Lecture 49: Transformation and Transduction

Question 1

Who demonstrated the transforming principle?

Answer 1

Griffith

Question 2

Describe Griffith’s experiment

Answer 2

• R cells (rough colonies) are non pathogenic
• S cells (smooth colonies) are pathogenic
• When mice were injected with R cells they were fine, because R cells are non-pathogenic
• When mice were injected with S cells they died, because S cells are pathogenic
• When mice were injected with heat-killed S cells, they were fine, because dead S cells are non-pathogenic.
• When mice were injected with R cells and heat killed S cells, the mice died
• This is because the DNA of the S cells was taken up by the R cells. This transformed the R cells into a pathogenic form

Question 3

What happened after the Griffith experiments?

Answer 3

This experiment led to the discovery that DNA is the genetic material (later confirmed by Avery, MacLeod, and McCarty in 1944).

Question 4

What do bacterial cells have to be in order to undergo transformation?

Answer 4

They have to be competent

Question 5

What is transformation?

Answer 5

• a process in which bacteria take up and incorporate foreign genetic material (usually DNA) from their environment into their own genome.
• This allows them to gain new traits, such as antibiotic resistance or virulence.
• It is a form of horizontal gene transfer, meaning genetic material is passed between organisms without reproduction.

Question 6

What is competence?

Answer 6

The ability of a bacterial cell to take up extracellular (‘naked’) DNA from the environment.

Question 7

Give some examples of bacteria that are naturally competent

Answer 7

Bacillus subtilis

Streptococcus pneumoniae

Neisseria gonorrhoeae

Haemophilus influenzae

Question 8

For bacteria that aren’t competent, what can happen?

Answer 8

Competence can be induced in bacteria that aren’t competent. For example, for biotech purposes with E. coli.

Question 9

What is recombination ?

Answer 9

Recombination is the process by which genetic material is exchanged between DNA molecules, leading to new genetic combinations.

Question 10

What is the advantage of recombination in bacteria?

Answer 10

Recombination result in more “fit” organisms and increases genetic diversity i.e. would serve similar purpose as eukaryotic recombination during meiosis

Question 11

What are the benefits of bacteria recombination?

Answer 11

Drug resistance (bacteria can survive antibiotics).

Novel virulence characteristics (bacteria can become more infectious).

Novel metabolic capabilities (bacteria can break down new substances for survival).

Question 12

What is horizontal gene transfer?

Answer 12

the process by which bacteria and other organisms transfer genetic material between each other, rather than passing it down from parent to offspring (which is vertical gene transfer). HGT is a major driver of bacterial evolution, allowing for rapid adaptation.

Question 13

What are the types of horizontal/lateral gene transfer?

Answer 13

• Transformation
• Transduction
• Conjugation

Question 14

Does horizontal gene transfer happen in eukaryotes?

Answer 14

It does happen, but it is rare

Question 15

When do bacterial cells usually gain competence?

Answer 15

Usually, competence happens just before entry to stationary phase i.e. around the time they stop growing

This is because this is the point where their resources are running out

Question 16

Describe differences in bacterial cell competence

Answer 16

Some e.g. Bacillus become competent for hours but only about 20% of the cells

Others e.g. Streptococcus all cells become competent but only for short amount of time

Most species not competent

Question 17

Why do bacteria become competent at the entry to stationary phase?

Answer 17

• Because as the cells enter stationary phase, they know they are at a risk of dying.
• So they use quorum sensing, the ability to regulate genes based on ppopulaiton density.

Question 18

How does B subtillis become competent?

Answer 18

• Cells secret ComX (small pheromone) so that its concentration increases in the environment
• As bacterial population density increases, ComX accumulates in the surroundings.
• Bacteria have ComP receptors and ComX will bind to these receptors.
• This will lead to a
  reaction of events leading to changes in gene expression.
• This change in gene expression will result in cells becoming competent and they can now undergo transformation, and take up foreign DNA from their environment.

(This system ensures that competence occurs only when bacterial population density is high, increasing the chances of successful gene transfer.)

Question 19

Apart from ComX, what is another pheromone that can bind to b. subtilis?

