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Applied and Environmental Microbiology, February 2001, p. 608-616, Vol. 67, No. 2
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.2.608-616.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Improvement and Optimization of Two Engineered Phage Resistance Mechanisms in Lactococcus lactis

Stephen McGrath,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 22 June 2000/Accepted 11 October 2000

Homologous replication module genes were identified for four P335 type phages. DNA sequence analysis revealed that all four phages exhibited more than 90% DNA homology for at least two genes, designated rep2009 and orf17. One of these genes, rep2009, codes for a putative replisome organizer protein and contains an assumed origin of phage DNA replication (ori2009), which was identical for all four phages. DNA fragments representing the ori2009 sequence confer a phage-encoded resistance (Per) phenotype on lactococcal hosts when they are supplied on a high-copy-number vector. Furthermore, cloning multiple copies of the ori2009 sequence was found to increase the effectiveness of the Per phenotype conferred. A number of antisense plasmids targeting specific genes of the replication module were constructed. Two separate plasmids targeting rep2009 and orf17 were found to efficiently inhibit proliferation of all four phages by interfering with intracellular phage DNA replication. These results represent two highly effective strategies for inhibiting bacteriophage proliferation, and they also identify a novel gene, orf17, which appears to be important for phage DNA replication. Furthermore, these results indicate that although the actual mechanisms of DNA replication are very similar, if not identical, for all four phages, expression of the replication genes is significantly different in each case.

* 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, February 2001, p. 608-616, Vol. 67, No. 2
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.2.608-616.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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