Extrachromosomal Replication
Eukaryotes – Prokaryotes
• Origins of Eukaryotes + evolutionary relationship with Archaea = unknown – 2 models
o Three primary domains (3D) scenario
▪
• Archaea and Eukarya have common ancestor
o Two primary domains (2D) scenario
▪
• Archaea and Bacteria are two primary domains
• Eukarya is a secondary domain that arose from the merging of an archaeon and a
bacterium
Horizontal or Lateral Gene Transfer
• Prokaryotic organisms:
o Can directly transfer DNA without reproduction
o Can pick up DNA from the environment
• Horizontal gene transfer
o Gene transfer that is separate from reproduction – non-sexual movement of genes between
genomes
o 3 main mechanisms
▪ Transformation
• Involves uptake of short fragment of naked DNA by
transformable bacteria
o Classical experiments of Griffith and Avery
▪ Pneumococcal bacteria
• S strain – smooth, pneumonia
• R strain – lab strain, rough, no
pneumonia
▪ Mice injected with:
• S strain = died of pneumonia
• S strain that was heat treated = survived
• R strain that was missing the capsular polysaccharide =
survived
• R strain that was missing the capsular polysaccharide + S
strain that was heat treated = died of pneumonia
▪ Conclusion
,Extrachromosomal Replication
• R-form bacteria transformed into stable S form –
transformation process required head treatment
▪ Conjugation
• Discovered by J. Lederberg and E. Tatum
o 2 strains of bacteria
▪ A: auxotroph for methionine
(met-) and biotin (bio-) +
prototroph for (thr+, leu+,
thi+)
▪ B: auxotroph for threonine
(thr-), leucine (leu-),
thiamine (thi-) + prototroph
(met+, bio+)
o Auxotroph = mutant strain of
microorganism that has nutritional
requirements – cannot grow on
minimal medium – requires certain supplements
o Prototroph = wild type strain of microorganism – grows in minimal medium
– does not require supplements
o Strain A was washed and plated on agar plates made with minimal medium
(MM) – that contains only inorganic salts + carbon source + water
▪ Results
• No colonies
• Backmutation cannot restore prototrophy
o Backmutation = process that causes reversion –
change in nucleotide pair in mutant gene that
restores the original sequence + original phenotype
o Strain B was washed and plated on agar plates made with minimal medium
▪ Results
• No colonies
• Backmutation cannot restore prototrophy
o Strain A and B are mixed for a few hours in medium containing all
supplements before plating on afar made with minimal medium (MM)
▪ Results
• Bacterial colonies at a frequency of 10-7
o Conclusion
▪ Either:
• Some form of recombination of genes occurred between
genomes of two strains → producing prototrophs (wild type
bacteria)
▪ Or:
• One cell leaked substances that the other picked up = cross-
feeding
• Davis – whether physical contact is needed to generate prototrophy – if cross-
feeding is a viable hypothesis
,Extrachromosomal Replication
o U tube
▪ Bacterial strain unable to synthesise certain nutrients is placed in
one arm
▪ Bacterial strain unable to synthesise different required metabolites
is placed in other arm
▪ Medium can be transferred between arms – through pressure or
suction
▪ Bacterial cells cannot pass through centre filter
o After hours of incubation – cells are washed and plated on agar plates with
minimal medium
▪ Results
• No colonies
o Conclusion
▪ Physical contact between two strains is necessary for recombination
• Conjugation needs bacteria to bacteria contact
▪ Transduction
• Involves transfer of DNA from one bacterium into another – via
bacteriophages
• Mechanisms that allow bacteria to exchange genetic information
o Fertility Factor
▪ Transfer of genetic information only occurred in one direction
• One cell acts as donor
o Fertility = ability to donate genetic material
• Other cell acts as recipient
o Cell that lost ability to transfer genetic material
▪ During bacterial gene transfer
• Recipient receives genetic information from donor = changed
▪ Fertility of E. coli
• Could be lost and regained easily
o Fertility factor (F) = ability to donate – a hereditary state
▪ Strains that carry F = can donate genetic information = F+ (male)
