General Microbiology - Schlegel, H. 1987

Viruses: Distribution and Structure
Bacterial Viruses (Bacteriophages)

Isolation and Detection. Isolating a bacteriophage is relatively simple. One merely needs to collect a sample from the natural habitat of the target bacterial species and incubate it with the Bacteria in a nutrient medium. If this enrichment culture is maintained under conditions favorable for the bacteria, any phages present in the sample will rapidly multiply. Viruses replicate exclusively within living, growing Cells. The remaining bacteria can then be removed by centrifugation or filtration, allowing the concentration of phages to be determined in the supernatant, or lysate. Plating a phage-free bacterial suspension onto a solid nutrient medium results in a confluent bacterial lawn across its surface. However, if a suspension containing a low concentration of phages is inoculated onto the lawn, clear, bacteria-free zones—known as plaques—will appear. At the site where a phage infects and destroys a bacterium, an expanding number of bacterial cells undergo lysis; after some time, this phage-infected region becomes visible to the naked eye as a clear spot against the opaque bacterial lawn (Fig. 4.1, C). Using appropriate techniques, Bacteriophages can be rapidly propagated on solid media using bacterial lawns or in liquid bacterial Suspensions. Bacterial cells are subsequently removed by centrifugation, and residual bacteria are eliminated using chloroform, yielding a phage lysate that typically contains from 10¹⁰ to 10¹³ phages per 1 ml.

Morphology of Bacteriophages. The Structure of bacteriophages has been investigated primarily using the T-series phages of Escherichia coli. Coliphage T2 consists of a polyhedral HEAD, approximately 100 nm in length, and a tail of roughly the same dimensions, leading to their Classification as complex viruses (Table 4.1). The head is composed of capsomeres and encloses the DNA molecule, with protein and DNA present in roughly equal proportions. The tail of phage T2 exhibits a sophisticated architecture comprising at least three distinct regions: a central hollow core, a contractile sheath surrounding the core, and a distal baseplate equipped with spikes and tail fibers (the latter mediate specific adsorption to the host Cell). Negative-staining electron micrographs reveal phage particles in two distinct states: in some particles, the head stands out sharply against an electron-dense Background and the tail sheath is extended, whereas in others, the head density is similar to the background and the sheath is contracted, as depicted schematically in Fig. 4.7. The first state (A) is characteristic of active phages containing DNA within the head, while the second state (B) represents phages that have injected their DNA into a bacterial host cell.

Many bacteriophages possess a simpler structure. Depending on the morphology of mature phage particles, several distinct types can be distinguished, as illustrated in Figures 4.8 and 4.9. The majority of phages contain double-stranded DNA; however, in recent years, several phages with single-stranded DNA and a few with single-stranded RNA have also been discovered. RNA-containing phages such as fr (Fig. 4.9, D), R17, Qß, and others possess some of the smallest known genomes, consisting of only 3,500 to 4,500 NUCLEOTIDES.

Class="center">

Fig. 4.7. Model of phage T2. A. Phage with extended sheath prior to adsorption. B. Phage with contracted sheath following adsorption and DNA injection. 1 - cross-section of the extended tail, showing 6 protein subunits of the sheath in a single plane; 2 - cross-section of the contracted sheath, showing 12 protein subunits of the sheath in a single plane; 3 - baseplate of an adsorption-competent phage with unfurled tail fibers.

4.2.1 Replication of Virulent Phages: The Lytic Cycle

Viral reproduction within a host cell is an exceptionally complex process. Its individual stages—from host cell infection to the release of mature infectious progeny—have been thoroughly characterized from biochemical, genetic, and morphological Perspectives using the T-series phages (T2, T4, T6). A major advantage facilitating these studies is that phage DNA contains 5-hydroxymethylcytosine instead of cytosine, enabling its synthesis to be readily tracked by monitoring the appearance of this unique base. Furthermore, mutant phage strains can be isolated in which specific steps of the replication cycle are blocked or occur only under restrictive conditions. Such mutants have helped elucidate the intracellular morphological development (morphopoiesis) of the phage, clarifying the precise temporal sequence in which various structural components are synthesized and assembled.

