Horizontal gene transfer; conjugation, transduction and natural transformation

Question 1

Types of gene transfer

Answer 1

Horizontal - DNA is passed from one bacterium to another (three processes Vertical - Through cell division, genetic information passed to progeny

Question 2

Whats the transforming principle

Answer 2

In 1928 griffin experiment showed heat killed streptococcus pneumonia can transform non virulent to virulent in the 1930s they proved this was because of DNA

Question 3

HGT leads to mosaic gene transfer

Answer 3

• Principle of formation of mosaic gene structures • An example is the penA gene in S. pneumoniae- responsible for penicillin resistance • HGT plays a major role in the spread of antibiotic resistance

Question 4

The three main mechanisms for HGT

Answer 4

1. Transduction (bacteriophage) 2. Conjugation (F pilus) 3. Natural transformation

Question 5

Zinder and Lederberg (1952): Generalized transduction in Salmonella typhimurium

Answer 5

• Mixing different auxotrophic strains of S. typhimurium • Transfer of genetic material did not require physical contact (rules out conjugation) • Transfering agent passes through a filter i.e. it is small • Process resistant to DNase treatment (rules out natural competence)

Question 6

Specialised Tranduction

Answer 6

• Differs from generalised transduction by

1. Transduced genes are specifically incorporated into the viral genome
2. The packaged virus carries a particular part of the infected bacterial chromosome

Question 7

Filamentous bacteriophage converts Vibrio cholerae to a more pathogenic form

Answer 7

• A bacteriophage, CTXphi, enocdes cholera toxin
• CTXphi uses type IV pili on the surface of V. cholerae as a receptor
• The infected V. cholerae acquires the toxin in vivo and hence becomes more virulent
• HGT can therefore be used to transfer virulence determinants, such as toxins

Question 8

Conjugation

Answer 8

• Discovered by Joshua Lederberg and Edward Tatum (1946)
• Also referred to as ‘mating’: transfer of genetic material from a donor to a recipient bacterial cell has parallels with sexual reproduction in higher organisms (but not very strong ones……)
• Donor cell contains F plasmid and hence is designated F+
• Recipient cell does not contain the plasmid, and is designated F-
• A common mechanism for transfer of antibiotic resistance (R plasmids)
• Requires direct cell-cell contact (not the case for transduction or natural competence)
• Uses the type IV secretion system (see following lectures on secretion systems)

Question 9

Hfr and chromosome mobilization

Answer 9

• F plasmid integrates into the chromosome at insertion sequence (IS) sites
• This becomes an Hfr strain
• Different Hfr strains are possible, with integration at different IS sites
• The apparatus for transfer of the integrated F plasmid is still expressed (type IV secretion)
• An Hfr cell therefore can donate integrated F plasmid DNA to an F- cell
• In doing so, some chromosomal DNA is also transferred

Question 10

Whats Hfr mapping

Answer 10

Using different HFr strains (with different insertion positions in the chromosome), it is possible to map the locations of different genes

• Before DNA sequencing, this enabled microbiologists to establish low resolution ‘maps’ of the locations of specific genes in a bacterial chromosome, through the interrupted mating experiment

Question 11

Whats interrupted mating

Answer 11

Hfr and an F- strain are combined, and allowed to ‘mate’

• After a set period of time, conjugation is interrupted by vigorous mixing (which breaks the F pilus connection)
• Bacteria are plated out on media which allow selection of specific genetic markers (eg antibiotic resistance)
• As the time of mating increases, more genes are transferred

this can be used to make a map as the first genes to be expressed and the ones following it are expressed near each other.

Question 12

Agrobacterium tumefaciens

Answer 12

Contains a tumour-inducing (Ti) plasmid which is transferred into plant cells by a type IV secretion mechanism

• DNA is secreted along a T-pilus, injected into the plant cell, and eventually incorporated into the plant genome
• Widely used in biotechnology, to incorporate foreign genes into plants

Question 13

Whats natural competence

Answer 13

• Occurs in Gram positive and Gram negative bacteria
• Up to ~80 species so far, but likely to be more widespread
• Can be constitutive, or induced by environmental conditions (eg nutritional privation)
• Takes up ‘naked’ DNA from outside the cell; some species have sequence-specific preferences
• Examples: G+ve Bacillus subtilis; Streptococcus pneumoniae (Griffith’s experiment) G-ve Neisseria meningitidis; Thermus thermophilus

Question 14

What are the functions of natural competence

Answer 14

• DNA as food
• DNA for repair of a damaged genome
• DNA to generate genetic diversity

Question 15

Whats the competence pathway in Gram + bacteria

Answer 15

Stage 1:

1. Competence pilus captures DNA
2. One DNA strand is degraded
3. A complex in the inner membrane transports DNA into the cytoplasm

Stage 2:

1. Some proteins (SsbB) protect the DNA from degradation
2. DprA interacts with RecA, which promotes homologous recombination into the chromosome

Question 16

Whats the competence pathway in Gram negative bacteria

Answer 16

1. Distinct stages are capture of DNA, uptake into the periplasm, nuclease activity to remove DNA strand, uptake into the cytoplasm
2. An additional portal or channel through the outer membrane is required
3. Some of the competence proteins are also used in type IV pilus biogenesis
4. You won’t be required to memorise all the protein components!