1. Transferencia del material genético. Conjugación, transformación y transducción. Mapeo genético

2. Mutations in Bacteria Mutations arise in bacterial populationsMutations arise in bacterial populations –Induced –Spontaneous Rare mutations are expressedRare mutations are expressed –Bacteria are haploid –Rapid growth rate Selective advantage enriches for mutantsSelective advantage enriches for mutants Gene transfer occurs in bacteriaGene transfer occurs in bacteria

3. Gene Mapping in Bacteria and Bacteriophages Mapping bacteria, 3 different methods: Conjugation Conjugation Transformation Transformation Transduction Transduction Bacteriophage mapping: Bacteriophage gene mapping Bacteriophage gene mapping Cis-trans complementation test Cis-trans complementation test

4. Bacteria transfer (or receive) genetic material 3 different ways Transfer always is unidirectional, and no complete diploid stage forms. 1.Conjugation 2.Transformation 3.Transduction

5. Mating types in bacteria –Donor F factor (Fertility factor)F factor (Fertility factor) –F (sex) pilus –Recipient Lacks an F factorLacks an F factor Donor Recipient

6. General Features of Gene Transfer in Bacteria UnidirectionalUnidirectional –Donor to recipient Donor does not give an entire chromosomeDonor does not give an entire chromosome –Merozygotes Gene transfer can occur between speciesGene transfer can occur between species

7. Conjugation 1.Discovered by Joshua Lederberg and Edward Tatum in 1946. 2.Unidirectional transfer of genetic material between donor and recipient cells by direct contact. 3.Segment (rarely all) of the donor’s chromosome recombines with the homologous recipient chromosome. 4.Recipients containing donor DNA are called transconjugants.

8. Lederberg & Tatum (1946) Experiment demonstrating recombination in E. coli. Recombination of 2 complimentary auxotrophs gives rise to a strain that can synthesize all nutrients.Recombination of 2 complimentary auxotrophs gives rise to a strain that can synthesize all nutrients.

9. Bernard Davis experiment demonstrated that physical contact is required for bacterial recombination.

10. E. coli conjugation

11. Conjugation-transfer of the sex factor F 1.William Hayes (1953) demonstrated that genetic exchange in E. coli occurs in only one direction. 2.Genetic transfer is mediated by sex factor F. 3.Donor is F+ and recipient is F-. 4.F is a self-replicating, circular DNA plasmid (1/40 the size of the main chromosome).

12. Conjugation-transfer of the sex factor F 5.F plasmid contains an origin sequence (O), which initiates DNA transfer. Also contains genes for hair-like cell surface (F-pili or sex- pili), which aid in contact between cells. 6.No conjugation can occur between cells of the same mating type. 7.Conjugation begins when the F plasmid is nicked at the origin, and a single strand is transferred using the rolling circle mechanism. 8.When transfer is complete, both cells are F+ double-stranded.

13. Transfer of the F factor

14. Conjugation of high- frequency recombinant strains 1.No chromosomal DNA is transferred by standard sex factor F. 2.Transfer of chromosome DNA is facilitated by special strains of F+ integrated into the bacteria chromosome by crossing over. 3.Hfr strains = high frequency recombination strains. 4.Discovered by William Hayes and Luca Cavalli-Sforza. 5.Hfr strains replicate F factor as part of their main chromosome.

15. Conjugation of high- frequency recombinant strains 5.Conjugation in Hfr strains begins when F+ is nicked at the origin, and F+ and bacteria chromosomal DNA are transferred using the rolling circle mechanism. 6.Complete F+ sequence (or complete chromosomal DNA) is rarely transferred (1/10,000) because bacteria separate randomly before DNA synthesis completes. 7.Recombinants are produced by crossover of the recipient chromosome and donor DNA containing F+.

16. Transfer of the Hfr F + factor

17. Excision of the F + factor also occurs spontaneously at low frequency. 1.Begin with Hfr cell containing F +. 2.Small section of host chromosome also may be excised, creating an F’ plasmid. 3.F’ plasmid is named for the gene it carries, e.g., F’ (lac)

18. Using conjugation to map bacterial genes 1.Begin with appropriate Hfr strains selected from F + x F - crosses and perform an interrupted mating experiment. 2. HfrHthr+ leu+ azi R ton R lac+ gal+str R F - thr leu azi S ton s lac galstr S 3.Mix 2 cell types in medium at 37°C.

19. Using conjugation to map bacterial genes 4.Remove at experimental time points and agitate to separate conjugating pairs. 5.Analyze recombinants with selective media. 6.Order in which genes are transferred reflects linear sequence on chromosomes and time in media. 7.Frequency of recombinants declines as donor gene enters recipient later.

