Premed · Premed · Microbiology
Lecture 16: Bacteriophages
Microbiology
Learning Objectives
By the end of this lecture, students will be able to:
- Describe the structure and classification of bacteriophages
- Explain the lytic and lysogenic replication cycles in detail
- Describe the molecular decision between lysis and lysogeny in phage lambda
- Explain lysogenic conversion and its role in bacterial virulence
- Discuss the applications of bacteriophages in molecular biology, phage therapy, and biotechnology
- Describe the ecological significance of phages
Lecture Content
I. Overview of Bacteriophages
Bacteriophages, also called phages, are viruses that infect bacteria. They are the most abundant biological entities on Earth, with an estimated 10^31 phage particles globally, and every bacterial species is likely infected by multiple phage types. Phages were discovered independently by Frederick Twort in 1915 and Felix d'Herelle in 1917. They have served as model systems for many of the most fundamental discoveries in molecular biology, including the Hershey-Chase experiment of 1952, which confirmed DNA as the genetic material, and the discovery of restriction enzymes, mRNA, the genetic code, and principles of gene regulation.
II. Bacteriophage Structure and Morphology
The most extensively studied phages have tailed morphology, formerly classified in the order Caudovirales. Three morphological families are traditionally recognized: Myoviridae (exemplified by T4), with an icosahedral head and a long contractile tail; Siphoviridae (exemplified by lambda), with an icosahedral head and a long non-contractile flexible tail; and Podoviridae (exemplified by T7), with an icosahedral head and a short non-contractile tail.
Other morphologies also exist. Filamentous phages such as M13 and fd are long flexible filaments with ssDNA genomes. Icosahedral phages without tails, such as phi X174, contain ssDNA. Lipid-enveloped phages like phi6 are rare and carry dsRNA genomes. Pleomorphic phages infecting archaea have also been described.
T4 phage is a particularly well-studied model. Its structure consists of an elongated icosahedral head (capsid) that contains 169 kb of linear dsDNA, and a tail comprising a contractile sheath surrounding a rigid tube, six tail fibers for host recognition, and a baseplate equipped with tail pins and lysozyme for penetrating the bacterial cell wall. The T4 genome of approximately 170 kb encodes roughly 300 proteins.
III. The Lytic Cycle
The lytic cycle results in the destruction (lysis) of the host cell and the release of progeny phages. Virulent phages, such as T4, can only undergo the lytic cycle.
The lytic cycle of T4 proceeds through several well-defined steps. Adsorption begins when the tail fibers recognize and bind to specific receptors on the bacterial surface, typically LPS or outer membrane proteins; this receptor specificity determines the phage's host range. Injection follows as the tail sheath contracts, driving the tail tube through the outer membrane and cell wall, and the DNA is injected into the cytoplasm while the capsid remains outside the cell as an "empty ghost."
Early gene expression involves an immediate takeover of the host: phage-encoded nucleases degrade the host DNA (T4's own DNA is protected from self-degradation because it contains hydroxymethylcytosine instead of cytosine). Anti-sigma factors redirect the host RNA polymerase to phage promoters, and enzymes for nucleotide biosynthesis and DNA replication are produced. During DNA replication, a phage-encoded DNA polymerase replicates the phage genome, producing concatemeric DNA.
Late gene expression leads to the synthesis of structural proteins -- head, tail, tail fibers, and baseplate components. Heads and tails are assembled separately, and DNA is packaged into preformed heads by a packaging motor using a "headful" mechanism. Heads and tails are then joined to form complete phage particles.
Lysis and release is orchestrated by a precise timing mechanism. Holin proteins form pores in the inner membrane at a programmed time, allowing endolysin (lysozyme) to gain access to the peptidoglycan, which it degrades. Spanins then disrupt the outer membrane in Gram-negative bacteria. The cell lyses, releasing approximately 100--200 progeny phages (the burst size).
The kinetics of the lytic cycle are described by the one-step growth curve: the eclipse period (during which no infectious phage can be detected intracellularly) is followed by the latent period (during which phages are assembled but not yet released), which culminates in the burst (cell lysis and a sudden increase in phage titer).
