A plasmid-borne gadRCB operon contributes to acid tolerance in Lactiplantibacillus plantarum ZR79

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.