Horizontal gene transfer in prokaryotes

Question 1

What is transformation?

What would enable this to occur more readily?

Through what process can a chromosome or plasmid be integrated into the genome?

How can you detect transformation? What are the steps involved?

What is the result?

How can E.coli become transferrable? (as they naturally aren’t)

How is the new chromosomal DNA sequence incorporated into the mutant locus?

Answer 1

DNA is incorporated into the chromosome in prokaryotes from the surroundings/environment

if new DNA contains sequences similar to the host genome (DNA must be homologous)

Homologous recombination

1. Take mutant strain deficient in a physiological function due to a genetic mutation (e.g auxotroph)
2. Take wild-type strain & isolate its DNA
3. Mix mutant cell with wild-type DNA & select for wild-type phenotype by plating on minimal medium

Cells survive because they have taken up the DNA sequence that complements the mutation

Pre-treatment with metal ions e.g Ca2+

1. Strand separation of DNA- and one strand of DNA breaks at 2 ends, so the single strand of donor DNA joins by base-pairing of homologous sequences
2. Then in replication, have 1 DNA giving rise to mutant, and 1 DNA giving rise to wild-type

Question 2

What are the main problems of taking up plasmids by transformation?

What are the 5 main characteristics of plasmids?

How can you use plasmid transformation for genetic engineering of prokaryotes?

Answer 2

Large plasmids less likely to be taken up, and not all plasmids can replicate in all hosts (host range)

Extrachromosomal DNA molecules
Circular or linear
Autonomous replication (doesn’t rely on the host)
Control their copy number
Ensure inheritance at each cell division with partioning

Insert DNA sequence into plasmid- then taken up by a prokaryote. Have prokaryotic strain producing novel gene product

Question 3

What is transduction?

What is the cycle/steps?

What does not occur at every infection?

How can you demonstrate transduction in a lab?

How often does transduction happen in nature?

Answer 3

Horizontal gene transfer mediated by bacteriophages

Lytic cycle:

1. Bacteriophage infects bacteria cell with its phage DNA
2. Phage DNA replicated & host DNA degrades, end up with DNA packed into heads of phages
3. Bacteriophage causes bacterial cell to lyse to release phages & go on to infect other cells

Sometimes host chromosomal DNA can be packaged into the phages, which mean that its next infection doesn’t result in cell lysis but instead delivers DNA fragment into the plasmid = homologous recombination (DNA must be homologous to occur)

1. Infect wild-type culture with phase
2. Isolate the progeny phage
3. Infect auxotrophic culture with progeny phage & look for survivors in the medium

1 in 10^7 cells- in bacteria and archaea to spread prokaryotic genes in oceans & gut

Question 4

What happens in plasmid conjugation?

What is required for conjugation?

What does donor bacterium produce to draw itself to the recipient?

What is the conjugative plasmid/transposon in e.coli? What region is dedicated to conjugation and what does it code for?

What is actually transferred to the recipient cell and how?

What is Tn1000 and Is2 and Is3?

What are the key steps involved?

Answer 4

Transfer of plasmid from donor to recipient bacterium

Conjugative plasmids & transposons, and physical contact between donor and recipient (Lederberg & Tatum 1946)

Sex pilus

Fertility (F) plasmid
tra region- sex pilus, conjugation genes and for the type 4 secretion system

A single strand of DNA from double strand is cut at oriT & the one strand & need conjugation genes in tra region for it to occur

Tn1000 = transposon for antibiotic resistance
IS2 & IS3 = insertion sequences (move like transposons but don’t tend to code antibiotic resistance genes- just have gene to allow to move)

1. Pilus attaches to recipient & retracts to bring them closer
2. Transfer one strand from F+ donor to F- recipient & is simultaneously replicated in F+
3. Synthesis of complimentary strand occurs in F-
4. Cells separate & end up with 2 F+

Question 5

Resistance R100 plasmid is a conjugative plasmid that can cause problems, why?

Where have R-plasmids been detected?

What is particularly special about F plasmid? What other type of conjugation can it undergo as a result?

How does this take place? What can sometimes happen with the host’s chromosomal genes?

What is an Hfr strain?

Answer 5

Carries multiple antibiotic resistance genes (transposons) & a 3rd of the plasmid is dedicated to conjugation

Old bacterial isolates (before use of antibiotics) & in non-pathogenic soil bacteria (so have been in existence for ages & its defence against antibiotic-producing soil microbes)

Episome (can integrate into main chromosome)- chromosomal genes

Integration done via insertion sequence IS3 on plasmid & homologous sequence on bacterial chromosome - end up with single crossover event (only 1 strand) with homologous recombination so plasmid is integrated into chromosome. Sometimes host chromosomal genes also end up in the recipient’s DNA.

F+ strain with high frequency of recombination- strains of E.coli with F plasmid incorporated into the chromosome