Page Banner

United States Department of Agriculture

Agricultural Research Service

Research Project: FUNCTIONAL FOOD DEVELOPMENT BY MICROBIAL BIOTECHNOLOGY

Location: Dairy and Functional Foods

Title: Molecular analysis of the bacteriocin-encoding plasmid pDGL1 from Enterococcus durans and genetic characterization of the durancin locus

Authors
item Du, Lihui -
item SOMKUTI, GEORGE
item RENYE, JOHN

Submitted to: Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: March 29, 2012
Publication Date: June 1, 2012
Citation: Du, L., Somkuti, G.A., Renye Jr, J.A. 2012. Molecular analysis of the bacteriocin-encoding plasmid pDGL1 from Enterococcus durans and genetic characterization of the durancin locus. Microbiology. 158:1523-1532.

Interpretive Summary: Most varieties of cheese and yogurt are produced by fermentation processes carried out by specific food grade bacterial cultures. The microbial population may also include bacteria that contribute flavor characteristics to the final food product. Some bacteria have the capacity to produce antimicrobial substances called bacteriocins that inhibit the growth of unwanted spoilage and disease causing bacteria. Tests on bacteria found in Mexican cheese samples identified a culture that produces a novel antimicrobial substance with activity against contaminating bacteria including Listeria that cause disease. The production machinery for the antimicrobial substance was found on a special molecule and its structural features were studied in detail. The producing culture was free of toxins, which implied that it may be suitable in food applications. The production machinery required for antimicrobial activity could be transferred to dairy cultures to produce the new bacteriocin. The antimicrobial effect of the new bacteriocin may help in protecting foods from disease causing bacteria and providing dairy foods with an improved margin of safety to consumers.

Technical Abstract: Enterococci constitute a significant component of lactic acid bacteria normally present in the intestinal microflora and include strains that produce bacteriocins. The genetic determinants for durancin GL in Enterococcus durans 41D were identified on the 8,347 bp plasmid pDGL1 by plasmid curing experiments. pDGL1 contained 9 putative open reading frames (ORFs) organized in three distinct functional regions. Region 1 encompassed two ORFs (orf1 and orf2): ORF1 apparently encoded the plasmid replication protein, while ORF2 displayed 85% sequence similarity to a putative plasmid replication protein from E. faecium. Region 2 contained two ORFs (orf4 and orf5), with ORF4 showing 74% identity to BacA, a bacteriocin produced by E. faecalis. The deduced DurA protein contained the conserved motif YYGNG, suggesting that durancin GL is a typical subclass IIa bacteriocin. ORF5 was shown to share 85% identity with the immunity protein BacB in E. faecalis. Region 3 encompassed five ORFs (orf3 and orf6 through orf9), with ORF3 and ORF6 exhibiting 99% identity with the transposase found in E. faecium and Macrococcus caseolyticus, respectively. ORF7 and ORF8 showed high similarity to a putative mobilization protein, while ORF9 displayed 87% sequence identity with a conserved hypothetical protein of unknown function. To further clarify the minimum requirement for durancin GL production, a 547-bp fragment containing durAB gene was translationally fused with the Streptococcus thermophilus P2201 promoter and then subcloned and heterologously expressed in S. thermophilus ST128. The result demonstrated that the cloned fragment included all the genetic components required for durancin GL production.

Last Modified: 9/29/2014