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Based on the organism list of the National Center for Biotechnology Information (NCBI) database (http://www.ncbi.nlm.nih.gov/taxonomy/) (15) and on phage counts from (3). Total phage numbers are lower than in table 2-1 because rRNA data are unavailable for some phage hosts. CFP, cubic, filamentous, and pleomorphic phages. aIncluding a phage-like particle.

so far, without phages. Most phages are found in easily grown and medically or industrially important bacteria:

1. Firmicutes with high G-C (coryneforms, mycobacteria, streptomycetes),

2. Firmicutes with low G-C (bacilli, lactobacilli, lacto-cocci, clostridia, staphylococci, streptococci),

3. Proteobacteria, especially of the g subdivision. The latter includes enterobacteria (over 800 phage observations) and pseudomonads.

Tailed phages predominate almost everywhere and occur in both eubacteria and Euryarcheota, suggesting that they originated before separation of these bacterial kingdoms. Siphoviridae are particularly frequent in actinomycetes, coryneforms, lactococci, and streptococci. Myoviruses and podoviruses are relatively frequent in enterobacteria, pseudomonads, bacilli, and clostridia. This distribution is probably related to features of bacterial speciation, for example to evolution of particular receptors or restriction endonucleases.

There is a chasm between the ubiquitous tailed phages and the other types of bacterial viruses. The latter are relatively rare and have narrow, particular host range. Inoviruses of the Plectrovirus genus are restricted to mycoplasmas, and Fuselloviridae, Lipothrixviridae, and Rudiviridae are limited to a particular archaeal subdivision, the extremely thermophilic Crenarcheota. This suggests that their hosts constitute ecological niches, in which these viruses originated. On the other hand, there are oddities in distribution: (i) filamentous inoviruses, though generally associated with enterobacteria and their relatives, occur in such diverse bacteria as Clostridium, Propionibacterium, and Thermus;

and (ii) tectiviruses are found in enterics, bacilli, and Thermus. As these phages are plasmid-dependent, their distribution may be explained by plasmid transfer (3).

Why Phage Classification?

To microbiologists focusing on a single microorganism, classification may appear a sterile exercise. Others, impressed by spectacular advances in limited fields, may consider that ''we know it all.'' Indeed, bacteriophages T4 and l are among the best-known viruses of all. This way of thinking is short-sighted and self-defeating. We have barely scratched the surface of virology. With respect to bacteriophages, (i) research has concentrated on a few viruses and neglected the 5000 others known, (ii) research is limited to about 15 countries in the world, and (iii) important habitats such as hot springs or fermentors have been little explored. My experience is that, armed with an electron microscope, I am certain to find scores of new phages in a bottle of sewage. At the same time, phages have practical applications that demand precise identification. Last but not least, microbiol-ogists want to understand the living world and many of them are actively engaged in teaching. For them, and their students, taxonomy is simplification. It is impossible and pointless to memorize the properties of 5000 individual tailed phages, but it is much more rewarding to study tailed phages as a group. The advantages of classification, especially with respect to phages, are many:

1. Classification is generalization and simplification.

a. It promotes comparison and thus virus research and better understanding of the viral world.

Figure 2-2 Selected myoviruses. A: Bacillus megaterium phage G, the largest myovirus known; note the spiral filament around the tail. B: Giant unknown bacteriophage found in macerated debris of Bombyx mori; note the wavy tail fibers. Phage heads of this size are easily deformed. C: Bdellovibrio bacteriovorus phage f1402, the smallest myovirus known (isolated by B. A. Fane, Department of Veterinary Science, University of Arizona, Tucson, AZ). x 297,000; bar indicates 100 nm. Phosphotungstate (2%, pH 7.2) (A, B)and uranyl acetate (2%, pH 4.0) (C).

Figure 2-2 Selected myoviruses. A: Bacillus megaterium phage G, the largest myovirus known; note the spiral filament around the tail. B: Giant unknown bacteriophage found in macerated debris of Bombyx mori; note the wavy tail fibers. Phage heads of this size are easily deformed. C: Bdellovibrio bacteriovorus phage f1402, the smallest myovirus known (isolated by B. A. Fane, Department of Veterinary Science, University of Arizona, Tucson, AZ). x 297,000; bar indicates 100 nm. Phosphotungstate (2%, pH 7.2) (A, B)and uranyl acetate (2%, pH 4.0) (C).

