Bacterial plasmids and transfer of genetic material

Question 1

Sizes of genomes

Answer 1

• A reduction in genome size is associated with parasitic lifestyles in all taxons
• Viruses are the “ultimate” parasites, and (most) have small genomes
• Exceptions: the genome of Pandoravirus is larger than that of some parasitic
eukaryotes (e.g. Microsporidia)

Question 2

Viruses have a large diversity in their genome organisations

Answer 2

• Viruses are simple structures
– protein coat containing either DNA or RNA
– inactive while extracellular
– inject or deliver their genomic material into cells
• RNA viruses
– Double or single stranded RNA genomes
– Includes influenza, HIV, coronavirus
• DNA viruses
– Double or single stranded DNA genomes
– Includes smallpox, herpesviruses, most bacteriophages
– Giant viruses (genome up to 2.5 Mb)

Question 3

Eukaryotic genomes - nuclear

Answer 3

Nuclear genomes
- Large (23 pairs of linear chromosomes, 3 billion bp in humans)
- Coding DNA: proteins, functional RNAs
- Pseudo-genes: no longer functional
- Non-coding regions: repetitive, e.g. centromere and telomeres
(~50%)

Question 4

Eukaryotic genomes - Organelle genome(s)

Answer 4

Organelle genome(s)

• Mitochondria genome
• Mitochondria are organelles involved in energy production by respiration
• Single circular DNA molecule (in humans: 16.5 kbp, 13 proteins
• Chloroplast genome in plant
• Apicoplast genome in parasitic eukaryotes

Question 5

Bacterial genome organisation

Answer 5

Most bacteria have a single circular chromosome
– 500 Kb – 10 Mb
– Escherichia coli
• Some have two circular chromosomes
• Some have linear chromosomes
– Borrelia burgdorferi a spirochaete which is the causative agent of Lyme disease
• Agrobacterium tumefaciens has both a circular and a
linear chromosome

Question 6

Diversity of bacterial genetic elements

Answer 6

Main chromosome
– Contains genes essential for all conditions
– One or two chromosomes, circular or linear
• Plasmids
– Extrachromosomal, genes not essential in all growth conditions
– Circular or linear
• Bacteriophages
– Viruses that infect bacteria
• Transposable elements
– Mobile DNA that can move in and out of chromosome
– Insertion sequences, composite transposons, some bacteriophage

Question 7

Bacterial plasmids

Answer 7

• Autonomously replicating pieces of double stranded DNA
– Exist independently of the main chromosome
• Generally smaller than the chromosome
– 1 kbp to 1 Mbp
• Often present in multiple copies per cell
– Anywhere from 1 to 500 copies per cell
• Do not encode functions essential for growth under all conditions
– May encode important functions
– May be essential in some niches

Question 8

Plasmid copy number

Answer 8

• Copy number is the average number of copies of a plasmid in a cell
– Copy number can vary between 1 and 100s
– Any genes on the plasmid will usually be present at multiple copies in the cell
• Plasmids have genes whose products regulate copy number per cell
– This number is constant during proliferation

Question 9

Plasmid-encoded functions

Answer 9

Plasmids carry genes encoding a wide variety of functions including
– Control of their replication (copy number control)
– Resistance mechanisms
• Antibiotics
• Heavy metals
– Virulence genes
• Toxin production, secretion systems
– Metabolic enzymes
– Production of antimicrobials (bacteriocins)
– Mobility (conjugation) factors

Question 10

Plasmids can alter bacterial phenotype

Answer 10

Shigella flexneri is the cause of dysentery and diarrhoea
– ~1 million deaths/year
• Virulence attributes include
– Attachment to and invasion through colonic epithelial cells
– induced death of macrophages
– Regulate number of immune cells
• Many of these functions on a 220-kb virulence plasmid
– Without this plasmid Shigella will not
cause disease
– Shigella is really E. coli with a virulence plasmid

Question 11

How can microbial DNA be transferred?

Answer 11

Vertical transfer (parent to daughter)
Horizontal transfer (cell to unrelated cell)

Question 12

Vertical transfer (parent to daughter)

Answer 12

– Chromosomes
• DNA on the chromosome including mobile DNA and some bacteriophage
– Plasmids

Question 13

Horizontal transfer (cell to unrelated cell)

Answer 13

– Transformation –>Uptake of free DNA
– Conjugation –>Direct transfer of DNA from cell to cell
– Transduction–>Transfer via a bacteriophage intermediate

Question 14

Transformation

Answer 14

Transformation: bacteria take up free DNA
from their surroundings
– DNA may be integrated into the chromosome or (if it is a plasmid) replicate freely
• A bacteria that can take up DNA is said to be “competent”
– Generally need to manipulate bacterial growth conditions to make bacteria competent
• Transformation is an important laboratory tool

