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United States Department of Agriculture

Agricultural Research Service

Title: Characterization of a Lytic Lactobacillus Plantarum Bacteriophage and Molecular Cloning of a Lysin Gene in Escherichia Coli

Authors
item Yoon, Sung-Sik - YONSEI UNIV., S. KOREA
item Kim, Jae-Won - NCSU
item Breidt, Frederick
item Fleming, Henry

Submitted to: International Journal of Food Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: November 19, 2000
Publication Date: January 1, 2001
Citation: Yoon, S., Kim, J., Breidt, F., Fleming, H.P. 2001. Characterization of a lytic Lactobacillus plantarum bacteriophage and molecular cloning of a lysin gene in Escherichia coli. Intern. J. Food Microbiol. 65:63-74.

Interpretive Summary: Some foods, such as cheeses, pickles and sauerkraut, are preserved by growth of certain types of desirable bacteria called lactic acid bacteria. These bacteria grow by fermenting sugars in the food to lactic acid, which results in preservation. These desirable bacteria are sometimes attacked and killed by bacterial viruses called bacteriophages. This may influence the quality of the food product, depending upon the type of lactic acid bacteria killed and characteristics desired in the product. We recently isolated a bacteriophage from kimchi, a fermented Korean food, which attacks a lactic acid bacterium important in vegetable fermentations. We characterized this bacteriophage as to a range of bacteria it is capable of killing, its morphology or appearance, and its biochemical and genetic makeup. We hope to exploit this bacteriophage in controlling the type of lactic acid bacteria involved in food fermentations, and thereby improve the quality of specific food products.

Technical Abstract: Bacteriophage SC921, which can infect Lactobacillus plantarum specifically, was isolated from a fermented vegetable source, Kimchi. This phage possesses a multiple host range, capable of lysing 6 of 11 strains of L. plantarum tested. The plaque formation was affected by the concentration of Ca2+, plating efficiency being highest at 25 mM CaCl2. Morphologically, this phage has an isometric head 60 nm in diameter and a non-contractile tail 260 nm long and 9 to 11 nm wide. Thus, it belongs to Bradley's group B or the Siphoviridae family according to bacteriophage classification. The bouyant density was measured about 1.58 g/cm3. SDS PAGE experimentation indicated that the phage particle contains two major structural proteins and several minor proteins. The phage genome was confirmed as a double stranded linear molecule. The length of the genome was estimated to be 66.5-kb by mapping genomic DNA digested with the restriction endonucleases: Kpn I, Sma I, and Xba I. The G + C content of the phage DNA is about 39.4%. For this lysin gene study, the 9.4-kb of Kpn I-digested phage DNA fragment were cloned into pUC19 and expressed in Escherichia coli. The Kpn I fragment was found to be related to the lysis gene of L. plantarum. The cloned fragment in pUC19 was hybridized to a 9.4-kb fragment generated by Kpn I digestion of phage SC921 as a probe. This confirmed that the fragment in pUC19 originated from phage DNA. In contrast to the lysin gene in coliphage lambda, this lysin gene was found to be near the middle of the phage genome.

Last Modified: 9/2/2014
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