Identification of a replication protein and repeats essential for DNA replication of the temperate lactococcal bacteriophage TP901-1

DNA replication of the temperate lactococcal bacteriophage TP901-1 was show n to involve the gene product encoded by orf13 and the repeats located with in 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 sho wn to confer a TP901-1 resistance phenotype when present in trans. Site-spe cific mutations were introduced into the replication protein and into the r epeats. The mutations were introduced into the TP901-1 prophage by homologo us recombination by using a vector with a temperature-sensitive replicon. S ubsequent analysis of induced phages showed that the protein encoded by orf 13 and the repeats within orf13 were essential for phage TP901-1 amplificat ion. In addition, analyses of internal phage DNA replication showed that th e ORF13 protein and the repeats are essential for phage TP901-1 DNA replica tion in vivo. These results show that orf13 encodes a replication protein a nd that the repeats within the gene are the origin of replication.