Lecture 7 - Chapter 6: Transformation & Homologous Recombination PDF

MICR 321: Advanced Microbiology Lecture 7: Chapter 6: Transformation Chapter 9: Homologous Recombination Transformation - Terms Wild type (WT) – normal members of the species Mutant (∆) – an organism that is genetically distinct from the normal members of a species...

MICR 321: Advanced Microbiology Lecture 7: Chapter 6: Transformation Chapter 9: Homologous Recombination Transformation - Terms Wild type (WT) – normal members of the species Mutant (∆) – an organism that is genetically distinct from the normal members of a species Transformation – direct uptake of “naked” DNA from the environment Often the best way to reintroduce experimentally altered DNA into cells DNA is derived from a donor cell and taken up by a recipient cell which is then called a transformant x https://blog.addgene.org/plasmids-101-transformation-transduction-bacterial-conjugation-and-transfection Types of transformation Natural Transformation Naturally transformable bacteria can take up DNA from their environment without special chemical or electrical treatments Naturally transformable bacteria are not always able to take up DNA only at certain stages in their life cycle Bacteria that are at a stage when they can take up DNA are competent Bacteria that can reach a competent state alone are naturally competent Ex: Bacillus subtilis, Streptococcus pneumoniae, Haemophilus influenzae, Neisseria gonorrhea Induced Transformation Bacteria need special chemical treatment or application of an electrical field to make them more permeable to take up DNA Ex: Escherichia coli, Salmonella spp. Discovery of Transformation Transformation was the first mechanism of gene exchange in bacteria to be discovered Who discovered that Streptococcus pneumoniae could be transformed into another form? Smooth type – form smooth colonies on agar plates because they excrete a polysaccharide capsule (also pathogenic in mice) Rough type – form small rough colonies on agar plates because they are mutants which lack the capsule (non-pathogenic in mice) Rough (R) Smooth (S)  Avery, McCarty, and MacLeod, 1944 Griffith’s Experiment Avery, McCarty, MacLeod The Transforming Principle: Protein RNA DNA Griffith determined dead pathogenic bacteria could give off a “transforming principle” that could change the live, non-pathogenic rough bacteria into pathogenic smooth bacteria  Proteobacteria Fig 6.2 Gram-negatives DNA Uptake DNA uptake can be species specific or nonspecific Uptake differs depending on whether bacteria are Gram-positive or Gram-negative For Gram negatives (Proteobacteria): 1) Binding of dsDNA to the outer cell surface of the bacterium 2) Movement of the DNA across the cell wall and outer membrane 3) Degradation of one of the DNA strands by nucleases 4) Translocation of the ssDNA across the inner membrane Firmicutes Once inside the cell the ssDNA may: Gram-positives Synthesize the complementary strand and reestablish itself as a plasmid Stably integrate in the chromosome by homologous recombination Be degraded For Gram-positives (Firmicutes) the steps are essentially the same except they don’t possess an outer membrane, so the DNA must only pass through the cell wall and cell membrane This series of images shows extension of a type IV pilus from a Vibrio cholerae cell (green) and contact being made between the tip of the pilus and a fragment of DNA (red). Retraction of the pilus brings the DNA to the cell surface and allows the DNA to enter the cell. Ellison et al., 2018. Nature Microbiology.  Fig 6.3 Visualization of DNA uptake using fluorescent labels. Competent B. subtilis (green) mixed with DNA (red). Bright red dots are seen in association with green cells but not with non-competent brown cells. Boonstra et al., 2018. mBio.  Fig 6.4 So why take up foreign DNA? Any DNA? Gram-positives (B. subtilis, S. pneumoniae) will take up any DNA Gram-negatives are picky. Some (Neisseria, Haemophilus) require a DNA uptake sequence (DUS) Nutrition DNA could serve as a nutrient source for cells, some produce DNases But taking up DNA from outside cell and degrading inside is harder than degrading outside and importing nucleotides. Then why do some only take up DNA from their own species? Repair Cells may take up DNA to repair damage to their own DNA But competence isn’t always induced in response to DNA damage Recombination Could serve the same function sex serves for higher organisms – allows for genetic reassortment to promote genetic diversity for survival. Combination? Competence Pheromones Most naturally transformable bacteria express their own competence genes. B. subtilis regulates competence through a two-component system A sensor kinase senses a signal and transmits that signal to a response regulator through a phosphorylation cascade. *more to come in Ch. 12 Global Regulation* What’s the signal?? Competence Pheromones – small peptides secreted by bacteria as they multiply which increase in concentration as bacterial numbers increase Cells become competent only in the presence of high concentrations of these pheromones Quorum sensing – small molecules produced by cells signal to other cells that density is high https://researchblog.duke.edu/2022/ 02/16/quorum-sensing-the-social- network-of-bacteria/  Fig 6.8 SK – sensor kinase RR – response regulator TA – transcriptional activator  Artificially Induced Competence Competence can be induced artificially in bacteria which aren’t naturally/ideally competent Plasmids are generally more efficient than linear DNA fragments Chemically-induced competence Treatment with calcium ions can make some bacteria competent by perturbing the cell surface (E. coli, Salmonella spp.) Electroporation Bacteria are exposed to DNA A brief, strong electric field alters the surface charge allowing DNA to pass through pores into the cell Requires special equipment  Chapter 9: Homologous Recombination  Box 9.2 Double-stranded break repair  Fig 9.1 Chapter 9: Homologous Recombination Fig 9.10  Fig 3.23 Molecular genetics at work We can introduce mutations into specific genes by engineering an engineered pLB altered gene product and transforming our bacterium of interest with the altered product WT Example: chromosome Gene is cloned into a plasmid vector vector recombines in containing Ampicillin resistance (Ampr). We engineer a Kanamycin resistance gene (Kanr) into a gene of interest. Transform this construct into the original strain Homologous recombination inserts the vector recombines plasmid into the gene and duplicates the out – taking ‘good’ region of insertion copy A second recombination event excises the plasmid leaving our mutated gene in the chromosome ∆ chromosome Cells will be Kanr and lack the functional gene Fig 3.22 Browse ahead: Chapter 5 Conjugation  Project Part 2: Due: Thursday, October 3rd by 11:00 am Group Leaders: 1. John Sirko 6. James Roberts 2. Ella Benjamin 7. Mary-Ann Cook 3. Joanna Chen 8. Madalyn Marshall 4. Hoa Nguyen 9. Seth Brewer 5. Eliana Safer 10. Madison Resch