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Applied and Environmental Microbiology, February 2001, p. 774-781, Vol. 67, No. 2
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.2.774-781.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Identification of a Replication Protein and Repeats Essential for DNA Replication of the Temperate Lactococcal Bacteriophage TP901-1

Solvej Østergaard,1 Lone Brøndsted,2,dagger and Finn K. Vogensen1,*

Department of Dairy and Food Science, The Royal Veterinary and Agricultural University, DK-1958 Frederiksberg C,1 and Department of Microbiology, Technical University of Denmark, DK-2800 Lyngby,2 Denmark

Received 25 September 2000/Accepted 27 November 2000

DNA replication of the temperate lactococcal bacteriophage TP901-1 was shown to involve the gene product encoded by orf13 and the repeats located within the gene. Sequence analysis of 1,500 bp of the early transcribed region of the phage genome revealed a single-stranded DNA binding protein analogue (ORF12) and the putative replication protein (ORF13). The putative origin of replication was identified as series of repeats within orf13 and was shown to confer a TP901-1 resistance phenotype when present in trans. Site-specific mutations were introduced into the replication protein and into the repeats. The mutations were introduced into the TP901-1 prophage by homologous recombination by using a vector with a temperature-sensitive replicon. Subsequent analysis of induced phages showed that the protein encoded by orf13 and the repeats within orf13 were essential for phage TP901-1 amplification. In addition, analyses of internal phage DNA replication showed that the ORF13 protein and the repeats are essential for phage TP901-1 DNA replication in vivo. These results show that orf13 encodes a replication protein and that the repeats within the gene are the origin of replication.

* Corresponding author. Mailing address: Department of Dairy and Food Science, The Royal Veterinary and Agricultural University, Rolighedsvej 30, DK-1958 Frederiksberg C, Denmark. Phone: 45 35 28 32 11. Fax: 45 35 28 32 31. E-mail: fkv{at}biobase.dk.

dagger Present address: Department of Dairy and Food Science, The Royal Veterinary and Agricultural University, DK-1958 Frederiksberg C, Denmark.

Applied and Environmental Microbiology, February 2001, p. 774-781, Vol. 67, No. 2
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.2.774-781.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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