Phages (Bacteriophages)

What is Phages?

Viruses that attack bacteria and archaea

No. of phage particles on planet

10^(31)

Genomes in phages consist of

DNA or RNA

Single-stranded (ss) or double-stranded (ds)

Host range of phages (Specialists v.s. Generalist)

Specialists: Species-specific (strain-specific) phages

Generalists: broad host range (salmonella species)

Harmful to humans or not

Harmless to humans → focus on bacteriophages

Where are phages found?

Present whenever bacteria are found

No. of phages v.s. bacteria

Outnumber bacteria by 10-fold

How do phages infect bacteria?

1. Absorbs into the bacterial cell wall, flagella or pili

2. Inject their genome into the cytoplasm of the bacterium

3 types of canonical phage infection cycles

• Chronic

• Lytic

• Lysogenic

Canonical phage infection cycles: What happen after phage infects bacteria?

1. Lay dormant - in a non-productive state

2. Enter productive state - produce progeny/new variants

Canonical phage infection cycles: Usual productive cycle

Upon entry into their host cell,

Phages can enter a productive replication cycle that results in the release of new virions (lysis), either without lysing the host (chronic cycle) or upon host lysis (lytic cycle)

Canonical phage infection cycles: Alternative non-productive cycle (Going dormant)

Phage genome (thick pink line) integrates into + replicated along with the host chromosome (lysogenic cycle)

Canonical phage infection cycles: Can phages exit the dormant state (Non-productive cycle)?

Either spontaneously or upon exogenous stimuli,

and switch to one of the productive cycle

Canonical phage infection cycles: Filamentous phages & cycles

Follow a productive chronic cycle

with some (not all) having the capacity to enter a non-productive lysogenic cycle

Canonical phage infection cycles: Temperate phages & cycles

(Any cycle: productive & lysogenic)

Characterized by their ability to be lysogenic (under non optimal conditions) and

Upon induction, they can produce new virions through either a chronic or a lytic cycle

Canonical phage infection cycles: Virulent phages & cycles

Replicate only through a lytic cycle

HT, horizontal transmission (other bacterial cells around infected by progeny released);

VT: vertical transmission (insert its genome into bacterial genomes).

What is Pseudolysgoeny ((double-stranded) dsDNA phage)? [in carrier state]

The stage of stalled phage development of a bacteriophage

→ in unfavorable growth conditions for the host cell, e.g., starvation

An unintegrated phage genome is asymmetrically passed (as not replicated) onto daughter cells [VT]

What happens to daughter cells in Pseudolysogeny?

Become resistant (indicated by red crosses) to secondary infections through the inheritance of the phage genome or, as in the case of phage P22, immunity factors

What is Superinfection exclusion in Pseudolysogeny?

A phenomenon where a host cell that is already infected by one virus becomes resistant to subsequent infections by similar viruses

(in intracellular phage particles)

What is phage peristence?

Phages remain within the cell without being released

TEM image of Pseudomonas aeruginosa with ssRNA phage

→ 3 days after bacterial population declared phage-free

An enlarged cell packed with viral particles in the center with an intact membrane (showing phage persistence)

Lytic phage infection cycle: Eclipse period

During the early phase of infection:

The host cell contains components of the phage, but no complete particles

(Phage particles produced)

Lytic phage infection cycle: Latent period

Eclipse + Intracellular Accumulation (Maturation)

The time between the attachment of a phage particle to the cell surface and the release of newly synthesized phages

End: Cell burst and phage release

Lytic phage infection cycle: Maturation period

Intracellular Accumulation (Maturation)

Lytic phage infection cycle: Burst Size

Difference between final full phage production and initial phage production

3 Phage characterization techniques

• Metagenomics (Meta sequencing) - but could lose info on species-specific gen. diversity

• Single-virus genomics- Cleaner way to analyze diversity and gen. interchangeability

• Culture and microscopy (lab-based) - Understand and characterise host range of phages

Plaque-forming unit (PFM/ml) equation

Example:

Phage density [PFU/ml] = 192 / (1.00E-03 * 0.01 ml) = 1.92E+07/ml

Phage assay: Plague + PFU meaning

Plague - clear zones on a bacterial lawn (Phage present there)

PFU = plague forming unit

Phage assay: Types?

