Learning Objectives Explain the regulation of gene expression in bacteria by induction and repression Classify mutations by type, define mutagen. Discuss two ways mutations can be repaired Outline the methods of direct and indirect selection of mutants Identify the purpose and outline the procedure for Ames test Compare the mechanisms of genetic recombination in bacteria: transformation, conjugation, and transduction
Regulation of Bacterial Gene Expression Constitutive enzymes are expressed at a fixed rate (i.e, they are on all the time) Other enzymes are expressed only as needed. They are usually under tight control – Repressible enzymes – Inducible enzymes
Groups of coordinately expressed and regulated genes are called operons Regulatory proteins bind to operators Transcription can be turned on or off Regulation of Transcription
Inducible Operons: Lac operon a.In absence of the substrate (lactose) the operon is off. b.When substrate is present the operon is on. 5
Repressible Operons: Arg operon a.Nutrient product (arginine) is being used by the cell. The operon is on. b.Nutrient product builds up, the operon is off.
Mutations Change in the genetic code Wild type: strain expressing natural, non-mutated characteristic Mutant: strain expressing mutated gene Mistakes during replication or damage to DNA Wrong bases incorporated – Transition A G or C T – Transversion A (C or T) or G (C or T) Insertion or deletion of bases Most are lethal, some are beneficial (e.g. drug resistance)
Point Mutations A single base is altered in the sequence Silent mutation: TAT to TAC >> Tyr to Tyr Missense mutation: TAT to TTT >> Tyr to Phe Nonsense mutation: TAT to TAA >> Tyr to stop
Potential Reading Frames Since triplet codons are read, there are three reading frames in the forward direction.
Frame Shift Mutations Addition or deletion of 1 or 2 bases knocks the sequence out of frame The whole amino acid sequence changes, usually results in a truncated (shortened) protein If the gene is essential, the mutation is lethal. Insertions or deletions in multiples of three may be tolerated These are back in frame again Can still be a big problem (Cystic Fibrosis, ∆508)
Causes of Mutations Spontaneous mutations during replication (1 in 10 5 - 10 10 ) Induced mutations Physical: Electromagnetic radiation – X-rays, gamma rays nick DNA – UV light causes T-T dimers to form Chemicals – Analogs of bases – Base-modifying chemicals Nitrosoguanidine, nitrous acid – Intercalators insert between bases Cause frameshift mutations
Quick test for mutagen strength. His - Salmonella typhimurium Mutagen reverts cells to His + The degree of mutagenicity is calculated Ames Test
Repair of Mutations Proofreading by DNA polymerase III Photoactivation or light repair by DNA photolyase Excision repair by DNA polymerase I and ligase.
DNA Recombination Horizontal gene transfer: Transformation (chromosomal DNA fragments) Conjugation (plasmids) Transduction (bacterial viruses)
Transformation Transfer of “naked” DNA. Griffith worked with Streptococcus pneumoniae Encapsulated: smooth colony appearance (S), virulent Lacking a capsule: rough colony appearance (R), non-virulent Dead virulent bacteria were able to “transform” live non-virulent bacteria
The Transforming Principle Dies Survives Effect Cell Type No capsule LiveR strain (b) Rough (R) Strain of Colony Effect Dies (a) Smooth (S) Strain of Colony Cell Type Capsule LiveS strain (c) Heat-killed S strain Survives (d) Heat-killed S strain Live R strain Live S and R strains isolated from dead mouse
Competence Non-specific ability to take up exogenous soluble fragments of DNA Some bacteria are always naturally competent Others regulate competence Still others need to be coaxed – Calcium chloride, chemicals, heat-shock, or electroporation
Sex pilus contracts, bringing cells together. F factor Bacterial chromosome Physical Conjugation Sex pilus makes contact with F* recipient cell. F+F+ F–F– F+F+ F–F– Conjugation Plasmid-directed transfer of plasmid DNA Requires cell contact via pilus
F Factor Donor (F + ) cell makes a copy of the F factor F factor is transferred into the recipient (F + ) cell via pilus Medical importance: R factors transfer resistance to antibiotics
Hfr Strains High frequency of recombination Integrated F factor (episome) Conjugal transfer Incorporation of new genes into the chromosome Used earlier to map the genome.
Transduction Bacterial virus (bacteriophage) serves as the carrier of DNA from the donor cell to a recipient cell Generalized transduction Specialized transduction
Generalized Transduction Phage injects its DNA into a cell Phage DNA serves as a template for new phage DNA and protein synthesis Packaging of a random fragment of bacterial DNA, and transfer to newly infected bacterial cells
Specialized Transduction Only certain genes transferred. Medical importance: toxins of Corynebacteria diphtheriae, Clostridium spp., and Streptococcus pyogenes are transferred by specialized transduction
Telling Them Apart Transformation – involves competency, transfers naked DNA (chromosomal or plasmid) Conjugation - involves cell contact through a pilus, transfers plasmid DNA Transduction - involves bacteriophage, transfers chromosomal DNA (specific or non-specific) DNase sensitivity (transformation) 0.2 µm membranes (conjugation)
Pathogenicity Islands Found in pathogens, improve pathogenicity: Yersinia pestis: ability to scavenge iron Staphylococcus aureus: ability to produce exotoxin Discovered because of the different G+C concentration and presence of bacteriophage or transpozon sequences.