🔬 Phage T4 Meets Microbial Diversity

Bacteriophage Ecology Group News (BEG News) was published mostly quarterly as an online newsletter for a total of 24 issues, July 1999 through April 2005. As follows is a reprint of an article from Volume 17. The newsletter’s successors are the ongoing Phage.org website, phage-therapy.org, and the Bacteriophage Ecology Group Facebook page.

Bacteriophages are extremely diverse in the range of microbial hosts that they infect and the chemical nature, size and geometry of their genetic materials. As the most abundant organisms on Earth [1], they contribute substantially to overall diversity of the gene pool in the microbial world. It is no surprise then that they have become prominent in the ongoing discussion on microbial diversity [2-4]. The interactions of phages with their hosts are not only important for maintenance of the ecological balance, they may also constitute a major component of the network for lateral transfer of genes among microorganisms. This notion is based on studies with all types of archived phages, which of course represent only a minute fraction of the estimated number of unique phage genomes in nature. Nevertheless, the extent to which phage may contribute to microbial diversity is becoming better appreciated because of an ongoing expansion of sequence databases for all types of organisms, including phage. In the US, funding agencies have teamed together to coordinate a significant increase in support for microbial genome sequencing projects, with a great deal of emphasis being placed on speed and economy of generating, assembling, annotating, and sharing sequence data. It is anticipated that the microbial genome database will continue to expand at a fast pace in the foreseeable future.

The most commonly used approach for sequencing a microbial genome involves genomic library construction (usually from randomly sheared genomic DNA), sequencing a large number of library clones (high-throughput sequencing), computer-assisted assembly of sequence data into contiguous segments (contigs), and carrying out more sequencing and data assembly to close gaps between contigs. The high-throughput stage is expected to yield data from overlapping clones for better accuracy of sequence reads and length of assembled contigs. In principle, the approach is straightforward and has the clearly defined goal of producing an accurate single contiguous sequence of the genome. In practice, it is riddled with bottlenecks that can be different for different genomes, depending on their sizes, states of modification, content of sequences that cannot be cloned and other factors. Personnel training and quality of operations in general are also critical factors to consider in such projects. It is very common for projects to use commercial outfits or collaborations with well-funded research institutes for operations that are impractical to support locally. Usually progress is very rapid during the early stages of a project, but gets bogged down in later stages. Some genomes remain 80-90% finished for months or years, but can still be mined for useful information, provided that this information is released to the scientific community. The technology continues to improve on several fronts, and we can expect that alternate approaches, e.g., circumventing cloning and more powerful computer programming, will cut down the time and expense required to produce a finished genome sequence and allow the sequencing of several genomes concurrently by the same team.

Phage oriented projects have so far completed the sequences of ~150 genomes (GenBank). In some cases, several members of the same phage family (Siphoviridae, Myoviridae or Podoviridae; ICTV nonenclature) are included in databases. Collectively, the data suggest that despite their vast differences in genetic composition, all dsDNA phages share similar genome architecture. The typical dsDNA phage genome consist of a mosaic of gene sets that are shared with other members of the same phage "genus" and gene sets that are unique to each genome and interspersed with the genus-specific sets. That is, dsDNA phage genomes seem to evolve by gathering genes from different sources, including genes that qualify the phages for membership in their particular genera. In some instances, lateral DNA transfer (by homologous or nonhomologous recombination) is suspected to be responsible for mosaic patterns that appear inside some phage genes. Since gene evolution by mutation (vertical change) and genome evolution by lateral DNA transfer probably occur independently of each other, it is difficult to relate whole genomes belonging to the same genus to one another in chronological order. Such timelines are more meaningful when sequences of shared (homologous) genes or gene clusters (or their protein products) are compared, e.g., divergence of an essential gene/protein within a phage genus. The framework represented by genomes of the T4-like phages is an excellent example of how vertical and horizontal evolution may drive diversity in a dsDNA phage genome type. The T4 genome type is large by viral standards and carries many genes that one usually finds in cellular rather than viral genomes. Among these are genes for some enzymes of intermediary metabolism, a multi-component DNA replisome, extensive machinery for genetic recombination, and certain types of mobile DNA elements (including homing endonuclease genes) that can move themselves and flanking DNA unidirectionally [5, 6]. There is also a well-studied prototype, phage T4 [7, 8], than can be used as reference when comparing nucleotide sequences and genome organization of different T4-like phages.

