Plasmids, Conjugation, and Generalised Transduction in Prokaryotes

Mechanisms of Genetic Exchange in Bacteria

1. Transformation – uptake of naked DNA

2. Conjugation – direct transfer via plasmid-mediated contact

3. Transduction – bacteriophage-mediated DNA transfer

These processes are forms of horizontal gene transfer (HGT)

Chromosome

Most bacteria (e.g. E. coli) possess:

• One essential circular double-stranded DNA chromosome

• Approx 4.6 Mbp in E. coli

• Mainly unique sequence DNA

Plasmids

Additional extrachromosomal DNA molecules:

• Circular dsDNA

• Much smaller than chromosome

• Non-essential for survival

• May provide selective advantage

General Features of Plasmids

• Replicate independently

• Can exist in multiple copies

• Often carry accessory genes

• May transfer between bacteria

Sex Plasmids (F Plasmid)

Plasmids encoding genes for bacterial conjugation

~35% of plasmid encodes:

• tra genes (transfer genes)

• Pilus formation proteins

• DNA transfer proteins

Contains insertion sequences:

• IS2

• IS3 (×2)

• IS1000 / γδ

These allow homologous recombination with chromosome

F (Fertility) Plasmid of E. coli

• ~100 kb

• Stringent replication → low copy number (1–2/cell)

• Self-mobile

• Encodes transfer machinery

F plasmid is an episome

Can exist:

1. Free in cytoplasm

2. Integrated into chromosome

R Plasmids (Resistance Plasmids)

Encode resistance to:

• Antibiotics

• Heavy metals

• Toxins

Features

• 30–100 kb

• Self-mobile

• Can transfer between unrelated species

Clinical Importance

Major contributor to:

• Spread of antimicrobial resistance

• Multidrug-resistant pathogens

Col Plasmids

Encode bacteriocins (e.g. colicins)

Colicins are Proteins that:

• Bind bacterial membranes

• Form pores

• Kill competing bacteria

Features

• Usually <25 kb

• Relaxed replication → high copy number (~30)

• Not self-mobile

Can be mobilized if F/R plasmid present.

Biotechnology Importance

Used as cloning vectors:

• Example: pGEM derivatives from ColE1

Discovery of Conjugation - Lederberg and Tatum (1946)

Experiment

Mixed two auxotrophic E. coli strains:

• Each lacked ability to synthesize different nutrients

Observation

• Prototrophic colonies appeared on minimal medium

Conclusion

Genetic exchange occurred between strains.

Discovery of Conjugation - U-Tube Experiment (Bernard Davis)

Setup

Two bacterial populations separated by filter:

• Allowed medium/DNA through

• Prevented cell contact

Result

• No prototrophic colonies

Conclusion

Physical contact is required for conjugation

F+ × F− Conjugation

F+ cells - Contains F plasmid

F- cells - Lack F plasmid

F+ × F− Conjugation mechanism

Step 1: Contact Formation

F+ cell produces:

• F pilus

Pilus attaches to F− cell.

Step 2: Bridge Formation

Pilus retracts:

• Pulls cells together

• Forms conjugation bridge

Step 3: Nicking at oriT

Relaxase/Tra proteins nick DNA at:

• oriT (origin of transfer)

Step 4: Rolling Circle Replication

• One strand displaced

• 5′ end transferred to recipient

Step 5: Complementary Strand Synthesis

Both donor and recipient synthesize complementary strands.

Outcome

• Donor remains F+

• Recipient becomes F+

Important Notes

• Transfer is unidirectional

• F+ generally does not transfer to F+

Hfr Strains (High Frequency Recombination) formation

Rare recombination event:

• F plasmid integrates into bacterial chromosome

Occurs via:

• Homologous recombination between insertion sequences

Hfr Strains (High Frequency Recombination) properties

Hfr cell:

• Has integrated F factor

• Still expresses tra genes

• Can initiate conjugation

Importance:

Because chromosomal genes transfer frequently during mating

Hfr × F− Conjugation

Step 1

• Nick occurs at integrated F plasmid oriT.

Step 2

• Transfer begins with part of F factor.

Step 3

• Transfer continues into adjacent bacterial chromosome.

Step 4

• Bridge usually breaks before full chromosome/F transfers.

