Bacterial genetics
byE. Börje Lindström
This learning object has been funded by the European Commissions FP6 BioMinE project
Definitions• Gene:
- the aa-sequence in a peptide/protein
- rRNA
- tRNA
- operator (O)
- promoter (P)
a DNA sequence that determines
• Allele:
- a different form of a gene (wt, mutant)
• Genotype: - the total amount of genes in an organism
• Phenotype:
- those properties that are expressed
Definitions, cont.• Mutation: - a permanent change in the genotype
- spontaneous (frequency 10-10—10-5)
- induced (frequency >10-5)
• 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)
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:
1) Enrichment with the e.g. penicillin-method
2) Screening with ’replica plating’
- e.g. Prototroph (wt) auxotroph (mutant)
Penicillin-method
MM-medium + pen
Inoculum
- Only prototrophs can grow are killed
Complex medium without penicillin
-All bacteria will grow
- both prototrophs and auxotrophs
- the auxotrophs are enriched
- Change the medium
Replica plating
Replica plating, cont.
Nomenclature
• Genotype: - trp- (trp+); (A, B, etc.)
• Phenotype: - Trp+; Trp-
Model system: tryptophan
• Amino acid:
try
Transfer of DNA between bacteria
Three processes:
1) Transformation:
2) Transduction:
3) Conjugation:
- f ree donor-DNA recipient
Donor Recipient
- donor-DNA within a virus (Phage) recipient
- cell-to-cell contact between donor and recipient
Transformation
Transformation, cont.The Donor
- lyse and cut the DNA into pieces
Free DNA (10 genes/ fragment)
The Recipient
- Cultivate, make competent cells
ds DNA - Uptake of DNA (ds or ss)
- homologue integration - transformed cell
TransductionTwo types:
• General transduction
• Specific transduction
-Model system: P1- E. coli
- P1 a virulent phage
- Model system: phage – E. coli
General Transduction
Specifik transduction
Specifik transduction, cont.
ConjugationThree different bacteria:
F-plasmid
FreeIntegrated
F- F+ Hfr (high frequency of recombination)
Recipient (female) Donors (males)
pili
Integration of the F-plasmid
oriTIS-element
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
Cross no. 1, cont.
Cross no. 1, cont.
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
Cross no. 2, cont.
Cross no. 2, cont.
A-B-
C-
A+ Ori T B+ C+
- Partial diploid40)
50)A+
B+
C-
A- Ori T B- C+
- Integration and exchange
Cross no. 2, cont.
Q: Why is streptomycin added to the plate?
Summary
• Obtaining a recombinant/conjugant require:
- genes must have been transferred from a donor to a recipient
- exchange of genes must occur