Bacteria and Archaea - plasmids and recombination

what is a plasmid

• Genetic information separate from the chromosomes of bacteria

• Can get incorporated into the genome

• passed down to daughter cells

are bacteria and archaea the only organisms with plasmids

• also found in fungi

• not all bacteria have plasmids

are plasmids essential

• considered non-essential

• but have important influence in metabolism and growth

genes in plasmids

• minimal genes (a few dozen at most)

• some may be adaptive

• purpose unclear

• ignored in research

plasmids vs DNA fragments

• plasmids are not fragments of DNA taken up from one bacteria (lysed) to another

• fragments must be incorporated into the chromosome

• plasmids are stably replicated and inherited not incorporated into the chromosome

4 essential components of plasmids

1. ori - origin or replication

2. rep - genes essential for replication

3. par - partitioning genes

4. Mobility elements

Ori - origin of replication

• determines copy number

• replication of plasmids occurs separately from replication of genome and chromosomes

rep - genes essential for replication

• start and regulate the process of replication

• ensures plasmid is passed to progeny

par - partitioning genes

ensures plasmid is passed to progeny

mobility elements

• for conjugation, DNA transfer, transposons and integrons
  

adaptive genes in plasmids (examples)

• antibiotic resistance genes

• metabolic genes

• virulence genes

• genes to handle unfavourable environments

metabolic genes

degrades threats - pesticides and nitrogen fixation

virulence genes

genes to make and secret toxic compounds = pathogenicity

why are plasmids so useful

• capable of niche specialization

• capable of horizontal gene transfer'

• increase diversity and resilience

• quick adaptations

what benefit do plasmids have in comparison to chromosomes

• chromosomes are large and require more energy to replicate = plasmids have an increased copy number advantage and control

• chromosomes are less flexible and are not capable of horizontal gene transfer

• chromosomes are slower = takes longer to adapt

disadvantages of plasmids

• can be maintained by individuals but not the entire species or population

• require energy to produce

• can be lost

advantages of more or less copies of plasmids

• more - high copies of antibiotic resistance genes have an advantage when pressure is high

• less - advantage when pressure is low

conjugation

• use sex pili

• donor and recipient are in direct contact during exchange

• Horizontal genetic information transfer from one bacterial cell to another

• occurs in both Gram positive and Gram negative bacteria

how does conjugation work

• donor cell has sex pilus

• fertility plasmid or DNA fragment forms in donor

• bridge forms between donor and recipient to transfer fertility plasmid to the recipient

Genetic exchange in Gram negative vs Gram positive bacteria

• Gram negative: use sex pilus and a fertility plasmid (F factor)

• Gram positive: Must interact using cell-to-cell contact, surface contact – cell adhesion because their thick peptidoglycan wall does not allow the sex pilus to pass through or for F factors to be used

F factor

• fertility plasmid

• carry the machinery for forming a sex pilus,

• enable conjugation method of exchange (genes -> proteins)

sex pilus

• specialized pili used in conjugation

• must recognise the bacterium using surface interactions

• the recognition sites can occur “cross species”, but are normally closely related

  
  

physical conjugation stage of genetic exchange

• donor is F+ meaning it has an F factor that codes for the sex pilus

• recipient is F- (lacking an F factor) and has receptors to stop other factors from preventing conjugation

• attachment accomplished by sex pilus (aka mating bridge)

• considered physical conjugation because the sex pilus physically opens a gateway between cell walls and membranes of two cells

F factor transfer (gene transfer stage of conjugation)

• mating bridge established

• origin of replication permits the breakage of double stranded DNA so that one DNA strand is transferred

• both cells make complementary strands

• both cells end up with a complete double stranded plasmid

• turns the F- bacteria to F+

Hfr gene transfer

• when the F factor is not a plasmid but part of the donor’s chromosome

• Only a piece of the donors chromosome is copied then donated because it usually breaks off when the mating bridge closes due to the length of the transfer

• recombination may occur in recipient

• recipient often not F+ in the end because only part of the F factor is transferred

what does Hfr stand for

High frequency transfer

conjugative plasmid

• has the machinery on a plasmid required to make sex pili

• F+ and F- bacteria

• the entire set of genes needed for conjugation are present (including the ori)

mobilized plasmids

• do not have the genes to make the machinery required to make a sex pili

• but can pass genes via hitchhiking

why release DNA fragments into the environment

• genetic exchange and adaptation

• biofilm formation

• communication

• source of food (energy) in a community

ways DNA fragments are made/released

• cell lysis

• phage infection

• competent cells

cell lysis and DNA fragment release

• viruses (bacteriophage) contribute to this during a lytic stage

• cells also lyse due to stress, imbalance, toxins, too
  high concentration of heavy metals, antibiotics …

phage infection and DNA fragment release

mispackaged phages can also bring about DNA fragments into a bacterium by transduction

competent cells

• bacterium who release DNA fragments during growth (rare)

• and uptake DNA fragments from the environment

• Can be controlled and adaptive

• Bacteria can release DNA fragments via vesicles (secretion systems)

• Can be a “fratricide” – a controlled sacrifice from few cells for the larger population

Griffith’s classic experiment

• live encapsulated strains with smooth (S) appearance are virulent and cause the mice to die

• live strains with rough (R) appearance are non-virulent and do not kill the mice

• Heat-killed S strain do not kill the mice

• Live R strain with Heat-killed S strain kill the mice

what does Griffith’s classic experiment tell us

the DNA fragments must have been transferred from the heat-killed S strain to the R strain that transformed the R strain into a virulent strain

industrial purpose of plasmids and recombination

• Plasmids are a vector for genes

• Can provide in-lab control over a population

• Creates assays – probes

• Confirmation of uptake and integration (eukaryotic + prokaryotic microbes)

• ex: chemical transformation and electroporation

steps of cloning GFP into a plasmid

• cut plasmid DNA with restriction enzyme (produces sticky ends)

• add GFP DNA obtained by PCR to the cut plasmid

• ligation occurs to produce continuous double stranded DNA

transformation

• non-specific acceptance of soluble DNA from the surrounding material by a bacterium (microbe)

• is facilitated by special DNA binding proteins

• competent cells undergo transformation

• DNA passes through cell wall + membrane and transformation is
  once the DNA is taken up – and therefore usable

competent cell lines

• able to take up DNA

• commercial (many different triggers), purchase

DNA binding proteins

required to recognise, interact, and uptake DNA

DNA uptake

one strand required

expression

microbial activity and functions, metabolism, defence systems, etc

industrial use of transduction

• employed in the lab to alter add genes to an organism

• Transposons and integrons can also be used for these purposes