Fig. 4.8. Various morphologies of bacteriophages (A–D) and geometric shapes of phage heads (E, F). A. Filamentous form (coliphage fd). B. Head (with a hexagonal outline) attached to a tail with a contractile sheath (e.g., coliphages T2, T4, and T6). C. Head with a long, flexible, non-contractile tail (e.g., coliphages T1 and T5). D. Head with a short tail (e.g., coliphages T3 and T7, Salmonella phage P22). E. Octahedron. F. Icosahedron. (Bradley D. E., Bacteriol. Rev., 31 [1967], 230.)

Fig. 4.9. A. Phage T2; 168,000×, negative contrast (phosphotungstic acid). B. Phage T2 with a contracted sheath and empty head; 168,000×, negative contrast (phosphotungstic acid). C. Lambda phage; 168,000×, negative contrast (uranyl acetate). D. Phage T7 with a short tail; 168,000×, negative contrast (uranyl acetate). E. Circular DNA molecule (replicative form) of phage fd; 50,000×, after spreading with cytochrome and shadowing. F. Phage fd with a single-stranded circular DNA molecule; 50,000×, shadowing at an angle. (Photographs by N. Frank.)

Like other viruses, phages are non-motile. When a suspension of free phages is mixed with bacterial cells, random collisions lead to the attachment of phage particles to the host cell surface (adsorption) and the subsequent injection of their DNA into The Cell. Following a period required for synthesis and maturation processes, the host cells undergo lysis, releasing the newly formed progeny phages (see Fig. 4.13).

Adsorption. Not every phage can adsorb to any bacterium. Host-phage Specificity is determined by the specificity of adsorption, which relies on specific receptors located within The Cell wall. For certain phages, these receptors reside in the lipoprotein layer, whereas for others, they are located in the lipopolysaccharide layer. The phage resistance of certain bacteria is likely due to the absence of these appropriate receptors. In the presence of excess bacteriophage, as many as 200–300 phage particles may adsorb onto a single cell.

Intracellular Development of the Phage. Adsorption is followed by injection, meaning the Introduction of phage DNA into the cell. During infection by phage T2, the baseplate appears to anchor to the host cell, the tail sheath contracts, and the hollow core is driven into the bacterial Cytoplasm. Experiments using phage with ³²P-labeled nucleic acid and ³⁵S-labeled protein demonstrated that only the nucleic acid enters the cell, while the protein coat remains outside. This capsid can be sheared off from the infected cell without impairing phage reproduction. During the so-called latent period, which averages about 25 minutes in Escherichia coli, no phages can be detected even if the bacterial cells are artificially disrupted. The injected phage DNA immediately triggers a complete reprogramming of the infected cell's METABOLISM (Fig. 4.10). Bacterial DNA Synthesis ceases almost instantly, followed within minutes by the cessation of bacterial RNA and Protein Synthesis, although total cellular protein content continues to rise steadily. DNA synthesis soon resumes at an accelerated rate, with initial phage DNA being synthesized using breakdown products of the degraded bacterial DNA. This metabolic shift and subsequent de novo synthesis of phage DNA can be quantified by monitoring the accumulation of 5-hydroxymethylcytosine, a base unique to the DNA of certain T-phages. Enzymes required for phage DNA synthesis are produced shortly after infection; these are termed "early Proteins." Conversely, "late proteins" include capsid proteins and phage lysozymes (endolysins), which are synthesized only during the latter half of the latent period.

Fig. 4.10. Temporal progression of BIOSYNTHETIC PROCESSES IN Escherichia coli following infection with phage T4. (Luria, Darnell, 1967.)

The final stage—maturation—involves the association of phage DNA with capsid proteins to form mature, infectious phage particles. The maturation of T-phages is a complex, multi-step pathway. First, empty capsids are assembled. Following the degradation of internal scaffolding proteins, the preformed heads are packed with DNA to a characteristic density and sealed. Subsequently, the tail components are attached in a precise sequence. The order of these events has been elucidated using conditional lethal mutants, in which all synthetic processes proceed normally at 25°C, whereas specific developmental stages are blocked at 43°C.

Fig. 4.11. Determination of the latent period and phage yield (illustrated by experiments on phage T2 propagation in Escherichia coli B cells). A young bacterial culture was infected with a phage suspension, and unadsorbed free phage particles were subsequently inactivated by The addition of anti-phage serum. After significant dilution, samples were taken at regular time intervals and plated on Agar along with an excess of phage-sensitive bacteria. For 25 minutes, the number of plaque-forming units remained constant at 5 (the latent period), after which it rose to 235 (reaching a plateau). According to these data, the average burst size—the number of phage particles produced per infected bacterial cell—was 47.

