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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)

Prescott−Harley−Klein: Microbiology, Fifth Edition VI. The Viruses 17. The Viruses: Bacteriophages © The McGraw−Hill Companies, 2002 the second -subunit receives an ADP-ribosyl group and this turns off some of the early T4 genes. The product of one early gene, motA, stimulates transcription of somewhat later genes, one of which produces the sigma factor gp55. This sigma factor helps RNA polymerase bind to late promoters and transcribe late genes, which become active around 10 to 12 minutes after infection. It is clear from the sophisticated control of RNA polymerase and the precise order in which events occur in the reproductive cycle that the expression of T4 genes is tightly regulated. Even the organization of the genome appears suited for efficient control of the life cycle. As can be seen in figure 17.6, genes with related functions—such as the genes for phage head or tail fiber construction—are usually clustered together. Early and late genes also are clustered separately on the genome; they are even transcribed in different directions—early genes in the counterclockwise direction and late genes, clockwise. Since transcription always proceeds in the 5′ to 3′ direction, the early and late genes are located on different DNA strands (see sections 11.5 and 12.1). Considerable preparation is required for synthesis of T4 DNA because it contains hydroxymethylcytosine (HMC) instead of cytosine (figure 17.7). HMC must be synthesized by two phageencoded enzymes before DNA replication can begin. After T4 DNA has been synthesized, it is glucosylated by the addition of glucose to the HMC residues. Glucosylated HMC residues protect T4 DNA from attack by E. coli endonucleases called restriction enzymes, which would otherwise cleave the viral DNA at specific points and destroy it. This bacterial defense mechanism is called restriction. Other groups also can be used to modify phage DNA and protect it against restriction enzymes. For example, methyl groups are added to the amino groups of adenine and cytosine in lambda phage DNA for the same reason. The replication of T4 DNA is an extremely complex process requiring at least seven phage proteins. Its mechanism resembles that described in chapter 11. Restriction enzymes and genetic engineering (pp. 320–21) 386 Chapter 17 The Viruses: Bacteriophages Hydroxymethylase Head filling polymerase RNA Head, neck, and modification Endolysin Tail tube Scaffolding protein DNA ligase Endonuclease rll (lysis) mot Head Tail baseplate and collar m Nucleotide etabolism Tail fiber Membrane T4 DNA synthesis, replication, DNA exonuclease DNA polymerase proteins Tail baseplate Figure 17.6 A Map of the T4 Genome. Some of its genes and their functions are shown. Genes with related functions tend to be clustered together. CH2 OH NH2 O N H N Figure 17.7 5-Hydroxymethylcytosine (HMC). In T4 DNA, the HMC often has glucose attached to its hydroxyl

Prescott−Harley−Klein: Microbiology, Fifth Edition VI. The Viruses 17. The Viruses: Bacteriophages © The McGraw−Hill Companies, 2002 T4 DNA shows what is called terminal redundancy; that is, a base sequence is repeated at both ends of the molecule (figure 17.8). When many DNA copies have been made, about 6 to 10 copies are joined by their terminally redundant ends with the aid of several enzymes (figure 17.8). These very long DNA strands composed of several units linked together with the same orientation are called concatemers. During assembly, concatemers are cleaved in such a way that the genome is slightly longer than the T4 gene set. The genetic map is therefore drawn circular (figure 17.6) because T4 DNA is circularly permuted (figure 17.9). The sequence of genes in each T4 virus of a population is the same but starts with a different gene at the 5′ end. If all the linear pieces of DNA were coiled into circles, the DNA circles would have identical gene sequences. The Assembly of Phage Particles The assembly of the T4 phage is an exceptionally complex selfassembly process. Late mRNA, or that produced after DNA replication, directs the synthesis of three kinds of proteins: (1) phage structural proteins, (2) proteins that help with phage assembly without becoming part of the virion structure, and (3) proteins involved in cell lysis and phage release. Late mRNA transcription begins 17.2 Reproduction of Double-Stranded DNA Phages:The Lytic Cycle 387 Y′ Z′ A′ B′ C′ D′ E′ F′ G′ Y′ Z′ A′ B′ C′ D′ E′ F′ G′ Y′ Z′ A′ B′ C′ D′ E′ F′ G′ A B C D A′ B′ C′ D′ A B C D E F