Week 4: Analysis of Bacterial Virulence

Question 1

Techniques of analysing bacterial virulence

Answer 1

• Targeted mutagenesis
• Genome analyisis + mapping by In-vitron Tranposition (GAMBIT)
• SIgnature Tagged Mutagenesis (STM)
• Invivo expression technology (IVET)

Question 2

Targeted mutagenesis

Answer 2

• Involves the insertion of a selective marker ie antibiotic resistance to help select for cells that have successfully taken up the plasmid
• This will plasmid will then help to inactivate the cell + produce a library of mutants
• May also include homolgous recombination

Question 3

GAMBIT

Answer 3

• Using transposons that contain genes for antibitotic resistance into amplified regions of chromosomes, then later selectively pressure the bacteria with antibiotics to select the bacteria surviving with the transposon
• Generally transposons will randomly insert themselves into the chromosomes
• This technique can help identify unknown virulence factors
• Genomic DNA is isolated from bacteria in pre- + post-selection pool + is subjected to PCR using a fixed chromosomal primer + a transposon primer

Question 4

Signature Tagged Mutagenesis

Answer 4

• A signature tag ie transposon is (sort of) randomly inserted
• Each tage has a unique 40bp central sequence, flanked by invariable 20bp arms, which are common to all tags
• These common sequences allow the tags to be amplified by PCR
• A complex pool of tags are then ligated to transposons
• These transposons are then used to mutagenise bacteria, creating a library of mutants, which are then used for infection models
• This library can help you check which mutants are missing after injection into the model, allowing you to thensequence the mutant + find which gene has been mutated by the transposon
• mutants not recovered are presumed to be avirulent

Question 5

In Vitro Expression Technology (IVET)

Answer 5

• Acts as a promoter trap
• Requires selective marker
• Needs to be carried out in an auxotrophic mutant
  1) A selective marker is inserted into the DNA, along with an auxotrop complementation gene that will switch on when promotion is involved + lacZY to help it to be expressed + identified
  2) Transcription of auxotroph complementation gene is now driven by trapped promoter
  3) Inoculated into the infection model