Eventually, the Bacterial cell wall is weakened by the action of phage-induced Lysozyme, leading to cell lysis and the release of progeny phages. This abrupt cellular destruction can be observed under a Microscope using dark-field illumination. The duration of the latent period and the burst size vary widely depending on the phage species, bacterial strain, and environmental conditions (Fig. 4.11). Researchers have successfully infected bacteria such as Haemophilus influenzae and Bacillus subtilis using native DNA extracted directly from bacteriophages. This type of infection, which is analogous to genetic transformation, is referred to as transfection.

4.2.2 Development of temperate Phages: Lysogeny

The bacteriophages described above typically lyse their host bacteria and are therefore classified as virulent. However, certain phages infect host bacteria without replicating autonomously or causing cell lysis; such phages are termed temperate. Their replication appears to occur synchronously with Bacterial Cell Division. Only on rare occasions—in roughly one out of every 10² to 10⁵ such "lysogenic" bacteria—does the phage spontaneously enter the lytic cycle, resulting in cell destruction. Detecting the release of infectious phage in such cases requires an indicator bacterial strain that is susceptible to the phage in question. Mixing lysogenic bacteria with an excess of indicator bacteria and plating the mixture on agar results in the growth of lysogenic colonies. Periodically, individual cells undergo lysis, and the released phages infect neighboring sensitive (indicator) bacteria, producing clear plaques within the confluent bacterial lawn. Nevertheless, a colony of lysogenic bacteria survives at the center of each such plaque (Fig. 4.12).

Lysogenic bacteria possess the latent capability to produce phages, yet this property cannot be detected by morphological or serological assays. In this non-infectious state—where the viral genome is transmitted exclusively to daughter cells during division—the phage is known as a prophage. Like other hereditary traits of the bacterial cell, the presence of a prophage is stably inherited. Because all descendants of a lysogenic cell are likewise lysogenic, the prophage must replicate synchronously and regularly alongside the host chromosome (Fig. 4.13).

Lysogenic bacteria are immune to superinfection by phages of the same type as their resident prophage. This Immunity is not mediated by a block in adsorption (as seen in resistance to virulent phages), but rather by the synthesis of a specific cytoplasmic repressor protein that prevents vegetative phage replication. This same repressor blocks the transition of the prophage back into a vegetative state and suppresses the synthesis of phage structural proteins. Thus, the ESTABLISHMENT OF THE lysogenic state is directly tied to The production of this repressor protein.

Fig. 4.12. Plaque formation by lysogenic bacteria and free phages. A dense suspension of non-lysogenic sensitive bacteria (Bacillus megaterium) containing a small number of cells of a lysogenic strain of the same species and a small amount of free phage particles was spread on the agar surface. Clear plaques are the result of the lytic activity of a single free phage particle; the small spots in the center of the plaques are colonies of lysogenic bacteria.

Spontaneously, without external influences, lysogenic bacteria rarely undergo lysis. However, a wide range of factors (ultraviolet rays, mitomycin C, or alkylating agents) can induce prophage development in each cell, leading to the formation and release of infectious phage. The success of such induction depends on the genetic constitution of the prophage, the physiological state of the host, and the cultivation conditions. Induction is evidently associated with the removal or inactivation of existing repressor molecules. Some mutants of temperate phages produce a thermolabile repressor, in which case a Temperature increase to 44°C is sufficient to trigger bacterial lysis.

Integration and induction of phage λ (lambda). Studies of phage lambda (λ), which is lysogenic for Escherichia coli K12, have clarified how the prophage is linked to the bacterial chromosome. The lysogenization of bacteria by this phage serves as a prime example of the temperate BACTERIOPHAGE LIFE CYCLE. The length of the lambda phage chromosome accounts for only 2% of the length of the bacterial chromosome.

