Genetics appt


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Genetics appt

  1. 1. Bacterial Genetics
  2. 3. Bacterial Genetics <ul><li>“ Acquiring genes through gene transfer provides new genetic information to microorganisms, which may allow them to survive changing environments.” </li></ul><ul><li>“ The major source of variation within a bacterial species is mutation.” </li></ul><ul><li>“ In mutations, usually only a single gene changes at any one time.” </li></ul><ul><li>“ In contrast, gene transfer results in many genes being transferred simultaneously, giving the recipient cell much more additional genetic information.” </li></ul>
  3. 4. Mutation <ul><li>Types of mutations </li></ul><ul><li>1. Deletion </li></ul><ul><li>2. Insertion sequences (transposons) </li></ul><ul><li>3. Frameshift mutations </li></ul><ul><li>4. Silent mutations </li></ul><ul><li>5. Missense mutations </li></ul><ul><li>6. Nonsense mutations </li></ul><ul><li>7. Mutagen induced </li></ul>
  4. 5. Overview <ul><li>Most bacteria are haploid which means that there is no such thing as dominance-recessive relationships among bacterial alleles. </li></ul><ul><li>Bacteria don’t have sex in the animal/plant sense of sex (i.e., mating followed by recombination of whole genomes). </li></ul><ul><li>Instead, bacteria acquire DNA from other bacteria through three distinct mechanisms: </li></ul><ul><ul><li>Transformation </li></ul></ul><ul><ul><li>Transduction </li></ul></ul><ul><ul><li>Conjugation </li></ul></ul>
  5. 6. Overview <ul><li>This DNA may or may not then recombine into the recipient’s genome. </li></ul><ul><li>We use phrases like “Lateral” or “Horizontal” Gene Transfer to describe these sexual interactions. </li></ul><ul><li>Bacterial DNA is also subject to mutation, damage (not the same thing as mutation), and natural selection . </li></ul>
  6. 7. <ul><li>Wild Type refers to the microorganism as isolated from the wild. </li></ul><ul><li>A mutated microorganism that has lost a metabolic function, particularly an ability to synthesize a specific growth factor, is called an Auxotroph. </li></ul><ul><li>The wild-type parent to an auxotroph is called a Prototroph. </li></ul><ul><li>A Mutation is found in a gene; a mutant is an organism harboring a Mutation. </li></ul>
  7. 8. <ul><li>We designate mutant phenotypes using three-letter abbreviations; the phenotype of a tryptophan- requiring auxotroph would be described as Trp - . </li></ul><ul><li>A bacterium that has mutated to resistance to an antibiotic (or other substance) is given the superscript “R”; thus, the phenotype ampicillin resistance is indicated as Amp R . </li></ul><ul><li>Mutants can be spontaneous or induced by a Mutagen; an agent that causes DNA to mutate. </li></ul>
  8. 9. Types of Mutations <ul><li>Base Substitution </li></ul><ul><li>Point mutation = single base is substituted. </li></ul><ul><li>Missense mutation = base change changes single amino acid to different amino acid. </li></ul><ul><li>Nonsense mutation = base change changes single amino acid to stop codon. </li></ul><ul><li>Null or Knockout mutation = mutation that totally inactivates a gene. </li></ul>
  9. 10. Types of Mutations <ul><li>Deletion or insertion mutation = change in number of bases making up a gene. </li></ul><ul><li>Frameshift mutation = insertion or deletion of something other than multiples of three bases. </li></ul><ul><li>Frameshifts typically radically change downstream codons, generating stop codons, and typically knocking out gene function. </li></ul><ul><li>Reversion mutation = mutated change back to that of wild type. </li></ul>
  10. 13. Suppressor Mutation <ul><li>Overcomes the effect of the first mutation. </li></ul><ul><li>The second mutation is within the same gene, the change may be called a second site reversion or intergenic suppression. </li></ul>
  11. 14. Rates of Mutation <ul><li>The mutation rate of different genes usually varies between 10 -4 and 10 -12 mutations per cell division (essentially equivalent to per cell). </li></ul><ul><li>10 -4 = one in 10,000; 10 -12 = one in one trillion. </li></ul><ul><li>To calculate the probability of two independent mutations we multiple the two mutation rates. </li></ul><ul><li>Thus, if streptomycin resistance occurs at a rate of 10 -6 mutations per cell division and the rate of mutation to resistance to penicillin is 10 -8 then the rate of mutation to both antibiotics is 10 -6 * 10 -8 = 10 -14 (note that the exponents are added). </li></ul>
