Genetic Adaptation II. Microbial Physiology Module 3

Transcription

1 Genetic Adaptation II Microbial Physiology Module 3

2 Topics Topic 4: Topic 5: Transposable Elements Exchange of Genetic Material Between Organisms Topic 5a: Protection Against Foreign DNA

3 Aims and Objectives By the end of this module you should be able to distinguish between IS and Tn elements understand the importance of transposable elements to the evolution and adaptation of microorganisms have a detailed understanding of the three main mechanisms of DNA transfer and be able to distinguish between them know how bacteria protect themselves against the introduction of foreign DNA

4 Last Time Look at How mutations arise Macrolesions Insertions, deletions, inversions, duplications Microlesions Frame-shift mutations Base substitutions Transitions Transversions Point mutations Silent Missense Nonsense

5 Last Time Mutations can allow organisms to acquire new functions over many generations, under suitable conditions (selective pressures)

6 This time Look at how organisms can acquire genetic material from other sources Transposable elements Other bacteria Transformation Transduction Generalised Specialised Conjugation

7 Recombination Central to all the mechanisms of DNA transfer via transposable elements, transduction, transformation and conjugation Recombination can be homologous or non-homologous non-homologous is also called site-specific reciprocal or non-reciprocal

8 Homologous Recombination Occurs between DNA sequences that are the same or very similar Results in a reciprocal exchange of genetic material between the two sources Best example: crossing over that occurs between pairs of chromosomes Bacterial example: Gene mapping using conjugation of Hfr with F - cells

9 Site-specific Recombination Exchange of material in sequences that show little or no sequence homology Exchange is non-reciprocal DNA transfer only occurs one way Can only happen at specific sites in the genome Example: Prophage formation in bacteriophage λ

10 Topic 4: Transposable Elements Elements within the bacterial genome (chromosome and accessory genetic elements) that are capable of translocating to new locations Jumping genes Transfer generally occurs through non-homologous recombination Some leave a copy in the original location Some move without duplicating the sequence Two types of transposable genetic elements Insertion sequences (IS) Transposons (Tn)

11 Insertion Sequences Simplest transposable elements Small Don t code for structural proteins Code for transposase Enzyme responsible for facilitating transposition Functions of the transposase Helps create the insertion site Joins the IS ends to the target site Fills all the gaps created by insertion

12 Structural Features of IS Inverted terminal repeats IS gene(s) Target site duplication (direct repeats)

13 Mechanism of Transposition Transposase TAGGGCAAA ATCCCGTTT IS AAACGGGAT TTTGCCCTA Inverted terminal repeats ACCGTCGGCATCA TGGCAGCCGTAGT Target site

14 Mechanism of Transposition Transposase TAGGGCAAA ATCCCGTTT IS AAACGGGAT TTTGCCCTA Transposase ACCGTCGGCATCA TGGCAGCCGTAGT DNA is cleaved at target site

15 Mechanism of Transposition TAGGGCAAA ATCCCGTTT IS AAACGGGAT TTTGCCCTA Transposase ACCGTCGGCATCA TG CA TGGCAGCCGTAGT

16 Mechanism of Transposition ACCGTCGGCATCA TG TAGGGCAAA ATCCCGTTT IS AAACGGGAT TTTGCCCTA CA TGGCAGCCGTAGT

17 Mechanism of Transposition ACCGTCGGCATCA TGGCAGCCGTAGT TAGGGCAAA ATCCCGTTT IS AAACGGGAT TTTGCCCTA ACCGTCGGCATCA TGGCAGCCGTAGT Transposase Transposase

18 Mechanism of Transposition Target site duplication (direct repeats) Inverted terminal repeats ACCGTCGGCATCA TGGCAGCCGTAGT TAGGGCAAA ATCCCGTTT IS AAACGGGAT TTTGCCCTA ACCGTCGGCATCA TGGCAGCCGTAGT

19 IS General Features Inverted terminal repeats Inverted repeats (generally) 9 to 40 bp Target site duplications Direct repeats 2 to 13 bp IS Size (bp) Target site duplication Terminal Repeats IS , inverted IS , inverted IS , inverted

20 Functions of IS Chromosomes may contain multiple copies of IS elements E. coli chromosome has 6-10 copies of IS1 May also be present on plasmids F plasmid has IS2 and IS3 When also present on chromosome Homologous recombination between like Is Integration of F plasmid into chromosome Hfr Higher frequency recombinant Promotes genetic exchange (including transposons)

