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Microbial Genetics Chapter 9 (p. 251-265) Copyright © The McGraw-Hill Companies, Inc) Permission required for reproduction or display. 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 Regulation of Transcription • Groups of coordinately expressed and regulated genes are called operons • Regulatory proteins bind to operators • Transcription can be turned on or off 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 AG or CT – 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) Replica Plating 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. 11 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 1051010) • 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 Ames Test • Quick test for mutagen strength. • His- Salmonella typhimurium • Mutagen reverts cells to His+ • The degree of mutagenicity is calculated 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 Strain of Colony Cell Type Effect Strain of Colony Effect Cell Type Capsule Smooth (S) (a) No capsule LiveS strain Rough (R) Dies (b) Heat-killed S strain Survives Live R strain Heat-killed S Live S and R strains strain Survives (c) LiveR strain (d) isolated from dead mouse Dies 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 Physical Conjugation F factor Bacterial chromosome F+ Sex pilus makes contact with F* recipient cell. F– F+ F– Sex pilus contracts, bringing cells together. 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) Transposons Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. • Jumping genes • Mobile genetic elements • Move from place to place in the genome, plasmids, and viral genomes • Disrupt genes when they land • May mobilize other genes (like antibiotic resistance) (1) (2) (3) (4) 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.