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Applied and Environmental Microbiology, January 2003, p. 41-48, Vol. 69, No. 1
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.1.41-48.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Use of Real-Time Quantitative PCR for the Analysis of LC3 Prophage Stability in Lactococci

Merete Lunde,^1* Janet Martha Blatny,¹ Dag Lillehaug,^1, Are Halvor Aastveit,² and Ingolf F. Nes¹

Laboratory of Microbial Gene Technology, Department of Chemistry and Biotechnology,¹ Department of Mathematical Sciences, Agricultural University of Norway, N-1432 Aas, Norway²

Received 29 July 2002/ Accepted 1 October 2002

Bacteriophages are a common and constant threat to proper milk fermentation. It has become evident that lysogeny is widespread in lactic acid bacteria, and in this work the temperate lactococcal bacteriophage LC3 was used as a model to study prophage stability in lactococci. The stability was analyzed in six LC3 lysogenic Lactococcus lactis subsp. cremoris host strains when they were growing at 15 and 30°C. In order to perform these analyses, a real-time PCR assay was developed. The stability of the LC3 prophage was found to vary with the growth phase of its host L. lactis IMN-C1814, in which the induction rate increased during the exponential growth phase and reached a maximum level when the strain was entering the stationary phase. The maximum spontaneous induction frequency of the LC3 prophage varied between 0.32 and 9.1% (28-fold) in the six lysogenic strains. No correlation was observed between growth rates of the host cells and the spontaneous prophage induction frequencies. Furthermore, the level of extrachromosomal phage DNA after induction of the prophage varied between the strains (1.9 to 390%), and the estimated burst sizes varied up to eightfold. These results show that the host cells have a significant impact on the lytic and lysogenic life styles of temperate bacteriophages. The present study shows the power of the real-time PCR technique in the analysis of temperate phage biology and will be useful in work to reveal the impact of temperate phages and lysogenic bacteria in various ecological fields.

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* Corresponding author. Mailing address: Laboratory of Microbial Gene Technology, Department of Chemistry and Biotechnology, Agricultural University of Norway, P.O. Box 5051, N-1432 Aas, Norway. Phone: 47 64 94 94 65. Fax: 47 64 94 14 65. E-mail: merete.lunde{at}ikb.nlh.no.

Present address: Dynal Biotech ASA, N-0309 Oslo, Norway.

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Applied and Environmental Microbiology, January 2003, p. 41-48, Vol. 69, No. 1
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.1.41-48.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

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