Bacterial plasmids and transfer of genetic material Flashcards
Sizes of genomes
• 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)
Viruses have a large diversity in their genome organisations
• 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)
Eukaryotic genomes - nuclear
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%)
Eukaryotic genomes - Organelle genome(s)
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
Bacterial genome organisation
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
Diversity of bacterial genetic elements
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
Bacterial plasmids
• 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
Plasmid copy number
• 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
Plasmid-encoded functions
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
Plasmids can alter bacterial phenotype
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
How can microbial DNA be transferred?
Vertical transfer (parent to daughter) Horizontal transfer (cell to unrelated cell)
Vertical transfer (parent to daughter)
– Chromosomes
• DNA on the chromosome including mobile DNA and some bacteriophage
– Plasmids
Horizontal transfer (cell to unrelated cell)
– Transformation –>Uptake of free DNA
– Conjugation –>Direct transfer of DNA from cell to cell
– Transduction–>Transfer via a bacteriophage intermediate
Transformation
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
Artificial transformation (in the lab) achieved by a variety of techniques
– 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
Use of antibiotic resistance plasmids
• 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
Conjugation
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+
Initiation of conjugation: forming the bridge
Initial steps
- Pilus tip of F+ cell binds to receptor on surface of F- cell
- Retraction of pilus brings F+ and F- cells close together
- Formation of the conjugative bridge, an opening between the cytoplasm of the two cells
DNA transfer steps - conjugation
- One strand of DNA is nicked at the oriT site
(only one of the two DNA strands is cleaved) - One DNA strand is pushed through the
conjugation bridge - F+ cell repairs plasmid
- F- recipient uses incoming DNA as a template
for DNA synthesis - DNA transfer/synthesis completed and cells
separate - Both cells are now F+
Transduction
Transduction = a means of genetic exchange whereby DNA is transferred from the donor cell to the recipient cell via a bacteriophage
Bacteriophage and transduction
• 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
Life cycles of bacteriophage
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
Generalised transduction
• 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
If DNA is transferred to a new cell it will only survive if it
– Can replicate by itself (e.g. a replicating plasmid)
– Is integrated into the chromosome (or into an already present plasmid)
How can DNA be integrated into the host chromosome?
– 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)
Transformation/Transfection
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)
How is Transformation/Transfection achieved?
• 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
Particle bombardment
- 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
Electroporation
• Increase in permeability of cell plasma membrane caused by applied electrical field
• Used for transformation of bacteria, yeast and
protoplasts
