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United States Department of Agriculture

Agricultural Research Service

Research Project: FUNCTIONAL FOOD DEVELOPMENT BY MICROBIAL BIOTECHNOLOGY

Location: Dairy and Functional Foods

Title: BlpC regulated bacteriocin production in Streptococcus thermophilus NRRL-B59671

Authors
item Renye, John
item Somkuti, George

Submitted to: Biotechnology Letters
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: November 9, 2012
Publication Date: November 27, 2012
Citation: Renye Jr, J.A., Somkuti, G.A. 2012. BlpC regulated bacteriocin production in Streptococcus thermophilus NRRL-B59671. Biotechnology Letters. 35:407-412.

Interpretive Summary: Streptococcus thermophilus is a lactic acid bacterium used in the production of yogurt and cheeses, and has been shown to produce small protein molecules called bacteriocins, which inhibit the growth of bacterial food contaminants. In some cases, the production of these biopreservative molecules in S. thermophilus will not function properly unless a small signal protein is artificially provided to activate production. ARS scientists developed a gene transfer molecule to remove the gene that makes the signal protein from S. thermophilus NRRL-B59671. Removal of the signal protein resulted in a bacterium which was unable to produce bacteriocin. A second gene ransfer molecule was developed to continuously make the signal protein and was shown to restore production of the bacteriocin. The results of this study are the first to show that this cell density signaling system can function properly in S. thermophilus and is responsible for the production of a natural bacteriocin by this food-grade microorganism. The findings can be applied to extending the shelf-life of fermented dairy foods by protecting them against undesirable bacteria with naturally produced biopreservatives.

Technical Abstract: Streptococcus thermophilus NRRL-B59671 was previously shown to produce a bacteriocin with anti-pediococcal activity, but the genetic components required for production were not characterized. Genome sequencing of several strains of S. thermophilus has identified a genetic locus which encodes a quorum sensing (QS) system to regulate the production of class II bacteriocins. However, for the strains possessing this gene cluster, the production of baceriocin like peptides (blp) was only observed if excess pheromone was provided, suggesting that the QS system was unable to function optimally under normal growth conditions. PCR analysis revealed that S. thermophilus NRRL-B59671 possessed blpC, which was reported to encode the 30-mer QS pheromone required for induction of the blp gene cluster. To investigate if BlpC regulated bacteriocin production in S. thermophilus NRRL-B59671, an integrative vector was designed to replace blpC with a gene encoding for kanamycin resistance. PCR analysis confirmed the removal of blpC from the S. thermophilus chromosome, and the resulting mutant was unable to inhibit the growth of Pediococcus acidilactici F. To confirm that the deletion of blpC was responsible for the loss of bacteriocin activity, a shuttle vector was constructed to constitutively express the BlpC pheromone. A fusion peptide was generated which utilized an alternative leader peptide to secrete the 30-mer pheromone peptide via the general secretory pathway of S. thermophilus. Constitutive expression of the pheromone allowed for the production of an active bacteriocin, confirming that the blp gene cluster encodes for the active bacteriocin produced by S. thermophilus NRRL-B59671.

Last Modified: 9/2/2014
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