1. Chapter 13 Microbial Genetics: Mechanisms of Genetic Variation 1

2. Study Objects 2 After reading this chapter, the student should be able to: discuss the nature of procaryotic recombination distinguish horizontal gene transfer from vertical gene transfer compare and contrast conjugation, transformation, and transduction discuss how transposable elements can move genetic material between bacterial chromosomes and within a chromosome to cause changes in the genome and the phenotype of the organism discuss the use of conjugation, transformation, and transduction to map the bacterial genome

3.  Initially characterized as altered phenotypes or phenotypic expressions.  Is a stable, heritable changes in sequence of bases in DNA  in procaryotes usually produce phenotypic changes  can occur spontaneously or be induced by chemical mutagens or radiation 3 Mutations [ mutare, to change]

4. 4 Examples of Mutagens Table 13.1

5.  arise without exposure to external agents.  This class of mutations may result from errors in DNA replication due to base tautomerization resulting in transition and transversion mutations due to insertion or deletion of nucleotides  may also result from the action of mobile genetic elements such as transposons 5 Spontaneous Mutations

6. Keto-enol Tautomerizations Tautomers are two forms of the same compound which differ by positions of a hydrogen atom and a pi bond. Ketones and enols are tautomers. The form which exists in highest concentration is determined by their relative stability. In most cases, the keto form is the more stable tautomer. 6 Keto-enol Tautomerizations

7. 7 Transition and Tautomerization Mutations

8. 8 Figure 13.1 (b)

9. 9 Additions and Deletions Figure 13.2

10.  alternative (and controversial) hypothesis proposed that some bacteria can select which mutations occur so they can better adapt to their environment  explanation may be that hypermutation occurs the random generation of multiple mutations with only those cells containing the favorable mutations able to survive 10 Directed or Adaptive Mutation

11.  caused by agents that directly damage DNA, alters its chemistry, or interferes with repair mechanisms base analogs structurally similar to normal bases mistakes occur when they are incorporated into growing polynucleotide chain DNA modifying agents alter a base causing it to mispair Intercalating agents distort DNA to induce single nucleotide pair insertions and deletions 11 Induced Mutations

12. 12 Base Analog – 5-Bromouracil Figure 13.3

13. 13 DNA Modifying Agent – Methyl-Nitrosoguanidine Figure 13.4

14.  results in formation of thymine dimers  the resulting DNA can no longer serve as a template 14 Ultraviolet (UV) damage of DNA Thymine Dimers – Produced by UV Radiation

15.  wild type most prevalent form of gene  forward mutation wild type  mutant form  reversion mutation mutant phenotype  wild type phenotype suppressor mutation occurs when the second mutation is at a different site than the original mutation 15 The Expression of Mutations

16.  in protein-coding genes can affect protein structure in a variety of ways  are named according to if and how they change the encoded protein  the most common types are: silent, missense, nonsense and frameshift mutations 1. silent mutation – change nucleoside sequence of codon – but not the encoded amino acid 2. missense mutation – a single base substitution that changes codon for one amino acid into codon for another amino acid 3. nonsense mutation - converts a sense codon to a stop codon 4. frameshift mutation – results from insertion or deletion of one or two bases pairs in the coding region of the gene 16 Point Mutations

17. 17 Types of Point Mutations – Forward Mutations

18. 18 Frameshift Mutation Figure 13.6

19.  conditional mutations – expressed only under certain environmental conditions  auxotrophic mutant – unable to make an essential macromolecule such an amino acid or nucleotide has a conditional phenotype wild-type strain from which it arose is called a prototroph 19 Other types of mutations

20.  mutations in regulatory sequences in lac operon, many of these mutations map in the operator site and produce altered operator sequences not recognized by repressor operon is always transcribed and  - galactosidase is always synthesized  Mutations in tRNA and rRNA genes protein synthesis is disrupted 20 More Types of Mutations

21.  mutations are generally rare one per 10 7 to 10 11 cells  finding mutants requires sensitive detection methods and/or methods to increase frequency of mutations Mutant Detection  observation of changes in phenotype  replica plating technique used to detect auxotrophic mutants 21 Detection and Isolation of Mutants

22. 22 Replica Plating Figure 13.7

23. 23 Mutant Selection use of environmental condition in which only desired mutant will grow e.g., selection for revertants from auxotrophy to prototrophy Figure 13.8

25.  based on observation that most carcinogens are also mutagens  tests for mutagenicity are used as screen for carcinogenic potential  e.g., Ames test for mutagenicity  Carcinogenicity Test mutational reversion assay 25 Carcinogenicity Testing

26. 26 reversion rate in presence of suspected carcinogen > reversion rate in absence of suspected carcinogen then, agent is a mutagen, and may be carcinogen All of the histidine auxotrophs will grow for the first few hours in the presence of the test compound until the His is depleted. Once the His is exhausted, only revertants that will grow. Ames Test

