Viruses that infect bacteria are known as phages or bacteriophages. Twort (19l5) described a degenerative alteration in staphylococcal colonies isolated from calf lymph that was transmissible serially via application of culture filtrates from the original growth. d’Herelle (1917) observed that the filtrates of dysentery patient faeces cultures produced transmissible lysis of a dysentery bacillus broth culture. He hypothesised that the lytic agent was a virus and designated it bacteriophage (phage: to eat). In nature, phages are commonly found in close interaction with bacteria. Phages are readily isolable from numerous habitats, including faeces, sewage, and other natural sources of mixed bacterial proliferation.
What are Bacteriophages?
- Bacteriophages are viruses of bacteria.
- They are the viruses that cause bacterial infection.
- They are obligate intracellular parasites that reproduce within bacteria by utilising some or all of the biosynthetic machinery of the host.
- Viruses are sometimes known as phages.
- Due to the presence of a nucleic acid genome encased in a protein coat, these extrachromosomal genetic components typically persist outside a host cell.
- Phages are prevalent in nature in intimate contact with bacteria and are widely disseminated in soil, excrement, and other environmental components.
- They are related with the transfer of genetic material between bacteria.
- Twort first reported the actions of bacteriophage in 1915, describing it as an infectious agent that altered the look of staphylococcal colonies.
- In 1979, d’Herelle proved the lytic activity of the culture filtrate on bacterial colonies. He hypothesised that the lytic agent was a virus and designated it bacteriophage (phage: to eat).
- The morphology of phages varies significantly. Also described are spherical or filamentous phages with DNA or RNA consisting of a single strand.
- The strong host specificity of viruses. They typically reside in the intestinal flora of humans and animals.
- They pass through screens that prevent bacteria from passing through.
- These are rendered inactive by boiling.
Role of Bacteriophages
- They play a key role in the translation of genetic information from one bacterium to another.
- In addition, they contribute to the evolution of bacterial kinds and the spread of certain virulence traits.
- In fact, phages may be helpful against bacterial infections, particularly those caused by antibiotic-resistant bacteria.
- Viruses have been utilised as cloning vectors in genetic engineering.
- They may have a role in bacterial population management in natural waterways.
Morphology of Bacteriophages
The majority of phages have a single, linear, double-stranded DNA genome. This genome is enveloped by a phage capsid protein coat. Typically, large phages consist of a head and a tail. The head encloses the genome, and the tail serves as both an attachment organ and a pathway for phage DNA to enter the host cell. The most thoroughly researched phages are the T-even phages (T2, T4, T6, etc.) that infect Escherichia coli. Traditionally, these phages serve as the model for characterising the morphology of bacteriophages. Phages have a hexagonal head and a cylindrical tail, like tadpoles.
- All phages possess a variable-sized and -shaped head structure.
- The form is icosahedral (20 sides) or filamentous.
- The length of the head ranges between 28 and 100 nanometers.
- In the majority of phages, the capsid of the bacteriophage head encloses a compartment containing the nucleic acid, which is a double-stranded DNA molecule.
- By virtue of its position within the bacteriophage head, this DNA is protected against destruction by environmental nucleases.
- However, a group of phages that infect solely male E. coli strains contains just RNA.
- The capsid or protein coat of a virus is formed of capsomeres, which are separate proteins.
- Many phages, although not all, have tails linked to their heads.
- During an infection, the nucleic acid flows via a hollow tube that comprises the tail.
- The tail of complex phages such as T4 is encircled by a contractile sheath and a terminal tail plate. The sheath contracts upon bacterial infection.
- The spikes and tail fibres come from the tail plate and bind particularly to receptors on the bacterial cell wall’s outer membrane.
Life Cycle of Bacteriophages
Phages have two distinct life cycle types. The virulent or lytic cycle concludes with the lysis of the host bacterium and the release of progeny virions. During the temprate or lysogenic cycle, the phage DNA integrates into the bacterial genome and replicates synchronously with it, without harming the host cell.
The steps of replication of a virulent phage are adsorption, penetration, synthesis of phage components, assembly, maturation, and release of offspring phage particles.
- The tail of phage particles attaches to virus-specific receptors on the host cell through random collision.
- Adsorption is a unique process dependent on the existence of complimentary chemical groups on the receptor sites on the bacterial surface and on the phage’s terminal base plate.
- Under perfect conditions, adsorption is a process that takes only minutes to complete.
- Any component on the bacterial surface can function as a phage receptor. Phage host specificity is determined at the adsorption level.
- Even bacterial strains that are resistant to infection by the complete phage can be experimentally infected by injecting phage DNA directly.
