Horizontal Gene Transfer & Molecular Identification of Pathogens

Griffith’s Experiment

discovery of bacterial transformation & that DNA is the transforming principle

• mice + streptococcus (R and S strains)

transformation

direct uptake of naked DNA, indirect gene transfer

• bacteria must be competent (able to uptake DNA)

  • ex: Bacillus spp. 

free donor DNA (fragment or plasmid); live, competent recipient cell

conjugation

transfer of DNA via direct connection (3 kinds)

1. physical conjugation

2. F Factor transfer

3. Hfr Transfer

Physical Conjugation

Pilus of donor cell (F+) attaches to receptor on recipient cell (F-) and retracts to draw the two cells together.

• for gram (-) bacteria

F Factor Transfer

• bridge between two cells made with a pilus 

• Transfer of the F factor, or conjugative plasmid

High frequency (Hfr) Transfer

• involves transmission of chromosomal genes from a donor cell to a recipient cell. 

• the donor chromosome is duplicated and transmitted in part to a recipient cell, where it is integrated into the chromosome 

• F factor is integrated into the chromosome 

transduction

indirect gene transfer, uses bacteriophages

• donor is lysed bacterial cell

• defective bacteriophage is carrier of donor DNA

• Live recipient cell of same species as donor

generalized transduction

1. Donor cell contains donor (host) chromosome; Phage DNA is inserted

2. host DNA is digested- pieces are separated (lytic cycle)

3. newly assembled phage incorporating piece of host DNA mistake   

4. phages are released

5. released phages infect recipient cell 

6. bacterial DNA gets integrated into the recipient cell’s chromosome 

see slides image

specialized transduction

1. prophage within the bacterial chromosome 

2. excised phage DNA contains some bacterial DNA 

3. new viral particles are synthesized from excised phage DNA

4. phages are released 

5. released phages infect recipient cell

see slides image 

transposons

• special DNA segments that have the capability of moving from one location in the genome to another (“jumping genes”) 

• cause rearrangement of the genetic material 

• can move from one chromosome site to another, from a chromosome to a plasmid, or from a plasmid to a chromosome 

• may be beneficial or harmful

searching/looking for a DNA sequence of a pathogen

• sequence DNA

• PCR

• ribotyping 

• fluorescence in situ hybridization (FISH)

small ribosomal subunit- 16 S (bacteria), 18 S (euk) rRNA gene

searching/looking for protein production

• ELISA

• Direct Fluorescence Antibody assay 

The polymerase chain reaction (PCR)

1. denaturation

2. primer annealing 

3. extension 

denaturation (PCR)

head DNA to 95 deg. celsius to separate DNA

primer annealing (PCR)

• 60 deg. celsius

• look for specific sequence of DNA, if find sequence → DNA is amplified

extension (PCR)

DNA copied 

restriction endonucleases 

recognize specific sequences of DNA and break phosphodiester bonds between adjacent nucleotides

restriction enzymes

• made by bacteria to protect themselves against bacteriophages 

  • protects its own DNA by methylating 

ribotyping

using restriction enzymes on 16 S rRNA gene to give us a “DNA fingerprint”

direct fluorescence antibody assay (DFA)

• antibodies recgonize certain antigens → fluorescent marker added

antigen

protein expressed by the pathogen

Fluorescence in situ hybridization (FISH)

• use fluorescent labeled primer that binds to complementary nucleic acid sequence → makes pathogen visible

• same idea as DFA 

transformation (general)

picks up free DNA

conjugation (general)

direct contact- only gram (+)

pilus → gram (-)

transduction (general)

bacteriophages to transfer DNA

1. general- random fragment of DNA

2. specialized- lysogenic (DNA next to prophage)