MICROBIAL GENETICS

1. What is Microbial Genetics?

Microbial genetics studies:

• Structure and function of microbial genomes

• How genes mutate

• How genes are transferred between cells

• How recombination creates new traits

Bacteria are excellent model systems because they grow fast and can be genetically manipulated easily.

2. Types of Genetic Variation in Microbes

2.1 Mutations

• Mutation = heritable change in DNA

• Creates genetic diversity

• Types:

o Point mutations (base substitutions)

o Insertions / deletions

o Frameshifts

o Silent, missense, nonsense mutations

Mutations can be:

• Spontaneous (DNA replication errors)

• Induced (UV, chemicals, mutagens)

3. Genetic Recombination

3.1 Meaning

Recombination = Exchange of DNA between two similar sequences → produces new genotypes.

3.2 Steps of Homologous Recombination

1. Endonuclease nicks DNA

 2. SSB proteins bind to prevent degradation

3. RecA-mediated strand invasion

4. Heteroduplex region forms

5. Resolution → patch or splice (Fig. 11.11)

Heteroduplex = DNA region where strands come from different sources.

Recombination is essential because transferred DNA survives only if recombined.

4. Horizontal Gene Transfer (HGT)

(Ref: Mechanisms of HGT: transformation, transduction, conjugation )

Definition

Transfer of genes between cells not by cell division.

(Ref: Vertical vs horizontal transfer diagram)

Importance

• Creates rapid evolution

• Spread of antibiotic resistance

• New metabolic pathways

• Virulence factors

• Genome diversification
5. Fates of Incoming DNA

1. Degraded by restriction enzymes

2. Replicates independently (possible only if it has its own ori, e.g., plasmid/phage)

3. Recombines into chromosome (via homologous recombination)

Only #2 and #3 lead to stable inheritance.

6. Modes of Gene Transfer in Bacteria

Three mechanisms:

1. Transformation – uptake of free DNA

2. Transduction – bacteriophage-mediated transfer
3. Conjugation – cell-to-cell contact; plasmid-mediated

1) Transformation — uptake of free DNA

Simple definition
Transformation = a bacterium takes up free (naked) DNA from its environment and incorporates it into
its genome, producing a heritable change.

Where it happens / why it matters

• Happens naturally in some species (e.g., Bacillus, Streptococcus, Neisseria, Haemophilus,

Acinetobacter, Thermus).
 • Important for acquiring new genes (antibiotic resistance, metabolic capabilities) and was
historically crucial in proving that DNA is the genetic material.

Key points

• Only competent cells can be transformed (competence is genetically regulated).

• Donor DNA fragments are typically ~10 kb (≈10 genes).

• DNA usually enters as single-stranded (one strand degraded during uptake).

• RecA mediates integration by homologous recombination.

• Natural competence can be controlled by quorum sensing, environmental signals (e.g., chitin for
Vibrio cholerae), or stress responses.

Mechanism — step by step

1. DNA release: Donor cells lyse and release fragmented chromosomal DNA into the environment.

2. Binding: Competent recipient expresses surface DNA-binding proteins (often pilus-like) that
capture double-stranded DNA.

3. Uptake: One DNA strand is degraded by nucleases and the other is transported into the
cytoplasm as single-stranded DNA.

4. Protection & pairing: Single-strand binding proteins protect it; RecA searches for homology
and mediates strand invasion into a homologous region of the recipient chromosome.

5. Recombination: Homologous recombination integrates donor sequence into chromosome →

stable transformant.

Artificial transformation (lab use)

• Chemical competence: treat E. coli with Ca²⁺ + cold shock → allows plasmid uptake.

• Electroporation: electric pulses produce temporary pores — works on many bacteria/archaea

and eukaryotic cells.

Outcomes & limits

• Usually only a few fragments are taken up in one event → limited genes transferred per event.

• If DNA does not recombine and is not a replicating element (plasmid), it will be degraded.

Applications

• Genetic mapping, gene cloning, making knockout strains, natural genetic engineering.
2. Conjugation — direct cell-to-cell transfer (plasmid-mediated)

Simple definition
Conjugation = DNA transfer between living bacterial cells by direct contact, usually via a conjugative
plasmid (e.g., the F plasmid).

Where it happens / why it matters

• Major route for spreading antibiotic resistance and metabolic plasmids across populations and
between species and genera.

• Very efficient and rapid under favorable conditions.

Key terms

• F plasmid (F, fertility plasmid) — ~100 kb circular plasmid in E. coli with:

o tra genes → pilus synthesis and transfer machinery

o oriT → origin of transfer

o IS elements → insertion sequences that allow integration into chromosome

• F+ = donor (has plasmid) ; F− = recipient (no plasmid)

 • Sex pilus = pilus that mediates cell pairing

Mechanism — step by step (F plasmid typical)

1. Pilus formation & contact: F+ donor produces a sex pilus that attaches to F− recipient; pilus
retracts to bring cells together.

2. Mating pair formation / coupling proteins stabilize contact.

3. Nick at oriT: TraI (a nicking enzyme with helicase activity) nicks one strand at the origin of
transfer (oriT).

4. Rolling-circle replication & transfer: Single strand is transferred to recipient while the donor
synthesizes a replacement complementary strand (rolling-circle replication in donor). The
recipient synthesizes the complementary strand as it receives the single strand.

5. Completion: Both cells end up with complete double-stranded plasmids → recipient becomes
F+ and can further donate.

Hfr strains (chromosome mobilization)

• If the F plasmid integrates into the chromosome (via IS elements), the cell becomes Hfr (high
frequency recombination).