<image>A diagram of the T4 bacteriophage lytic cycle. Panel A: T4 phage structure with labeled head (dsDNA inside), contractile tail sheath, baseplate, and tail fibers. Panel B: Step-by-step cycle within an E. coli cell: (1) Tail fibers bind to LPS receptors; (2) Tail sheath contracts, injecting DNA; (3) Host DNA degraded; early gene expression; (4) Phage DNA replication; (5) Late gene expression -- capsid and tail assembly; DNA packaging; (6) Holin-endolysin system lyses the cell; progeny phages released. Panel C: One-step growth curve graph showing eclipse period, latent period, and burst (rise period) with the burst size indicated.</image>
IV. The Lysogenic Cycle
Temperate phages, such as lambda, can either lyse the host or integrate into the host chromosome. In lysogeny, the phage genome integrates into the bacterial chromosome as a prophage, where it is replicated passively with the host DNA during cell division. The lysogenic cell (lysogen) is immune to superinfection by the same phage type because phage genes are mostly silenced by a repressor.
Phage Lambda -- the Lysogeny-Lysis Decision
Upon infection, lambda DNA circularizes via complementary cos (cohesive) ends. Two competing regulatory pathways then determine the outcome. In the lytic pathway, the gene N product (an antiterminator) and gene Q product (a late antiterminator) promote transcription of replication and lysis genes. In the lysogenic pathway, the gene cI encodes the lambda repressor (CI), which binds to operators OL and OR, blocking transcription of lytic genes while maintaining its own transcription through positive autoregulation from the PRM promoter. Lambda integrase then catalyzes site-specific recombination between the phage attP site and the bacterial attB site, inserting the prophage into the chromosome flanked by attL and attR.
The decision between these pathways hinges on several factors. The CII protein activates transcription of cI and integrase, but CII is inherently unstable and is degraded by the host FtsH protease. High multiplicity of infection (MOI) and poor host nutritional status favor CII accumulation, tipping the balance toward lysogeny. Low MOI and vigorous host growth favor lysis. Environmental signals that cause DNA damage, such as UV light, trigger the SOS response, which leads to RecA-mediated cleavage of the CI repressor and prophage induction -- the switch from lysogeny back to the lytic cycle.
V. Lysogenic Conversion
Prophage genes can alter the phenotype of the host bacterium, frequently by increasing its virulence, a phenomenon known as lysogenic conversion. Many important bacterial toxins are encoded on prophages. Corynebacterium diphtheriae produces diphtheria toxin from the tox gene carried by phage beta. Clostridium botulinum carries botulinum toxin genes on prophages. Vibrio cholerae produces cholera toxin (CTX) encoded by phage CTX-phi. Streptococcus pyogenes harbors erythrogenic (pyrogenic) toxin genes on prophages. Escherichia coli O157:H7 carries Shiga-like toxin genes (stx1, stx2) on lambdoid prophages. Staphylococcus aureus acquires Panton-Valentine leukocidin (PVL) and staphylokinase from phages. Lysogenic conversion is thus a form of horizontal gene transfer that plays a central role in driving the evolution of bacterial pathogens.
VI. Phage Defense and Counter-Defense
Bacteria and phages are locked in an evolutionary arms race, with each side deploying an array of offensive and defensive strategies. Bacterial anti-phage defenses include restriction-modification (R-M) systems, which cleave foreign DNA at unmethylated recognition sites while protecting the cell's own DNA by methylation; CRISPR-Cas, an adaptive immune system that stores spacers from past infections and guides Cas nucleases to cleave matching phage DNA; abortive infection (Abi) systems, in which the infected cell dies before phage replication is complete, sacrificing itself to protect the population ("altruistic suicide"); receptor modification or loss through mutations that alter or eliminate phage receptors; and superinfection immunity, whereby lysogenic cells are resistant to reinfection by the same phage type.