- Transfection is the infection of a bacteria by the nucleic acid of a naked phage.
- Following adsorption, the majority of phages inject their nucleic acid into the bacterial cytoplasm while leaving their protein capsid outside.
- Similar to an injection with a syringe, penetration resembles injection. Six tail pins establish contact with the host cell surface and securely bind the phage plate to it upon adsorption.
- The contractile tail sheath then contracts, driving the tail tube’s hollow interior into the bacterial cell wall.
- The phage DNA then traverses the tail’s interior hollow tube.
- The phage’s empty head and tail stay outside the bacterium as the shell or ‘ghost’ following penetration.
- The presence of lysozyme on the phage tail, which creates a hole in the bacterial cell wall for the entry of the phage core, may promote bacterial penetration.
- When bacteria are mixed with phage particles at a high multiplicity (that is, a very large number of phages per bacterial cell), many holes are formed on the bacterial cell, resulting in the loss of cell contents. Without viral replication, lysis of bacteria occurs. This is referred to as external lysis.
iii. Synthesis of Phage Nucleic Acid and Proteins
- The manufacturing of phage components begins as soon as the phage nucleic acid penetrates the host cell.
- The earliest products to be generated (referred to as early proteins) are the enzymes required for the construction of the complex compounds that are unique to the phage.
- Subsequently, late proteins such as the protein subunits of the phage head and tail are produced.
- During this time, bacterial protein, DNA, and RNA synthesis ceases, and the cell is pushed to produce viral components.
- Late genes are expressed only after phage DNA replication has occurred.
- The structural components of new phage particles, including their heads, tails, and fibres, are encoded by late gene products.
- Phage lysozyme, which degrades the bacterial cell wall to release the mature phage particles, is also a late product.
iv. Assembly and Maturation
- In order to create the mature progeny phage particle, the structural proteins and nucleic acid of the phage assemble along certain paths.
- In the bacterial cell, phage DNA, head protein, and tail protein are generated independently.
- The DNA is compacted into a polyhedron and ‘packed’ into the head; the tail structures are then inserted.
- This process of assembling phage components into an infectious mature phage particle is known as maturation.
- At the end of the intracellular phase, many phages lyse their host cells.
- Typically, the adult progeny phages are released by the lysis of the bacterial cell.
- During phage replication, the bacterial cell wall is weakened and develops a spherical form.
- The weakening cell wall is ruptured or lysed by phage enzymes, resulting in the release of mature daughter phages.
- The time between the entry of the phage nucleic acid into the bacterial cell and the appearance of the first infectious intracellular phage particle is referred to as the eclipse phase.
- It is the time necessary for the synthesis of phage components and their assembly into mature phage particles.
- Latent period refers to the time between the infection of a bacterial cell and the initial release of infectious phage particles (20 to 40 minutes).
- Immediately following the latent period, the number of phage particles released grows until the maximum number of daughter phages is reached.
- The period in which the quantity of infectious phages released increases is referred to as the rising phase.
- The average number of offspring phages produced per infected bacterial cell is referred to as the burst size (100 to 300 phages).
- Experiments are conducted in which one phage is used to infect one bacteria, and the discharge of infected phage particles is measured serially over a period of time.
- The plotted results of such an experiment are referred to as the one-step growth curve.
- In contrast to virulent phages, which result in the lysis of the host cell, temperate phages form symbiotic relationships with their hosts without harming them.
- After entering the host cell, the nucleic acid of a temperate phage becomes incorporated into the bacterial chromosome.
- The nucleic acid of the integrated phage is known as the prophage.
- The prophage replicates synchronously with the host chromosome, mimicking its behaviour.
- This behaviour is known as lysogeny, and a bacterium that harbours a prophage within its genome is known as a lysogenic bacterium or lysogen, as the prophage retains the ability to lyse its host bacterium. Phages capable of entering into this relationship are known as temperate phages.
- In most cases, the prophage is integrated into the bacterial genome, but it can also live independently. The lysogenization process has little effect on bacterial metabolism.
- In some instances, temperate phages contribute to the pathogenicity of gram-positive and gram-negative bacteria isolated from clinical settings.
- The prophage imparts new characteristics to the lysogenic bacterium. This process is referred to as lysogenic or phage conversion.
- The existence of prophage b in Corynebacterium diphtheriae determines its toxin production. Elimination of this prophage eliminates the toxigenicity of the bacillus, whereas lysogenization can transform nontoxigenic strains into toxigenic ones.
- Types C and D of Clostridium botulinum only generate toxin when infected with phages CE b and DE b, respectively.
