes to Efficient GABA Synthesis toward Cytosolic Near-Neutral pH.Front. Microbiol. 8:206. doi: 10.3389/fmicb.2017.00206

Received: 10 November 2016;Accepted: 30 January 2017;
Published: 14 February 2017.

Copyright © 2017 Wu, Tun, Law, Khafipour and Shah. This is an open-access article distributed under the terms of theCreative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

*Correspondence: Nagendra P. Shah,bnBzaGFoQGhrdS5oaw==