20. Interrupted mating experiment

21. Genetic map-results of interrupted E. coli mating experiment.

22. Generating a map for all of E. coli 1.Location and orientation of the Hfr F + in the circular chromosome varies from strain to strain. 2.Overlap in transfer maps from different strains allow generation of a complete chromosomal map.

23. Circular genetic map of E. coli Total map units = 100 minutes ~time required for E. coli chromosome to replicate at 37°C.

24. Significance Gram - bacteriaGram - bacteria –Antibiotic resistance –Rapid spread Gram + bacteriaGram + bacteria –Production of adhesive material by donor cells

25. Transformation Unidirectional transfer of extracellular DNA into cells, resulting in a phenotypic change in the recipient.Unidirectional transfer of extracellular DNA into cells, resulting in a phenotypic change in the recipient. First discovered by Frederick Griffith (1928).First discovered by Frederick Griffith (1928). DNA from a donor bacteria is extracted and purified, broken into fragments, and added to a recipient strain.DNA from a donor bacteria is extracted and purified, broken into fragments, and added to a recipient strain. Donor and recipient have different phenotypes and genotypes.Donor and recipient have different phenotypes and genotypes. If recombination occurs, new recombinant phenotypes appear.If recombination occurs, new recombinant phenotypes appear.

26. More about transformation Bacteria vary in their ability to take up DNA.Bacteria vary in their ability to take up DNA. Bacteria such as Bacillus subtilis take up DNA naturally.Bacteria such as Bacillus subtilis take up DNA naturally. Other strains are engineered (i.e., competent cells).Other strains are engineered (i.e., competent cells). Competent cells are electroporated or treated chemically to induce E. coli to take up extracellular DNA.Competent cells are electroporated or treated chemically to induce E. coli to take up extracellular DNA.

27. Bacteria known to be capable of transformation Natural transformationNatural transformation –Gram positive bacteria Streptococcus pneumoniae, S. sanguis, B. Subtilis, B. Cereus, B. StearothermophilusStreptococcus pneumoniae, S. sanguis, B. Subtilis, B. Cereus, B. Stearothermophilus –Gram negative bacteria Neisseria gnonorrheae, Acinetobacter calcoaceticus, Moraxella osloensis, M. urethansNeisseria gnonorrheae, Acinetobacter calcoaceticus, Moraxella osloensis, M. urethans Psychrobacter sp., Azotobacter agilis, Haemophilus influenzae, H. Parainfluenzae, Pseudomonas stutzeriPsychrobacter sp., Azotobacter agilis, Haemophilus influenzae, H. Parainfluenzae, Pseudomonas stutzeri Artificial transformationArtificial transformation Escherichia coli, Salmonella thyphimurium, Pseudomonas aeruginosas y muchas otras.Escherichia coli, Salmonella thyphimurium, Pseudomonas aeruginosas y muchas otras.

28. Transformation –Recombination Legitimate, homologous or generalLegitimate, homologous or general recA, recB and recC genesrecA, recB and recC genes Significance Significance – Phase variation in Neiseseria – Recombinant DNA technology StepsSteps –Uptake of DNA Gram +Gram + Gram -Gram -

29. Heteroduplex DNA Transformation of Bacillus subtilis

31. Hanahan and Bloom, 1996, Chapter 132, Escherichia coli and Salmonella, ASM Press Chemical competence In some bacteria, including E. coli, treatment of cells with divalent cations at low temperature, facilitates the uptake of plasmid DNA into the cell (linear DNA can be taken up, but is shredded by cytoplasmic DNases before it can do anything)In some bacteria, including E. coli, treatment of cells with divalent cations at low temperature, facilitates the uptake of plasmid DNA into the cell (linear DNA can be taken up, but is shredded by cytoplasmic DNases before it can do anything) Remains unclear how this worksRemains unclear how this works Uptake channels made of polyP, PHB, and Ca

32. High field strengths result in very transient holes in the cellular envelope Under the appropriate conditions, DNA leaks in and DNA leaks out. A high concentration of plasmid outside results in a rapid influx of plasmids into the cell. Electroporationcuvette Cells go here High voltage shock Electroporation

33. Transformation efficiency Saturating cells (# of transformants/  g of DNA) 10 6 -10 9 /  g of pBR322 app. 10 11 plasmids/  g pBR322 can also be analyzed as % of cells that receive plasmid Saturating DNA % of DNA molecules that successfully transform cells How well has your transformation worked? ProtocolSat. cellsSat. DNA Chemical1%12% Electro10%90%