• Conjugation from Hfr transfers chromosomal genes starting at oriT and proceeding linearly.
Transfer often breaks before complete F plasmid transfers → recipient usually remains F− but
may acquire chromosomal genes if recombination occurs.

• Different Hfr strains (integration site/orientation) transfer genes in different orders — useful for
gene mapping.

F′ (F-prime) plasmids

• If integrated F excises incorrectly, it can carry adjacent chromosomal genes → F′ plasmid.

• F′ transfers those chromosomal genes at high frequency and can create merodiploids (partial
diploids) in recipients — useful for complementation tests.

Outcomes & significance

• Rapid spread of plasmids in populations → epidemiological importance (resistance).

• Mobilization of nonconjugative elements possible (mobilizable plasmids or genomic islands).

Applications

Genetic mapping (Hfr mapping), creating merodiploids, studying plasmid biology, biotechnology.
3) Transduction — phage-mediated gene transfer

Simple definition
Transduction = bacteriophage (phage) accidentally packages host DNA and transfers it to another
bacterium.

Two major types

A. Generalized transduction

• What it is: During the lytic cycle, phage packaging machinery sometimes packages random
fragments of host chromosomal DNA instead of phage DNA.

• Result: A transducing particle (defective; cannot produce new phage) injects donor DNA into
a recipient.

• Features: Any gene from donor can be transferred — but frequency is low (~1 in 10⁶–10⁸ phage
particles for a given gene).

• Fate: Donor DNA must recombine with recipient chromosome to be stably maintained.

Classic examples: phage P22 in Salmonella, P1 in E. coli.

B. Specialized transduction

• What it is: Occurs with temperate phages that integrate into the host chromosome (lysogeny),
e.g., λ phage.

• Mechanism: When prophage excises incorrectly during induction, it may take adjacent host
genes with it → packaged into phage genomes.

• Result: Efficient transfer of specific host genes located near the integration site (limited set).

• Example: λ phage occasionally carries gal genes from E. coli.

Phage conversion

• Lysogenization can change host phenotype (phage-encoded genes expressed in host).

• Examples:

o Corynebacterium diphtheriae gains diphtheria toxin gene by lysogeny with β-phage.

o Changes in surface polysaccharides (serotype) in Salmonella after lysogeny.

Gene Transfer Agents (GTAs)

• Bacterial-produced, virus-like particles packaging random small pieces of host DNA; they are
not true viruses (no phage genes packaged to create more GTAs). GTAs are significant in some
marine bacteria for gene flow.

Outcomes & significance

• Transduction can shuttle antibiotic resistance, virulence factors, metabolic genes.

• Because phages are abundant, transduction is an important ecological gene transfer mechanism.

Important common mechanistic concepts

• RecA: central protein for homologous recombination and strand exchange — necessary for
integration of transferred DNA into chromosome.

• Fates of incoming DNA: (1) degraded (restriction systems, nucleases), (2) replicate
independently (if has ori), (3) recombine into chromosome.

• Selection: Geneticists use selectable markers (antibiotic resistance, nutritional markers) to

recover rare recombinants.

• Ecological impact: HGT speeds adaptation — antibiotic resistance, toxin genes, metabolic
pathways spread rapidly across populations.
7. Mobile DNA Elements (Transposable Elements)

Mobile DNA elements are pieces of DNA that can move from one location to another within a genome.
They play major roles in genome evolution, mutation, and gene transfer.

There are two main types:

7.1 Insertion Sequences (IS elements)

Definition:
Small, simple mobile DNA elements found in bacteria.

Characteristics

• Very small (usually 700–2500 bp).

• Contain only one gene → transposase, the enzyme that enables movement.

• Flanked by inverted repeat sequences.

Functions / Effects

• Can insert into genes → cause mutations (gene inactivation).

• Can cause genome rearrangements (deletions, inversions, duplications).

• Provide homology sites allowing plasmids (like F plasmid) to integrate into chromosome → this
forms Hfr strains.

Simple meaning:
IS elements are tiny “jumping genes” that move around and disrupt DNA.

7.2 Transposons (Tn elements)

Definition:
Larger mobile DNA elements that carry extra genes in addition to transposase.

Characteristics

• Larger than IS elements.

• Contain transposase + other genes, often antibiotic resistance genes (e.g., ampicillin,
kanamycin resistance).

• Often surrounded by two IS elements.

Mechanisms of movement

1. Conservative transposition (“cut and paste”)

o Transposon leaves old site → inserts into new site.

2. Replicative transposition (“copy and paste”)

o Original stays; copy moves elsewhere.

Importance

• Spread of antibiotic resistance genes between bacteria.

• Important for pathogenesis (virulence genes on transposons).

• Drive evolution by reshaping genome structure.

Simple meaning:
Transposons are big jumping genes that often carry resistance genes and help bacteria evolve.

8. CRISPR-Cas Defense System

CRISPR = Clustered Regularly Interspaced Short Palindromic Repeats

Cas = CRISPR-associated proteins

This is the adaptive immune system of bacteria and archaea.

How CRISPR works (simple steps)

1. Virus infects the cell

→ Bacterial cell cuts and saves a small piece of the viral DNA.

2. Spacer addition
→ Viral fragment inserted into the CRISPR locus as a “memory.”

3. CRISPR RNA (crRNA) produced

→ Contains the stored viral sequence.

4. crRNA + Cas protein attack

→ Guide RNA recognizes matching foreign DNA
→ Cas enzyme cuts and destroys it.

What CRISPR protects against

• Bacteriophages

• Conjugative plasmids
 • Any DNA previously encountered by the cell

Key point

CRISPR is sequence-specific → only targets DNA matching its stored spacers.