Molecular analysis of the glutamate decarboxylase locus in Streptococcus thermophilus ST110

Authors: Somkuti, George; Renye, John; Steinberg, Dennis

Submitted to: Journal of Industrial Microbiology and Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/25/2012
Publication Date: 7/1/2012
Citation: Somkuti, G.A., Renye Jr, J.A., Steinberg, D.H. 2012. Molecular analysis of the glutamate decarboxylase locus in Streptococcus thermophilus ST110. Journal of Industrial Microbiology and Biotechnology. 39:957-963.

Interpretive Summary: Consumer interest has increased in functional foods that provide extra health benefits in addition to nutritional value and resulted in a substantial growth in the variety of such foods in the marketplace. Gamma-aminobutyric acid (GABA) is a bioactive amino acid that is widely distributed in nature and found in microbes, plants and animals. The beneficial activities of GABA include a soothing effect on the nervous system, promoting lower blood pressure, and even preventing diabetic conditions. Among microbes, several types of lactic acid bacteria (LAB) that are used in the industrial production of fermented dairy foods such as cheeses and yogurt have the capacity to produce GABA from glutamic acid which is another amino acid set free from proteins by special enzymes. ARS researchers examined a group of 42 yogurt fermentation cultures and found that 40% of cultures carried the gene complex necessary to produce GABA. The results demonstrated the importance of selecting LAB cultures with high GABA-producing capacity that may have potential for applications in the development of functional foods enriched with GABA. Researchers also worked out methods to transfer the GABA-producing capacity to cultures that lack the gene complex needed for of GABA production which increases the range of their usefulness in food development.

Technical Abstract: GABA ('-aminobutyric acid) 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 the 5.48-kb pMEU5a shuttle vector, which was used to transform Escherichia coli DH5a. 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 for possible applications in the development of functional foods benefiting human health.