  12. 15. Rates of Mutation <ul><li>That is, we would have to have a population of one-hundred trillion cells to have one double mutant, which even for bacteria is a lot of cells. </li></ul><ul><li>This is the basis for Combination Therapy, e.g., the use of more than one chemotherapeutic against tuberculosis, HIV, cancer, etc. </li></ul><ul><li>The odds of sufficiently multiply resistant mutants drops with each new chemotherapeutic added to the mix. </li></ul>
  13. 16. Biochemical Mutations <ul><li>Prototrophs – can grow on minimal media </li></ul><ul><li>Resistant mutant – is a particular type of biochemical mutant that acquires resistance to some pathogen, chemical, or antibiotic. </li></ul><ul><li>Easy to select for and very useful in microbial genetics. </li></ul>
  14. 17. Biochemical Mutations <ul><li>Cause a change in the biochemistry of the cell. </li></ul><ul><li>Inactivate a biosynthetic pathway </li></ul><ul><li>Auxotrophs – cannot grow on minimal medium </li></ul>
  15. 18. The Expression of Mutations <ul><li>The expression of a mutation will only be noticed if it produces a detectable, altered phenotype. </li></ul><ul><li>Wild type  forward mutation </li></ul><ul><li>Reversion mutation  back mutation </li></ul>
  16. 19. Mutant Selection <ul><li>Replica plating – direct and indirect method. </li></ul><ul><li>Ames Test – developed in the 1970s by Bruce Ames, used to detect carcinogens </li></ul>
  17. 21. Direct Selection for Mutants
  18. 23. Indirect Selection: Isolation of ts Mutants This is one example of isolation of mutants carrying conditionally lethal mutations found in essential genes.
  19. 24. Indirect Selection: Replica Plating
  20. 25. Indirect Selection: Penicillin Enrichment
  21. 27. Microbial Genetics: Recombination and Plasmids <ul><li>Recombination is the process in which a new recombinant chromosome, one with a genotype different from either parent, is formed by combining genetic material from two organisms. </li></ul>
  22. 28. Self-Transmissible R Plasmid Resistance Transfer Factor (conjugation genes) Note multiple resistance genes.
  23. 29. Transfer Protection: R-M Systems <ul><li>Not all incoming DNA is necessarily good for the receiving bacterium (i.e., DNA can be parasitic). </li></ul><ul><li>Bacteria employ Restriction Enzymes to protect themselves from the foreign DNA. </li></ul><ul><li>Restriction enzymes recognize specific, palindromic (same spelling backward and forward) DNA sequences of 4 to 8 base pairs in length that are known as Recognition Sequences. </li></ul><ul><li>Bacteria also employ Modification Enzymes that modify DNA to protect it from Restriction Enzymes. </li></ul><ul><li>Together these are called Restriction-Modification Systems. </li></ul><ul><li>Restriction enzymes are crucial components of genetic engineering. </li></ul>
  24. 30. Replicons <ul><li>Is a DNA molecule or sequence that has a replication origin and is capable of being replicated. </li></ul><ul><li>Plasmids and bacterial chromosomes are separate replicons. </li></ul><ul><li>Plasmids – less than 30 genes, single or multiple copies </li></ul>
  25. 31. Curing <ul><li>Plasmids can be eliminated from host cells in a process known as curing. </li></ul><ul><li>May occur spontaneously or be induced by treatments that inhibit plasmid replication. </li></ul><ul><li>Acridine mutations, UV and ionizing radiation, thymine starvation, and growth above optimal temperature. </li></ul>
  26. 32. Bacterial Plasmids <ul><li>Conjugation, the transfer of DNA between bacteria involving direct contact, depends on the presence of an extra piece of circular DNA – plasmid DNA. </li></ul>
  27. 33. Classified: mode of existence and spread <ul><li>Episome – is a plasmid that can exist either with or without being integrated into the host’s chromosome. </li></ul>
  28. 34. Fertility Factors <ul><li>A plasmid called the fertility factor or F factor plays a major role in conjugation in E. coli . </li></ul><ul><li>About 94.5 kilobases long and bears genes responsible for cell attachment and plasmid transfer between specific bacterial strains during conjugation. </li></ul>
  29. 35. Resistance Factors <ul><li>R factors – often confer antibiotic resistance on the bacteria that contain them </li></ul><ul><li>Code for enzymes capable of destroying or modifying antibiotics. </li></ul><ul><li>Usually not integrated into host chromosome </li></ul><ul><li>Resistance genes are within a transposon </li></ul>
  30. 36. Bacterial Recombination <ul><li>Transformation </li></ul><ul><li>Transduction </li></ul><ul><li>Conjugation </li></ul>
  31. 37. Griffith’s Experiment
  32. 38. DNA as the Genetic Material The early work of Fred Griffith in 1928 on the transfer of virulence in the pathogen S. pneumoniae set the stage for DNA research.