34. Dubnau. 1999. Ann. Rev. Microbiol. 53:217 Natural transformation in Gram positives Examples: Streptococcus pneumoniae Bacillus subtilis no base specificityno base specificity limited # of uptake sites (30-75)limited # of uptake sites (30-75) nicked internallynicked internally complement is degraded during transportcomplement is degraded during transport recombines in recipientrecombines in recipient

35. Dubnau. 1999. Ann. Rev. Microbiol. 53:217 Natural transformation in Gram negatives Examples: Haemophilus influenzae Neisseriae gonorrhoeae sequence specific – uptake sequencessequence specific – uptake sequences 4-8 sites/cell4-8 sites/cell no cell bound intermediateno cell bound intermediate import of ds DNA to periplasmimport of ds DNA to periplasm complement is degraded during transport into cytoplasmcomplement is degraded during transport into cytoplasm recombines in recipientrecombines in recipient

36. Dubnau. 1999. Ann. Rev. Microbiol. 53:217 the reverse of a conjugal transfer system - some components similar to Tra functions Gram positive uptake machinery -dedicated machinery for the transport of DNA into the cell

37. - must cross periplasm and outer membrane Dubnau. 1999. Ann. Rev. Microbiol. 53:217 Gram-negative uptake machinary

38. Energy for driving the process? Intracellular ATP hydrolysisIntracellular ATP hydrolysis pH gradient – PMF?pH gradient – PMF? Complement degradationComplement degradation

39. Function for natural transformation NutritionNutrition DNA repairDNA repair Genetic diversificationGenetic diversification

41. Mapping using transformation Recombination frequencies are used to infer gene order. p+q+ o+x p q o 1.If p+ and q+ frequently cotransform, order is p-q-o. 2.If p+ and o+ frequently cotransform, order is p-o-q.

42. Transduction 1.Bacteriophages (bacterial viruses) transfer genes to bacteria (e.g., T2, T4, T5, T6, T7, and ). 1.Generalized transduction transfers any gene. 1.Specialized transduction transfers specific genes. 2.Phages typically carry small amounts of DNA, ~1% of the host chromosome. 3.Viral DNA undergoes recombination with homologous host chromosome DNA.

43. Transduction Genetic exchange mediated by bacterial viruses (bacteriophage)Genetic exchange mediated by bacterial viruses (bacteriophage) Two basic types of bacterial virusesTwo basic types of bacterial viruses Lytic viruses – infect cells, multiply rapidly, lyse cellsLytic viruses – infect cells, multiply rapidly, lyse cells Lysogenic viruses – infect cells, can integrate into genome and go dormant (a prophage)Lysogenic viruses – infect cells, can integrate into genome and go dormant (a prophage) - at some point, can excise, multiply and lyse cells.- at some point, can excise, multiply and lyse cells.

44. Phage Composition and Structure CompositionComposition –Nucleic acid Genome sizeGenome size Modified basesModified bases –Protein ProtectionProtection InfectionInfection Structure (T 4 )Structure (T 4 ) –Size –Head or capsid –Tail Tail Tail Fibers Base Plate Head/Capsid Contractile Sheath

45. Infection of Host Cells by Phages Adsorption Adsorption – LPS for T4 Irreversible attachmentIrreversible attachment Sheath ContractionSheath Contraction Nucleic acid injectionNucleic acid injection DNA uptakeDNA uptake

46. Types of Bacteriophage Lytic or virulent – Phage that multiply within the host cell, lyse the cell and release progeny phage (e.g. T4)Lytic or virulent – Phage that multiply within the host cell, lyse the cell and release progeny phage (e.g. T4) Lysogenic or temperate phage: Phage that can either multiply via the lytic cycle or enter a quiescent state in the bacterial cell. (e.g.,  )Lysogenic or temperate phage: Phage that can either multiply via the lytic cycle or enter a quiescent state in the bacterial cell. (e.g.,  ) –Expression of most phage genes repressed –Prophage –Lysogen

47. Brock Biology of Microorganisms, vol. 9, Chapter 8 Bacteriophage have a range of morphologies from simple filaments to large complex structures May contain either RNA or DNA associated with a protein coatMay contain either RNA or DNA associated with a protein coat Almost all bacteria have phage associated with themAlmost all bacteria have phage associated with them

48. Smithsonian (Oct 2000) Attach to specific receptors on the surface of their host bacteria T4 bacteriophage on the surface of an E. coli cell Transfer their nucleic acid into the host cell

49. Life cycle of phage Life cycle of phage

50. Generalized transduction of E. coli by phage P1

51. Transduction mapping is similar to transformation mapping Gene order is determined by frequency of recombinants. Gene order is determined by frequency of recombinants. If recombination rate is high, genes are close together. If recombination rate is high, genes are close together. If recombination rate is low, genes are far apart. If recombination rate is low, genes are far apart.

