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The glutamate decarboxylase (GAD) system catalyzed the conversion of L-glutamate to gamma-aminobutyric acid (GABA) in a proton-consuming reaction, which played a critical role to maintain intracellular pH homeostasis. However, the genetic organization and functional roles of GAD system in Lactiplantibacillus plantarum remain incompletely understood. In this study, L. plantarum ZR79, a GABA-producing strain, was successfully screened from 120 L. plantarum strains based on gas production and pH increase after 48-h fermentation. Comparative genomic analysis revealed that only L. plantarum ZR79 harbors two glutamate decarboxylases encoded by gadA and gadB , respectively. Insertional inactivation of the gadB gene abolished the ability to synthesize GABA, suggesting that the gadB gene plays a critical role in GABA biosynthesis in L. plantarum ZR79. The complete genome sequencing analysis combined with RT-PCR revealed that the gadB gene was located on the plasmid pZR79, which was co-transcribed with gadR and gadC . Plasmid stability assays revealed that pZR79 was stably maintained in ZR79 over 200 generations. Furthermore, acid stress survival assays confirmed that gadB -mediated GABA production contributes to the acid tolerance of L. plantarum ZR79. This study provides the first evidence of a plasmid-encoded gadRCB operon in L. plantarum , offering new insights into the strain-specific genetic basis of GABA biosynthesis and its physiological role in acid stress resistance. Key points • L. plantarum ZR79 harbors two distinct glutamate decarboxylase-encoding genes, gadA and gadB, with the gadB gene cluster located on its plasmid pZR79. • Insertional inactivation of the gadB gene abolished the ability of L. plantarum ZR79 to synthesize GABA. • The gadB gene increased the survival ability in L. plantarum ZR79 under acidic conditions.

Psychobiotics are a novel class of probiotics with potential to confer mental wellness via production of neuroactive compounds such as gamma-aminobutyric acid (GABA). The demand for new biological sources of GABA has increased steadily. Therefore, the current study reports the isolation of 17 presumptive lactic acid bacteria (LAB) from marine samples and their screening for GABA synthesis from monosodium glutamate (MSG) using thin-layer chromatography (TLC). The isolate SH9 was selected as a high GABA producing strain. The GABA content of SH9 cell free supernatant (CFS) was quantitatively determined by high performance liquid chromatography (HPLC) to be 0.97 g/L. SH9 was identified biochemically and molecularly as Enterococcus faecium (identity 99%). Moreover, SH9 demonstrated promising probiotic potentials; it gave no signs of hemolysis and could survive at low pH values and high bile salt concentrations. It also exhibited antimicrobial activity against highly pathogenic strains and the ability to grow at 6.5% NaCl. In addition, SH9 CFS showed anti-inflammatory and antioxidant properties. The glutamate decarboxylase (GAD) gene was detected in SH9 by using specific primers. Product of 540 bp was obtained, sequenced, and analyzed (accession number: MW713382). The inferred amino acid sequence was 99.3% identical to Lactobacillus plantarum M-6 gadB gene. The findings of this study suggest that the marine isolate E. faecium SH9 could be used as a novel psychobiotics in the development of GABA rich healthy products.

Gamma-aminobutyric acid (GABA) is a non-protein amino acid playing a significant role in the central nervous system and the gut–brain axis. This study investigated the potential to produce GABA by lactic acid bacteria (LAB) isolated from different varieties of organic tomatoes. The isolated LAB were taxonomically identified by 16S rRNA gene sequencing, the presence of the gadB gene (glutamate decarboxylase) was detected, and GABA production was quantified using HPLC. Levilactobacillus brevis CRAI showed the highest GABA production under optimised fermentation conditions with 4% monosodium glutamate (MSG). The genome sequencing of L. brevis CRAI revealed the presence of gadA and gadB isoforms and assessed the strain’s safety profile. The gene expression analysis revealed that the gadA and gadB genes were upregulated in the presence of 4% MSG. The probiotic potential of L. brevis CRAI was also assessed by functional assays. The strain showed strong antimicrobial activity against representative enteropathogens, i.e., Escherichia coli ETEC, Salmonella choleraesuis, and Yersinia enterocolitica, and anti-inflammatory effect, reducing nitric oxide production in LPS-stimulated RAW264.7 macrophages. In addition, its ability to adhere to intestinal epithelial Caco-2 cells was demonstrated. These results highlight L. brevis CRAI as a promising candidate for the development of GABA-enriched functional foods or probiotic supplements with the perspective to modulate the gut-brain axis.