  33. 39. Original Transformation Exp. F. Griffith (1928) using pneumococci
  34. 43. Transformation
  35. 45. Transduction <ul><li>Bacterial viruses participate in this mode of bacterial gene transfer. </li></ul><ul><li>Generatlized transduction </li></ul><ul><li>Specialized transduction </li></ul>
  36. 48. Generalized Transduction
  37. 49. Specialized Transduction
  38. 52. Generalized Transduction Bacteriophages are viruses that only infect (and can kill) bacteria.
  39. 57. Bacterial Conjugation <ul><li>F + x F - </li></ul><ul><li>HFr </li></ul><ul><li>F’ </li></ul>
  40. 59. Conjugation: Sex or F Pilus
  41. 67. DNA-Mediated Transformation Note that DNA is taken up naked from the environment.
  42. 68. DNA-Mediated Transformation
  43. 69. Artificial Competence by Electroporation Competence denotes the ability to take up DNA naked from the environment. Most bacteria are not naturally competent but many can be made artificially so. Artificially induced competence is very important to gene cloning.
  44. 70. Transfer of non-R Virulence Factors <ul><li>Genes that can make bacteria more virulent (able to cause disease) are called Virulence Factor genes. </li></ul><ul><li>Virulence factors include fimbriae that allow attachment to host tissues, exotoxins, etc. </li></ul><ul><li>Virulence factor genes may be transferred by transformation, transduction, or conjugation. </li></ul><ul><li>Virulence factor genes tend to congregate on bacterial chromosomes in regions known as Pathogenicity Islands. </li></ul><ul><li>New bacterial pathogens can emerge via the uptake of entire pathogenicity islands transferred intact from unrelated bacteria. </li></ul>
  45. 71. Restriction Endonuclease Action
  46. 72. Restriction Endonuclease Action Note in particular that DNA is cut at palindromic regions.
  47. 73. DNA Modification: RE Protection
  48. 76. Conjugation: F Plasmid Transfer
  49. 77. F and Other Plasmids <ul><li>F plasmids encode genes that allow both their replication and transfer. </li></ul><ul><li>They are thus known as Self-Transmissible Plasmids. </li></ul><ul><li>There are other plasmids that can take advantage of conjugation but don’t encode the the necessary genes. These are non-self transmissible plasmids. </li></ul><ul><li>Transduction is also capable of transferring smaller plasmids. </li></ul><ul><li>R plasmids are named not for their mode of transmission but instead for the resistance genes that they encode such as to antibiotics. </li></ul><ul><li>Some plasmids are present in bacteria in low copy numbers (1 or 2/bacterium) whereas other plasmids are present in high copy numbers (such 100s/bact.) . </li></ul><ul><li>Plasmids, R and otherwise, can have very wide host ranges allowing easy transfer of already evolved genes between bacterial species. </li></ul>
  50. 81. Generalized Transduction
  51. 84. Ames Salmonella Test