52. Mapping genes of bacteriophages 1.Infect bacteria with phages of different genotypes using two-, three-, or four-gene crosses  crossover. 2.Count recombinant phage phenotypes by determining differences in cleared areas (no bacteria growth) on a bacterial lawn. 3.Different phage genes induce different types of clearing (small/large clearings with fuzzy/distinct borders).

53. Fine structure gene-mapping of bacteriophages Same principles of intergenic mapping also can be used to map mutation sites within the same gene, intragenic mapping. 1.First evidence that the gene is sub-divisible came from C. P. Oliver ‘s (~1940) work on Drosophila. 2.Seymour Benzer’s (1950-60s) study of the rII region of bacteriophage T4.

54. Seymour Benzer’s (1950-60s) study of the rII region of T4 1.Studied 60 independently isolated rII mutants crossed in all possible combinations. 2.Began with two types of traits: plaque morphology and host range property. 1.Growth in permissive host E. coli B; all four phage types grow. 1.Growth in non-permissive host E. coli K12(); rare r+ recombinants grow (rare because the mutations are close to each other and crossover is infrequent).

55. Seymour Benzer’s (1950-60s) study of the rII region of T4 3.Benzer also studied 3000 rII mutants showing nucleotide deletions at different levels of subdivision (nested analyses). 4.Was able to map to T4 to level equivalent to 3 bp. 5.Ultimately determined that the rII region is sub- divisible into >300 mutable sites by series of nested analyses and comparisons.

56. Benzer’s method for identifying recombinants of two rII mutants of T4.

57. Benzer’s map of the rII region generated from crosses of 60 different mutant T4 strains.

58. Benzer’s deletion analysis of the rII region of T4 No recombinants can be produced if mutant strain lacks the region containing the mutation.

59. Benzer’s deletion map divided the rII region into 47 segments.

60. Benzer’s composite map of the rII region indicating >300 mutable sites on two different genes. Small squares indicate point mutations mapping to a given site.

61. Seymour Benzer’s cis-trans complementation test 1.Used to determine the number of functional units (genes) defined by a given set of mutations, and whether two mutations occur on the same unit or different units. 2.If two mutants carrying a mutation of different genes combine to create a wild type function, two mutations compliment. 3.If two mutants carrying a mutation of the same gene create a mutant phenotype, mutations do not compliment.

62. Seymour Benzer’s cis-trans complementation test.

63. Example of complementation in Drosophila

64. Transposable Genetic Elements Definition: Segments of DNA that are able to move from one location to anotherDefinition: Segments of DNA that are able to move from one location to another PropertiesProperties –“Random” movement –Not capable of self replication –Transposition mediated by site-specific recombination TransposaseTransposase –Transposition may be accompanied by duplication

65. Transposase ABCDEFG GFEDCBA Types of Transposable Genetic Elements Insertion sequences (IS)Insertion sequences (IS) –Definition: Elements that carry no other genes except those involved in transposition –Nomenclature - IS1 –Structure –Importance – Mutation –Plasmid insertion –Phase variation

66. IS H1 gene H2 gene H1 flagella H2 flagella Phase Variation in Salmonella H Antigens

67. Types of Transposable Genetic Elements Transposons (Tn)Transposons (Tn) –Definition: Elements that carry other genes except those involved in transposition –Nomenclature - Tn10 –Structure Composite TnsComposite Tns – Importance Antibiotic resistance IS Resistance Gene(s) IS Resistance Gene(s)

68. Plasmids Definition: Extrachromosomal genetic elements that are capable of autonomous replication (replicon)Definition: Extrachromosomal genetic elements that are capable of autonomous replication (replicon) Episome - a plasmid that can integrate into the chromosomeEpisome - a plasmid that can integrate into the chromosome

69. Classification of Plasmids Transfer propertiesTransfer properties –Conjugative –Nonconjugative Phenotypic effectsPhenotypic effects –Fertility –Bacteriocinogenic plasmid –Resistance plasmid (R factors)

70. Structure of R Factors RTFRTF –Conjugative plasmid –Transfer genes Tn 9 Tn 21 Tn 10 Tn 8 RTF R determinant R determinantR determinant –Resistance genes –Transposons