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Molecular phylogeny of phi29-like phages and their evolutionary relatedness to other protein-primed replicating
1Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Flemingovo 2, CZ-16637 Prague 6, Czech Republic.
Abstract:
The phi29-like phage genus of Podoviridae family contains phages B103, BS32, GA-1, M2, Nf, phi15, phi29, and PZA that all infect Bacillus subtilis. They have very similar morphology and their genomes consist of linear double-stranded DNA of approximately 20 kb. The nucleotide sequences of individual genomes or their parts determined thus far show that these phages evolved 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 mechanism of DNA replication is used by the Cp-1 related phages (also members of the Podoviridae family) and by the phage PRD1 (member of the Tectoviridae family). Based on the complete or partial genomic sequence data of these phages it was possible to analyze the evolutionary relationship within the phi29-like phage genus as well as to other protein-primed replicating phages. Noncoding regions containing origins of replication were used in the analysis, as well as amino acid sequences of DNA polymerases, and with the phi29-like phages also amino acid sequences of the terminal proteins and of the gene 17 protein product, an accessory component of bacteriophage DNA replicating machinery. Included in the analysis are also results of a comparison of these phage DNAs with the prophages present in the Bacillus subtilis genome. Based on this complex analysis we define and describe in more detail the evolutionary branches of phi29-like phages, one branch consisting of phages BS32, phi15, phi29, and PZA, the second branch composed of phages B103, M2, and Nf, and the third branch having phage GA-1 as its sole member. In addition, amino acid sequences of holins, proteins involved in phage lysis 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.
Insights
This study analyzes the evolution of phi29-like phages, revealing distinct evolutionary branches within the genus. Findings also highlight potential issues in current bacteriophage classification systems.
Area of Science:
- Molecular Biology
- Virology
- Evolutionary Biology
Background:
- The phi29-like phage genus, part of the Podoviridae family, infects Bacillus subtilis and shares similar morphology and genome structure.
- These phages utilize a terminal protein (TP) for DNA replication priming, a mechanism also observed in Cp-1 related phages and phage PRD1.
- Previous studies suggest these phages evolved from a common ancestor, necessitating detailed evolutionary analysis.
Purpose of the Study:
- To analyze the evolutionary relationships within the phi29-like phage genus.
- To investigate evolutionary connections between phi29-like phages and other protein-primed replicating phages.
- To refine bacteriophage classification by examining holin protein sequences.
Main Methods:
- Comparative genomic analysis of nucleotide sequences, noncoding regions (origins of replication), and amino acid sequences (DNA polymerases, terminal proteins, gene 17 product).
- Inclusion of prophage DNA sequences from Bacillus subtilis in the analysis.
- Analysis of amino acid sequences of holins to extend evolutionary insights to other Gram-positive bacterial phages.
Main Results:
- Identified three distinct evolutionary branches within the phi29-like phage genus: (BS32, phi15, phi29, PZA), (B103, M2, Nf), and (GA-1).
- Confirmed evolutionary links with other protein-primed replicating phages through sequence comparisons.
- Holin protein analysis revealed inconsistencies in current bacteriophage classification.
Conclusions:
- The study provides a detailed evolutionary framework for phi29-like phages, defining specific clades.
- The findings underscore the utility of protein-primed replication mechanisms and holin sequences in understanding phage evolution.
- The research suggests a need for re-evaluation of existing bacteriophage classification systems based on molecular data.