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《环境微生物学与实验》课程教学资源（文献资料）The Viruses - Bacteriophages

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Prescott−Harley−Klein: Microbiology, Fifth Edition VI. The Viruses 17. The Viruses: Bacteriophages © The McGraw−Hill Companies, 2002 382 Chapter 17 The Viruses: Bacteriophages You might wonder how such naive outsiders get to know about the existence of bacterial viruses. Quite by accident, I assure you. Let me illustrate by reference to an imaginary theoretical physicist, who knew little about biology in general, and nothing about bacterial viruses in particular.... Suppose now that our imaginary physicist, the student of Niels Bohr, is shown an experiment in which a virus particle enters a bacterial cell and 20 minutes later the bacterial cell is lysed and 100 virus particles are liberated. He will say:“How come, one particle has become 100 particles of the same kind in 20 minutes? That is very interesting. Let us find out how it happens! . . . Is this multiplying a trick of organic chemistry which the organic chemists have not yet discovered? Let us find out.” —Max Delbrück Chapter 16 introduces many of the facts and concepts underlying the field of virology, including information about the nature of viruses, their structure and taxonomy, and how they are cultivated and studied. Clearly the viruses are a complex, diverse, and fascinating group, the study of which has done much to advance disciplines such as genetics and molecular biology. Chapters 17 and 18 focus on virus diversity. This chapter is concerned with bacterial viruses or bacteriophages; the next surveys animal, plant, and insect viruses. The taxonomy, morphology, and reproduction of each group are covered. Where appropriate, the biological and practical importance of viruses is emphasized (Box 17.1), even though viral diseases are examined in chapter 38. Since the bacteriophages (or simply phages) have been the most intensely studied viruses and are best understood in a molecular sense, this chapter is devoted to them. 17.1 Classification of Bacteriophages Although properties such as host range and immunologic relationships are used in classifying phages, the most important are phage morphology and nucleic acid properties (figure 17.1). The genetic material may be either DNA or RNA; most known bacteriophages have double-stranded DNA. Most can be placed in one of a few morphological groups: tailless icosahedral phages, viruses with contractile tails, viruses with noncontractile tails, and filamentous phages. There are even a few phages with envelopes. The most complex forms are the phages with contractile tails, for example, the T-even phages of E. coli. 1. Briefly describe in general terms the morphology and nucleic acids of the major phage types. 17.2 Reproduction of Double-Stranded DNA Phages: The Lytic Cycle After DNA bacteriophages have reproduced within the host cell, many of them are released when the cell is destroyed by lysis. A phage life cycle that culminates with the host cell bursting and reMicrobiologists have previously searched without success for viruses in marine habitats. Thus it has been assumed the oceans probably did not contain many viruses. Recent discoveries have changed this view radically. Several groups have either centrifuged seawater at high speeds or passed it through an ultrafilter and then examined the sediment or suspension in an electron microscope. They have found that marine viruses are about 10 times more plentiful than marine bacteria. Between 106 and 109 virus particles per milliliter are present at the ocean’s surface. It has been estimated that the top one millimeter of the world’s oceans could contain a total of over 3 1030 virus particles! Although little detailed work has been done on marine viruses, it appears that many contain double-stranded DNA. Most are probably bacteriophages and can infect both marine heterotrophs and cyanobacteria. Up to 70% of marine procaryotes may be infected by phages. Viruses that infect diatoms and other major algal components of the marine phytoplankton also have been detected. Marine viruses may be very important ecologically. Viruses may control marine algal blooms such as red tides (p. 580), and bacterioBox 17.1 An Ocean of Viruses phages could account for 1/3 or more of the total aquatic bacterial mortality or turnover. If true, this is of major ecological significance because the reproduction of marine bacteria far exceeds marine protozoan grazing capacity. Virus lysis of procaryotic and algal cells may well contribute greatly to carbon and nitrogen cycling in marine food webs. It could reduce the level of marine primary productivity in some situations. Bacteriophages also may greatly accelerate the flow of genes between marine bacteria. Virus-induced bacterial lysis could generate most of the free DNA present in seawater. Gene transfer between aquatic bacteria by transformation (see pp. 305–7) does occur, and bacterial lysis by phages would increase its probability. Furthermore, such high phage concentrations can stimulate gene exchange by transduction (see pp. 307–9). These genetic exchanges could have both positive and negative consequences. Genes that enable marine bacteria to degrade toxic pollutants such as those in oil spills could spread through the native population. On the other hand, antibiotic resistance genes in bacteria from raw sewage released into the ocean also might be dispersed (see section 35.7)

