6.3 Bacteriophages
Overview and Biological Significance of Bacteriophages
Definition and Host Range:
Bacteriophages (commonly referred to simply as phages) are a specialized class of viruses that exclusively infect bacterial cells.
They are incapable of infecting animal cells and therefore pose no direct medical threat to human health.
Microflora Interactions:
The human body hosts approximately 10x as many bacterial cells as human cells.
Bacteriophages continuously interact with and infect this indigenous human microflora within the body.
Influence on Bacterial Evolution:
During infection, phages introduce foreign genetic material into host bacteria.
This genetic exchange drives bacterial evolution and leads to novel genetic combinations within host-associated bacterial strains.
Therapeutic Potential (Phage Therapy):
Because phage replication cycles often culminate in the destruction of prokaryotic cells, phages serve as potential therapeutic agents.
They provide an alternative mechanism to combat severe bacterial infections that have developed resistance to standard antibiotics or antimicrobial drugs.
Molecular Biology and Genetic Engineering Applications:
The natural capacity of bacteriophages to insert foreign nucleic acids into host cells makes them valuable tools in modern biotechnology, molecular biology, and genetic research.
Complex Structural Anatomy of Bacteriophages
Morphological Classification:
Bacteriophages possess a complex viral structure composed of multiple functional sub-units.
Primary Structural Components:
Capsid (Head):
A 20-sided polygonal protein shell exhibiting icosahedral symmetry.
Houses the viral genome, which can consist of either DNA or RNA.
Collar:
Attaches the capsid head to the rest of the phage body.
Functions in sensing environmental conditions.
Sheath:
Composed of contractile protein.
Produces an internal tube through which viral nucleic acids are translocated from the capsid directly into the host cell cytoplasm.
Base Plate:
Situated at the distal end of the sheath.
Mediates crucial structural interactions with the surface of host cells during infection.
Tail Fibers:
Specialized protein appendages that initiate attachment by recognizing and binding to specific cell surface receptors on the target host.
Lack of Independent Motility:
Bacteriophages are non-living entities that possess no mechanisms for self-propulsion or directed movement.
Collisions between virions and host bacterial cells occur purely by random physical chance.
Microscopic Visualization:
Phage anatomy—specifically the distinct icosahedral capsid and contractile sheath—can be directly visualized using Transmission Electron Microscopy (TEM).
The Lytic Replication Cycle: Attachment and Direct Entry
Definition of Lytic Pathway:
A destructive viral replication pathway that terminates in host cell lysis and host death.
Obligate lytic phages are traditionally categorized using the letter T followed by an even number (such as the T4 phage).
Step 1: Attachment:
Tail fibers make physical contact with specific receptor proteins located on the host bacterial outer membrane.
The interaction between tail fibers and target host proteins confers high host cell specificity.
Because phages cannot move independently, attachment relies entirely on random collisions with target bacteria.
Step 2: Direct Entry (Hypodermic Needle Mechanism):
Entry of the phage genome is categorized as direct entry (one of three major mechanisms of viral cell entry).
Mechanics of entry:
Tail fibers bend upon binding target membrane proteins.
Bending lowers the base plate onto the cell envelope.
The contractile protein sheath contracts, forcing an internal tube through the cell wall and injecting viral nucleic acids into the cytoplasm.
Structural Distinction from Animal Viruses:
In animal virus entry, the entire virion (capsid and genome) is internalized by the host cell.
In bacteriophage direct entry, the structural protein components (capsid, sheath, base plate, tail fibers) remain outside the host cell; only the naked viral genome enters the host interior.
The Lytic Replication Cycle: Biosynthesis, Assembly, and Release
Step 3: Replication (Biosynthesis):
Host DNA Degradation:
Bacteriophages encode or carry an enzyme termed DNase.
DNase cleaves and degrades host bacterial chromosomal DNA into individual nucleotides.
Viral Component Synthesis:
The viral genome hijacks host transcription and translation machinery to synthesize viral structural proteins and replicate viral nucleic acids.
Step 4: Assembly:
Cytoplasmic pool of components: Required viral elements accumulate, including viral genomes, capsids, contractile sheaths, and tail fibers.
Packaging and Maturation:
Replicated phage genomes are actively packaged into newly formed capsids.
