1. Bacterial genetics by E. Börje Lindström This learning object has been funded by the European Commissions FP6 BioMinE project

2. 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

3. 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)

4. 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)

5. 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

6. Replica plating

7. Replica plating, cont.

8. Nomenclature Genotype: - trp - (trp + ); (A, B, etc.) Phenotype: - Trp + ; Trp - Model system: tryptophan Amino acid: try

9. 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

10. Transformation

11. 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

12. Transduction Two types: General transduction Specific transduction -Model system: P1- E. coli - P1 a virulent phage - Model system: phage – E. coli

13. General Transduction

14. Specifik transduction

15. Specifik transduction, cont.

16. Conjugation Three different bacteria: F-plasmid Free Integrated F-F- F+F+ Hfr (high frequency of recombination) Recipient (female) Donors (males) pili

17. Integration of the F-plasmid oriT IS-element

18. 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

19. Cross no. 1, cont.

21. 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

22. Cross no. 2, cont.

23. A-A- B-B- C-C- A+A+ Ori T B+B+ C+C+ - Partial diploid 40)40) 50)50) A+A+ B+B+ C-C- A-A- Ori T B-B- C+C+ - Integration and exchange

24. Cross no. 2, cont. Q: Why is streptomycin added to the plate?

25. Summary Obtaining a recombinant/conjugant require: - genes must have been transferred from a donor to a recipient - exchange of genes must occur