• Spotting assay - Serial dilutions of phage lysate (fewer phage with increasing dilution)

• Plague assay - Only single dilution on the lawn - count plaques to determine no. of phages

Types of dsDNA phages: Tailed (name just for ref)

Myoviridae (T4) and Herelleviridae

Podoviridae (T7)

Ackermannviridae (AG3)

Siphoviridae (Lambda)

Types of dsDNA phages: Non-tailed (names just for ref.)

Corticoviridae (PM2)

Tectiviridae (PRD1)

Plasmaviridae (MVlL2)

Types of dsDNA phages: Most prevalent dsDNA in human microbiota

Crassviridae - present in 73% of human fecal samples

→ important in gut microbiota

2 types of ssDNA phage

Microviridae (with icosahedral capsids) and Inoviridae (filamentous phages)

Type of dsRNA phage + how to attach to host?

Cystoviridae

• ✅ external lipids in membrane

→ Pseudomonas phage phi6 (Spike protein, 3 segments included (not 1 linear))

————————————————————

• DON’T BIND TO BACTERIA BODY - attach to appendices (e.g. to type IV pilus of host)

Type of ssRNA Phage (simple)

Class Leviviricetes (ICTV 2023 Release)

→ Escherichia phage MS2

• just maturation proteins + coat proteins + ssRNA

[f-pili (appendices) attach to phages]

What phage genomes do?

Encode as few as three genes and as many as hundreds of genes

Why do RNA viruses have high mutation rates?

Their RNA-dependent RNA polymerase LACK proofreading activity

Phage diversity: Virus-Host DB - updates till 26/02/2026

VERY diverse

21,579  dsDNA phages

448  ssDNA phages

13  dsRNA phages

27  ssRNA phages

NCBI data = 883 ssRNA phage ref. genomes (11 phages w known host)

Why is research on phages with dsDNA genomes more focused on than RNA?

There are biases in experimental procedures and computational analysis:

• Protocols optimized for the isolation and characterization of dsDNA phages

• DNA sequencing is cheaper than RNA sequencing

• RNA phages have higher genetic diversity than DNA phages - harder to do?

Arms race of: Bacteria-phage (host-parasite) coevolution

Both species must constantly adapt each other (like Red Queen hypothesis)

Arms race of Bacteria-phage coevolution: What they evolve? and lead to?

Bacteria evolve resistance to phages

Phages evolve resistance-countering mutations

→ specialize on specific host genotypes

Leads => selection of rare host genotypes that are resistant to the dominant phage population

5 antiphage defense mechanisms and strategies in bacteria

• Restriction-modification systems

• BREX (Bacteriophage Exclusion)

• CRISPR-Cas

• Toxin-antitoxin systems

• Abortive infection

Antiphage defense mechanisms and strategies in bacteria: Restriction-modification systems usage

• Protect bacterial host DNA by methylation

• Restriction enzymes degrade foreign (phage) DNA (not methylated)

(by. adding methyl groups (a small chemical group) to specific sites on their own DNA. This methylation acts as a protective marker. When foreign DNA enters the cell, it lacks these methylation markers and is recognized as invader DNA. The system then cuts up the foreign DNA to protect the cell.)

Antiphage defense mechanisms and strategies in bacteria: BREX (Bacteriophage Exclusion) usage

• Protect bacterial host DNA by methylation

• Other part of system blocks phage replication

Antiphage defense mechanisms and strategies in bacteria: Toxin-antitoxin systems usage

Proteins w diff. enzymatic activity

Range of mechanisms (intrfere w DNA rep. degrade RNA, inhibit cell synthesis) to defend against phages

Antiphage defense mechanisms and strategies in bacteria: Abortive infection usage (based on Toxin antitoxin system)

✅MOST bacterial pop. depends on this

Strategy to contain phage infection, mediated by toxin-antitoxin systems

• When bacteria alr infected by phage

• Cascade of signals happens

• Trigger Abortive infection →bacterial cell destroy itself

✅ Advantageous for population as the infected cell won’t replicate more variants

Antiphage defense mechanisms and strategies in bacteria: Restriction-modification systems v.s. BREX

R-M system (kill):

relies on restriction enzymes to degrade foreign DNA

BREX system (defense):

blocks phage replication through epigenetic modifications and other mechanisms

CRISPR - full name

Clustered Regularly Interspaced Short Palindromic Repeats

Cas - full name

CRISPR-associated proteins

Classes and types of Cas

2 classes: Class 1 and Class 2 and 6 types (I to VI)

Cas: Identified in where

Identified in at least half of the available bacterial genomes

What is a CRISPR loci?