• F+ cells do not attach other F+ cells
▪ Strains that lack F = cannot donate = F- (female)
•
o 2 bacteria that are in contact – conjugated
▪ Conjugation by the pilus
• F episome
o Plasmid
• Any extrachromosomal hereditary particle
• Found in all domains of life
• Common in prokaryotes
o Eubacteria (true bacteria)
o Archaea
• Common in eukaryotes (yeasts)
o Eukarya
▪ Episome
• Any form of plasmid that can move in and out of chromosomes
, Extrachromosomal Replication
▪ F episome
• 100kb in length
• Two origins of replication (red)
o oriV – vegetative origin
▪ Used when plasmid is free in the
cell
▪ One copy per bacterial
chromosome
o oriT – transfer origin
▪ Used when plasmid is transferred
into another cell
▪ Cis-acting element located at the beginning of the transfer region
▪ Location where transfer of F plasmid is initiated
▪ Large (33 kb) region with 40 genes
• Transposable sequences (yellow)
o Allow integration of plasmid in different places of the bacterial chromosome
– via homologous recombination with identical sequences
o Transfer of F Episome by conjugation
▪ Tra region
• Most genes in the transfer region are involved in DNA transfer and replication
o At least 12 tra genes are needed for modification and assembly of pilus
molecule + stabilisation of the type 4 secretion system (T4SS)
▪ T4SS – system that allows transfer of DNA from one bacterial cell to
the next
• Contains a number of genes – arranged in
loci named tra and trb
o traA – encodes pilin
o traI – encodes relaxase
o traD – encodes coupling protein
ATPase
o traY – encodes transcription factor – DNA bending
o traM – interacts with DNA and numerous proteins
▪ Pilus
• Hairlike structure 2-3 um long
• Hollow cylinder
• 8 nm diameter with 3 nm axial hole
• F+ cell 2-3 pili
• Pili are involved in all conjugations between
gram-negative bacteria
o Stages
▪ Pilus makes contact recipient cell
▪ Pilus retracts
• Cells come closer together
▪ Cell pairs stabilised
• F plasmid nicked in one strand
o Factors form mating pair formation
and stabilisation
▪ Components of
lipopolysaccharide
▪ OmpA protein
Eukaryotes – Prokaryotes
• Origins of Eukaryotes + evolutionary relationship with Archaea = unknown – 2 models
o Three primary domains (3D) scenario
▪
• Archaea and Eukarya have common ancestor
o Two primary domains (2D) scenario
▪
• Archaea and Bacteria are two primary domains
• Eukarya is a secondary domain that arose from the merging of an archaeon and a
bacterium
Horizontal or Lateral Gene Transfer
• Prokaryotic organisms:
o Can directly transfer DNA without reproduction
o Can pick up DNA from the environment
• Horizontal gene transfer
o Gene transfer that is separate from reproduction – non-sexual movement of genes between
genomes
o 3 main mechanisms
▪ Transformation
• Involves uptake of short fragment of naked DNA by
transformable bacteria
o Classical experiments of Griffith and Avery
▪ Pneumococcal bacteria
• S strain – smooth, pneumonia
• R strain – lab strain, rough, no
pneumonia
▪ Mice injected with:
• S strain = died of pneumonia
• S strain that was heat treated = survived
• R strain that was missing the capsular polysaccharide =
survived
• R strain that was missing the capsular polysaccharide + S
strain that was heat treated = died of pneumonia
▪ Conclusion
,Extrachromosomal Replication
• R-form bacteria transformed into stable S form –
transformation process required head treatment
▪ Conjugation
• Discovered by J. Lederberg and E. Tatum
o 2 strains of bacteria
▪ A: auxotroph for methionine
(met-) and biotin (bio-) +
prototroph for (thr+, leu+,
thi+)
▪ B: auxotroph for threonine
(thr-), leucine (leu-),
thiamine (thi-) + prototroph
(met+, bio+)
o Auxotroph = mutant strain of
microorganism that has nutritional
requirements – cannot grow on
minimal medium – requires certain supplements
o Prototroph = wild type strain of microorganism – grows in minimal medium
– does not require supplements
o Strain A was washed and plated on agar plates made with minimal medium
(MM) – that contains only inorganic salts + carbon source + water
▪ Results
• No colonies
• Backmutation cannot restore prototrophy
o Backmutation = process that causes reversion –
