Bacterial genetics

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Bacterial genetics

• by
• E. Börje Lindström

This learning object has been funded by the
European Commissions FP6 BioMinE project
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Definitions

• Gene

a DNA sequence that determines

• the aa-sequence in a peptide/protein
• rRNA
• tRNA
• operator (O)
• promoter (P)

• Allele

- a different form of a gene (wt, mutant)

• Genotype

- the total amount of genes in an organism

• Phenotype

- those properties that are expressed
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Definitions, cont.

• Mutation

• a permanent change in the genotype
• spontaneous (frequency 10-1010-5)
• induced (frequency gt10-5)

• Mutant

- an organism that has a changed genotype

• Two types of mutants

• Gain of a property
• (possible to select for)

• Loss of a property
• (not selectable)

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How to find the mutant?

• Those who are selectable

• e.g. Antibiotic sensitive ? resistant
• streak out the culture on a plate containing the
  antibiotic.

• Those who are not selectable

- e.g. Prototroph (wt) ? auxotroph (mutant)

• Enrichment with the e.g. penicillin-method
• Screening with replica plating

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Penicillin-method
Inoculum
- Only prototrophs can grow ? are killed
MM-medium pen
- Change the medium

• All bacteria will grow
• both prototrophs and auxotrophs
• the auxotrophs are enriched

Complex medium without penicillin
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Replica plating
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Replica plating, cont.
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Nomenclature
Model system tryptophan

• Genotype

- trp- (trp) (A, B, etc.)

• Phenotype

- Trp Trp-

• Amino acid

try
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Transfer of DNA between bacteria
Donor
Recipient
Three processes

• Transformation

- f ree donor-DNA ? recipient
2) Transduction
- donor-DNA within a virus (Phage) ? recipient
3) Conjugation
- cell-to-cell contact between donor and recipient
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Transformation
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Transformation, cont.
The Donor
The Recipient
- Cultivate, make competent cells
- lyse and cut the DNA into pieces
Free DNA (?10 genes/ fragment)
ds DNA
- Uptake of DNA (ds or ss)
- homologue integration
- transformed cell
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Transduction
Two types

• General transduction

• Model system P1- E. coli
• P1 a virulent phage

• Specific transduction

- Model system phage l E. coli
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General Transduction
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Specifik transduction
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Specifik transduction, cont.
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Conjugation
Three different bacteria
F-plasmid
Integrated
pili
Free
Hfr (high frequency of recombination)
F-
F
Recipient (female)
Donors (males)
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Integration of the F-plasmid
oriT
IS-element
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Cross no. 1

• F x F-

F- ? F (all)

• The F-plasmid is transferred
• a ss-DNA is transferred
• new DNA synthesis occur in both cells
• the complete F-plasmid is transferred
• rapid spread of the F-plasmid

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Cross no. 1, cont.
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Cross no. 1, cont.
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Cross no. 2

• Hfr x F-

F- ? F- (all)
Course of events
1) Cell-to-cell contact
- pili promote a conjugation bridge (Week)
2) ss-brake at oriT in Hfr
3) One of the Hfr-strands is transferred ori T
first

• new synthesis in both cells
• constant transfer speed (takes 100 min for E.
  coli)

4) Pairing of homologue DNA-segments
5) Integration and exchange of genetic material
6) Brake down of linear extra-chromosomal DNA
7) A conjugant/ recombinant has been constructed
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Cross no. 2, cont.
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Cross no. 2, cont.
40)
- Partial diploid
50)
A-
B-
C
Ori T
- Integration and exchange
A
B
C-
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Cross no. 2, cont.
Q Why is streptomycin added to the plate?
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Summary

• Obtaining a recombinant/conjugant require

• genes must have been transferred from a donor to
  a recipient
• exchange of genes must occur