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Applied and Environmental Microbiology, December 1998, p. 4883-4890, Vol. 64, No. 12
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Purification and Genetic Characterization of Enterocin I from Enterococcus faecium 6T1a, a Novel Antilisterial Plasmid-Encoded Bacteriocin Which Does Not Belong to the Pediocin Family of Bacteriocins

Belén Floriano, José L. Ruiz-Barba, and Rufino Jiménez-Díaz*

Departamento de Biotecnología de Alimentos, Instituto de la Grasa, Consejo Superior de Investigaciones Científicas, 41012 Seville, Spain

Received 12 March 1998/Accepted 11 August 1998

Enterocin I (ENTI) is a novel bacteriocin produced by Enterococcus faecium 6T1a, a strain originally isolated from a Spanish-style green olive fermentation. The bacteriocin is active against many olive spoilage and food-borne gram-positive pathogenic bacteria, including clostridia, propionibacteria, and Listeria monocytogenes. ENTI was purified to homogeneity by ammonium sulfate precipitation, binding to an SP-Sepharose fast-flow column, and phenyl-Sepharose CL-4B and C2/C18 reverse-phase chromatography. The purification procedure resulted in a final yield of 954% and a 170,000-fold increase in specific activity. The primary structure of ENTI was determined by amino acid and nucleotide sequencing. ENTI consists of 44 amino acids and does not show significant sequence similarity with any other previously described bacteriocin. Sequencing of the entI structural gene, which is located on the 23-kb plasmid pEF1 of E. faecium 6T1a, revealed the absence of a leader peptide at the N-terminal region of the gene product. A second open reading frame, ORF2, located downstream of entI, encodes a putative protein that is 72.7% identical to ENTI. entI and ORF2 appear to be cotranscribed, yielding an mRNA of ca. 0.35 kb. A gene encoding immunity to ENTI was not identified. However, curing experiments demonstrated that both enterocin production and immunity are conferred by pEF1.


* Corresponding author. Mailing address: Departamento de Biotecnología de Alimentos, Instituto de la Grasa, Consejo Superior de Investigaciones Cientificas, Aptdo. 1078, 41012 Seville, Spain. Phone: 34-5-4692516. Fax: 34-5-4691262. E-mail address: rjimenez{at}cica.es.


Applied and Environmental Microbiology, December 1998, p. 4883-4890, Vol. 64, No. 12
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.



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