G Y Z A′ B′ C′ D′ E′ F′ G′ Y′ Z′ Exonuclease activity E F G Y Z E′ F′ G′ Y′ Z′ A B C D A′ B′ C′ D′ Y Z A B C D E F G Y Z A B C D E F G Y Z A B C D E F G Figure 17.8 An Example of Terminal Redundancy. The gene sequences in color are terminally redundant; they are repeated at each end of the DNA molecule. This makes it possible to join units together by their redundant ends forming a concatemer. For example, if the 3′ ends of each unit were partially digested by an exonuclease, the complementary 5′ ends would be exposed and could base pair to generate a long chain of repeated units. The breaks between terminal sequences indicate that the DNA molecules are longer than shown here. A′ B′ C′ D′ E F G H I A′ B′ C′ D′ A B C D E F G H I Y Z E′ F′ G′ H′ I′ Y′ Z′ C D E F G H I Y Z A B G′ H′ I′ Y′ Z′ A′ B′ C′ D′ Y Z I′ Y′ Z′ A B C D E′ F′ E′ F′ G′ H′ Y Z A B C D E F G H I Y′ Z′ A′ B′ C′ D′ E′ F′ G′ H′ I′ Figure 17.9 Circularly Permuted Genomes Cut from a Concatemer. The concatemer formed in figure 17.8 can be cut at any point into pieces of equal length that contain a complete complement of genes, even though different genes are found at their ends. If each piece has single-stranded cohesive ends as in figure 17.8, it will coil into a circle with the same gene order as the circles produced by other pieces

Prescott−Harley−Klein: Microbiology, Fifth Edition VI. The Viruses 17. The Viruses: Bacteriophages © The McGraw−Hill Companies, 2002 about 9 minutes after T4 DNA injection into E. coli. All the proteins required for phage assembly are synthesized simultaneously and then used in four fairly independent subassembly lines (figure 17.10). The baseplate is constructed of 15 gene products. After the baseplate is finished, the tail tube is built on it and the sheath is assembled around the tube. The phage prohead or procapsid is constructed separately of more than 10 proteins and then spontaneously combines with the tail assembly. The procapsid is assembled with the aid of scaffolding proteins that are degraded or removed after construction is completed. A special portal protein is located at the base of the procapsid where it connects to the tail. The portal protein is part of the DNA translocating vertex, a structure that helps initiate head assembly and aids in DNA movement into and out of the head. Tail fibers attach to the baseplate after the head and tail have come together. Although many of these steps occur spontaneously, some require special virus proteins or host cell factors. 388 Chapter 17 The Viruses: Bacteriophages Head proteins Tail fiber proteins Baseplate proteins Baseplate Tube Tube and sheath Collar Whiskers and neck Mature head with DNA Prohead DNA Prohead Tail fibers Figure 17.10 The Assembly of T4 Bacteriophage. Note the subassembly lines for the baseplate, tail tube and sheath, tail fibers, and head. DNA packaging within the T4 head is still a somewhat mysterious process. In some way the DNA is drawn into the completed shell so efficiently that about 500 m of DNA are packed into a cavity less than 0.1 m across! It is thought that a long DNA concatemer enters the procapsid in an ATP-dependent process until it is packed full and contains about 2% more DNA than is needed for the full T4 genome. The concatemer is then cut, and T4 assembly is finished. The first complete T4 particles appear in E. coli at 37°C about 15 minutes after infection. Release of Phage Particles Many phages lyse their host cells at the end of the intracellular phase. The lysis of E. coli takes place after about 22 minutes at 37°C, and approximately 300 T4 particles are released. Several T4 genes are involved in this process. One directs the synthesis of an endolysin that attacks the cell wall peptidoglycan. Another phage protein called a holin produces a plasma membrane lesion that stops respiration and allows the endolysin to attack the peptidoglycan. Presumably it forms holes in the membrane. 