Plasmids II

Question 1

Transformation

Answer 1

Uptake of short fragment of naked DNA by transformable bacteria

Question 2

Transduction

Answer 2

Bacteriophages -< transfer of DNA from 1 bacteria to another

Question 3

What is an auxotroph and prototroph?

Answer 3

Auxotroph has nutrients missing, prototroph can synthesis own material

Question 4

F episome

Answer 4

A plasmid that can also traffic in and out of chromosomes

Question 5

Characteristics of F episome

Answer 5

5% of E.coli genomic size
2 oriR’s:
- oriV : ‘free’ plasmid one copy/bacterium
- oriT: cis-acting element at beginning of transfer region where transfer f plasmid initiated
tra region: 33kb with 40 genes -> allow integration via homologous recombination

Question 6

Tra operon

Answer 6

mostly transfer + replication

• 12 genes for modification + assembly for piin in sex pilus
• stabilisation of T4SS
• arranged in loci as tra +trb

Question 7

What do the different tra’s do?

Answer 7

traA - pilin
traI - relaxase
traD - coupling protein ATPase
traY - DNA binding TF
traM - protein interactions 
tra N,G, ompK -> formation and stabilisation of mating pair

Question 8

Mechanism of conjugation

Answer 8

1. Pilus from F+
2. Pilus retracts -> proximity
3. Cell pairs stabilise
4. Relaxosome formation + concomitant rolling circle synthesis
5. DNA transfers through T4SS at beginning of rolling circle rep.
6. Transferred DNA becomes double stranded

Question 9

What does the relaxosome consist of?

Answer 9

Relaxase (traI) + accessory proteins (traY, traM, IHF)

Question 10

How is the relaxosome formed?

Answer 10

It forms at the oriT of -ve supercoiled DNA -> monomer relaxase covalently links 5’ end generated during phosphodiester bond hdrolysis

Question 11

What happens after the relaxosome is formed?

Answer 11

1. Relaxase, dimer, nicks oriT at nic
2. relaxase also unwinds DNA
3. relaxase-bounds DNA is bound by coupling protein traD + traM
4. TraM mediates relaxosome-transferase contact transport through T4SS

Question 12

A Hfr strain forms when

Answer 12

F episome integrates host chromosome through insertion sequences

Question 13

How does f episome replicate in hfr strain?

Answer 13

Uses tra operon but can’t replicate fully. Conjugation starts at oriT but paired cells are not as stable so rolling circle only partially transfers chromosome.

Question 14

When you receive from an hfr strain, you’re still…

Answer 14

F - , + partial chromosome can’t replicate so it’s either lost/recombines with host

Question 15

In antibiotic resistance, instead of f factor it’s

Answer 15

r factor e.g. shigella

Question 16

Bacteriocins are

Answer 16

bacterial secretions that inhibit the growth of closely related bacterial strains

Question 17

What is an example of a bacteriocin?

Answer 17

Co1E1 plasmid inhbiits shigella -> it’s a channel forming TM protein with copy # 10-15 (high) since plasmid rep. not necessarily coupled to chr. rep.

Question 18

CoIEI is non-transferrable by bacterial mating since…

Answer 18

mating pair formation genes absent

Question 19

mob+ can be transferred if

Answer 19

combined with conjugate plasmid (F factor. R factor)

Question 20

Why is it the normal frequency of recombination higher in hfr strains?

Answer 20

F factor integrated into chromosomal DNA so contains oriT which can be nicked to allow recombination

Question 21

Give the name of the vectors in increasing size limit:

Answer 21

Plamid, phage, cosmid, BAC, YAC, MAC