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Cell and Systems Biology
William Navarre

Lecture 22 Bacterial Genetics (Transformation) 2 1. 3 mechanisms involved in horizontal gene trasnfer 2. transduction: how genes hop from one cell to another on a phage particle 3 3. transformation: bacteria that is competent to take up DNA from the envmt will take up free DNA and through recombination, integrate it into their own chromosome 4. transduction: a phage (virus that infects a cell) that infects a cell pops open the cell and every once in a while, they reproduce 1. when they package their own DNA into their protein shell, they sometimes accidentally grab a pieces of the host chromosome and they inject this into new host bacteria 5. conjugation: selfish mobile genetic elements 1. can spread from cell to cell and they themselves encode their own apparatus for injecting their own DNA into neighbouring cells 4 6. transformation is particularly important in the discovery of DNA as the genetic material 1. they took a smooth strain (virulent) of streptococcus pneumonia and put into mice – mice died 2. there are mutants of strep that lack the slime capsule – rough strain that doesn’t kill mice 3. if kill S strain, it doesn’t kill mice 4. surprisingly, when killed S strain was mixed w R strain and injected into mice, the mice died 1. the R strain regained the ability to produce capsule 5. they purified the heat-killed S strains to find what was conferring virulence to the R strains 1. capsule? – (purified capsule and mixed it w the R strain) didn’t improve virulence of the R strain 2. nucleic acid? – (purified DNA and mixed w R strain) mice were killed 3. thus, virulence was stably inherited in the chromosome 5 6. looking at the rough and smooth colonies, they found that R cells mixed w S cell DNA produced virulent colonies 7. thus, DNA was concluded to have a biological property and is the genetic material 8. R cells took up DNA from the S cells and incorporated that into their own DNA 1. the gene that was mutated in the R cell was replaced w the wt copy from the S cell DNA 6 9. genetic transformation is a process by which free DNA is incorporated into a recipient cell and brings about genetic change 1. transformation = genetic change 10. a cell that is competent is a cell that can take up DNA 1. not all bacterial cells can take up DNA 11. natural competence are cells in the wild that can take up DNA 12. artificially-induced competence is like E. coli where Ca2l is used to make it take up DNA 13. Griffith et al. used streptococcus pneumonia which is competent 1. if they used staphylococcus aureus, their experiment would have never worked 8 14. in gm+ bacteria, competence in the ability to take up DNA is not expressed all the time but is regulated w growth phase 15. competence relies on culture density 16. this is caused by quorum sensing and a quorum sensing-activated pathway 9 17. the competence-stimulating pheromone (a peptide) is synthesized in the cytoplasm as a precursor form 18. it is processed and exported out of the cell 19. the whole culture is now able to sense the concentration of this using a sensor kinase (comD), which then phosphorylates comE 20. phosphorylated comE turns on the gene comX 21. comX is a sigma factor 22. it activates the transcription of late competence genes which is the machinery required to take up the DNA in the envmt 23. it also encodes a negative regulatory protein in the late competence genes that eventually turns off expression of the com operon – so that it isn’t expressed all the time 24. bacteria that were previously not competent placed in another condition, they can become competent 1. there are many bacteria that have competence genes in their DNA that aren’t activated 2. one example is vibrio cholerae 10 25. when vibrio cholerae is grown in the presence of chitin (exoskeleton of some animals), they will form biofilm on the chitin and turn competent 26. non-pathogenic vibrio species probably turned pathogenic bc it obtained virulence genes through HGT using that mechanism 11 27. gm+ and gm- have differences in the competence apparatus 28. gm- bacteria has has to get a large molecule of DNA that across two membranes 29. in gm+ cell, there is sequence-independent binding of DNA (doesn’t matter what the seq is) 1. there are more 50+ binding sites per cell 2. DNA is fragmented into ~18-20kb 3. one strand of DNA is degraded, one is transported inside the cell 30. gm- bacteria have fewer binding sites 1. there is sequence-specific binding – looks for particular seq motifs that directs it to take in certain DNA 2. DNA is taken into periplasm then one strand is degraded while the other is imported into the cytoplasm 12 31. a macromolecular machine is required for this process 1. NRG is required 2. multiple proteins forming a complex in the membrane is required 1. this complex is responsible for pumping in the DNA 32. gm+ competence system contains a short pseudopilus (comG), a nuclease (NucA) responsible for fragmenting the DNA, DNA-binding protein (comEA), ATPase helicase (comFA) helps unwind the DNA and is a motor protein, transmembrane pore (ComEC) 1. inside the cell, there is the ssDNA binding proteins 13 33. the pseudopilus it thought to grab onto DNA and brings it to the comEA DNA-binding protein where NucA can act on it to fragment it 14 34. 3’ end of the DNA is brought into the cell after fragmentation by NucA 15 35. the 5’ end gets degraded 36. once inside, the DNA gets locked up by the ssDNA-binding protein – prevents it from being a reversible reaction and sliding back out 16 37. the DNA then gets engaged to the recombination complex 17 38. one of the key differences in the gm- bacteria is that it only imports DNA when it has a particular sequence (DUS) 39. diff bacteria recognize diff sequences 40. when look at the genomes of the bacteria, there is a fairly large DNA motif 1. you only find these motifs a couple of times in bacterial genome, these sequences are found at frequencies much higher than would be expected by chance in their respective genomes
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