17.3 Reproduction of Single-Stranded DNA Phages Thus far, only double-stranded DNA phage reproduction has been discussed, with the lytic phage T4 as an example. The reproduction of single-stranded DNA phages now will be briefly reviewed. The phage of X174, family Microviridae, is a small ssDNA phage using E. coli as its host. Its DNA base sequence is the same as that of the viral mRNA (except that thymine is substituted for uracil) and is therefore positive; the genome contains overlapping genes (see figure 11.20b). The phage DNA must be converted to a double-stranded form before either replication or transcription can occur. When X174 DNA enters the host, it is immediately copied by the bacterial DNA polymerase to form a double-stranded DNA, the replicative form or RF (figure 17.11). The replicative form then directs the synthesis of more RF copies, mRNA, and copies of the DNA genome. The phage is released by host lysis through a different mechanism than used by the T4 phage. The filamentous ssDNA bacteriophages behave quite differently in many respects from X174 and other ssDNA phages. The fd phage, family Inoviridae, is one of the best studied and is shaped like a long fiber about 6 nm in diameter by 900 to 1,900 nm in length (figure 17.1). The circular ssDNA lies in the center of the filament and is surrounded by a tube made of a small coat protein organized in a helical arrangement. The virus infects male E. coli cells by attaching to the tip of the pilus; the DNA enters the host along or possibly through the pilus with the aid of a special adsorption protein. A replicative form is first synthesized and then transcribed. A phage-coded protein then aids in replication of the phage DNA by use of the rolling-circle method (see section 11.3). The filamentous fd phages do not kill their host cell but establish a symbiotic relationship in which new virions are continually released by a secretory process. Filamentous phage coat

Prescott−Harley−Klein: Microbiology, Fifth Edition VI. The Viruses 17. The Viruses: Bacteriophages © The McGraw−Hill Companies, 2002 proteins are first inserted into the membrane. The coat then assembles around the viral DNA as it is secreted through the host plasma membrane (figure 17.12). The host bacteria grow and divide at a slightly reduced rate. 1. How is a one-step growth experiment carried out? Summarize what occurs in each phase of the resulting growth curve. Define latent period, eclipse period, rise period or burst, and burst size. 2. Be able to describe in some detail what is occurring in each phase of the lytic dsDNA phage life cycle: adsorption and penetration, nucleic acid and protein synthesis, phage assembly, and phage release. Define the following terms: lytic cycle, receptor site, early mRNA, hydroxymethylcytosine, restriction, restriction enzymes, concatemers, replicative form, late mRNA, and scaffolding proteins. 3. How does the reproduction of the ssDNA phages X174 and fd differ from each other and from the dsDNA T4 phage? 17.4 Reproduction of RNA Phages Many bacteriophages carry their genetic information as singlestranded RNA that can act as a messenger RNA and direct the synthesis of phage proteins. One of the first enzymes synthesized is a viral RNA replicase, an RNA-dependent RNA polymerase 17.4 Reproduction of RNA Phages 389 Figure 17.12 Release of the Pf1 Phage. The Pf1 phage is a filamentous bacteriophage that is released from Pseudomonas aeruginosa without lysis. In this illustration the blue cylinders are hydrophobic α-helices that span the plasma membrane, and the red cylinders are amphipathic helices that lie on the membrane surface before virus assembly. In each protomer the two helices are connected by a short, flexible peptide loop (yellow). It is thought that the blue helix binds with circular, single-stranded viral DNA (green) as it is extruded through the membrane. The red helix simultaneously attaches to the growing virus coat that projects from the membrane surface. Eventually the blue helix leaves the membrane and also becomes part of the capsid. Figure 17.11 The Reproduction of X174, a Strand DNA Phage. See text for details. + DNA Bacterial DNA polymerase Replication ± DNA (replicative form) Transcription + mRNA + DNA Proteins New virions Translation

Prescott−Harley−Klein: Microbiology, Fifth Edition VI. The Viruses 17. The Viruses: Bacteriophages © The McGraw−Hill Companies, 2002 (figure 17.13). The replicase then copies the original RNA (a plus strand) to produce a double-stranded intermediate (RNA), which is called the replicative form and is analogous to the DNA seen in the reproduction of ssDNA phages. The same replicase next uses this replicative form to synthesize thousands of copies of RNA. Some of these plus strands are used to make more RNA in order to accelerate RNA synthesis. Other RNA acts as mRNA and directs the synthesis of phage proteins. Finally, RNA strands are incorporated into maturing virus particles. The genome of these RNA phages serves as both a template for its own replication and an mRNA. MS2 and Q, family Leviviridae, are small, tailless, icosahedral ssRNA phages of E. coli, which have been intensely studied (figure 17.1). They attach to the F-pili of their host and enter by an