Answer 19

CSF

Question 20

What type of DNA is taken up for transformation?

Answer 20

• DNA is released during bacteria cell lysis
• Competent bacteria can bind and import these DNA fragments, potentially incorporating them into their genome through homologous recombination.

Question 21

What type of DNA will b subtillis take up for transformation?

Answer 21

• B. subtilis will take up DNA of any origin

Question 22

Why so some bacteria only take up DNA from their own species for transformation?

Answer 22

Because there is the risk of bacteria obtaining harmful phage DNA

Question 23

Which bacteria will only take up DNA from their own species? (transformation)

Answer 23

Neisseria gonorrhoeae and Haemophilus influenzae will only take up from same species

Question 24

How can the bacteria distinguish between DNA from another bacteria of the same species and from a different species?

Answer 24

Because it recognises specific DNA sequences. A short sequence of nucleotides is recognised by the receptor on bacteria.

Question 25

Describe the general mechanism of transformation

Answer 25

DNA bind to surface proteins on the cell Depending on species single or double stranded DNA enters the cell The DNA will then Bind to competence-specific protein whilst inside the cell. This prevents the DNA from getting broken down. The DNA will then recombine with the DNA of the bacteria.

Question 26

Is it true that recombination may convey new traits like lactose intolerance?

Answer 26

Yes

Question 27

How can new traits be acquired?

Answer 27

Gene fragment usually big enough to contain several genes Recombination may convey new traits

Question 28

What are phages?

Answer 28

Viruses that kill bacteria

Question 29

What is tranduction?

Answer 29

genetic exchange in bacteria mediated by bacteriophages

Question 30

Describe phages

Answer 30

Phage genomes can be single or double stranded DNA or RNA Genome can be small – a few kbp or big – hundreds of kbp Can be circular or linear

Question 31

What are possibly the most abundant organisms on earth?

Answer 31

Phages

Question 32

Do phages co-exist with hosts in all environments?

Answer 32

Yes

Question 33

What are phages influenced by?

Answer 33

characteristics of host population dynamics long term evolution

Question 34

Phage therapy timeline...

Question 35

Are phages very specific?

Answer 35

Yes, they usually recognise specific species or strains (need to know bacteria first)

Question 36

Can phage therapy be used as treatment for many diseases?

Answer 36

Yes, but more research on phages needs to be conducted

Question 37

Do phages work well on biofilm?

Answer 37

Yes

Question 38

Are phages safe?

Answer 38

Yes, but only a few studies on its safety, so more research needs to be done

Question 39

Does T2 phage infect and kill e.coli?

Answer 39

Yes

Question 40

What does the head of lambda phage contain?

Answer 40

Head contains double stranded linear genome Size ~48 kbp

Question 41

What is the tail of lambda phage important for?

Answer 41

Tail important for interacting with E. coli

Question 42

How can lambda phage infect bacteria?

Answer 42

Infection can be either lytic or lysogenic (not unique to 𝝺 but only applies to some phages)

Question 43

Describe the lytic cycle (phages; transduction)

Answer 43

- The phage attaches to the bacteria and inserts its DNA into the bacteria - The once-linear DNA circularises. - The circular DNA now undergoes replication, transcription and translation so that new virions of phage are produced. - The bacterial cell lyses and releases the virions of phage - The released phages can then infect other bacterial cells, continuing the cycle.

Question 44

What is the phage restriction system?

Answer 44

a bacterial defense mechanism against bacteriophage (viral) infection.

Question 45

Describe the phage restriction system

Answer 45

Bacteria have a defense system called the restriction-modification system. When a bacteriophage injects its DNA into the bacterial cell, the bacterium can cut the foreign DNA using restriction enzymes These enzymes recognize specific DNA sequences and cleave phage DNA, preventing infection. Host DNA protected because it is modified by methylation (many enzymes insensitive to methylation).

Question 46

why is the phage restriction system important?

Answer 46

Restriction enzymes are now widely used in molecular biology for DNA cutting and genetic engineering. The discovery of these enzymes helped develop recombinant DNA technology.

