The genome of the tailed temperate coliphage P2 (Myoviridae) contains some genes that probably are horizontally transferred additions to the genome. One of these genes, the Z/fun gene, was recently found intact in the genome of Neisseria meningitidis. We have investigated the presence of P2-like phages, and the genetic variation at the position corresponding to the phage P2 Z/fun locus, in the Escherichia coli reference collection (ECOR). P2-like phages are common in E. coli since they are present in about 30% of the ECOR strains. Hybridizations and PCR amplifications indicate that the overall variation among these phages is small. Amplification of the region corresponding to the phage P2 Z/fun locus in 11 prophages revealed that this is a multivariable locus. Sequencing of the region resulted in 10 completely different sequences but with a similar high AT-content as the Z/fun gene. All sequences contained at least one open reading frame with good transcription and translation signals. All sequences were also surrounded by a highly similar, previously undiscovered, inverted repeat (IR). We also found this IR in genetically unstable regions in pathogenic enterobacteria. This demonstrates that P2-like phages are important factors in the evolution of bacteria, not only because they carry a diversity of lysogenic conversion genes but also because they can act as vectors for single genes. The genes found between the IRs have unknown functions, and only a few clearly similar genes have been found in other bacteria.

The amount and distribution of variation in the genomic region containing the genes in the lytic-lysogenic genetic switch and the sequence that determines the integration site into the host chromosome were analyzed for 38 P2-like phages from Escherichia coli. The genetic switch consists of two convergent mutually exclusive promoters, Pe and Pc, and two repressors, C and Cox. The immunity repressor C blocks the early Pe promoter, leading to the establishment of lysogeny. The Cox repressor blocks expression of Pc, allowing lytic growth. Phylogenetic analyses showed that the C and Cox proteins were distributed into seven distinct classes. The phylogenetic relationship differed between the two proteins, and we showed that homologous recombination plays a major role in creating alterations in the genetic switch, leading to new immunity classes. Analyses of the host integration site for these phages resulted in the discovery of a previously unknown site, and there were at least four regular integration sites. Interestingly, we found no case where phages of the same immunity class had different host attachment sites. The evolution of immunity and integration sites is complex, since it involves interactions both between the phages themselves and between phages and hosts, and often, both regulatory proteins and target DNA must change.P2-like phages are a group of related temperate phages that grow on ␥-proteobacteria and share common traits such as morphology, control of lytic versus lysogenic growth, and noninducibility by UV light (for a recent review, see reference 26). Temperate phages have the ability to reproduce by two alternative life cycles: the lytic or the lysogenic cycle. In the latter life cycle, the phage genome integrates into a specific location on the host chromosome, and most phage genes are turned off by the phage-encoded immunity repressor C. P2-like phages are prevalent in Escherichia coli strains; about 30% of the strains in the ECOR collection (28) contain P2-like prophages (27). P2-like phages that are found in other ␥-proteobacteria are more distantly related to P2 than those found in E. coli, and it seems as if the evolution of the P2-like phages tracks the evolution of their respective hosts (26; A. S. Nilsson, unpublished data). An analysis of the DNA sequence of the late structural genes of 18 P2-like isolates that grow on E. coli showed that these genes are at least 96% identical to the genes of P2 (25). Thus, these P2-like coliphages might be considered different isolates of P2, but they have been shown to have different immunities, based on their capacity to grow on bacteria lysogenized with different P2-like phages (6,9,14) and to integrate at at least two different sites in the host chromosome (22,38). Phage 186 is a more distantly related E. coli phage, and its immunity repressor, cI, differs in size and sequence from the C repressors of the P2-like coliphages in this study. In fact, both cI and Apl (the equivalent of Cox) of phage 186 are more related to the cI and Cox repressors of Ha...