unknown mechanism. These phages have only three or four genes and are genetically the simplest phages known. In MS2, one protein is involved in phage adsorption to the host cell (and possibly also in virion construction or maturation). The other three genes code for a coat protein, an RNA replicase, and a protein needed for cell lysis. Only one dsRNA phage has been discovered, the bacteriophage 6 of Pseudomonas phaseolicola (figure 17.1). It is also unusual in possessing a membranous envelope. The icosahedral capsid within its envelope contains an RNA polymerase and three dsRNA segments, each of which directs the synthesis of an mRNA. It is not yet known how the dsRNAs are replicated. 1. How are ssRNA phages reproduced, and what role does RNA replicase play in the process? 2. What is peculiar about the structure of phage 6? 17.5 Temperate Bacteriophages and Lysogeny Up to this point many of the viruses we have discussed are virulent bacteriophages; these are phages that lyse their host cells during the reproductive cycle. Many DNA phages also can establish a different relationship with their host. After adsorption and penetration, the viral genome does not take control of its host and destroy it while producing new phages. Instead the viral genome remains within the host cell and replicates with the bacterial genome to generate a clone of infected cells that may grow and divide for long periods while appearing perfectly normal (see figure 13.18). Each of these infected bacteria can produce phages and lyse under appropriate environmental conditions. They cannot, for reasons that will become clear later, be reinfected by the same virus—that is, they have immunity to superinfection. This relationship between the phage and its host is called lysogeny. Bacteria having the potential to produce phage particles under some conditions are said to be lysogens or lysogenic, and phages able to enter into this relationship are temperate phages. The latent form of the virus genome that remains within the host but does not destroy it is called the prophage. The prophage usually is integrated into the bacterial genome but sometimes exists independently. Induction is the process by which phage reproduction is initiated in a lysogenized culture. It leads to the destruction of infected cells and the release of new phages—that is, induction of the lytic cycle. Lysogeny was briefly described earlier in the context of transduction and genetic recombination, but will be discussed in more detail here. Generalized and specialized transduction (pp. 307–9) Most bacteriophages that have been studied are temperate, and it appears that there are advantages in being able to lysogenize bacteria. Consider a phage-infected culture that is becoming dormant due to nutrient deprivation. Before bacteria enter dormancy, they degrade their own mRNA and protein. Thus the phage is faced with two problems: it can only reproduce in actively metabolizing bacteria, and phage reproduction is usually permanently interrupted by the mRNA and protein degradation. This predicament can be avoided if the phage becomes dormant (lysogenic) at the same time as its host; in fact, nutrient deprivation does favor lysogeny. Temperate phages also have an advantage in situations where many viruses per cell initiate an infection—that is, where there is a high multiplicity of infection (MOI). When every cell is infected, the last round of replication will destroy all host cells. Thus there is a risk that the phages may be left without a host and directly exposed to environmental hazards for months or years. This prospect is avoided if lysogeny is favored by a high MOI; some bacteria will survive, carry the virus genome, and synthesize new copies as they reproduce. Not surprisingly a high MOI does stimulate lysogeny. A temperate phage may induce a change in the phenotype of its host cell that is not directly related to completion of its life cycle. Such a change is called a lysogenic conversion or a conversion and often involves alterations in bacterial surface characteristics or pathogenic properties. For example, when Salmonella is infected by an epsilon phage, the structure of its outer lipopolysaccharide layer (see pp. 58–60) may be modified. The phage changes the activities of several enzymes involved in con- 390 Chapter 17 The Viruses: Bacteriophages + RNA RNA replicase (mRNA activity) ± RNA (replicative form) Replication RNA replicase + RNA mRNA activity Virus proteins + RNA genomes New virions Translation ( ( Figure 17.13 The Reproduction of Single-Stranded RNA Bacteriophages.