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Bacterial genetics

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  • Allolactose = isomer of lactose, inactivates the repressor P lease O ffer Z ach Y our A pple
  • Allolactose = isomer of lactose, inactivates the repressor P lease O ffer Z ach Y our A pple
  • Repressor
  • Repressor
  • Repressor
  • Repressor
  • Repressor
  • Transcript

    • 1. The Genetics of BacteriaBacterial GenomeControl of Gene Expression
    • 2. Bacterial Genome Circular, double stranded Nucleoid region  where DNA is tightly packed  Similar to nucleus in eukaryote but not surrounded by a membrane Genome contains one chromosomal DNA and many plasmids
    • 3. Bacterial Replication One origin of replication Bidirectional
    • 4. Asexual Bacterial Reproduction Binary fission Asexual form of reproduction rapid Bacterial colony = identical clone
    • 5. Origins of Mutation Although new mutations are individually rare (low probability of spontaneous mutation) bacterial proliferation (growth rate) is high Thus, new mutations can have a significant impact on genetic diversity when reproduction rate is high Contrast with slow reproductive organisms (e.g. humans) where heritable variation is not due to new mutations but due to sexual recombination of existing genetic information
    • 6. Mathematical Mutation E.coli reproduction  ~ 2 x 1010 new cells per day Spontaneous mutation rate  ~ 10-7 mutations per division (1 in 10 million) E. coli mutation rate  2 x 1010 divisions per day x 10-7 mutations per division  2000 bacteria with mutations per day
    • 7. Evidence of GeneticRecombination in Bacteria Even though new mutations are a major source of genetic variation in bacteria, genetic recombination adds more diversity Experiments:  Lederberg and Tatum  Griffith
    • 8. Lederberg & Tatum Experiment (1946) http://biology-forums.com/gallery/33_27_06_11_10_48_52.jpeg
    • 9. Lederberg & Tatum Experiment (1946) http://biology-forums.com/gallery/33_27_06_11_10_48_52.jpeg
    • 10. Lederberg & Tatum Experiment (1946) http://biology-forums.com/gallery/33_27_06_11_10_48_52.jpeg
    • 11. Lederberg & Tatum Experiment (1946) http://biology-forums.com/gallery/33_27_06_11_10_48_52.jpeg
    • 12. Example in textbook: Fig 18.12 Mutant Mutant E. coli need both arg makes arg Mixture of makes trp not trp mutants not arg and trp to survive Mutants that can’t make arg or trp will die unless provided in the medium Add both mutants together into same Grow on minimal medium minimal medium (solution of glucose and salts without arg and trp)  None should live… but some did!!! No colonies Colonies No colonies
    • 13. Recall: Griffith’s Experiment Recombinant
    • 14. Evidence of GeneticRecombination in Bacteria Spontaneous mutation alone could not explain why the mixture of cells could survive in minimal medium Cells that survived must have acquired genes from the other mutant strain
    • 15. Mechanism of GeneticRecombination Eukaryote:  Sexual reproduction / fertilization  Crossing over / meiosis Prokaryotes:  Conjugation  Transformation  Transduction
    • 16. Comparing Mechanism ofBacterial Genetic Recombination State of Mechanism Requirement State of donor recipient Physical contactConjugation via sex pili; Living Living F plasmid Free DNA in theTransformation Dead Living environment Killed byTransduction Bacteriophage Living bacteriophage
    • 17. Sexual Bacterial Reproduction Conjugation: direct transfer of genetic material between two joined bacterial cells One way DNA transfer:  “male” donor  “female” recipient Mechanism  Donor extends sex pili to recipient  Sex pilus retracts, pulling cells together  DNA transferred through cytoplasmic bridge http://static.open.salon.com/files/bacterial_conjugation21228227521.jpg
    • 18. Plasmids Small, circular, self-replicating pieces of DNA (separate from the bacterial chromosome) Contain a small number of genes Can incorporate themselves into the bacterial chromosome Episome: genetic elements that can exist either as a plasmid or as part of the bacterial chromosome
    • 19. Advantage of Plasmids Plasmids are not required for bacterial cells to survive under normal conditions Under stress, genes on plasmids can confer advantages (e.g. resistance) Plasmids increase genetic variation and thus the likelihood of survival in bacteria
    • 20. Important Plasmids F (fertility) plasmid facilitates genetic recombination  Genes for production of sex pili  Fig 18.15a (male = F+, female = F-)  female become male if receive F plasmid from male R (resistance) plasmid  genes that make bacteria resistant to antibiotics  Also carries genes encoding the sex pili
    • 21. Conjugation http://www.pc.maricopa.edu/Biology/rcotter/BIO%20205/LessonBuilders/Chapter%209%20LB/Ch9b9.html
