Bacterial genetics

by

E. 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