L10: Plasmids, conjugation and generalised transduction

Question 1

How do prokaryotes achieve genetic exchange?

Answer 1

• Transformation (uptake of ‘naked’ DNA)
• Conjugation (utilises plasmids)
• Transduction (utilises bacteriophages)
  *no sexual cycle
  *no meiosis

Question 2

Types of plasmids

Answer 2

• Sex plasmids
• R plasmids
• col plasmids

Question 3

E.coli genome facts

Answer 3

4.6 Mbp
One essential circular dsDNA chromosome

Question 4

Sex plasmids example

Answer 4

e.g. F plasmid (fertility)
- ~100 Kbp
- stringent replication (copy no. 1-2)
- self mobile
- episome

Question 5

Insertion sequence elements in elements in F plasmid

Answer 5

• 1x IS2
• 2x IS3
• 1xIS1000 aka gamma-delta

Question 6

R plasmids

Answer 6

• Encode resistance (R)
• Smaller than F plasmid at ~30-100 Kbp
• Promiscuous
• Typically encode resistance to one or more antibiotics, heavy metals or toxins

Question 7

col plasmids

Answer 7

• Small; <25 Kbp
• Encoding biological factors (e.g. colicin - toxic to other E.coli )
• Relaxed replication (copy no. ~30)
• Don’t encode functions permitting transfer between individual bacteria (may be transferred if F or R plasmids present in same cell encoding functions required for contact and transfer)
• Useful in cloning
  e.g. pGEM3zf derived from colE1 plasmid

Question 8

Lederberg and Tatum, Hayes

Answer 8

Lederberg, Tatum
- Mixed two auxotrophic strains of bacteria
- Observed some prototrophic colonies when mixture plated on minimal media
- Colonies resulted from genetic exchange between original strains
Hayes
- U-tube expt confirmed physical contact between strains required

Question 9

Bacterial conjugation

Answer 9

• Unidirectional DNA transfer (bacterial ‘mating’
• F+, F- cell establish contact by long tubular F- pilus; cytoplasmic bridge
• F plasmid carries tra (transfer) genes for contact and mobilisation functions - encodes pilin protein to build pilus

Question 10

F plasmid transfer: genes and process

Answer 10

• tra genes encode contact and DNA transfer functions
• Transfer initiated by introducing a nick in DNA at OriT (Origin of transfer)
  -> process carried out by complex including TraI, TraY
• 5’ end of ssDNA transferred to recipient
  -> in case of F plasmid, takes a few minutes and all can be transferred before bridge breaks
  -> result is 2 F+ cells
• Rolling circle replication forms ssDNA from F plasmid
• DNA synthesis in F- recipient restores second strand

Question 11

Hfr strains (integration of F plasmid into bacterial chr.)
- What are they
- How do they occur (features)

Answer 11

• Hfr: High frequency recombination
• F factor becomes integrated into bacterial chromosome
• Rare event
• Can be integrated in either orientation according to orientation of recombining sequences
• Integrated via recombination between insertion sequences on F plasmid and chr.
• Reversible
• Unidirectional conjugation
  -> fragile bridge; very rare more than a fraction of chr. is exchanged
• Not species specific; allows horizontal gene transfer (success of promiscuous conjugation dependent on DNA homology, recombination required to integrate transferred fragment

Question 12

Jacob and Wollman

Answer 12

Interrupted mating experiment
- Donor HfrH (thr⁺, leu⁺, azi^R, ton^R, lac⁺, gal⁺, str^S): Protortophic, resistant to sodium azide, infection w/ bacteriophage T1, sensitive to streptomycin
- Recipient F- (thr^-, leu^-, azi^S, ton^S, lac^-, gal^-, str^R): Auxotrophic for threonine, leucine, sensitive to sodium azide and infection by bacteriophage T1, resistant to streptomycin
-> mix bacteria and then at various times after mating commences, w/draw samples, break mating cells apart and plate bacteria on selective media to determine which genes have been transferred from Hfr to F-
-> used to prove that E.coli genetic map is circular

Question 12

F’ plasmids (definition, formation process)

Answer 12

• Produced as a result of imprecise excision of F plasmid from bacterial chr.s; carries bacterial genes
  1. F plasmid integrated into bacterial chr. (e.g. adjacent to lac⁺ region)
  2. F factor loops out incorrectly, including a piece of the chr.
  3. Single crossover generates F’lac
  4. F’lac+ can transfer to recipient. As circular DNA can be maintained w/out integrating into recipient chr., recipient becomes lac+/lac- partial diploid (merodiploid)
  -> used in genetic analysis of gene regulation (sexduction)

Question 13

Gene transfer by generalised transduction

Answer 13

• Mediated by virulent bacteriophages e.g. P1 (transfer of any part of bacterial genome)