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Journal of Bacteriology, December 2004, p. 8010-8017, Vol. 186, No. 23
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.23.8010-8017.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Essentiality of the Early Transcript in the Replication Origin of the Lactococcal Prolate Phage c2

Anja H. Schiemann,¹ Jasna Rakonjac,¹ Michael Callanan,^1, James Gordon,¹ Kayla Polzin,^2, Mark W. Lubbers,² and Paul W. O'Toole^1*

Institute of Molecular BioSciences, Massey University,¹ Fonterra Research Centre, Palmerston North, New Zealand²

Received 22 May 2004/ Accepted 18 August 2004

The genome of the prolate-headed lytic lactococcal bacteriophage c2 is organized into two divergently oriented blocks consisting of the early genes and the late genes. These blocks are separated by the noncoding origin of DNA replication. We examined the functional role of transcription of the origin in a plasmid model system. Deletion of the early promoter P_E1 abolished origin function. Introduction of mutations into P_E1 which did not eliminate promoter activity or replacement of P_E1 with an unrelated but functional promoter did not abolish replication. The A-T-rich region upstream of P_E1, which is conserved in prolate phages, was not required for plasmid replication. Replacement of the P_E1 transcript template sequence with an unrelated sequence with a similar G+C content abolished replication, showing that the sequence encoding the transcript is essential for origin function. Truncated transcript and internal deletion constructs did not support replication except when the deletion was at the very 3' end of the DNA sequence coding for the transcript. The P_E1 transcript could be detected for all replication-proficient constructs. Recloning in a plasmid vector allowed detection of P_E1 transcripts from some fragments that did not support replication, indicating that stability of the transcript alone was not sufficient for replication. The data suggest that production of a transcript of a specific length and with a specific sequence or structure is essential for the function of the phage c2 origin in this model system.

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* Corresponding author. Present address: Alimentary Pharmabiotic Centre, Department of Microbiology, University College Cork, Cork, Ireland. Phone: 353 21 490 3997. Fax: 353 21 490 3101. E-mail: pwotoole{at}ucc.ie.

Supplemental material for this article may be found at http://jb.asm.org/.

Present address: Teagasc, Moorepark, Fermoy, Co. Cork, Ireland.

Present address: Land O Lakes Inc., St. Paul, MN 55164-0101

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Journal of Bacteriology, December 2004, p. 8010-8017, Vol. 186, No. 23
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.23.8010-8017.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

• Rakonjac, J., O'Toole, P. W., Lubbers, M. (2005). Isolation of Lactococcal Prolate Phage-Phage Recombinants by an Enrichment Strategy Reveals Two Novel Host Range Determinants. J. Bacteriol. 187: 3110-3121 [Abstract] [Full Text]