Figure 2-3 Myoviruses (A-C) and siphoviruses (D, E). A: Staphylococcus hyicus phage Twort. B: Coliphage RB69. The head of the left particle is slightly deformed and the tail of the right particle is contracted. C: "Killer particle'' of Bacillus subtilis; note the small, bacterial DNA-containing head. D: Phage NM1 of Sinorhizobium meliloti with transverse tail disks and globular fixation structures. E: Bacillus subtilis phage f105. x 297,000; bar indicates 100 nm. Uranyl acetate (A, C, E) and phosphotungstate (B, D).

Figure 2-3 Myoviruses (A-C) and siphoviruses (D, E). A: Staphylococcus hyicus phage Twort. B: Coliphage RB69. The head of the left particle is slightly deformed and the tail of the right particle is contracted. C: "Killer particle'' of Bacillus subtilis; note the small, bacterial DNA-containing head. D: Phage NM1 of Sinorhizobium meliloti with transverse tail disks and globular fixation structures. E: Bacillus subtilis phage f105. x 297,000; bar indicates 100 nm. Uranyl acetate (A, C, E) and phosphotungstate (B, D).

b. It is indispensable for teaching. No textbook is conceivable without it and students, as teacher well know, want certitude. Taxonomy is also a basic ingredient in theses. Without classification, virology is just a magma for the beginner.

2. Classification is essential for phylogenetic studies. It identifies the very biological groups that are to be compared. For example, isolated genes or genome sequences are of limited interest, but gain immensely in significance when traced to other categories of organisms. Without phage classification, we would probably not know that such things as horizontal gene transfer exist.

3. Classification is required for proper identification of:

a. New phages.

b. Harmful phages in biotechnology and industrial fermentations.

c. Industrially important phages in patent applications.

d. Therapeutic phages. Now that phage therapy is making a comeback, they must be identified. It is indeed inconceivable that people should be treated (even injected) with something unknown.

4. Classification is a great research aid. For example, the DNA size of tailed phages can be predicted with accuracy from capsid dimensions.

Outlook

Phage taxonomy is bound to evolve. New phage families are likely to be discovered in unusual habitats, such as volcanic springs, hypersaline lagoons, or the mammalian

Figure 2-5 Selected isometric and filamentous phages. A: E. coli microvirus fX174, x 297,000. B: Tectivirus 37-64 of Thermus sp.; note inner membrane and deformed particles; x 297,000 C: E. coli levivirus R17 adsorbed to pili, x 148,500 D: Inovirus H75 of Thermus thermophilus, x 92,400. Bars indicate 100 nm. Uranyl acetate (A) and phosphotungstate (B-D).

Figure 2-4 Siphoviruses (A, B) and podoviruses (C-F). A: Staphylococcus aureus phage 6. B: Bacillus subtilis phage BS5. C: Lactococcus lactis ssp. cremoris phage KSY1. D: Salmonella typhimurium phage P22. E: Bacillus sp. phage GA-1. F: Coliphage Esc-7-11. x 297,000; bar indicates 100 nm. Uranyl acetate (A, B, F) and phosphotungstate (C, D, E).

Figure 2-5 Selected isometric and filamentous phages. A: E. coli microvirus fX174, x 297,000. B: Tectivirus 37-64 of Thermus sp.; note inner membrane and deformed particles; x 297,000 C: E. coli levivirus R17 adsorbed to pili, x 148,500 D: Inovirus H75 of Thermus thermophilus, x 92,400. Bars indicate 100 nm. Uranyl acetate (A) and phosphotungstate (B-D).

rumen. Indeed, strange phage-like particles that probably represent novel families have been reported many times, for example arrow-shaped, apparently archaebacteria-related particles in saline environments (18). Families are the most stable part of the present edifice of viral taxonomy. Genera and species are more fluid because there are no universally accepted or applicable criteria for defining them. Mayr's "biological species definition" (11), based on interbreeding with production of fertile offspring, was devised for songbirds and is almost inapplicable to viruses. The polythetic species concept (13), which is in principle applicable to genera, is a concept and does not provide concrete guidelines for definitions of phage species or genera. I contend that no biologist (or virologist) can certify what a species is. Virus (or phage) classification is therefore still an art.