Question 15

Artificial transformation (in the lab) achieved by a variety of techniques

Answer 15

– Chemicals such as CaCl2 make membranes permeable
– Electroporation uses voltage to generate pores in membrane
• Transformation of plasmid DNA is used in many recombinant techniques.
– plasmid DNA is not easily degraded
– plasmids replicate autonomously so genes
are expressed without recombination into chromosome
– can introduce foreign DNA/genes into cells

Question 16

Use of antibiotic resistance plasmids

Answer 16

• Plasmids with antibiotic resistance markers are extensively used in molecular biology research
• The antibiotic resistance marker allows selection of the recombinant bacteria
• Plasmids are used to express genes for purification or
functional analysis

Question 17

Conjugation

Answer 17

Conjugation: bacterial DNA is transferred by direct cell
to cell contact
• To be able to initiate conjugation a bacteria must have
– DNA which encodes a sex pilus
– Express proteins required for DNA transfer
– DNA with a region that can begin transfer (origin of transfer oriT)
• These factors may be expressed
– From plasmids such as the F plasmid
– Can also be expressed from genes on the chromosome
• Conjugation positive cells often designated F+

Question 18

Initiation of conjugation: forming the bridge

Answer 18

Initial steps

1. Pilus tip of F+ cell binds to receptor on surface of F- cell
2. Retraction of pilus brings F+ and F- cells close together
3. Formation of the conjugative bridge, an opening between the cytoplasm of the two cells

Question 19

DNA transfer steps - conjugation

Answer 19

1. One strand of DNA is nicked at the oriT site
   (only one of the two DNA strands is cleaved)
2. One DNA strand is pushed through the
   conjugation bridge
3. F+ cell repairs plasmid
4. F- recipient uses incoming DNA as a template
   for DNA synthesis
5. DNA transfer/synthesis completed and cells
   separate
6. Both cells are now F+

Question 20

Transduction

Answer 20

Transduction = a means of genetic exchange whereby DNA is transferred from the donor cell to the recipient cell via a bacteriophage

Question 21

Bacteriophage and transduction

Answer 21

• Bacteriophage (or phage) are viruses that infect bacteria
• Bacteriophages can exist outside cells but are inactive/inert while extracellular
• When they contact a susceptible cell they
– Attach
– Inject their DNA
– Replicate or integrate into genome

Question 22

Life cycles of bacteriophage

Answer 22

Bacteriophage can replicate in two ways
• Lytic cycle (lytic and temperate phage)
– Phage injects DNA, replicates, lyses the host cell and
releases progeny phage
• Lysogenic cycle (temperate phage only)
– Phage injects its DNA and this integrates into the
chromosome (no replication or release of progeny)
– Phage DNA is then replicated along with the host
chromosome

Question 23

Generalised transduction

Answer 23

• Occurs when a bacteriophage incorrectly packages bacterial DNA into the phage particle
– The incorrectly packaged DNA must fit into the phage head (<100 kbp)
• These bacteriophage can still infect a new bacteria
– But the injected DNA will not result in the production of viable phage
• Transferred genes will only be stably maintained in the new cell if they recombine into the chromosome

Question 24

If DNA is transferred to a new cell it will only survive if it

Answer 24

– Can replicate by itself (e.g. a replicating plasmid)

– Is integrated into the chromosome (or into an already present plasmid)

Question 25

How can DNA be integrated into the host chromosome?

Answer 25

– The DNA molecule may encode its own proteins for integration (integrases, recombinases)
• Usually the case with mobile DNA (transposons)
– The DNA may recombine into the chromosome by homologous recombination
• Requires some level of DNA sequence similarity (homology)

Question 26

Transformation/Transfection

Answer 26

Transfection is the introduction of foreign DNA into animal cells
(similar to transformation of prokaryotic cells and other eukaryotes)
– Stable (integrated into chromosome)
– Transient (expressed from a replicating vector/plasmid)

Question 27

How is Transformation/Transfection achieved?

Answer 27

• Modification of the eukaryotic membrane by
– Incubation of cells in certain chemicals
– Application of an electric pulse (electroporation)
• Addition of the DNA in liposomes
– Relies on liposome/membrane fusion
• Physical introduction using gene gun
– DNA coupled to an inert solid is fired directly into nucleus

Question 28

Particle bombardment

Answer 28

• Also known as the gene gun
• Coat plasmid with gene of interest on tungsten or gold particles (microprojectiles)
• Shoot - Penetration of cell wall
• Microprojectiles enter the cells, transgenes may incorporate into chromosomal DNA
• Selectable markers used to identify cells that take up transgene
• Regenerate plant cells

Question 29

Electroporation

Answer 29

• Increase in permeability of cell plasma membrane caused by applied electrical field
• Used for transformation of bacteria, yeast and
protoplasts