Bacterial Genetics And Gene Transfer

Front 1

Describe the Griffiths experiment.

Back 1

Transformaiton proved using streptococcus pneumonia:

1. Sterlie bacteria to become avirulent and inject a mouse - mouse lives

2. Inject regular - virulent - bacteria into a mouse - mouse dies

3. Injection heat killed virulent bacteria into a mouse - mouse lives

4. Inject both, heat killed virulent and avirulent bacteria at the same time = MOUSE DIES

Front 2

What did the Griffiths experiment prove?

Back 2

Proved the bacterial phenomenon of transformation.

The virulent heat killed strain's DNA was transferred to the avirulent strain to recombine into a live virulent strain.

Front 3

In what state will bacteria transform spontaneously?

How can this state be induced in a lab?

Back 3

1. Competence - in late log phase

2. i) With divalent metal cations - e.g. CaCl2 followed by a heat shock at 42 degrees or electroporation

Front 4

Describe electroporation.

Back 4

Electrical field is applied to cells to increase the permeability of the membrane - allowing DNA to be introduced to the cell.

Front 5

Why does electroporation induce competence?

Back 5

Voltage application disturbs the phospholipid bilayer.

Front 6

What is the name of the structure which gram positive organisms use to transform DNA?

What does it consist of?

Give a brief description of each component.

Back 6

1. The translocasome complex

2.

i) DNA binding protein that captures extracellular DNA from the environment

ii) Proteins which form a transmembrane pore through which DNA can pass through

iii) Nuclease which degrades the DNA into a single strand

Front 7

Describe in detail the function of the nuclease in the translocasome complex.

Back 7

The nuclease degrades one strand of a double stranded DNA molecule which pulls the other strand intact through the pore and into the cell.

Front 8

What causes the expression of the translocasome complex in Streptococcus?

Back 8

1. Competence factor - activates sensor kinases on bacterial surface membrane which

2. Phosphorylates a 2nd messenger - ComE which stimulates the transcription of SigH.

3. SigH directs transmission of transformase components

Front 9

Describe the Tatum + Lederberg experiment and what it showed.

Back 9

1. Leucine vital for bacterial growth. Needs ability to produce by itself or source methionine, biotin, threonine and proline.

2. Two mutant bacterial types were sources , one which could metabolise only threonine, proline by itself and one which could metabolise only methionine and biotin by itself

3. Each type placed on their own onto plates which lack met, bio, thr and pro and on one plate both types were mixed

4. Bacterial colonies able of producing all amino acid types grew on the mixed plate

This showed gene transfer had occured from one bacterial strain to another

Front 10

What part of natural bacterial growth allows conjugation?

Back 10

Creation of biofilms which allows cross species DNA transfer to occur.

Front 11

What is conjugation?

Back 11

Conjugation is the donation of genes from one cell to another which is conjugated alongside the donatee DNA

Front 12

What is absolutely critical for conjugation to be able to occur?

Back 12

1. Conjugation requires transferable plasmids that have all the genes required for pilus formation and DNA export

Front 13

What are the names of the origins of replication on the E.coli plasmid?

Back 13

OriV and OriT

Front 14

Describe the conjugation process of the E.coli F-plasmid.

Back 14

1. Donor cell (F+) and recipient cell (F-) come into contact via an extending sex pilus from the F+ cell

2. Sex pilus connects to receptors on the F- cell

3. Pilus contracts - drawing the two cells together to form a conjugation bridge where the cell membranes fuse together

4. The bridge formation triggers synthesis of F-Factor helicase

5. F-Factor helicase nicks the phosphodiester backbone at 'oriT'

6. DNA polymerase III is recruited to oriT and DNA replication begins

7. The 5' end of the DNA strand begins to transfer across the conjugation bridge

8. Remaining strand in the donor cell is replicated

9. The DNA has been transfered and the F- cell is now converted to an F+ cell

Front 15

How does kanamycin resistance work?

Back 15

PO4- added

Front 16

How does trimethoprim resistance work?

Back 16

'New' enzyme made to avoid action

Front 17

How does Tetracylcine resistance work?

Back 17

Exportation

Front 18

How does Chloramphemicol resistance work?

Back 18

Degraded

Front 19

2 plasmids in the ____ compatibility group ______ coexist?

Back 19

1. Same

2. Cannot

Front 20

What are incompatibility groups based on?

Back 20

Replication machinery.

Front 21

Why can't two plasmids in the same incompatibility group coexist?

Back 21

1. Plasmids of the same incompatibility groups share sequences at their 'ori' sites

2. Since they share the same molecular mechanisms for the control of their replication and hence share the same copy number

3. Therefore if a bacteria selects for one of the plasmids e.g through being smaller / faster to conjugate then the other will disappear from the population

Front 22

What are the possible fates for an F factor which enters an F- cell?

Back 22

Once inside F factor can either:

i) Stay as a plasmid

ii) integrate into the chromosome - becomes an episome

Front 23

What is required for host DNA integration?

Back 23

Requires recombination between two circular DNA duplexes.

Front 24

When an F-Factor integrates into a bacterial cell what is it termed?

Back 24

A high frequency recombination (Hfr) strain

Front 25

What is the principle of gene mapping by conjugation?