    • 22. Transformation Alteration of a bacterial cell’s genotype by the uptake of naked foreign DNA When two different strains of bacteria are mixed together, eventually they will show characteristics of both strains
    • 23. Transformation
    • 24. Conditions for Transformation Some bacteria have surface proteins that recognize and transport DNA from closely related species Some bacteria only transform when placed in a specific type of environment  E.g. E.coli in high calcium concentration
    • 25. Control of Gene Expression Bacteria depend on their environment to survive If an essential nutrient is not present in the environment, bacteria must be able to synthesize the nutrient on its own However, bacteria do not need to produce the essential nutrient if it is already present in the environment
    • 26. Levels of Metabolic Control The amount of cellular products can be controlled by regulating:  Enzyme activity: alters protein function  Transcription: affects whether a gene is turned on or off
    • 27. Example:E. coli in human intestine No tryptophan present, needs to synthesize Tryptophan present, does not need to synthesize Feedback inhibition on the production of trp
    • 28. Levels of Metabolic Control Protein function level: adjust activity of enzymes already present Short term, quick onset regulation
    • 29. Levels of Metabolic Control Transcription level: control the expression of genes which affects number of enzymes produced Long term, slow onset regulation
    • 30. Gene Expression Constitutive expression:  genes which are always turned on  known as housekeeping genes  Example: regulatory genes Induced expression:  genes which are only turned on as needed  Example: structural genes
    • 31. Bacterial transcription: DNAcomponents Structural genes  Genes to be transcribed by RNAP Promoter  Region on DNA where RNAP binds to start transcription  acts as the on/off switch for genes Operator  region on DNA that controls access of RNAP to the genes  Can activate or repress transcription
    • 32. Operon A group of genes that share a single promoter Contains an promoter, operator and structural genes Results in production of proteins that have related functions. Example: enzymatic proteins that catalyze reactions in a metabolic pathway
    • 33. Operon Regulation Regulatory gene  region on DNA that codes for the production of the regulatory protein  upstream of the operon  constitutive expression: transcribed continuously Regulatory protein  Binds to operator and blocks attachment of RNAP  Allosteric: alternates between active and inactive forms
    • 34. Operon Regulation Effector molecule: any molecule that can regulate the activity of a protein Corepressor:  activates the repressor  causing regulatory protein to bind to the operator  inactivates gene expression Inducer:  inactivates the repressor  prevents regulatory protein from binding to the operator  activates gene expression
    • 35. trp operon Components trp operon Promoter Structural Genes Final product is tryptophan Regulatory Operator Gene Fig. 18.20a
    • 36. trp operon Components Found in E. coli Structural gene:  5 genes making 5 polypeptides that combine to make 3 enzymes  Enzymes participate in a sequence of steps to make tryptophan (trp) an amino acid
    • 37. trp operon Components trp operon Promoter Structural Genes Regulatory Operator Final product is tryptophan Gene Fig. 18.20a
    • 38. trp operon Regulation Regulatory gene (trpR)  Codes for an inactive trp repressor protein  Trp repressor binds to operator when activated by effector molecule  Constitutive expression
    • 39. trp operon Regulation Q: If regulatory protein (trp repressor) is being constitutively expressed how would that affect transcription? Q: Why is this a problem? How do you think the cells solves this problem? trp operon Promoter Structural Genes Regulatory Operator Final product is tryptophan Gene Fig. 18.20a
    • 40. trp operon Regulation Q: When do you want the repressor to bind to the trp operon? Q: Will the effector molecule repress or induce gene expression? trp operon Promoter Structural Genes Regulatory Operator Final product is tryptophan Gene Fig. 18.20a
    • 41. trp operon Regulation Effector molecule: tryptophan (trp)  A corepressor trp binds to regulatory protein (trp repressor) which binds to operator  prevents RNA polymerase from transcribing genes  Stops trp production
    • 42. trp operonAbsence of corepressor Inactive repressor Active transcription Fig. 18.20a
    • 43. trp operonPresence of corepressor Activated repressor by corepressor No transcription Negative feedback Fig. 18.20b
    • 44. Repressible operon Gene is normally “on” Transcription is inhibited when a molecule binds allosterically to a regulatory protein Negative feedback / feedback inhibition Anabolic pathways Example: Trp operon is turned off when tryptophan (corepressor) binds to repressor
