L13

Question 1

explain gene transfer

Answer 1

• Transfer of plasmids can transfer pathogenic characteristics or antibiotic resistance genes
  
  • Between cells of SAME generation
    ○ Recombination
  • Between generations of cells
    ○ Replication
  • Within a cell
    ○ Transcription
    Translation

Question 2

4 methods of horizontal gene transfer

Answer 2

conjugation, transformation, transduction, vesicle mediated transfer

Question 3

explain conjugation

Answer 3

○ Transfer of DNA from a LIVE bacterium to another via pilus/chemical signalling, which draws the bacteria together
○ Direct cell to cell contact
○ For conjugative/mobilizable plasmids
○ Most common for natural transfer
Fastest rate

Question 4

explain transformation

Answer 4

Uptake of DNA released from DEAD bacteria (lysed)
Must be closely related
Rare naturally, about 1%

Artificially makes cell
Depends on level of competence of recipient cells (rare in nature)
Competent cells- have modified cell walls + specific receptor sites on plasma membrane that binds DNA
Ideal for rapid amplification’
Recipient cells cut out part of its own chromosome, and replaces it with the new fragment

Question 5

explain transduction

Answer 5

○ Bacteriophage (viruses that infect bacteria) picks up genetic material from 1 bacterial cell, and deposits it in another
Use host factors for replication
○ Donor + recipient must both be in host range of the bacteriophage
○ 3rd fastest rate

Question 6

explain vesicle mediated transfer

Answer 6

○ Only specific to particular organisms
○ Very uncommon
○ Extracellular vesicles
○ Vesicle production + uptake rates differ between species
○ Slowest rate

Question 7

what are the steps of vesicle-mediated transfer

Answer 7

• Mediated by OMV (outer membrane vesicles)
  1. DNA is released through OMV from donor cells into surrounding environment
  2. OMVS merge with outer membrane of recipient cell
  3. Genetic material migrates into cytoplasm
  4. giving bacterium new adaptive capacities through expression of genetic material

Question 8

define plasmids

Answer 8

• Circular molecule of extrachromosomal DNA found in bacteria, carries genes
  
  • All plasmids contain atleast 1 origin of replication, so can multiple independently of chromosome
  • Several plasmid types can exist in 1 cell, but closely related cannot (must be different)
  • Compact due to supercoiling
  • Copy no. Ranges from 1-50
    Can be transferred

Question 9

what can plasmids be classified depending on

Answer 9

○ Antibiotic resistance
○ Colicin genes
§ Colicins are proteins that can be toxic for some strains of E.coli (used to kill related bacteria)
○ Degradative plasmids
○ Virulence plasmids
§ Help bacteria infect humans
○ Conjugative plasmids
§ Used for horizontal transfer
§ Conjugation- ability to promote the transfer of DNA between 2 bacterial cells, resulting in transfer of plasmid from 1 cell to another, is one way

Question 10

explain F plasmids and what donor cells should have

Answer 10

• Type of conjugative plasmid
  
  • Donors (F+ cell) must have a plasmid carrying genetic info for DNA transfer (controlled by a set of tra genes/F factors)
  • Donor cell have pili (tubes of protein) on surface
    ○ Pili make contact with receptors on surface of recipient (F- cell) , forming a mating pair
    Pili retract, pulling the 2 cells together for contact, forming conjugation bridge through which DNA is transferred

Question 11

explain the process of conjugative replication

Answer 11

1. Nicking protein makes a nick in the double stranded DNA o plasmid, at origin site of transfer (oriT site)
2. Helical unwinded the DNA
3. The strand is transferred across the conjugation bridge, with the 5’ end leading
4. At same time, 3’ end extends to replace the removed strand of DNA
5. Once strand arrived, DNA synthase regrows so double stranded (so F- cel becomes F+ cell, is identical)

*refer to onenote

Question 12

different between F plasmids vs Hfr plasmids

Answer 12

• F plasmids are sperate from the bacterial chromosomes
  Hfr cells possess a plasmid that is part of the chromosome, must be ripped off to go into new cell, a copy grows in donor

*refer to onenote

Question 13

what does a competent recipient cell have (and which method of horizontal transfer requires a competent cell)

Answer 13

transformation

• modified cell walls
• specific receptor sites on membrane that binds closely related DNA

Question 14

explain how to create chemical competence

Answer 14

§ Wash cell with cold CaCl2
§ Add DNA
§ Heat shock to induce DNA to go through permeable cell wall

Question 15

explain how to create electrical competence

Answer 15

§ Mix DNA + cells
§ Induce high voltage, produces pores in cell wall
§ Transient pores suck DNA in

Question 16

explain lytic bacteriophages

Answer 16

§ No integration
Replicates by making progeny phage + lysing cell for release to reinfect (horizontal transfer)

Question 17

explain lysogenic bacteriophage

Answer 17

§ Integration into host genome
§ Replication along with chromosomal DNA (vertical transfer)
□ Phage then called prophage
□ Bacteria called lysogeny
□ Relationship called lysogeny
□ Lytic cycle can then be triggered leading to horizontal transfer
□ Some bacteriophages have obligate lytic lifecycles

Question 18

what are the steps of the lysogenic cycle

Answer 18

1. DNA molecule injected
   
   2. Brief transcription period, repressor and an integrase are transcribed
   3. Repressor turns off, so transcription of viral DNA occurs
   4. Integration into host chromosome (exception is P1 phage’s that do not integrate, but enter cell + exist as a plasmid)
   5. Replication along with host

Question 19

explain generalised transduction

Answer 19

§ Transfer fragments of host DNA ONLY
§ Phage only contains hsot bacterial DNA, NOT phage DNA
§ Unusual
§ Transducing particles can ifnect recipient cells
Viability of the transducing phage dependent on ability to integrate into a new host

Question 20

what is the process of generalised transduction

Answer 20

lytic with recombination

Question 21

explain specialised transduction

Answer 21

§ Can occur with lysogenic phages
§ Transduction of host DNA which flanks the prophage
Results from excision error of phage DNA on lytic trigger, can entail the loss of some phage genes

Question 22

process of specialised transduction

Answer 22

1. A phage infects the donor bacterial cell
2. inserts phage genome in bacteriums nucleoid
3. removes itself + a little portion of donors nucleoid
4. replicates
5. repeats, replicates alongside host

Question 23

explain transposition and its 2 mechanisms

Answer 23

○ Ability of a genetic sequence to move from 1 location to another (1 DNA molecule to another, chromosome to a plasmid)
○ Mechanisms
§ Cute + paste (conservative)
□ Transposon is cut out of DNA where it residues, inserted into a new location
§ Replicative
□ Transposon replicated, 1 copy remains at original location, other located at a new one

Question 24

what are the 2 transposable elements of transposons

Answer 24

insertion and composite transposons

Question 25

insertion vs composition sequences

Answer 25

- Short piece of DNA, carries no known genes except that required for transposition - Simplest /// - Carry 1 or more genes flanked by matching insertion sequences

Question 26

explain the possible consequences of insertion sequence

Answer 26

○ Mutation § Introduction of a inserted sequence into a bacterial gene, resulting in inactivation of the gene ○ Plasmid insertion into chromosomes § Site at which plasmid inserts into bacterial chromosome at or near insertion sequence in the chromosome ○ Gene expression § Some regulated by IS § E.g. Flagellar antigens in salmonella, changes in response to immune system attack