27.  Replication errors and a variety of mutations can alter the nucleotide sequence, a microorganism must be able to repair changes in the sequence that might be lethal.  proofreading correction of errors in base pairing made during replication errors corrected by DNA polymerase  other DNA repair mechanisms also exist 27 DNA Repair

28.  corrects damage that causes distortions in double helix  two types of repair systems are known nucleotide excision repair base excision repair both remove the damaged portion of the DNA strand and use the intact complementary strand as a template to synthesize new DNA 28 Excision Repair

29. 29 Nucleotide Excision Repair in E. coli UvrABC endonuclease

30. 30 Base Excision Repair Figure 13.11

31.  photoreactivation used to directly repair thymine dimers thymines separated by photochemical reaction catalyzed by photolyase  direct repair of alkylated bases catalyzed by alkyltransferase or methylguanine methyltransferase 31 Direct Repair

32.  type of excision repair  e.g., mismatch repair system in E. coli mismatch correction enzyme scans newly synthesized DNA for mismatched pairs mismatched pairs removed and replaced by DNA polymerase and DNA ligase 32 Mismatch Repair

33.  used by E. coli mismatch repair system to distinguish old DNA strands from new DNA strands old DNA (template strand) methylated; new DNA not methylated the repair system cuts out the mismatch from the unmethylated strand  catalyzed by DNA methyltransferases 33 DNA methylation DNA adenine methyltransferase (DAM)

34.  repairs DNA with damage in both strands  involves recombination with an undamaged molecule in rapidly dividing cells, another copy of chromosome is often available  RecA protein catalyzes recombination events 34 Recombinational Repair

35.  Despite having multiple repair systems, sometimes the damage to an organism’s DNA is so great the normal repair mechanisms can’t repair all the damage.  DNA synthesis stops completely and SOS response is activated.  inducible repair system  a global control network  used to repair excessive damage that halts replication, leaving many gaps recA protein initiates recombination repair recA protein also acts as protease, destroying a repressor protein and thereby increasing production of excision repair enzymes 35 The SOS Response

36.  mutations are subject to selective pressure Each mutant form that survives becomes an allele, an alternate form of a gene  recombination is the process in which one or more nucleic acids are rearranged or combined to produce a new nucleotide sequence 36 Creating Genetic Variability

37.  during meiosis, crossing over between homologous chromosomes creates new combinations of alleles  this is followed by chromosomal segregation into gametes and zygote formation  transfer of genes from parents to progeny is called vertical gene transfer 37 Recombination in Eucaryotes Recombination During Meiosis

38.  occurs in the three mechanisms evolved by bacteria to create recombinants  genes can be transferred to the same or different species  transfer of genes donor to recipient  exogenote DNA that is transferred to recipient  endogenote genome of recipient  merozygote recipient cell that is temporarily diploid as result of transfer process 38 Horizontal (Lateral) Gene Transfer (HGT) in Procaryotes

39. 39 Figure 13.16

40.  three types homologous recombination site specific recombination transposition 40 Recombination at the Molecular Level

41. 41 Homologous Recombination – Double-Strand Break Model Figure 13.17

42. Nonreciprocal Homologous Recombination incorporation of single strand of DNA into chromosome, forming a stretch of heteroduplex DNA proposed to occur during bacterial transformation 42 Nonreciprocal General Recombination

43.  important in insertion of viral genome into host chromosomes  there is only a small region of homology between inserted genetic material and host chromosome 43 Site-specific recombination

44.  segments of DNA that move about the genome in a process called transposition can be integrated into different sites in the chromosome  are sometimes called “jumping genes”  the simplest transposable elements are insertion sequences  transposable elements which contain genes other than those used for transposition are called composite transposons 44 Transposable Elements

45. 45 Types of Transposable Elements Figure 13.19

46. Insertion Sequences and Transposons Transposable element or transposon 1.Do not require extensive areas of homology 2.Behave somewhat like lysogenic prophage except that transposon originated in one chromosomal location and can move to a different location in the same chromosome. 3. Unable to reproduce autonomously and to exist apart from the chromosome

47. 47

48. 48 Simple Transposition Figure 13.20

49. 49 Replicative Transposition Figure 13.21

50. 50 The Tn3 Composite Transposon within an R Plasmid Figure 13.22

51.  small, autonomously replicating DNA molecules that can exist independently or, as episomes, integrate reversibly into the host chromosome  conjugative plasmids such as the F plasmid can transfer copies of themselves to other bacteria during conjugation 51 Bacterial Plasmids

52. 52 The F Plasmid Figure 13.23

53. Bacterial Conjugation The transfer of genes between bacteria that depends on Direct cell to cell contact mediated by the F pilus 53 Figure 13.24

54. 54 F Plasmid Integration Figure 13.25

55. 55 Evidence for Bacterial Conjugation Figure 13.26 Conjugation-Cell-Cell contact 에 의한 conjugation bridge 형성에 의하여 donor cell (F+) 의 ss-DNA 가 recipient (F-) cell 로 transfer 되는 process : Rolling circle model 에 의한 DNA 전달 Joshua Lederberg 와 Edward Tatum- 1946