Fig. 4.13. Life cycles of a temperate phage (using phage lambda as an example). Following the infection of Escherichia coli by phage lambda, either phage reproduction with subsequent lysis occurs (lytic cycle) or the bacterium becomes lysogenized. The phage DNA is represented as a linear double helix, which circularizes upon entering the bacterium. This circle can remain autonomous or integrate into the bacterial DNA. In the first case, the lytic cycle unfolds. The circularized DNA replicates; through "rolling-circle" replication, a concatemer of four phage DNA copies is produced. Phage genes direct the synthesis and assembly of head and tail proteins, and the packaging of a single DNA copy into each phage head. The heads spontaneously attach to the tails. Upon lysis of the host cell, about a hundred mature phages are released, which can in turn infect other cells. However, the circular phage DNA can also lose its autonomy and become incorporated (integrated) into the host DNA. IN THIS case, the cell becomes lysogenic. The latent phage, or "prophage," replicates along with the host cell chromosome. The lysogenic bacterium can divide indefinitely without undergoing lysis. The excision of the phage DNA from the chromosome, leading to cell lysis, can occur spontaneously or under The Influence of an inducing factor, such as irradiation or a mutagen.

In free phage particles, DNA is present as a linear (non-circular) double helix (Fig. 4.14). Each strand extends 12 nucleotides beyond the duplex at one end. These two single-stranded ends are complementary to each other and can join together via base pairing; hence they are referred to as "sticky" ends. If such DNA molecules are placed in vitro in a solution, an equilibrium between linear and circular DNAs is established due to the interaction between the complementary bases of the single-stranded ends. The same circularization occurs after phage lambda infects a cell. In this process, both gaps between the strand ends are sealed by polynucleotide ligase. The function of this bacterial enzyme is to repair single-strand breaks in DNA Double helices by joining nucleotides. Thus, no phage-encoded enzymes are required to circularize the linear phage DNA.

Fig. 4.14. Integration (insertion) of phage lambda into the Escherichia coli chromosome and its release from the chromosome (excision). In the phage particle, the DNA is presented as a linear double helix with unpaired complementary ends. In solution or within the bacterial cell, the "sticky" complementary ends bind to each other, and the nick in each strand is sealed by ligase. Subsequently, the closed double-stranded circle approaches the chromosome (between the gal and bio genes), both double helices break, and the resulting free ends rejoin crosswise. As a result, the phage DNA becomes incorporated (inserted or integrated) into the host chromosome. The phage has now transformed into a prophage, and the cell has become lysogenic (in this case, for phage lambda). The reverse process can lead to the excision of the phage DNA and its transition back to an autonomous state.

In lysogenic cells, the prophage is firmly linked to the host cell chromosome. During cell conjugation, the prophage is transferred along with the host chromosome from the donor cell to the recipient cell. Genetic experiments demonstrate that phage lambda is attached to the host chromosome at a specific site (between the galactose Operon and the biotin locus). It was initially assumed that the bacteriophage DNA merely attaches to the bacterial chromosome in this region. However, genetic mapping of the phage and recombination experiments revealed that during lysogenization, the phage DNA is not merely attached to the bacterial DNA, but is actually incorporated into it.

The incorporation (integration) of prophage DNA into the host cell chromosome evidently occurs through breakage and cross-recombination (Fig. 4.14). An enzyme called lambda integrase is responsible for this reaction. It recognizes two distinct, non-homologous nucleotide sequences—one in the chromosomal DNA and one in the phage DNA—and brings the two double helices into close alignment; the helices are then cleaved and rejoined crosswise. The individual steps of this Site-Specific Recombination are illustrated in Fig. 4.14.

In the integrated state, the phage DNA replicates along with the bacterial DNA and is subject to the same regulatory controls as the duplication of bacterial Chromosomes. The information contained within the phage DNA remains dormant during this time. Only when the prophage transitions to the vegetative state is the autonomy of the phage DNA restored, and phage multiplication begins. This reverse process can occur spontaneously or as a result of induction (e.g., upon exposure to ultraviolet irradiation). The excision of the phage DNA from the bacterial chromosome presumably occurs via the Reversal of the processes that led to its integration, and it proceeds with high precision: over 99% of phage particles released from lysogenic cells are identical to the parental (infectious) phage. This means that upon excision, the phage DNA is excised precisely at the same site where integration took place. Only in rare instances (one in 100,000) does the excision of phage DNA occur abnormally (see Section 15.3.3 regarding Transduction).

Once the prophage has transitioned to the vegetative state via excision, it becomes autonomous again and can multiply within the bacterial cell like a virulent phage. Thus, excision leads to the lysis of the bacterium and the release of phage lambda.