This study aimed to enhance γ-aminobutyric acid (GABA) in pigeon pea milk (CCM). The drink was prepared from germinated pigeon pea and fermented using the probiotic Lactiplantibacillus plantarum Dad-13. Various nutrients significantly increased the GABA content in pigeon pea milk, i.e., sucrose 3% (4409 mg/L), monosodium glutamate (MSG) 1% (59,562 mg/L), and whey 4% (5283 mg/L), respectively. Glutamate decarboxylase (GAD)-encoding genes were identified in the genome of the strain. The strain carried only one gadB gene, and no other gad genes were found in the genomes when compared with other strains. During fermentation, various metabolites, including organic acids, amino acid derivatives, and flavonoids, were detected. These metabolites may promote anti-inflammatory activity in cytokines such as TNF-α and IL6. In conclusion, the development of fermented pigeon pea enriched with GABA using probiotic L. plantarum Dad-13 shows promising potential as a functional food that can promote health benefits and help prevent diseases.

Functional diversity within isogenic spatially organised bacterial populations has been shown to trigger emergent community properties such as stress tolerance. Considering gadB gene encoding a key glutamate decarboxylase involved in E. coli tolerance to acidic conditions, we investigated its expression in hydrogels mimicking the texture of some structured food matrices (such as minced meat or soft cheese). Taking advantage of confocal laser scanning microscopy combined with a genetically-engineered dual fluorescent reporter system, it was possible to visualise the spatial patterns of bacterial gene expression from in-gel microcolonies. In E. coli O157:H7 microcolonies, gadB showed radically different expression patterns between neutral (pH 7) or acidic (pH 5) hydrogels. Differential spatial expression was determined in acidic hydrogels with a strong expression of gadB at the microcolony periphery. Strikingly, very similar spatial patterns of gadB expression were further observed for E. coli O157:H7 grown in the presence of L. lactis . Considering the ingestion of contaminated foodstuff, survival of E. coli O157:H7 to acidic stomachal stress (pH 2) was significantly increased for bacterial cells grown in microcolonies in acidic hydrogels compared to planktonic cells. These findings have significant implications for risk assessment and public health as they highlight inherent differences in bacterial physiology and virulence between liquid and structured food products. The contrasting characteristics observed underscore the need to consider the distinct challenges posed by these food types, thereby emphasising the importance of tailored risk mitigation strategies.

Functional diversity within isogenic spatially organized bacterial populations has been shown to trigger emergent community properties such as stress tolerance. Taking advantage of confocal laser scanning microscopy combined with a transcriptional fluorescent fusion reporting at single cell scale the expression of the glutamic acid decarboxylase gadB in E. coli O157:H7, it was possible to visualize for the first-time spatial patterns of bacterial gene expression in microcolonies grown in a gelled matrix. The gadB gene is involved in E. coli tolerance to acidic conditions and its strong over-expression was observed locally on the periphery of embedded microcolonies grown in acidic hydrogels. This spatialization of gadB expression did not correlate with live/dead populations that appeared randomly distributed in the colonies. While the planktonic population of the pathogens was eradicated by an exposition to a pH of 2 (HCl) for 4h, mimicking a stomachal acidic stress, bacteria grown in gel-microcolonies were poorly affected by this treatment, in particular in conditions where gadB was spatially overexpressed. Consequences of these results for food safety are further discussed.Competing Interest StatementThe authors have declared no competing interest.