We have determined the sequence and genome structure of the broad-host-range, virulent Listeria myovirus A511. The nonredundant unit (informational) genome spans 134,494 bp. Genes are arranged in two major and clearly distinct clusters, encoding structural components and DNA replication functions. Such modular genome organization is very common among tailed phages and can be regarded as a universal paradigm in bacteriophage biology.
Other myoviruses of low-G+C-content host bacteria, such as Staphylococcus aureus
phage K (47
), Lactobacillus plantarum
bacteriophage LP65 (9
), and Enterococcus faecalis
), infect noncompatible hosts and should therefore be genetically isolated from each other and from A511. Therefore, the similarities regarding overall genome composition and the sequence homologies found among these phages are intriguing. Comparison of the genomic maps of A511, K, and LP65 revealed a flexible organization comprising life cycle-specific gene clusters of these virulent phage genomes, even more flexible than those of the temperate phages (36
). For example, cell lysis genes and tRNA gene clusters of A511, K, and LP65 are located at completely different linear coordinates within their genomes, highlighting the mosaicism among these phage genomes. However, the overall conserved organizations in large parts of the early and late gene clusters of A511, LP65, and K suggest a modular distribution and acquisition of these elements during the evolution of these viruses.
The A511 phage DNA molecule features two identical 3,125-bp terminal sequences, amounting to a total unit genome of 137,619 bp packaged into the phage capsid. The genes located within the redundancy are the first to enter and be expressed in the infected cell. This agrees well with the observation that they appear to be optimized for efficient early expression in the host environment, since the transcriptional units and individual genes feature near-consensus bacterial promoter sequences and ribosome binding sites, respectively. For SPO1, it has been proposed that the corresponding genes represent a “host-takeover module” (57
), which paves the way for efficient phage replication. However, A511 gp1 to gp9 show no homology to SPO1 products or any other known proteins, and their precise role during the initial stages of the infection process remains to be determined.
Phages A511, P100, and K are virtually indistinguishable by morphology (Fig. ), and, with respect to dimensions, mode of tail contraction, and base plate structure, also match with LP65 (9
). This underlines the relationship of SPO1-like phages with host ranges including different bacterial genera and agrees well with the finding that morphology correlates well with similarities among the structural-component-related genes and proteins. In contrast, only little similarity was seen among the genes directing DNA replication and many other unknown functions, which suggests a more host-specific adaptation of these elements when we consider divergent evolution of these viruses alongside their host bacteria. Interestingly, six of these genes feature weak to moderate homologies to coliphages T4 and T5. Similar findings have also been reported for LP65 (9
). These genes represent the only link of A511 to phages of gram-negative bacteria, underlining the relatively isolated position of the SPO1-like phages within the Myoviridae
Clearly, high-frequency recombination among phages infecting common hosts occupying similar ecological niches will result in a high degree of mosaicism in local phage populations. Horizontal exchange of genetic modules of different sizes also takes place across the entire phage population, although at a much lower frequency (4
). Homologous recombination certainly plays an important role in module exchange among viruses infecting related host bacteria. This is common in lysogens, from which the incoming phage can draw from the gene pool of prophages or cryptic phage remnants residing in the bacterial genomes (4
The increasing numbers of the available phage sequences, genome maps, and comparative alignments indicate that promiscuous mix-and-match of genetic information is a common feature also among virulent phages such as those in the SPO1 group, which are able to exchange genetic material only during the first stages of the infection process. Considering that these phages are generally able to infect only one genus of host bacteria but mostly feature a rather broad host range within this taxonomic group, one has to conclude that their relationship is based on divergence from a common ancestor infecting less-diversified host bacteria and/or from sporadically occurring intergeneric transduction events, followed by subsequent (illegitimate) recombination. Interestingly, A511 appears more closely related to Staphylococcus phages than to phages infecting Bacillus; the phylogeny of the host bacteria would suggest a different situation. However, the number of available sequences is much too limited for a reasonable phylogenetic analysis.
None of the known SPO1-like phages infects bacteria from more than one genus, although their hosts are all grouped into the phylum Firmicutes and are therefore more or less genetically linked. This is especially true for the hosts of the phages described in this paper, which all belong to the class Bacilli and the orders Bacillales (Listeria, Staphylococcus, and Bacillus) and Lactobacillales (Lactobacillus). Interestingly, it is not known whether SPO1-like phages for other classes within this phylum (e.g., Clostridia) exist.
It has been demonstrated that bacteriophages with circularly permuted genomes can transduce genetic markers, whereas phages featuring invariable genome ends do not (22
). The inability of A511 to transduce can now be explained by the structure of its genome, and this explanation likely holds true for other SPO1-like phages. Initiation of DNA packaging by terminase is probably dependent on recognition of a specific (yet unidentified) DNA sequence motif (pac
), which prevents accidental packaging of nonphage bacterial or plasmid DNA.
The presence of terminally redundant, nonpermuted genomes in myoviruses infecting gram-positive bacteria was reported for Bacillus
phage SPO1 (deduced from restriction maps and DNA-DNA hybridization) (50
) and supposed for phage SP82 (10
). The similarity of Lactobacillus
phage LP65 to SPO1-like myoviruses was mentioned previously (9
), but the authors of that study did not investigate the genome structure. We have shown that, besides that of A511, the DNA molecules of both P100 and K have redundant end regions. Interestingly, the extent of terminal redundancy in these otherwise similar phages varies greatly, from 3.1 kb in A511 to approximately 20 kb in K. In fact, our data indicate that this is a general feature of the SPO1-like phages. The sequence of Enterococcus faecalis
EF24C has been published very recently (60
) and features significant homologies to A511 (47% overall nucleic acid identity and 64 proteins with >33.7% identity [expectation values of <0.001]). Although
EF24C was classified as being a SPO1-like phage, the authors of that publication assumed that it has a circularly permuted genome. Considering the findings presented here, however, it is not unlikely that this phage also features redundant genome ends. In contrast, the giant Bacillus thuringiensis
phage 0305ϕ8-36, which also features 6.5-kb terminal redundancy (59
), probably represents a lineage of its own. It is clearly different from the SPO1-like phages with respect to morphology, genome size, and its complete lack of sequence homologies.
In conclusion, it can be anticipated that many of the yet-uncharacterized myoviruses mentioned in the literature, those infecting not only Listeria
, and Bacillus
but also other genera such as Enterococcus
), will fall into this group of broad-host-range, virulent SPO1-like phages featuring large, terminally redundant genomes. A511 represents a typical example and suitable model to study this evolutionarily very successful class of bacterial viruses.