Prescott−Harley−Klein: Microbiology, Fifth Edition VI. The Viruses 17. The Viruses: Bacteriophages © The McGraw−Hill Companies, 2002 leasing virions is called a lytic cycle. The events taking place during the lytic cycle will be reviewed in this section, with the primary focus on the T-even phages of E. coli. These are doublestranded DNA bacteriophages with complex contractile tails and are placed in the family Myoviridae. They are some of the most complex viruses known. The structure of T-even coliphages (p. 376) The One-Step Growth Experiment The development of the one-step growth experiment in 1939 by Max Delbrück and Emory Ellis marks the beginning of modern bacteriophage research. In a one-step growth experiment, the reproduction of a large phage population is synchronized so that the molecular events occurring during reproduction can be followed. A culture of susceptible bacteria such as E. coli is mixed with bacteriophage particles, and the phages are allowed a short interval to attach to their host cells. The culture is then greatly diluted so that any virus particles released upon host cell lysis will not immediately infect new cells. This strategy works because phages lack a means of seeking out host cells and must contact them during random movement through the solution. Thus phages are less likely to contact host cells in a dilute mixture. The number of infective phage particles released from bacteria is subsequently determined at various intervals by a plaque count (see section 16.4). A plot of the bacteriophages released from host cells versus time shows several distinct phases (figure 17.2). During the latent period, which immediately follows phage addition, there is no release of virions. This is followed by the rise period or burst, when the host cells rapidly lyse and release infective phages. Finally, a plateau is reached and no more viruses are liberated. The total number of phages released can be used to calculate the burst size, the number of viruses produced per infected cell. The latent period is the shortest time required for virus reproduction and release. During the first part of this phase, host bacteria do not contain any complete, infective virions. This can be shown by lysing them with chloroform. This initial segment of the latent period is called the eclipse period because the virions 17.2 Reproduction of Double-Stranded DNA Phages:The Lytic Cycle 383 dsDNA dsRNA Cystoviridae Leviviridae 100 nm Fuselloviridae Tectiviridae Rudiviridae Plasmaviridae Lipothrixviridae Microviridae Inoviridae Plectrovirus Inoviridae Inovirus Podoviridae Siphoviridae Myoviridae, elongated head Myoviridae, isometric head Corticoviridae DNA RNA ssDNA ssRNA "SNDV-like viruses" Figure 17.1 Major Bacteriophage Families and Genera. The Myoviridae are the only family with contractile tails. Plasmaviridae are pleomorphic. Tectiviridae have distinctive double capsids, whereas the Corticoviridae have complex capsids containing lipid

