Molecular phylogeny of phi 29-like phages and their evolutionary relatedness to other protein-primed replicating phages and other phages hosted by Gram-positive bacteria

The phi 29-like phage genus of Podoviridae family contains phages B103, BS3 2, GA-1,M2, Nf, phi 15, phi 29, and PZA that all infect Bacillus subtilis. They have very similar morphology and their genomes consist of linear doubl e-stranded DNA of approximately 20 kb. The nucleotide sequences of individu al genomes or their parts determined thus far show that these phages evolve d from a common ancestor. A terminal protein (TP) that is covalently bound to the DNA 5'-end primes DNA replication of these phages. The same mechanis m of DNA replication is used by the Cp-l related phages (also members of th e Podoviridae family) and by the phage PRD1 (member of the Tectoviridae fam ily). Based on the complete or partial genomic sequence data of these phage s it was possible to analyze the evolutionary relationship within the phi 2 9-like phage genus as well as to other protein-primed replicating phages. N oncoding regions containing origins of replication were used in the analysi s, as well as amino acid sequences of DNA polymerases, and with the phi 29- like phages also amino acid sequences of the terminal proteins and of the g ene 17 protein product, an accessory component of bacteriophage DNA replica ting machinery. Included in the analysis are also results of a comparison o f these phage DNAs with the prophages present in the Bacillus subtilis geno me. Based on this complex analysis we define and describe in more detail th e evolutionary branches of phi 29-like phages, one branch consisting of pha ges BS32, phi 15, phi 29, and PZA, the second branch composed of phages B10 3, M2, and Nf, and the third branch having phage GA-1 as its sole member. I n addition, amino acid sequences of holins, proteins involved in phage lysi s were used to extend the evolutionary study to other phages infecting Gram positive bacteria. The analysis based on the amino acid sequences of holins showed several weak points in present bacteriophage classification.