Sheaths, base plates, and tail fibers attach to capsids to yield fully formed virions.
A single infected bacterial cell typically produces hundreds to thousands of complete phage particles.
Step 5: Release (Lysis):
Enzymatic Degradation:
Phages express an enzyme called lysozyme.
Lysozyme breaks down peptidoglycan within the bacterial cell wall, compromising its structural integrity.
Cell Lysis:
Osmotic pressure destroys the weakened bacterial host wall, resulting in complete cell lysis and host death.
Viral Dispersal:
Newly assembled phages escape into the extracellular matrix to randomly contact and infect adjacent host cells.
The Lysogenic Replication Cycle and Prophage Dynamics
Definition of Lysogenic Pathway:
A replication pathway wherein the phage genome integrates directly into the host bacterial chromosome without inducing immediate cell lysis.
Phages capable of executing this pathway are termed temperate phages (such as the lambda phage)
Temperate phages possess the capacity to enter either the lytic or lysogenic cycle.
Shared Initial Steps:
Step 1 (Attachment) and Step 2 (Direct Entry) are identical in both lytic and lysogenic replication cycles.
Step 1 of Lysogeny: Integration:
Following nucleic acid entry, viral DNA is integrated directly into the circular chromosome of the host bacterium.
The integrated viral genome residing within the host chromosome is called a prophage.
Step 2 of Lysogeny: Lysogenic Cell Division:
The host bacterial cell carries out normal growth and binary fission.
Whenever the bacterium replicates its own chromosome prior to division, it passively replicates the integrated prophage.
Both daughter cells inherit a copy of the prophage genome without undergoing cell death.
Multiplication Strategy: A single initial infection event allows the viral genome to propagate exponentially into millions of daughter cells across generations.
Step 3 of Lysogeny: Excision and Re-Entry into Lytic Cycle:
Induction via Cellular Stress:
External environmental or internal physiological stressors that threaten host survival trigger prophage activation.
The "Abandon Ship Strategy":
If the bacterial host cell perishes while harboring a passive prophage, the integrated viral genome dies with the host.
To prevent destruction, the prophage detects host distress and excises itself from the bacterial chromosome.
Completion of Lytic Re-Entry:
Once excised, the viral DNA initiates host DNA degradation (via DNase), viral component biosynthesis, capsid packaging, assembly, and final host cell lysis via lysozyme release.
Culturing Methods and Quantitative Analysis of Phages
Laboratory Culturing Conditions:
Bacteriophages are simple to culture in laboratory environments because they only require appropriate host bacterial cultures and initial phage samples.
Phages can be grown in either liquid media (broth) or solid media (agar plates), dependent upon the growth requirements of the specific host strain.
Viral Titer (Viral Load):
Defined as the quantitative concentration or total number of infectious viral particles present in a given liquid sample.
Assessing Titer via Turbidity:
Turbidity Definition: The measure of cloudiness or opacity in a liquid suspension.
Physical Scale Disparity: Bacterial cells are orders of magnitude larger in size and volume than individual viral virions.
Interpretation of Cloudiness:
High Turbidity (Cloudy): Indicates a high concentration of intact, living host bacterial cells relative to viral particles.
Low Turbidity (Clear): Indicates widespread phage infection and cell lysis, leaving fewer intact bacterial cells in suspension.
Host Specificity Testing: Changes in suspension clarity after phage inoculation can determine whether a specific bacterial strain serves as a viable host for a particular phage.
Spectrophotometric Quantification:
Visual inspection is insufficient for precise lab measurements; quantitative optical instruments are required.
Spectrophotometer Function:
Measures the amount of light that passes through or is absorbed by a liquid culture tube.
Absorbance: The proportion of light scattered or absorbed by intact suspended bacterial cells.
Transmittance: The proportion of light passing unimpeded through the liquid sample.
Experimental Protocol:
A liquid suspension of bacterial host cells is prepared in test tubes.
Baseline turbidity (absorbance and transmittance) is measured using a spectrophotometer prior to phage introduction.
Bacteriophages are added to the bacterial culture.
After incubation, turbidity is re-measured.
Active phage infection lyses host cells, reducing bacterial numbers, lowering solution turbidity, decreasing light absorbance, and increasing light transmittance.