Back 25

It takes around 100 minutes for a conjugation event of the entire chromosome to occur then we can map each gene by timing how long it takes to transfer each gene to the recipient.

Front 26

What points must be considered to map effectively?

Back 26

1. Requires E coli High frequency recombination strain with a recipient which has a mutation in the target gene

2. Gene must have a phenotype in order to measure when repair by conjugation has occurred.

3. The F- strain must also be resistant to antibiotics to allow selection for it.

4. The Hfr cell must be sensitive to the antibiotic

Front 27

Using F-proA and Streptomycin as an example. Describe the process of conjugation mapping.

Back 27

F-proA is required to synthesise proline.

1. Streptomycin sensitive Hfr strain mixed with streptomycin F-proA minus mutant

2. At timed intervals, samples are retrieved and conjugation is interrupted by blending

3. At each interval, the disrupted mixture is plated onto a selective media:

i) lacks proline

ii) contains streptomycin

4. Until the proA gene has been transferred, no colonies will grown on the media

Front 28

What is the principle of conjugation mapping?

Back 28

Because bacterial conjugation in an Hfr strain will begin at the nicked oriT site in the reverse (5') direction of oriT, the F factor is the last gene to be transfered and genes closest to it transfer first. The further away a gene is from the F-Factor the longer the transfer will take.

Front 29

How is conjugation mapping measured?

Back 29

Measured in 'map minutes' away from the location of the F-insertion

Front 30

Describe the process of F-Factor excision.

Back 30

If F-factor is fully integrated into the hos cell it can be excised via host recombination mechanisms.

Rarely, the excision screws up and takes some E.coli chromosome out with the F factor plasmid. This forms an F-prime (F') plasmid / factor.

Front 31

What is 'aggregation substance'?

Back 31

Aggregation substance is a bacterial 'pheromone' which promotes conjugation in Enterococus faecalis.

Front 32

What is the function of aggregation substance?

Back 32

1.Catalyses cell-cell contact and the formaiton of the conjugation complex

-Recipients try to entice donation by producing these pheromone peptides which enter donors stimulating RAD1 transfer genes to be expressed.

Front 33

Describe the basic proceessof bacteriophage transduction

Back 33

Bacteriphages can accidentally move genes between cells as an offshoot of their phage lytic life cycle.

Front 34

What types of transduction are there?

Back 34

1. Generalised transduction

2. Specialised transduction

Front 35

Why do some types of bacteriophage perform generalised transduction?

Back 35

These bacteriophages have trouble distinguishing their own DNA from host DNA when packaging DNA.

Front 36

What is the overall event of generalised transduction?

Back 36

Random pieces of host DNA get packaged into the phage head.

Front 37

Describe in detail how the Salmonella bacteriphage - P22 carries out generalised transduction.

Back 37

1. Recognises 'Pac site' in Salmonella genome

2. Uses pac site to align many P22 genomes into contactomeres - Where the pac site defines the end of the genome

3. the P22 phage cuts its genome where it lines up with the pac site to begin packaging.

4. HOWEVER, there are some very similar sites which resemble the pac site, elsewhere in the Salmonella genome

5. Sometimes these pac similar sites get cleaved by P22 and packaged into a phage head instead of P22 phage DNA

Front 38

When a P22 phage undergoes transduction what are they called?

Back 38

Transducing particles

Front 39

What process occurs when a transducing particle infects a new cell?

Back 39

When a transducing particle injects its DNA it has no phage DNA in it so instead causes a change in genetic makeup of the recipient cell

Front 40

Describe how the E.coli phage - lambda phage carries out a process described as specialised transduction.

Back 40

1. Upon initial entry Lambda phage (LP) has a linear DNA strand

2. It forms a 'circular strand' at cohesive ends, called 'cos' sites

3. Part of the E.coli chromosome called attB can recombine with a small piece of LP DNA called attP - a process catalysed by the phage's integrase protein.

4. The product is an E.coli chromosome with an integrated phage genome

Front 41

What is a prophage?

Back 41

A bacterial genome with an integrated phage genome

Front 42

Describe the makeup of the Lp / E.coli prophage.

Back 42

The prophage is flanked by the hcimeric att sites called attL and attR.

Front 43

Describe the process of prophage DNA.

Back 43

1. Reactivated recomination mechanisms excise the lambda phage exactly. Leaving the E.coli genome intact.

2. Viral DNA can then replicate to produce more phage particles

Front 44

How can the activation of prophage DNA mess up to cause specific transduction?

Back 44

1. During the excision phae some errors can occur between the host DNa sequences with lay next to the original cos sites

2. In E.coli this is the Gal gene which allows the cell to utilise galactose

3. This gene can be excised - leaving the phage genes behind.

Front 45

What is the particle resulting from specific transduction termed?

What can it do?

Back 45

Transducing phage - can recombine to the host chromosome of a new recipient cell ath the attB site again - creating a meroploid.

Front 46

What is a meroploid?

Back 46

A type of bacteria which has been transformed by a transducing phage which has two copies of the same gene.

Front 47

How can genes other than Gal be moved in erroneous transduction?

Back 47

If the host strain has a mutated attB site the lamda phage is able to integrate at other sites on the chromosome - secondary sites.