Chapter 4
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Chapter 4. Genetic Approaches to Studying Bacterial Pathogenesis. Chapter 4. Genetic Approaches to Studying Bacterial Pathogenesis. Genetic complementation. Identify Yersinia pseudotuberculosis genes that confer invasiveness on E. coli. Gentamycin kills E. coli that do not invade.

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Chapter 4

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Chapter 4

Chapter 4

Genetic Approaches to Studying Bacterial Pathogenesis


Chapter 4

Chapter 4

Genetic Approaches to Studying Bacterial Pathogenesis

Genetic complementation

Identify Yersinia pseudotuberculosis genes that confer invasiveness on E. coli


Chapter 4

Gentamycin kills E. coli that do not invade.

Gentamycin does not penetrate mammalian cells.

Positive selection

Pick and test individually


Chapter 4

After identification of

gene for invasion factor:

Generate DNA sequence

= inv gene

Deduce protein coding region

= invasin protein

Manipulate gene further to prove that invasin really does promote cell invasion.

“Loss-of-function mutation”

Plasmid cannot replicate in Y. pseudotuberculosis (but it can replicate in E. coli)


Chapter 4

Suicide plasmid containing inv loss-of-function mutation transferred from E. coli to Y. pseudotuberculosis

Need 2 recombination events to replace the inv gene in Y. pseudotuberculosis with the loss-of-function inv allele. (See previous slide)

Test Y. pseudotuberculosis inv mutants and show that they do not invade.


Chapter 4

Transposon-based methods

simple transposon

composite transposon

Insertion of a transposon in a gene most often creates a loss-of-function mutation.

Transposon marks the site of the mutation (sequence and antibiotic resistance)


Chapter 4

Many types of engineered transposons

phoA gene

Encodes a periplasmic

phosphatase

engineered phoA gene lacks N-terminus so expression depends on fusion to an adjacent gene after transposition

PhoA+ colonies turn blue (cut X-P dye)

Vibrio cholerae virulence genes maximally expressed at pH 6.5 and high osmolarity

Result?

Decreased virulence


Chapter 4

Genetic screen versus genetic selection.

Positive selection

Negative selection

This is actually a screen for a negative trait: inability to grow in spleen.

Studying mouse model for typhoid fever


Chapter 4

Unique marker put into each transposon

Create a library of S. typhimurium mutants that each contain a mini-Tn5 insertion

(suicide plasmid used to move mini-Tn5 from E. coli into S. typhimurium)


Chapter 4

Promoter-trapping

Look for genes of S. typhimurium thatare expressed in infection but not in the laboratory

This is only one strategy but it is doable.


Chapter 4

4


Chapter 4

Antibody-based approach


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