Question 47

What is a lysogen?

Answer 47

strain of bacteria carrying a lysogenic phage e.g. E. coli (𝝺)

Question 48

What is a prophage?

Answer 48

phage in lysogenic state

Question 49

Describe the lysogenic cycle

Answer 49

- Phage attaches to bacteria and inserts its DNA - The DNA becomes integrated into the host bacteria's genome and new daughter cells will inherit this phage, - No new virions are produced. - E coli will live normally and healthily until something triggers the lytic cycle, but it is very rare and unlikely that something will trigger the lytic cycle.

Question 50

When a bacteria has undergone the lysogenic cycle, how is the lytic cycle suppressed?

Answer 50

The lytic cycle is suppressed by expression of phage protein that stops lysis This protein also suppresses lysis by other phage of the same type infecting the cell This is perhaps because carrying a prophage is probably an advantage to the host

Question 51

What decided whether or not a bacteria goes through the lytic or lysogenic cycle?

Answer 51

Depends on genetic and environmental factors: Nutritional state, UV light etc.

Question 52

Is it true that most phages dont carry genes that are damaging to humans?

Answer 52

Yes, which is why phage therapy should have few side effects.

Question 53

Is it true that only some bacteria can undergo transduction?

Answer 53

Yes

Question 54

What are the 2 types of transduction?

Answer 54

Generalised transduction and specialised transduction

Question 55

What is generalised transduction?

Answer 55

Transfer of any DNA to the recipient cell by lytic or lysogenic phage

Question 56

What is Specialised transduction?

Answer 56

Transfer of specific genes via lysogenic phages

Question 57

Describe generalised transduction

Answer 57

- Phage infects e coli cell. - The phage DNA is replicated, and the host cell DNA is degraded. - Virions are packaged, and occasional bots of host DNA are packed by mistake. - The bacterial cell bursts (lyses), releasing the newly formed phage particles. Some of these phages contain bacterial DNA instead of viral DNA. - The defective phage infects a new bacterial cell, but instead of injecting viral DNA, it injects the previous host’s bacterial DNA. - If this DNA is incorporated into the new host’s genome via recombination, it can lead to genetic changes in the recipient cell.

Question 58

What are some key points to remember about generalised transduction?

Answer 58

This process does not produce new phages because the transducing phages lack phage DNA. The transferred bacterial DNA may carry new traits, like antibiotic resistance or virulence factors. Generalized transduction is a random process, meaning any part of the bacterial genome can be transferred. The probability of a specific genetic marker being transferred is very low (1 in a million to 1 in 100 million cells).

Question 59

Describe specialised conduction

Answer 59

- specialized transduction only transfers genes near the phage’s integration site. - A bacteriophage infects a bacterial cell - Instead of immediately killing the host, the phage integrates its DNA into the bacterial genome at a specific site. - The integrated phage DNA is called a prophage, and the bacterium continues to grow and divide, copying the phage DNA along with its own. - Under certain conditions (e.g., stress, UV exposure), the prophage excises itself from the bacterial genome to enter the lytic cycle (where it produces new virus particles). - However, sometimes the excision is imprecise, and the phage accidentally removes nearby bacterial genes along with its own DNA. At the same time, some of the phage’s own genes may be lost, making it defective. - The defective phage DNA (which now includes some bacterial genes) is replicated and packaged into new virus particles. Since some essential viral genes are missing, these new phages cannot complete a full infection cycle. - The defective phages are still capable of infecting another bacterium. - They inject their DNA (which includes the bacterial genes from the previous host) into the new bacterial cell. - The bacterial gene carried by the phage may integrate into the recipient’s genome through homologous recombination. - If successful, the recipient bacterium gains new genetic traits, such as antibiotic resistance or the ability to metabolize new substances. -

Question 60

Key messages

Answer 60

Horizontal gene transfer is a natural process that allows bacteria to diversity their genes and essential for our biotech exploitation Genetic material taken up is inserted into the genome via recombination: Transformation, transduction and conjugation Phages are viruses that prey on bacteria, but they can defend themselves Phages are lytic or lysogenic (know the lifestyles!)