Molecular analysis of the glutamate decarboxylase locus in Streptococcus thermophilus ST110

Abstract

γ-aminobutyric acid (GABA) is generated from glutamate by the action of glutamic acid decarboxylase (GAD) and characterized by hypotensive, diuretic, and tranquilizing effects in humans and animals. The production of GABA by lactic acid starter bacteria would enhance the functionality of fermented dairy foods including cheeses and yogurt. The survey of 42 strains of the yogurt starter culture Streptococcus thermophilus by PCR techniques indicated the presence of a glutamate decarboxylase gene (gadB) in 16 strains. DNA sequencing data indicated that the GAD/GABA antiporter locus (gadB/gadC) in GAD⁺ S. thermophilus strains is flanked by transposase elements (5′ and 3′) and positioned between the luxS (5′) and the HD-superfamily hydrolase genes (3′). The PCR amplification product of a ca. 2-kb genomic fragment that included the gadB and its putative promoter region was inserted into a shuttle vector, which was used to transform Escherichia coli DH5α. Subsequently, the recombinant plasmid pMEU5a-1/gadB (7.24 kb) was electrotransformed into the GAD-negative strain S. thermophilus ST128. The ST128 transformants carrying the plasmid-encoded gadB produced functional GAD enzyme as evidenced by the conversion of glutamate to GABA at a rate similar to strains with the gadB/gadC operon located on the chromosome. The results demonstrated the potential to impart to non-GABA-producing strains of S. thermophilus and other lactic acid bacteria the GAD⁺ phenotype that improves their appeal in possible applications in the development of health-promoting functional foods.