Prescott−Harley−Klein: Microbiology, Fifth Edition VI. The Viruses 17. The Viruses: Bacteriophages © The McGraw−Hill Companies, 2002 Binding is probably due to electrostatic interactions and is influenced by pH and the presence of ions such as Mg2 and Ca2. After the baseplate is seated firmly on the cell surface, conformational changes occur in the baseplate and sheath, and the tail sheath reorganizes so that it shortens from a cylinder 24 rings long (see p. 376) to one of 12 rings. That is, the sheath becomes 384 Chapter 17 The Viruses: Bacteriophages were detectable before infection but are now concealed or eclipsed. The number of completed, infective phages within the host increases after the end of the eclipse period, and the host cell is prepared for lysis. The one-step growth experiment with E. coli and phage T2 provides a well-studied example of this process. When the experiment is carried out with actively growing cells in rich medium at 37°C, the growth curve plateau is reached in approximately 30 minutes. Bacteriophage reproduction is an exceptionally rapid process, much faster than animal virus reproduction, which may take hours. Adsorption to the Host Cell and Penetration Bacteriophages do not randomly attach to the surface of a host cell; rather, they fasten to specific surface structures called receptor sites. The nature of these receptors varies with the phage; cell wall lipopolysaccharides and proteins, teichoic acids, flagella, and pili can serve as receptors. The T-even phages of E. coli use cell wall lipopolysaccharides or proteins as receptors. Variation in receptor properties is at least partly responsible for phage host preferences. The structure of cell walls, flagella, and pili (pp. 55–61, 62–66) T-even phage adsorption involves several tail structures (see figure 16.19). Phage attachment begins when a tail fiber contacts the appropriate receptor site. As more tail fibers make contact, the baseplate settles down on the surface (figures 17.3 and 17.4). Time (minutes) Phage count Burst size Latent period Rise period Eclipse Figure 17.2 The One-Step Growth Curve. In the initial part of the latent period, the eclipse period, the host cells do not contain any complete, infective virions. During the remainder of the latent period, an increasing number of infective virions are present, but none are released. The latent period ends with host cell lysis and rapid release of virions during the rise period or burst. In this figure the blue line represents the total number of complete virions. The red line is the number of free viruses (the unadsorbed virions plus those released from host cells). When E. coli is infected with T2 phage at 37°C, the growth plateau is reached in about 30 minutes and the burst size is approximately 100 or more virions per cell. The eclipse period is 11–12 minutes, and the latent period is around 21–22 minutes in length. Landing Attachment Tail contraction Penetration and unplugging DNA injection Figure 17.3 T4 Phage Adsorption and DNA Injection. See text for details. Figure 17.4 Electron Micrograph of E. coli Infected with Phage T4. Baseplates, contracted sheaths, and tail tubes can be seen (36,500).

Prescott−Harley−Klein: Microbiology, Fifth Edition VI. The Viruses 17. The Viruses: Bacteriophages © The McGraw−Hill Companies, 2002 shorter and wider, and the central tube or core is pushed through the bacterial wall. Finally, the DNA is extruded from the head, through the tail tube, and into the host cell. The tube may interact with the plasma membrane to form a pore through which DNA passes. The penetration mechanisms of other bacteriophages often appear to differ from that of the T-even phages but have not been studied in much detail. Synthesis of Phage Nucleic Acids and Proteins Since the T4 phage of E. coli has been intensely studied, its reproduction will be used as our example (figure 17.5). Soon after phage DNA injection, the synthesis of host DNA, RNA, and protein is halted, and the cell is forced to make viral constituents. E. coli RNA polymerase (see section 12.1) starts synthesizing phage mRNA within 2 minutes. This mRNA and all other early mRNA (mRNA transcribed before phage DNA is made) direct the synthesis of the protein factors and enzymes required to take over the host cell and manufacture viral nucleic acids. Some early virusspecific enzymes degrade host DNA to nucleotides, thereby simultaneously halting host gene expression and providing raw material for virus DNA synthesis. Within 5 minutes, virus DNA synthesis commences. Promoters and transcription (pp. 261–63) Virus gene expression follows an orderly sequence because of modifications of the RNA polymerase and changes in sigma factors. Initially T4 genes are transcribed by the regular host RNA polymerase and the sigma factor 70. After a short interval, a virus enzyme catalyzes the transfer of an ADP-ribosyl group from NAD to an -subunit of RNA polymerase. This helps inhibit the transcription of host genes and promotes virus gene expression. Then 17.2 Reproduction of Double-Stranded DNA Phages:The Lytic Cycle 385 DNA injection 0 min Early mRNA made 2 min Host DNA degraded mRNA 3 min Phage DNA made 5 min Late RNA made 9 min Heads and tails made 12 min 13 min Heads filled 15 min Virions formed 22 min Host cell lysis Host chromosome Figure 17.5 The Life Cycle of Bacteriophage T4. (a) A schematic diagram depicting the life cycle with the minutes after DNA injection given beneath each stage. mRNA is drawn in only at the stage during which its synthesis begins. (b) Electron micrographs show the development of T2 bacteriophages in E. coli. (b1) Several phages are near the bacterium, and some are attached and probably injecting their DNA. (b2) By about 30 minutes after infection, the bacterium contains numerous completed phages. (a) (b1) (b2)

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