In a collaborative project with Henry Krisch (CNRS, Toulouse, France), we have been sequencing the genomes of a number of T4-like Myoviridae that diverge in host range and/or other characteristics, as determined by preliminary genetic and genomic scanning. Thanks to the efforts of Hans Ackermann, a number of these phages that infect bacterial hosts other than E. coli have been archived at LaValle University (Quebec, CA) and made available for our studies. The sequences of 2 Aeromonas phages, Aeh1 (A. hydrophila) and 44RR2.8t (A. salmonicida) and 2 coliphages RB69 and RB49 are now posted on a publicly accessible web site (http://phage.bioc.tulane.edu) and are in the process of being submitted to GenBank. Although the available data probably represent only a very tiny sampling of what must exist in nature for this type of phage genome, certain predictions can already be made with regards to the kind of diversity one may encounter if a much more extensive collection of T4-like phages is analyzed. For example, whereas genome size appears to be rather fixed for some dsDNA phages, T4-like genomes can vary in length over a wide range. Currently, the observed range is ~164Kbp (for phage RB49) to ~233Kbp (for phage Aeh1). So it appears that genomes of the T4 kind can recruit variable amounts of DNA to go with a certain core that is common to all. Reversible gain and loss of genes and homologues may occur depending on composition of the gene pool where exchanges take place. Based on what we know from T4 studies, the highly recombinogenic character of this genetic system may allow it to be an effective scavenger of DNA from microbial hosts. . The Aeh1 genome carries 23 tRNA genes (19 amino-acid specificities), which is one indication of DNA acquisition from cellular sources. Matches to bacterial sequences in databases account for 2-5% of the predicted ORFs for any of the genomes sequenced so far. This is probably a vast underestimate of the contribution of bacterial DNA to T4-like genomes. More likely, much of the other nonT4-like DNA we observe for these phages has its matches in microorganisms that have yet to be discovered. The combinatorial potential of the genome framework acquired by the T4-like phages might underlie a potential for these phages to cross species barriers between bacteria. If this is happening in nature, then the T4-like population and unrelated phage populations with similar potential [4] could be dynamically affecting microbial diversity on a global scale.

It is still unclear what constitutes the "core" DNA of a T4-like genome. It could be >100 ORFs. Because morphological criteria have figured significantly in the classification of phages into families and genera, it has not been surprising to find homologues of the T4 morphogenesis genes in all the genomes examined so far in the "T4-Like Genome Project" (http://phage.bioc.tulane.edu). On the other hand, homologues of the T4 DNA replication/recombination gene clusters are consistently being observed to coexist with the morphogenesis clusters. Functional coupling between replication and morphogenesis has been documented in T4 studies, and could conceivably be required for natural selection of this type of phage genome. It remains to be seen if phages of the T4 morphotype exist in nature which utilize a different mode of replication from the T4 paradigm, or vice versa. To find out, one would have to utilize specific probes to access a much larger set of genomes than exists today in laboratory archives. It is particularly important to be able to screen environmental sources for genomes of phages that cannot be isolated through traditional plaque assays. T4-like phages that have significantly larger genomes than T4 and those that grow on bacterial hosts other than E. coli (or the enterobacteria in general) are underrepresented in laboratory collections [9]. Also, no phages of this genus have been reported whose heads/genomes are much smaller than T4. Finding more of T4's relatives in a variety of environmental niches and sequencing them would boost our understanding of the pathways leading to microbial diversity. In addition, such phages/genomes would constitute a treasure chest of genes and proteins for all types of studies in basic and applied molecular biology. I beg the BEG to undertake the search for more T4-like phages.

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Pedulla, P. L., Ford, M.E., Houtz, J.M., Karthikeyan, T., Wadsworth, C., Lewis, J.A., Jacobs-Sera, D., Falbo, J., Gross, J., Pannunzio, N.R., Brucker, W., Kumar, V., Kandasamy, J., Keenan, L., Bardarov, S., Kriakov, J., Lawrence, J.G., Jacobs jr., W.R., Hendrix, R.W., Hatfull, G.F. (2003) Origins of highly mosaic Mycobacteriophage genomes. Cell 113: 171-182.

Belle, A., Landthaler, M., and Shub, D. A. (2002). Intronless homing: site-specific endonuclease SegF of bacteriophage T4 mediates localized marker exclusion analogous to homing endonucleases of group I introns. Genes Dev 16, 351-362.

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Miller, E. S., Kutter, E., Mosig, G., Arisaka, F., Kunisawa, T., and Ruger, W. (2003). Bacteriophage T4 Genome. Microbiol Mol Biol Rev 67, 86-156.

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Jim D. Karam
Department of Biochemistry, Tulane University Health Sciences Center, New Orleans, Louisiana

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