CRISPR loci are a cluster of short DNA repeats (white boxes) separated by equally short spacer sequences of phage and plasmid origin (coloured, numbered boxes).

How are CRISPR arrays created?

A small piece of the virus's DNA was captured when bacteria were infected by the virus

The captured DNA is inserted into the bacterium's own DNA

→ creating CRISPR arrays

With CRISPR arrays, what happens if the virus attacks again?

Bacterium produces RNA segments from the CRISPR arrays.

• RNA segments attach to specific regions of the virus's DNA.

• Enzyme used to cut the DNA apart → disabling the virus

What is the CRISPR array preceded by, and what does the preceder contain?

Preceded by a leader sequence (grey box) containing the promoter for its expression.

What does the repeat/spacer array contain and do?

This repeat/spacer array is flanked by an operon of CRISPR-associated (cas) genes (blue-tone arrows)

→ encode the machinery for the immunization and immunity stages of the system.

Stages of CRISPR-Cas immunity: Immunization stages

Immunization stage: Bacterial cells haven’t met the phage yet

• Spacer sequences are captured upon entry of the foreign DNA into the cell (Genome chopped into small pieces of sequences)

• Integrated into the first position of the CRISPR array (incorporated in the CRISPR area of the bacterial genome)

Stages of CRISPR-Cas immunity: Immunity stages

Immunity stage: Spacer is used to target invading DNA that carries a cognate sequence for destruction (killer)

• Spacers are transcribed and processed into small CRISPR RNAs (crRNAs) in the ‘crRNA biogenesis’ phase.

• CRISPR array induced

• small guide RNAs (antisense guides for Cas) produced

• RNA-guided nucleases (form a complex)

• Locate and cleave the sequence (black arrowhead) in the phage genome (‘targeting’ phase)

Phage counter-defenses: Binding bacterial immune proteins (How it works)

Using (anti-CRISPR, Anti-restriction proteins, etc.)

for protein-protein interaction

Phage counter-defenses: Post-translational modifications of immune proteins (How it works)

(If Toxin-antitoxin system)

Chemical modification of proteins that are part of the bacterial defense machinery

Phage counter-defenses: Targeting secondary messengers that activate immunity mechanisms (How it works)

Cleave signalling molecules

→ disrupt immunity regulation of bacteria

(Secondary messengers = nonprotein molecules or ions that bind to specific target proteins, and disseminate information received by cellular receptors)

What phage in nature do?

Phages shape the taxonomic and functional composition of microbial communities as well as their stability

VLP = virus-like particle

→ indirectly interfere w nutrient cycling in nature

How do phages affect bacterial communities (diversity)?

By integrating in their genomes and replicating within their host

How do phages affect bacterial communities (Abundance)?

Change the abundance of bacteria by inducing bacterial death

How do phages affect bacterial communities (Physiology)?

Modify the physiology of bacteria by modifying host metabolism

How do phages affect bacterial communities (Virulence)?

Change the virulence of bacteria by transferring relevant genes

Phages in biomedicine and agriculture: Phage therapy

Used to target and destroy specific bacteria without harming human cells

Phages in biomedicine and agriculture: Phage therapy - Combatting antimicrobial resistance (AMR)

Phages offer a promising solution to the growing problem of AMR by providing an alternative to traditional antibiotics.

Phages in biomedicine and agriculture: Biocontrol in agriculture

To control bacterial pathogens in crops, reducing the need for chemical pesticides

Phages in biomedicine and agriculture: Food safety

Applied to food products to eliminate harmful bacteria, ensuring food safety and extending shelf life

What phages used as model system in research?

• MS2 - first sequenced genome

• Lambda - well-studied for its lysogenic cycle for understanding gene regulation in bacteria

• T4 - studying DNA replication mechanisms due to its complex and efficient replication machinery

• M13 (a filamentous phage) - studying protein-protein interactions and phage display technology

• ΦX174 - understanding viral genome structure

How to identify protein-ligand interaction in research?

Phage display technology: Engineering phage particles to display specific peptides or proteins

Knowledge gap on phage diversity need on

• species diversity (genome diversity)

• host range

• bacterial defense mechanisms against phages

• phage counter-defense mechanisms in ssDNA, ssRNA, and dsRNA phages.