Phage counter-defenses include anti-restriction strategies such as modifying their own DNA (for example, glucosylation of hydroxymethylcytosine in T4) or encoding anti-restriction proteins; anti-CRISPR proteins that inhibit Cas nuclease activity; and mutations in receptor-binding proteins that allow adaptation to altered host receptors. These arms race dynamics drive ongoing co-evolution between phages and their bacterial hosts.
<image>A diagram comparing the lytic and lysogenic cycles of phage lambda. Starting point: lambda phage infects E. coli and injects its linear dsDNA, which circularizes. Decision point (fork in the pathway): Left branch (Lytic cycle) -- DNA replication, head and tail assembly, cell lysis, release of progeny phages. Right branch (Lysogenic cycle) -- integrase catalyzes integration into the bacterial chromosome at attB, forming a prophage; CI repressor maintains lysogeny; lysogen divides normally with prophage replicated passively. An arrow from the lysogenic branch back to the lytic branch shows prophage induction (triggered by UV/DNA damage, RecA-mediated cleavage of CI). Lysogenic conversion examples annotated: diphtheria toxin, cholera toxin, Shiga toxin.</image>
VII. Applications of Bacteriophages
A. Phage Therapy
Phage therapy is the use of phages to treat bacterial infections. Originally explored by d'Herelle in 1919, it fell out of favor in Western medicine after the discovery of antibiotics, though it continued to be used in Eastern Europe, particularly in Georgia and Poland. The growing antibiotic resistance crisis has renewed interest in phage therapy worldwide. Its advantages include high specificity (narrow host range minimizes disruption of the normal microbiome), self-amplification at the site of infection, the ability to penetrate biofilms, and low toxicity. Challenges include the need to match specific phages to the infecting pathogen due to narrow host range, the development of bacterial resistance to phages, regulatory hurdles, and immune clearance of phage particles. Recent case reports and clinical trials have shown promise, particularly for multidrug-resistant infections.
B. Phage Display
In phage display, peptides or proteins are genetically fused to phage coat proteins (such as pIII of M13) and displayed on the phage surface. Libraries containing millions of phage variants are screened for binding to specific targets through a process called biopanning. This technology is used to discover antibodies, peptide ligands, and enzyme inhibitors. George Smith and Gregory Winter received the Nobel Prize in Chemistry in 2018 for their development of phage display.
C. Other Applications
Phages find numerous additional applications. Phage typing identifies bacterial strains based on their susceptibility to specific phages. Reporter phages are engineered to express luciferase or fluorescent proteins upon infecting specific bacteria, enabling rapid pathogen detection. Phages have contributed essential molecular biology tools, including lambda vectors for cloning, the T7 promoter system for protein expression, and M13 for single-stranded DNA production. In biocontrol, phage preparations are used to control foodborne pathogens, such as ListShield for combating Listeria in food products.
VIII. Ecological Significance of Phages
Phages are major regulators of bacterial populations in all environments. Through the viral shunt, phage-mediated lysis of bacteria releases dissolved organic matter (DOM) and nutrients back into the microbial loop, profoundly influencing global carbon and nutrient cycling. Phages drive bacterial diversity through "kill the winner" dynamics, in which the most abundant bacterial populations are preferentially targeted for lysis, preventing any single species from dominating. Additionally, phages facilitate horizontal gene transfer through transduction and shape the composition and function of microbial communities across virtually every ecosystem on Earth.
<image>An ecological diagram of the phage-mediated "viral shunt" in an aquatic environment. Panel A: A marine food web showing phytoplankton, bacteria, protozoan grazers, and higher trophic levels. Panel B: Bacteriophages infecting and lysing bacteria, releasing dissolved organic matter (DOM), dissolved nutrients (N, P), and viral particles back into the water. Arrows show how DOM is re-utilized by other bacteria (the microbial loop), effectively short-circuiting carbon transfer to higher trophic levels. The magnitude of the viral shunt is annotated (estimated 20-40% of marine bacteria lysed daily by phages). Panel C: "Kill the winner" model -- bar graph showing that the most abundant bacterial species is disproportionately targeted by phages, maintaining community diversity.</image>