56. 56 The U-Tube Experiment The U-tube experiment used to show that genetic recombination By conjugation requires direct physical contact between bacteria. Bernard David discovered that physical contact of the cells was necessary for gene transfer

57. 57 Figure 13.28(a) F + x F - Mating a copy of the F factor is transferred to the recipient and does not integrate into the host chromosome donor genes usually not transferred

58. 58 Figure 13.28 (b) and (c) HFr Conjugation donor HFr cell has F factor integrated into its chromosome donor genes are transferred to recipient cell a complete copy of the F factor is usually not transferred

59.  result when the F factor incorrectly leaves the host chromosome  some of the F factor is left behind in the host chromosome  some host genes have been removed along with some of the F factor these genes can be transferred to a second host cell by conjugation 59 F’ Conjugation

60. 60 F’ x F - Mating Figure 13.30

61. The mechanism of Bac. Conjugation. F+ x F- mating Hfr x F- mating Hfr: F factor 가 chromosome 의 특 정 site 에 crossover 에 의해 interation 된 donor cell. Hfr 에서 F-factor 의 interation 은 가 역적이다. 즉, interation 과 detachment 는 비 슷하게 일어난다

62. 그림 13-15 F’ conjugation Due to an error in excision, the A gene of an Hfr cell is picked up by the F factor and transferred to a recipient during conjugation

63.  uptake of naked DNA by a competent cell followed by incorporation of the DNA into the recipient cell’s genome 63 Bacterial Transformation

64. 64 Bacterial Transformation in S. pneumoniae Figure 13.32

65. 65 DNA Uptake System Figure 13.33

66. 그림 13-16 DNA transformation Griffith 가 Streptococcus pneumonia 를 통해 형질 전환시키는 active substance (transforming principle) 임을 증명 Avery, Macleod 가 transforming principle 이 DNA 임을 밝힘 Competent cell: transforming DNA 를 받아 들일수있는 cell 로 cell life cycle 동안 특정 시기에 출현

67. The mechanism of transformation: A long dsDNA binds to the surface with the aid of a DNA- binding protein and nicked by the nuclease. One strand is degraded by the nuclease. The single strand enters the cell and is integrated into the host chromosome in place of the homologous region of the host DNA

68.  the transfer of bacterial genes by viruses  viruses (bacteriophages) can carry out the lytic cycle in which the host cell is destroyed or the viral DNA can integrate into the host genome, becoming a latent prophage 68 Transduction

69. 69 Lytic and Lysogenic Cycles of Temperate Phages Figure 13.34

70.  any part of bacterial genome can be transferred  occurs during lytic cycle  during viral assembly, fragments of host DNA mistakenly packaged into phage head generalized transducing particle 70 Generalized Transduction

71.  carried out only by temperate phages that have established lysogeny  only specific portion of bacterial genome is transferred  occurs when prophage is incorrectly excised 71 Specialized Transduction

72. Lytic versus Lysogenic Infection by Phage Lambda Transduction: donor bac. 에서 recipient bac. 로 bac. Chromosome 의 일부가 bacteriophage 에의해 전달되는 gene transfer 과정

73. 73 The Mechanism of Transduction for Phage Lambda and E. coli Figure 13.37

74.  locating genes on an organism’s chromosomes  mapping bacterial genes accomplished using all three modes of gene transfer Hfr mapping  used to map relative location of bacterial genes  based on observation that chromosome transfer occurs at constant rate  interrupted mating experiment Hfr x F - mating interrupted at various intervals order and timing of gene transfer determined 74 Mapping the Genome

75. 75 Mapping the Genome The Interrupted Mating Experiment Figure 13.38 (a)

76. 76 Mapping the Genome The Interrupted Mating Experiment Figure 13.38 (b)

77. E. coli genetic map gene locations expressed in minutes, reflecting time transferred made using numerous Hfr strains 77 Figure 13.40

78.  used to establish gene linkage  expressed as frequency of cotransformation  if two genes close together, greater likelihood will be transferred on single DNA fragment 78 Transformation mapping

79. 79 Mapping the Genome A cotransduction Experiment Figure 13.39

80.  used to establish gene linkage  expressed as frequency of cotransduction  if two genes close together, greater likelihood will be carried on single DNA fragment in transducing particle 80 Generalized transduction mapping

81.  provides distance of genes from viral genome integration sites  viral genome integration sites must first be mapped by conjugation mapping techniques 81 Specialized transduction mapping

82.  viral genomes can also undergo recombination events  viral genomes can be mapped by determining recombination frequencies  physical maps of viral genomes can also be constructed using other techniques 82 Recombination and Genome Mapping in Viruses

83. Genetic Recombination in Bacteriophages recombination frequency determined when cells infected simultaneously with two different viruses 83

84. 84 The Formation of Phage Plaques Figure 13.42