Two putative structural genes, orf tmp (tape measure protein) and orf bpp (baseplate protein), of the temperate lactococcal phage TP901-1 were examined by introduction of specific mutations in the prophage strain Lactococcus lactic ssp. cremoris 901-1. The adsorption efficiencies of the mutated phages to the indicator strain L. lactic ssp. cremoris 3107 were determined and electron micrographs were obtained. Specific mutations in orf tmp resulted in the production of mostly phage head structures without tails and a few wild-type looking phages. Furthermore, construction of an inframe deletion or duplication of 29% in orf tmp was shown to shorten or lengthen the phage tail by approximately 30%, respectively. The orf tmp is proposed to function as a tape measure protein, TMP, important for assembly of the TP901-1 phage tail and involved in tail length determination. Specific mutations in orf bpp produced phages which were unable to adsorb to the indicator strain and electron microscopy revealed particles lacking the baseplate structure. The orf bpp is proposed to encode a highly immunogenic structural baseplate protein, BPP, important for assembly of the baseplate. Finally, an assembly pathway of the TP901-1 tail and baseplate structure is presented.

A complete analysis of the entire genome of the temperate lactococcal bacteriophage TP901-1 has been performed and the function of 21 of 56 TP901-1-encoded ORFs has been assigned. This knowledge has been used to propose 10 functional modules each responsible for specific functions during bacteriophage TP901-1 proliferation. Short regions of microhomology in intergenic regions present in several lactococcal bacteriophages and chromosomal fragments of Lactococcus lactis are suggested to be points of exchange of genetic material through homologous recombination. Our results indicate that TP901-1 may have evolved by homologous recombination between the host chromosome and a mother phage and support the observation that phage remnants as well as prophages located in the Lactococcus chromosome contribute significantly to bacteriophage evolution. Some proteins encoded in the early transcribed region of the TP901-1 genome were more homologous to proteins encoded by phages infecting gram-positive hosts other than L. lactis. This protein homology argues for the occurrence of horizontal genetic exchange among these bacteriophages and indicates that they have access to a common gene pool.

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.Strains of Lactococcus lactis are industrially important members of the lactic acid bacteria. They are commonly used as starter cultures in the production of fermented milk products, but their susceptibility to bacteriophage attack, which causes fermentation failure, continues to be a significant problem in industrial practice. This has led to a number of studies aimed at understanding the physical and genetic organization of lactococcal phages (for review, see reference 17). The interest in DNA replication of lactococcal bacteriophages has increased in recent years, since this knowledge may lead to development of new phage resistance mechanisms for lactococcal strains. So far, only a few features of the lactococcal phage origins of replication have been described. The presence of series of repeats has been identified in all lactococcal phage origins described until now. In two cases, phage c2 and sk1, these repeats were shown to be able to function as plasmid origins of replication in Lactococcus (9, 45). Another feature of the repeats is the Per (phage-encoded resistance) phenotype, which has been shown for the lactococcal phages 50, 31, and Tuc2009 (21,33,35). The Per effect is observed as resistance against phage infection of the host harboring the phage origin of replication on a plasmid in trans. Per has been suggested to function by titrating out factors required for replication of the infecting phage (21, 35). Binding of the putative replication initiation protein to the repeats of the origin has been shown for the lactococcal phage Tuc2009 (33).In this work we investigate the biological importance of a replication protein and the origin of replication in DNA replication of the temperate lactococcal bacteriophage TP901-1. The putative replication initiator protein (ORF13) and the origin of replication, identified as series of repeats wi...