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Applied and Environmental Microbiology, May 1999, p. 1891-1899, Vol. 65, No. 5
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Molecular Characterization of a Phage-Encoded Resistance System in Lactococcus lactis

Stephen McGrath,1 Jos F. M. L. Seegers,1 Gerald F. Fitzgerald,1,2,3 and Douwe van Sinderen2,*

National Food Biotechnology Centre,1 Department of Microbiology,2 and Department of Food Science and Technology,3 University College Cork, Cork, Ireland

Received 30 November 1998/Accepted 1 March 1999

A specific fragment of the genome of Tuc2009, a temperate lactococcal bacteriophage, was shown to contain several open reading frames, whose deduced protein products exhibited similarities to proteins known to be involved in DNA replication and modification. In this way, a putative single-stranded binding protein, replisome organizer protein, topoisomerase I, and a methylase were identified. When the genetic information coding for the putative replisome organizer protein of Tuc2009, Rep2009, was supplied on a high-copy-number plasmid vector, it was shown to confer a phage-encoded resistance (Per) phenotype on its lactococcal host UC509.9. The presence of this recombinant plasmid was shown to cause a marked reduction in Tuc2009 DNA replication, suggesting that the observed phage resistance was due to titration of a factor, or factors, required for Tuc2009 DNA replication. Further experiments delineated the phage resistance-conferring region to a 160-bp fragment rich in direct repeats. Gel retardation experiments, which indicated a protein-DNA interaction between this 160-bp fragment and the Rep2009 protein, were performed. UC509.9 strains harboring plasmids with randomly mutated versions of this fragment were shown to display a variable phage resistance phenotype, depending on the position of the mutations.

* Corresponding author. Mailing address: Department of Microbiology, University College Cork, Cork, Ireland. Phone: 353 21 902811. Fax: 353 21 903101. E-mail: douwe{at}ucc.ie.

Applied and Environmental Microbiology, May 1999, p. 1891-1899, Vol. 65, No. 5
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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