Bacterial genetics by E Brje Lindstrm This learning

Bacterial genetics by E. Börje Lindström This learning object has been funded by the European Commissions FP 6 Bio. Min. E project

Definitions • Gene: a DNA sequence that determines - the aa-sequence in a peptide/protein - r. RNA - t. RNA - 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

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: - e. g. Prototroph (wt) auxotroph (mutant) 1) Enrichment with the e. g. penicillinmethod 2) Screening with ’replica plating’

Inoculum Penicillin-method - Only prototrophs can grow are killed MM-medium + pen - Change the medium -All bacteria will grow - both prototrophs and auxotrophs Complex medium without penicillin - the auxotrophs are enriched

Replica plating

Replica plating, cont.

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

Transfer of DNA between bacteria Donor Recipient Three processes: 1) 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

Transformation

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

Transduction Two types: • General transduction -Model system: P 1 - E. coli - P 1 a virulent phage • Specific transduction - Model system: phage l – E. coli

General Transduction

Specifik transduction

Specifik transduction, cont.

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

Integration of the F-plasmid IS-element ori. T

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- (all) Course of events: 1) Cell-to-cell contact - pili promote a conjugation bridge (Week) 2) ss-brake at ori. T in Hfr 3) One of the Hfr-strands is transferred – ori T first 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 -new synthesis in both cells - constant transfer speed (takes 100 min for E. coli)

Cross no. 2, cont.

Cross no. 2, cont. 40) A+ B+ C+ Ori T A- 50) Ori T B- - Partial diploid C- AA+ B+ BC- 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