21 Transposons (Tn) Also called composite transposons More complex than IS involve genes other than those required for transposition larger Formed by the proximity of two ISs on a chromosome sequence between the ISs is captured an transposed along with the IS elements

22 Tn9 Direct repeats of IS1 768 bp 768 bp Tn9 IS1 cam R IS1 23 bp IS1 terminal inverted repeats

23 Tn5 Inverted repeats of LS bp 1533 bp IS50L kan R ble R str R IS50R 9 bp terminal inverted repeats

24 Regulation of Tn5 transposition Different IS elements at each end IS50L and IS50R IS50R produces 2 proteins Transposase Repressor translated from start site within the transposase gene more abundant than the transposase

25 Regulation of Tn5 transposition IS50L kan R ble R str R IS50R Transposase Repressor Phage DNA containing Tn5 Phage DNA containing Tn5 Tn5 Tn5 Repressor > Transposase: Tn5 occasionally transposes Repressor >>> Transposase: Tn5 seldom transposes Tn5 Genomic DNA without Tn5 Genomic DNA containing Tn5

26 Tn3 Elements Larger than IS elements Have inverted terminal repeats Produce target site duplication Carry genes other than other required for transposition Not flanked by other IS elements Transposition is a two-step process cointegration resolution

27 Tn3 Structure 38 bp inverted terminal repeats tnpa tnpr bla Transposase Resolvase / Repressor B-lactamase

28 Tn3 Transposition: Cointegration Tn3 Donor Plasmid Transposase Recipient Plasmid Cointegrate

29 Tn3 Transposition: Resolution Cointegrate Resolvase Tn3 Tn3 Donor Plasmid Recipient Plasmid

30 Conservative and Replicative Transposition Conservative transposition transposon is excised from one location and inserted into another no duplication of transposon DNA eg. Tn5 Replicative transposition transposon DNA is duplicated during the transposition process source transposon remains in its original location eg. TnA transposon such as Tn3

31 Important Note re Transposition Transposition IS (essentially) a recombination event Generally does not involve homologous sequences i.e. is non-homologous Does not use the general recombination system Happens at specific DNA sequences Site-specific recombination transposase used instead of RecA

32 Properties of IS and Tn elements Name Size Direct Terminal Repeats Genes (bp) Repeats Carried IS bp 23 bp, inverted None IS bp 32 bp, inverted None IS bp 32 bp, inverted None Tn bp 38 bp, inverted amp R Tn bp 1400 bp (IS50), inverted kan R, ble R, str R Tn bp 768 (IS1), direct cam R Tn bp 1400 bp (IS10), inverted tet R

33 Significance of Transposable Elements Medical significance transmission of antibiotic resistance genes passage of genes from chromosome to plasmid from plasmid to other cells cells need not be the same strain process occurs in pathogenic strains of Staphylococcus, Enterococcus, Neisseria, Shigella and Salmonella bacteria can acquire resistance to multiple antibiotics MDR strains multiple drug resistant

34 Phase Variation Phenomenon observed in Salmonella Associated with flagella formation Flagella are usually composed of a single protein flagellin Phase variation results in flagella proteins being one of two different types Which protein is produced depends on the orientation of an invertible DNA segment

35 Topic 5: Exchange of Genetic Material between Organisms Mutations are essential for the generation of microbial diversity The ability to test combinations of mutations under selective pressure accelerates the evolutionary process Process accelerated by the ability of bacteria to acquire DNA from other sources Three mechanisms Transformation Transduction Conjugation

36 Transformation The uptake of naked DNA from the environment into a recipient cell free DNA is usually the result of lysis of the donor cell Only competent cells can be transformed secrete competency factors which induce 8-10 proteins required for transformation Competency depends on growth stage best if dividing at maximal rate competency can be increased by cations and temperature

37 Mechanism of Transformation in Streptococcus Naked DNA Membrane-Associated DNA binding protein Nuclease Chromosome

38 Mechanism of Transformation in Streptococcus DNA binds to the binding protein Nuclease nicks and degrades on strand

39 Mechanism of Transformation in Streptococcus Nucleotides released

40 Mechanism of Transformation in Streptococcus

41 Mechanism of Transformation in Streptococcus Integration of single stranded DNA into chromosome

42 Mechanism of Transformation in Streptococcus Heteroduplex chromosome

43 Mechanism of Transformation in Streptococcus Replication

44 Successful Transformation DNA degraded by recipient: Unsuccessful Transformation DNA incorporated into recipient genome: Successful Transformation