The most important present challenge is the interpretation of horizontal gene transfer in tailed phages. It may be argued that any classification of these viruses is impossible because of gene shuffling and the occurrence of certain genes in apparently unrelated viruses. This attitude is a dead end which can only lead to forfeiting the benefits of classification. It overlooks major realities of viral biology: (i) Common genes do not necessarily indicate close relationships. Horizontal gene transfer is frequent and ubiquitous. Some (all?) genes move through the living world; for example T4 lysozyme surfaces in goose eggs and human tears (1). (ii) Recombination can mask or simulate relationships. (iii) In viruses related by evolution, genomic relationships between different taxa are to be expected. The question of devising genera or species in tailed phages thus becomes a quantitative one. We may have to follow the bacteriologists who, arbitrarily, fixed the threshold value for species delineation at 60-70% DNA homology (8).

References

1. Ackermann, H.-W. 1998. Tailed bacteriophages—The order Caudovirales. Adv. Virus Res. 51:135-201.

2. Ackermann, H.-W. 1999. Bacteriophages, pp. 398-411. In J. P. Lederberg (ed.-in-chief), Encylopedia of Microbiology, 2nd edn, vol. 1. Academic Press, New York.

3. Ackermann, H.-W. 2001 Frequency of morphological phage descriptions in the year 2000. Arch. Virol. 146: 843-857.

4. Bradley, D. E. 1967. Ultrastructure of bacteriophages and bacteriocins. J. Bacteriol. 31:230-314.

5. D'Herelle, F. 1918. Technique de la recherche du microbe filtrant bacteriophage (Bacteriophagum intestinale). C. R. Soc. Biol. 81:1160-1162.

6. Duckworth, D. H. 1976. Who discovered bacteriophage? Bacteriol. Rev. 40:793-802.

7. Huber, K. E., and M. K. Waldor. 2000. Filamentous phage integration requires the host recombinases XerC and XerD. Nature 417:656-659.

8. Johnson, J. L. 1984. Nucleic acids in bacterial classification, p. 8-11. In N. T. Krieg and J. G. Holt (eds.), Bergey's Manual of Systematic Bacteriology, vol. 1. Williams & Wilkins, Baltimore, Md.

9. Lwoff, A., R. W. Horne, and P Tournier. 1962. A system of viruses. Cold Spring Harbor Symp. Quant. Biol. 27:51-62.

10. Maniloff, J., and H.-W. Ackermann. 1998. Taxonomy of bacterial viruses: establishment of tailed virus genera and the order Caudovirales. Arch. Virol. 143:2051-2063.

11. Mayr, E. 1942. Systematics and the Origin of Species from the Viewpoint of a Zoologist, p. 119. Columbia University Press, New York.

12. Skalka, A., and P. Hanson. 1972. Comparisons of nucleo-tides and common sequences in deoxyribonucleic acid from selected bacteriophages. J.Virol. 9:583-593.

13. Van Regenmortel, M. H. V. 1990. Virus species, a much overlooked, but essential concept in virus classification. Intervirology 31:241-254.

14. Van Regenmortel, M. H. V, C. M. Fauquet, D. H. L. Bishop, E. B. Carstens, M. K. Estes, S. M. Lemon, J. Maniloff, D. J. McGeoch, C. R. Pringle, and R. B.Wickner (eds.). 2000. Virus Taxonomy, pp. 63-136, 267-284, 389-393, 645-650. Seventh Report of the International Committee on Taxonomy of Viruses. Academic Press, San Diego, Calif.

15. Vander Byl, C., and A. M. Kropinski. 2000. Sequence of the genome of Salmonella bacteriophage P22. J. Bacteriol. 182:6472-6481.

16. Wheeler, D. L., C. Chappey, A. E. Lash, D. D. Leipe, T. L. Madden, G. D. Schuler, T. A. Tatusova, and B. A. Rapp. 2000. Database resources of the National Center for Biotechnology Information. Nucleic Acids Res. 28:10-14.

17. Wildy, P. 1971. Classification and nomenclature of viruses. First Report of the International Committee on Nomenclature of Viruses. Monographs in Virology, vol. 5. Karger, Basel.

18. Zillig, W., A. Kletzin, C. Schleper, I. Holz, D. Janekovic, J. Hain, M. Lanzendörfer, and J. K. Kristjansson. 1994. Screening for Sulfolobales, their plasmids, and their viruses in Icelandic solfataras. Syst. Appl. Microbiol. 16:609-628.

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