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Journal of Bacteriology, May 2005, p. 3110-3121, Vol. 187, No. 9
0021-9193/05/$08.00+0     doi:10.1128/JB.187.9.3110-3121.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Isolation of Lactococcal Prolate Phage-Phage Recombinants by an Enrichment Strategy Reveals Two Novel Host Range Determinants

Jasna Rakonjac,^1* Paul W. O'Toole,^1, and Mark Lubbers²

Institute of Molecular BioSciences, Massey University, Palmerston North, New Zealand,¹ Fonterra Research Centre, formerly the New Zealand Dairy Research Institute, Palmerston North, New Zealand²

Received 9 July 2004/ Accepted 24 January 2005

Virulent lactococcal prolate (or c2-like) phages are the second most common phage group that causes fermentation failure in the dairy industry. We have mapped two host range determinants in two lactococcal prolate phages, c2 and 923, for the host strains MG1363 and 112. Each phage replicates on only one of the two host strains: c2 on MG1363 and 923 on 112. Phage-phage recombinants that replicated on both strains were isolated by a new method that does not require direct selection but rather employs an enrichment protocol. After initial mixed infection of strain 112, two rotations, the first of which was carried out on strain MG1363 and the second on 112, permitted continuous amplification of double-plating recombinants while rendering one of the parent phages unamplified in each of the two rotations. Mapping of the recombination endpoints showed that the presence of the N-terminal two-thirds of the tail protein L10 of phage c2 and a 1,562-bp cosR-terminal fragment of phage 923 genome overcame blocks of infection in strains MG1363 and 112, respectively. Both infection inhibition mechanisms act at the stage of DNA entry; in strain MG1363, the infection block acts early, before phage DNA enters the cytoplasm, and in strain 112, it acts late, after most of the DNA has entered the cell but before it undergoes cos-end ligation. These are the first reported host range determinants in bacteriophage of lactic acid bacteria required for overcoming inhibition of infection at the stage of DNA entry and cos-end ligation.

Present address: Department of Microbiology and Alimentary Pharmabiotic Centre, University College Cork, Ireland.

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