45 Transformation Conclusion Transformed plasmid DNA need not recombine to be maintained by the cell Natural Transformation Gram Positive Gram Negative Artificial Transformation Streptococcus pneumoniae Bacillus subtilus Bacillus cereus Neisseria gonorrheae Haemophilus influenzae Pseudomonas stuzeri Escherichia coli Salmonella Typhimurium Pseudomonas aeruginosa

46 Transduction Transfer of DNA from a donor to a recipient via a bacteriophage (bacterial virus) Uses errors in the life cycle of the bacteriophage to transfer the DNA Two classes of transduction generalised specialised

47 Bacteriophage Lifecycles: Lytic

48 Bacteriophage Lifecycles: Lysogeny

49 Generalised Transduction Replication cycle of virulent bacteriophage has 3 phases invasion replication release During infection host DNA is fragmented occasionally packaged instead of phage DNA defective phage can be any DNA fragment Defective phage can infect can t replicate or induce lysis

50 Generalised Transduction Mechanism Virus infects cell Viral DNA Host DNA

51 Infection Viral DNA enters cell

52 Degradation of Host DNA Fragmentation of host DNA by viral enzymes

53 Replication Virus coat protein

54 Assembly Wild-type phage Transducing phage

55 Lysis

56 Infection by Transducing Phage Virus infects cell Donor DNA Host DNA

57 Infection Donor DNA enters cell

58 Fate 1: Degradation X Unsuccessful gene transfer X Donor DNA is degraded by host enzymes

59 Fate 2: Not Replicated X Abortive transduction X Donor DNA is stable but does not integrate into the chromosome

60 Fate 3: Integration Stable gene transfer Donor DNA replace homologous region in recipient chromosome

61 Specialised Transduction Utilises the lysogenic cycle of some bacteriophage eg. bacteriophage λ Phage DNA is propagated along with the cell s chromosome Integration into the chromosome is a site-specific recombinational event DNA transfer is the result of errors in the bacteriophage lifecycle Only DNA around the site of insertion can be transferred

62 Prophage Prophage: Integration of phage DNA into host chromosome at a specific site Host chromosome

63 Induction Prophage excision error incorporates some host DNA with the phage DNA

64 Prophage Initiation of the Lytic Cycle

65 Replication

66 Assembly Specialised transducing phage

67 Lysis

68 Infection by Transducing Phage Virus infects cell Donor DNA Host DNA

69 Infection

70 Fate 1: Recombination Recombination between homologous sequences

71 Fate 1: Recombination Bacterial chromosome contains donor DNA

72 Fate 2: Integration Normal bacteriophage

73 Fate 2: Integration

74 Fate 2: Integration Prophage DNA Donor DNA Recipient DNA Two copies of genes around the insertion point

75 Conjugation Sex in bacteria Direct transfer of DNA from one cell to another across a protein bridge pilus rolling-circle replication Requires an F-plasmid transfer of plasmid DNA from F + to F - cell when an episome can get Hfr transfer of chromosomal DNA

76 Bacterial Mating F + cell F - cell

77 Pilus Forms and Replication F + cell F - cell Rolling Circle Replication

78 Plasmid Transferred F + cell F + cell

79 Disrupt Mating F + cell F + cell

80 Higher Frequency Recombinants Hfr cell F - cell

81 Rolling-Circle Replication Hfr cell F - cell Rolling Circle Replication

82 Transfer Continues Hfr cell F - cell

83 Disrupt Mating Donor Recipient Recombination between homologous sequences

84 Successful Recombinant Donor Recipient

85 Distinguishing between Mechanisms of DNA Transfer Transfer Process Transformation Transduction Conjugation Cell-Cell contact required? No No Yes Sensitive to Dnase? Yes No No

86 Topic 5a: Protection Against Foreign DNA Repair mechanisms protect against mutation Mechanism exists to protect cells against foreign DNA Restriction endonucleases cleave DNA at specific sequences eg. EcoRI from Escherichia coli RY13 cuts at the sequence GAATTC 5 GAATTC 3 3 CTTAAG 5

87 Restriction Enzymes and the Bacterial Immune System Any DNA sequence that matches the recognition sequence of the enzyme will be cleaved eg. EcoR I cuts λ DNA 5 times What protects the cell from it s own REs? Methylation Specific sequences that would be recognised by the host RE are methylated RE can t digest methylated DNA bacterial immune system

88 Suggested Reading and Learning Exercises Atlas: Chapter 7, Sections 7-2 to 7-4 Learning Exercises Rolling-circle replication Bacteriophage Mu Module 3 question sheet

89 Next Week Module 4 Physiological Adaptation