    • 45. trp operon animationTutorial http://bcs.whfreeman.com/thelifewire/content/chAnimation http://highered.mcgraw-hill.com/olc/dl/120080/bi http://www.as.wvu.edu/~dray/219files/TrpAttenu (ignore information on attenuation)
    • 46. Models of Negative Regulation Repressible Operon (e.g. trp)  Repressor made in inactive form  activated by corepressor  Turns gene off Inducible Operon (e.g. lac)  Repressor made in active form  inactivated by inducer  Turns gene on
    • 47. lac operon Recall: Lactose = glucose + galactose
    • 48. lac Operon Simulation WebSite:  Spongelab.com  Explore  Biology  Games and Simulation  “Gene Machine: The Lac Operon”
    • 49. lac operon Components lac operon Promoter Structural Genes Regulatory Operator Gene Hydrolyze Lactose Adds acetyl lactose into transporter group to glucose and (into cell) galactose galactosePlease Offer Zach Your Apple Fig. 18.21
    • 50. lac operon Components Found in E. coli Structural Gene codes for 3 enzymes  β-galactosidase (lacZ gene): lactose hydrolysis into glucose and galactose  permease (lacY gene): membrane protein that transports lactose into the cell  transacetylase (lacA gene): adds acetyl group to galactose (significance in lactose metabolism unclear)
    • 51. lac operon Components lac operon Promoter Structural Genes Regulatory Operator GenePlease Offer Zach Your Apple Fig. 18.21
    • 52. lac operon Regulation Regulatory gene (lacI)  Codes for an active lac repressor protein  lac repressor binds to operator  Constitutive expression
    • 53. lac operon Regulation Q: What affect does the lac repressor have on gene expression? Q: Why would it be beneficial to the cell to have a lac repressor that is constitutively expressed? RNAP Transcription TranslationRepressor Fig. 18.21a
    • 54. lac operon Regulation Q: What would the effector molecule do when it binds to the lac repressor? What effect does that have on gene expression? Q: What molecule would be an appropriate effector molecule for the lac operon and why? RNAP Transcription Translation Repressor Fig. 18.21a
    • 55. lac operon Regulation Effector molecule: allolactose  An isomer of lactose  An inducer allolactose binds to regulatory protein (lac repressor)  Inactivates lac repressor preventing it from binding to the operator  Allows RNA polymerase to transcribe genes  Produces enzyme β-galactosidase which hydrolyzes lactose
    • 56. lac operon Regulation E. Coli cells mainly use glucose as a source of energy When living in a system with lactose, bacteria can hydrolyze lactose to produce glucose The lac operon is only turned on when lactose is present
    • 57. lac operonAbsence of Inducer Active repressor No transcription RNAP Transcription TranslationRepressor Fig. 18.21a
    • 58. lac operonPresence of Inducer: allolactose Inactivated repressor by inducer Active transcription RNAP Transcription Translation Inducer Molecule Repressor A molecule that can bind to the repressor to prevent the repressor from binding to the operator. A “derepressor” that induces transcription. Fig. 18.21b
    • 59. lac operonPresence of Inducer: allolactose Translation RNAP Transcription TranslationRepressor Fig. 18.21b
    • 60. lac operonPresence of Inducer: allolactose Fig. 18.21b
    • 61. Inducible operon Gene normally “off” Transcription is stimulated when a molecule binds allosterically to a regulatory protein Proteins made “on demand” Catabolic pathway (breakdown substances) Example: Lac operon turned on when allolactose (inducer) binds to repressor
    • 62. lac operon animationTutorial http://bcs.whfreeman.com/thelifewire/content/chp13/13 http://www.sumanasinc.com/webcontent/animations/co (looks the same as above)Animation http://vcell.ndsu.nodak.edu/animations/lacOperon/movi http:// www.dartmouth.edu/~cbbc/courses/movies/LacOperon. (a little low tech)
    • 63. Negative Gene Regulation Operons are switched off by the active form of the regulatory protein (repressor)  Trp operon: repressor activated by corepressor, binds to operator  Lac operon: repressor made in active form, binds to operator
    • 64. Overview of Gene Regulation Bacterial Gene Regulation Negative Gene Regulation Positive Gene Regulation (e.g. allolactose on lac operon) (e.g. cAMP on lac operon) An active regulatory protein An active regulatory protein turns gene expression OFF turns gene expression ON (repressed) (activated)Repressible Operon (e.g. trp) Inducible Operon (e.g. lac)Has the ability to be turned Has the ability to be turned ONOFF by a corepressor by an inducerGene expression normally ON Gene expression normally OFF
    • 65. Positive Gene Regulation Active form of the regulatory protein turns on or increases the transcription of the operon Animation of positive and negative regulation of the lac operon: http://highered.mcgraw-hill.com/olc/dl/120080/bi