- Salmonella phages in temperate environments can alter the antigenic characteristics of somatic O antigen. When Salmonella is infected by an epsilon phage, the outer lipopolysaccharide layer may undergo structural changes. S. Anatum has an antigenic formula of 3, 10: e, h: 1,6; however, when lysogenized by a temperate phage, its antigenic formula changes to 3, 15: e, h: 1,6.
- During the proliferation of lysogenic bacteria, the prophage may become “excised” from some cells.
- The excised prophage commences lytic replication and releases daughter phage particles that infect and lysogenize additional bacterial cells. This is characterised as spontaneous prophage induction.
- Certain physical and chemical agents can encourage all lysogenic bacteria in a population to switch to the lytic cycle, although this is an uncommon occurrence.
- Such agents include ultraviolet light, hydrogen peroxide, and nitrogen mustard. A lysogenic bacterium is resistant to reinfection by identical or similar phages. The term for this is superinfection immunity.
Examples of phage conversions
The examples of phage conversions are as follows:
- Phage-mediated conversion of Salmonella somatic antigens: This occurs when a significant number of temperate Salmonella phages alter the antigenic characteristics of the somatic O antigen. When lysogenized by a temperate phage, Salmonella Anatum (antigenic formula: 3,10:e, h:1,6) reveals a new antigenic formula (3,15:e, h:1,6), which is an antigenic formula of Salmonella Newington.
- Corynebacterium diphtheriae’s toxicity: Toxin synthesis in C. diphtheriae is attributable to the presence of prophage beta (). By removing the prophage, the bacteria become nontoxigenic. Similarly, nontoxigenic C. diphtheriae can be rendered toxic via phage-mediated lysogenization.
- Toxicity of Clostridium botulinum: Clostridium botulinum toxicity is dictated by the presence of phages CE and DE in C. botulinum types C and D, respectively. Elimination of the phages eradicates the bacillus’s toxigenicity.
- Transduction: Bacteriophages can transport genes from one bacterium to another via transduction. This is referred to as transduction. This behaviour has been observed in numerous bacterial species and is considered as one of the primary routes by which genetic material is transferred between bacteria in nature.
Significance of Phages
1. Virulent Phage
i. Phage Assay
- When a phage is given to a sensitive bacterial lawn culture, clearance occurs following incubation. These lysis zones are known as plaques.
- The size, shape, and composition of plaques are unique to each phage. Plaque assay can be used to determine the amount of viable phages in a sample since, under optimal conditions, a single phage particle can produce one plaque.
- Plaquing is also useful for purifying phages since plaques are similar to bacterial colonies.
ii. Phage Typing
- Typing of bacteriophages (phages) is determined by the specificity of phage surface receptors for cell surface receptors.
- Many phages can be employed as epidemiological markers to distinguish between biochemically or serologically indistinguishable bacterial strains due to their restricted host range.
- Staphylococcus aureus, Salmonella typhi, and Vibrio cholerae outbreaks have been traced using this technique.
- The to-be-typed strain is inoculated onto a nutrient agar plate to form a grass culture. After phages have been dried, a predetermined dose is administered to squares that have been designated (routine test dose).
- The maximum dilution of the phage preparation that achieves confluent lysis is referred to as the standard test dose (RTD).
- Some phages will lyse the culture after an overnight incubation, whereas others will not.
- The designation of the phage(s) that lyse the strain expresses the phage type of the strain. Plaque refers to the area of lysis caused by a phage.
- Since a single phage particle is capable of forming a single plaque, the plaque assay can be used to quantify the amount of viable phages in a sample.
- The most crucial use of phage typing is intraspecies typing of bacteria, such as the phage typing of S. typhi and staphylococci.
- There are phages that lyse all members of a bacterial genus (e.g., Salmonella), all members of a species (e.g., B. anthracis), and all members of a biotype or subspecies (e.g., Mukerjee’s phage IV, which lyses all strains of classical V. cholerae but not V. cholerae biotype EI Tor).
2. Temperate Phage
- Bacteriophages can operate as gene carriers from one bacterium to the next. This is referred to as transduction.
- There are two recognised types of transduction: generalised transduction, in which any piece of the donor DNA can be transmitted, and specialised transduction, in which only a particular set of genes can be transported to a recipient cell.
ii. Toxin Production
- The synthesis of toxins by Corynebacterium diphtheriae and Clostridium botulinum types C and D is determined by prophage DNA-carrying genes.
iii. Antigenic Property
- Salmonella phages in temperate environments can alter the antigenic characteristics of somatic O antigen.
- This acquisition of new capabilities by infected bacterial cells is known as “phage conversion.”
iv. Cloning Vector
- In genetic modifications, bacteriophages have been utilised as cloning vectors.