Result

Recipient typically remains:

• F−

Because:

• Entire F factor rarely transferred

Fate of Transferred DNA from Hfr × F− Conjugation

Transferred chromosomal fragment:

• Linear

• Must recombine with recipient chromosome

• Non-recombined DNA degraded

significance of Hfr × F− Conjugation

Enables:

• Chromosomal gene transfer

• Mapping of bacterial genes

Interrupted Mating and Gene Mapping - Jacob and Wollman Experiment

Principle: Genes closer to oriT enter first.

Method:

1. Mix Hfr + F−

2. Allow conjugation

3. Interrupt mating at intervals

4. Plate on selective media

5. Determine transferred markers

Interpretation:

• Earlier transferred genes - Closer to oriT

• Later transferred genes - Further from oriT

Key Finding:

• Different Hfr strains - Different integration sites/orientations

• Combined maps showed - E. coli chromosome is circular

F′ (F Prime) Plasmids and Sexduction

Formation:

Imprecise excision of integrated F plasmid:

• Removes nearby chromosomal genes with F plasmid

• e.g. If F integrates near lac operon:

  • Excision may produce F′lac

Transfer:

• F′ plasmid transferred like normal F plasmid.

Outcome in Recipient:

• Recipient becomes Partial diploid (merodiploid)

Example:

• lac+/lac−

Importance of F′ (F Prime) Plasmids and Sexduction

Used to study:

• Dominance/recessivity in bacteria

• Gene regulation (e.g. lac operon)

Transduction

Transfer of bacterial DNA via bacteriophage

Virulent Bacteriophage Lytic Cycle

• Attachment to bacterial receptor

• DNA injection

• Host DNA degradation

• Phage genome replication

• Phage assembly

• Cell lysis

• Release of progeny phages

Generalised Transduction

Generalised: Because any bacterial gene may be transferred

Random bacterial DNA transferred by phage

Step 1

• P1 infects donor bacterium.

Step 2

• Host chromosome fragmented during lytic cycle.

Step 3

• Packaging error occurs: Bacterial DNA packaged into phage head instead of phage DNA

• Produces Transducing phage

Step 4

• Transducing phage infects recipient.

Step 5

• Injected donor bacterial DNA recombines with recipient chromosome.

Outcome

• Stable transductant formed.

Bacteriophage P1

Classic generalized transducing phage of E. coli.

Linkage Mapping by Cotransduction

• A phage head has limited DNA capacity.

• Therefore Only genes physically close together can fit into same transducing fragment

Cotransduction

Transfer of two linked genes together by same phage

Cotransduction Frequency

High - Genes very close

Low - Genes further apart

Zero - Genes too far apart

Cotransduction mapping rule

Genes closer together are cotransduced more frequently

Limit of Cotransduction

Approx:

• 100 kb

• Corresponds to P1 packaging size

Transformation

• doesn’t require contact

• mediator is naked DNA

• dna source is enviornment

• Random uptake

• Usually requires recombination

• Mapping use - cotransformation

Conjugation

• requires contact

• mediator is Pilus/plasmid

• dna source is Donor bacterium

• Ordered transfer

• requires recombination for chromosomal transfer

• Interrupted mating mapping use

Transduction

• doesn’t require contact

• mediator is phage

• dna source is Donor bacterium

• Random (generalized)

• requires recombination

• Cotransduction mapping use

Why Do Hfr Recipients Usually Stay F−?

Entire chromosome + full F factor rarely transferred before bridge breaks

Why Is Chromosomal Transfer Ordered?

• Transfer begins at fixed oriT in integrated F plasmid

• Proceeds linearly through chromosome

Why Must Transduced DNA Recombine?

• Delivered DNA is linear fragment

• Linear DNA degraded unless integrated

Why Does Cotransduction Reflect Distance?

• Phage packages fixed-size DNA fragment

• Closer genes more likely in same fragment

Episome

DNA element able to exist:

• Independently OR integrated into chromosome

Merodiploid

Partial diploid bacterium containing:

• Two copies of some genes

Transconjugant

Recipient bacterium after conjugation

Transductant

Recipient bacterium after transduction

Prototroph

Wild-type; grows on minimal medium

Auxotroph

Requires nutrient supplementation