Lecture 10 Flashcards
How does evolution occur?
Genetic diversity by recombination and mutations
What do eukaryotes use to effect recombination?
Sexual cycle + meiosis
What do prokaryotes use to effect recombination?
Transformation - uptake of ‘naked’ DNA
Conjugation - Utilises plasmids
Transduction - Utilises bacteriophages
What do many bacteria, including E. coli, have as a genome?
Single circular dsDNA chromosome
3 distinct types of plasmid
Sex plasmids
R plasmids
col plasmids
Sex plasmids
F plasmid of E. coli ~100kbp - stringent replication (copy number 1-2), self mobile
Approx 35% sequences encode transfer between bacteria
Remaining sequences contain 4 insertion sequences (1 x 1S2, 2 x 1S3, 1 x 1S1000 aka gammadelta)
-Mediate transfer of bacterial genes by conjunction
- F plasmid is an episome - may exist as free circular plasmid or integrated to chromosome
R plasmids
~30-100kbp
Self mobile
Encode resistance to drugs, heavy metals, toxins
Evolved a lot over last 60 years with antibiotic use
R plasmids spread through environment
col plasmids
Small (mostly below <25kbp)
Encode biological factors
Relaxed replication (copy number ~30)
Don’t encode functions permitting transfer between individual bacteria
- Transfer may occur if F and R plasmids present in same cell encoding functions for contact and transfer
- Manipulated for useful vectors e.g. pGEM3zf from colE1 plasmid
How was F plasmid mediated conjunction discovered
Lederberg and Tatum - 1946
- 2 auxotrophic strains mixed
- Some phototrophic colonies observed when mixture plated on minimal media
- Caused by genetic exchange
- U-tube established physical contact required
Bacterial conjugation
F+ cells contacts F- cell - connection established by long F- pilus
F+ pilus contracts forming a bridge
Genetic material transferred by cytoplasmic bridge
F plasmid carries tra genes for contact and mobilisation functions - encodes pilin protein to build pilus
F-plasmid transfer
- tra genes encode contact and DNA transfer functions
- Transfer initiated by introducing a nick in DNA at oriT
- 5’ end of ssDNA transferred to recipient
- Rolling circle replication forms ssDNA from F-plasmid
- DNA synthesis in F- recipient restores second strand
Transfer of F plasmid by conjunction in E. coli
Transfer of F factor from donor F+ cell to recipient F- cell during F+ x F- matings
All F plasmid transferred and replicated = DNA circularised and original F- cell becomes F+ cell
What does transfer of bacterial chromosomes require?
F factor becomes integrated into bacterial chromosome
Chromosomal integration of F-plasmid
- Can be integrated in either orientation according to orientation of recombining sequences
- Integrated by recombination between insertion sequences of F plasmid and chromosome
-Reversible
Hfr transfer
High frequency transfer
F plasmid integrated into chromosome where it encodes transfer functions
F plasmid oriT nicked
F factor initiates transfer to recipient
F plasmid transferred followed by bacterial chromosome
Transferred chromosomal DNA recombines with recipient chromosome
Hft conjugation
- Conjugate with f- strains
- Unidirectional, ordered transfer of chromosomal genes from donor to recipient
- Part of F transferred first - bridge fragile and breaks
- Conjugation not species specific - Horizontal gene transfer
- DNA homology determines success of promiscuous conjugation
Donors in mapping bacterial genes by conjugation
Donor: HfrH, thr+, aziR, tonR, lac+, gal+, strS
Phototrophic
Resistant to sodium azide
Infection with bacteriophage T1
Sensitive to streptomycin
Recipients in mapping bacterial genes
F-, thr-, leu-, aziS, tonS, lac-, hal-, strR
Auxotrophic for threonine and leucine
Sensitive to sodium azide
Sensitive to infection by bacteriophage T1
Resistant to streptomycin
Process of mapping bacterial genes
Mix bacteria
Withdraw samples at time intervals after mating commences
Break apart mating cells
Plate bacteria on selective media to determine which genes transferred from Hfr to F-
Imprecise excision of F plasmid from bacterial chromosome
- F plasmid integrated into bacterial chromosome
- F factor loops out incorrectly, including a piece of the chromosome
- Single crossover generates F’lac
- F’lac+ can transfer to recipient (lac-)
- Circular DNA can be maintained without integration into chromosome
- Recipient become lac+/lac- partial diploid - Used in genetic analysis of gene regulation
Life cycle of virulent bacterophage
- T4 bacteriophage uses tail fibres to stick to receptor sites on surface of E. coli
- Phage injects it’s DNA after sheath of tail constructs a hollow core through wall
- Empty capsid left as ghost, DNA hydrolysed
- Phage proteins and nucleotides produced to copy phage genome
Phage parts come together to form head, tail and tail fibres
- Phage directs production of lysozyme to hydrolyse cell wall and burst cell by osmosis, releasing new bacteriophages