change in nucleotide pair in mutant gene that
restores the original sequence + original phenotype
o Strain B was washed and plated on agar plates made with minimal medium
▪ Results
• No colonies
• Backmutation cannot restore prototrophy
o Strain A and B are mixed for a few hours in medium containing all
supplements before plating on afar made with minimal medium (MM)
▪ Results
• Bacterial colonies at a frequency of 10-7
o Conclusion
▪ Either:
• Some form of recombination of genes occurred between
genomes of two strains → producing prototrophs (wild type
bacteria)
▪ Or:
• One cell leaked substances that the other picked up = cross-
feeding
• Davis – whether physical contact is needed to generate prototrophy – if cross-
feeding is a viable hypothesis
,Extrachromosomal Replication
o U tube
▪ Bacterial strain unable to synthesise certain nutrients is placed in
one arm
▪ Bacterial strain unable to synthesise different required metabolites
is placed in other arm
▪ Medium can be transferred between arms – through pressure or
suction
▪ Bacterial cells cannot pass through centre filter
o After hours of incubation – cells are washed and plated on agar plates with
minimal medium
▪ Results
• No colonies
o Conclusion
▪ Physical contact between two strains is necessary for recombination
• Conjugation needs bacteria to bacteria contact
▪ Transduction
• Involves transfer of DNA from one bacterium into another – via
bacteriophages
• Mechanisms that allow bacteria to exchange genetic information
o Fertility Factor
▪ Transfer of genetic information only occurred in one direction
• One cell acts as donor
o Fertility = ability to donate genetic material
• Other cell acts as recipient
o Cell that lost ability to transfer genetic material
▪ During bacterial gene transfer
• Recipient receives genetic information from donor = changed
▪ Fertility of E. coli
• Could be lost and regained easily
o Fertility factor (F) = ability to donate – a hereditary state
▪ Strains that carry F = can donate genetic information = F+ (male)
• F+ cells do not attach other F+ cells
▪ Strains that lack F = cannot donate = F- (female)
•
o 2 bacteria that are in contact – conjugated
▪ Conjugation by the pilus
• F episome
o Plasmid
• Any extrachromosomal hereditary particle
• Found in all domains of life
• Common in prokaryotes
o Eubacteria (true bacteria)
o Archaea
• Common in eukaryotes (yeasts)
o Eukarya
▪ Episome
• Any form of plasmid that can move in and out of chromosomes
, Extrachromosomal Replication
▪ F episome
• 100kb in length
• Two origins of replication (red)
o oriV – vegetative origin
▪ Used when plasmid is free in the
cell
▪ One copy per bacterial
chromosome
o oriT – transfer origin
▪ Used when plasmid is transferred
into another cell
▪ Cis-acting element located at the beginning of the transfer region
▪ Location where transfer of F plasmid is initiated
▪ Large (33 kb) region with 40 genes
• Transposable sequences (yellow)
o Allow integration of plasmid in different places of the bacterial chromosome
– via homologous recombination with identical sequences
o Transfer of F Episome by conjugation
▪ Tra region
• Most genes in the transfer region are involved in DNA transfer and replication
o At least 12 tra genes are needed for modification and assembly of pilus
molecule + stabilisation of the type 4 secretion system (T4SS)
▪ T4SS – system that allows transfer of DNA from one bacterial cell to
the next
• Contains a number of genes – arranged in
loci named tra and trb
o traA – encodes pilin
o traI – encodes relaxase
o traD – encodes coupling protein
ATPase
o traY – encodes transcription factor – DNA bending
o traM – interacts with DNA and numerous proteins
▪ Pilus
• Hairlike structure 2-3 um long
• Hollow cylinder
• 8 nm diameter with 3 nm axial hole
• F+ cell 2-3 pili
• Pili are involved in all conjugations between
gram-negative bacteria
o Stages
▪ Pilus makes contact recipient cell
▪ Pilus retracts
• Cells come closer together
▪ Cell pairs stabilised
• F plasmid nicked in one strand
o Factors form mating pair formation
and stabilisation
▪ Components of
lipopolysaccharide
▪ OmpA protein
