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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 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. Gentamycin does not penetrate mammalian cells. Positive selection Pick and test individually
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)
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.
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)
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
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
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)
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.
