Bacterial genetics Flashcards
What are the three mechanisms used by bacteria to achieve genetic exchange?
Transformation (taking up naked DNA from environment)
Transduction (use of bacteriophage)
Conjugation (use of plasmids)
What type of DNA sequence is predominant in bacterial genome?
unique (very little repetitive DNA sequences)
What are the three distinct types of plasmid?
col plasmids
R plasmids (resistance)
Sex (E.g. F) plasmids
Describe the bacterial Sex plasmids
E.g. F plasmid of E.coli = unidirectional transfer of genetic information from donor to recipient cell
- relatively LARGE (~100 kpb)
- precise replication (1-2 COPY NUMBER)
- SELF MOBILE
- about 35% encodes for FUNCTIONS PERMITTING TRANSFER between bacteria
- remaining sequence contains four TRANSPOSONS (insertion sequence)
- mediate transfer of bacterial genes by CONJUGATION
- an EPISOME = free circular plasmid or integrated into chromosome
Describe bacterial R plasmids
- variable size but relatively large
- self mobile - can transfer between unrelated species
- encodes resistance to antimicrobials, heavy metals and toxins
- evolved parallel with use of widespread antibiotics
Describe bacterial col plasmids
- variable size but small (roughly 10 kbp)
- encode biological factors (e.g. colicin)
- relaxed replication (~30 COPY NUMBER)
- do not encode functions permitting transfer
- may be transferred if F or R plasmids present that encode functions required for transfer
- extensively manipulated by molecular biologist to generate cloning vectors.
Describe the experiment that discovered the F plasmid mediated conjugation in E. coli
Lederberg and Tatum
- mixed two multiply auxotrophic strains of E.coli (A and B - what the A strain was auxotrophic for, the B strain was wildtype and vice versa) and plated out Strain A, strain B and the mixture of both strains onto minimal media
- observed some phototrophic colonies on the minimal media with the mixture which resulted from genetic exchange between the A and B strains.
What was the experiment that proved transfer of genetic material was being observed in the Lederberg and Tatum experiment?
Some people believed that the reason for colony growth when the two strains were mixed was due to the production of substances in the medium by the two strains that permitted growth.
Therefore: the U-tube experiment was performed to prove that this was not the case:
- the two strains were placed into either end of a U tube with a fine pored filter between the strains at the base of the tube, which allowed medium to move between the two sides, but not bacteria.
- then separated out the two strains and plated then on minimal media
- saw no growth which proved that the bacterial cells required contact to transfer genetic material and facilitate growth of prototrophic colonies
Describe the process of bacterial conjugation in F+ x F- matings
1) F+ cell contains an F plasmid which contains the tra genes, such as that encoding pilin required for the formation of the F-pilus (tra genes encode contact and DNA transfer functions)
2) the F-pilus contacts an F- cell, forming a bridge
3) transfer intitiated by introducing a nick at OriT (Origin of transfer) and rolling circle replication occurs from the 3’ OH, displacing the 5’ end of the strand, which is then transferred into the recipient through the cytoplasmic bridge (the 5’ end of ssDNA leads into recipient cell)
4) As the DNA moves into the recipient cell, synthesis of the complementary strand occurs in a 5’-3’ direction
- the results are two F+ cells
Describe the process of bacterial conjugation in Hfr x F- matings
1) rare, low frequency generation of a Hfr strain by F plasmid integration into the bacterial chromosome through recombination between specific transposons on the F plasmid and the chromosome - reversible insertion
2) F plasmid encodes for transfer functions such as formation of the cytoplasmic bridge
2) OriT is nicked and rolling circle DNA synthesis occurs from the 3’ OH, displacing the 5’ end causing it to lead into the recipient cell - the F plasmid is transferred into the recipient, followed by bacterial chromosome.
3) within the recipient cell the complementary strand is synthesised in 5’ to 3’ direction and homologous recombination can occur between the transferred chromosomal DNA and the recipient chromosome
Why is it unusual to get transfer of the entire chromosome or F plasmid?
the cytoplasmic bridge is fragile so it usually breaks before compete transfer is achieved
What is horizontal gene transfer? why does conjugation permit this?
transfer between species
conjugation permits this because it is not species specific
Describe the experiment that used conjugation to map bacterial genes
Jacob and Wollman = interrupted mating experiment
Donor HfrH - phototrophic, resistant to sodium azide, resistant to infection with bacteriophage T1, sensitive to streptomycin
Recipient F- - auxotrophic for Leu and Thr, sensitive to sodium azide, sensitive to bacteriphage T1 infection, resistant to streptomycin
- Bacteria mixed and vortex at various times after mating to break the cytoplasmic bridge and prevent transfer of anymore genetic material
- plate on selective media and determine which genes have been transferred from Hfr to F- stain = shows the order of genes relative to OriT
What did interrupted mating experiments with a variety of Hfr strains (different sites of insertion) show about the E.coli chromosome?
- showed it was circular
What are F’ plasmids and how are F’ plasmids formed?
F’ plasmids are free circular F plasmids that carry bacterial genes
F’ plasmids are formed by imprecise excision of the F factor from the bacterial chromosome resulting in adjacent genes being taken with it
What can be the result of conjugation involving an F’ plasmid?
- full F’ plasmid is transferred into the recipient cell, which means that the recipient cell now become a PARTIAL DIPLOID for the specific gene being carried (as it contains one in its chromosome, and one in the F’ plasmid)
Describe the process of gene transfer by generalised transduction (using bacteriophage P1 and E.coli as an example)
- virulent P1 bacteriophage infects E.coli
- P1 contains genes that encode for proteins that digests the bacterial chromosome and allow phage genome replication, translation and assembly (some E.coli genome may be packed into a P1 phage head to produce a TRANSDUCING PHAGE)
- phage produces lysozyme to destroy the bacterial cell wall and cause lysis and release of bacteriophage into the surroundings
The transducing phage can then infect another E.coli cell and there may be recombination between the bacterial DNA carried by the phage and the chromosome of the new host. The recipient allele is degraded and there is a PHENOTYPE CHANGE.
Give an example of an F’ plasmid?
F’lac = critical in understanding the lac operon
- used in sexduction = transfer F’ into a recipient cell
