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Journal of Virology, October 2002, p. 10234-10244, Vol. 76, No. 20
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.20.10234-10244.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Characterization of the Putative Replisome Organizer of the Lactococcal Bacteriophage r1t

Manuel Zúñiga, Blandine Franke-Fayard, Gerard Venema, Jan Kok,^* and Arjen Nauta

Department of Genetics, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, 9751 NN Haren, The Netherlands

Received 29 March 2002/ Accepted 11 July 2002

Analysis of the nucleotide sequence of the genome of the lactococcal bacteriophage r1t showed that it may encode at least two proteins involved in DNA replication. On the basis of its similarity with the G38P protein encoded by the Bacillus subtilis phage SPP1, the product of orf11 (Pro11) is thought to be involved in the initiation of phage DNA replication. This protein was overexpressed in Lactococcus lactis and partially purified. Gel retardation analysis using various r1t DNA fragments indicates that Pro11 specifically binds to a sequence located within its cognate gene. DNase I footprinting showed that Pro11 protects a stretch of DNA of 47 bp. This region spans four 6-bp short direct repeats, which suggests that the region contains four binding sites for Pro11. 1,10-Phenanthroline-copper footprinting confirmed the protection of the hexamers. An asymmetric protection pattern of each strand was observed, suggesting that Pro11 contacts each DNA strand separately at contiguous hexamers. We propose a model for the binding of Pro11 to its target sites that may account for the torsion strain required for strand opening at the origin of replication.

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* Corresponding author. Mailing address: Department of Genetics, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands. Phone: 31 50 3632111. Fax: 31 50 363 2348. E-mail: j.kok{at}biol.rug.nl.

Present address: Instituto de Agroquímica y Tecnología de los Alimentos (C.S.I.C.), Department of Biotechnology, 46100-Burjassot (Valencia), Spain.

Present address: University of Leiden, Department of Parasitology, 2300 RC Leiden, The Netherlands.

Present address: Corporate Research FCDF, 7400 AB Deventer, The Netherlands

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Journal of Virology, October 2002, p. 10234-10244, Vol. 76, No. 20
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.20.10234-10244.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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