17 Transductionטרנסדוקציה DNA מועבר מחיידק אחד לשני ע"י וירוס. בקטריופאז' – וירוס התוקף תאי חיידקים בלבד.
18 בקטריופאג' או פאג' - Bacteriophage or phage טרנסדוקציהבקטריופאג' או פאג' - Bacteriophage or phage= וירוס התוקף חיידקlytic or virulent phage lysogenic or temperate phageBacterial DNA transfer Generalized transduction Restricted transduction
40 ConjugationLederberg & Tatum, auxotrophs in E. coli auxotrophs mixed – 108 cells plated on minimal medium reversion vs. ? shown cell contact required
41 ConjugationHayes, et. al., 1950s discovered family of mutants with a high frequency of recombination – Hfr mutants no conversion of F- in mating bacterial gene transfer highplasmid integrated into host genome specific locus ‘cross-over’
42 ConjugationPilus needed for cell contactDNA synthesis needed for transfer rolling circle replication begins at origin (ori) one strand nicked
43 ConjugationPlasmid exit from genome: Hfr → F+ Hfr → F'
55 Microbial GeneticsGenetic notation Three letter designation for each gene lac gal ara his lacZ, hisG, araCGenome sum total of all the genes of the organism all of the ‘chromosomes’ of the organism, the DNA bacteria are haploidGenotype vs. Phenotype Genotype: genetic makeup of the organism Phenotype: appearance; visible characteristics Does phenotype always reflect the genotype?
56 Microbial GeneticsGene - unit of inheritance structural gene - information for a protein regulatory gene - control protein formation operon – structural genes plus regulatory genes
57 Recombination bringing new gene combinations together Eukaryotes - crossing over during meiosis reciprocal exchange
58 Recombination bringing new gene combinations together Eukaryotes - crossing over during meiosis reciprocal exchangeProkaryotes - transfer of genes from one cell to another one-way transfer of genes DNA transferred: exogenote recipient DNA: endogenote partial diploid may be formed
59 Recombination bringing new gene combinations together Eukaryotes - crossing over during meiosis reciprocal exchangeProkaryotes - transfer of genes from one cell to another one-way transfer of genes DNA transferred: exogenote recipient DNA: endogenote partial diploid may be formed Transformation Conjugation Transduction Limited transfer: one gene to a few genes closely related cells
60 Transformation. Griffiths, 1928 - vaccine for pneumonia Transformation Griffiths, vaccine for pneumonia Streptococcus pneumoniae smooth vs. rough strains ‘transforming principle’Avery, McCarty & MacLeod, 1944
61 Transformation naked DNA released by cell binds to competent recipient cell competence is physiological state competence requires protein synthesis DNA binding protein on cell surface – receptor DNA must be dsDNA of suitable size (450-15,000 bp) one strand degraded ssDNA enters cell pairs with genome heteroduplex formed by recA
64 ConjugationHayes, et. al., 1950s transfer one-way, not reciprocal donor and recipient strains (+ and – strains) donor: F recipient: F- mating of F+ x F- → 2 F F is F plasmidbacterial gene transfer rare
65 TransductionRestricted transduction only genes adjacent to insertion site phage λ - att locus gal or bio loci
66 TransductionRestricted transduction only genes adjacent to insertion site phage λ - att locus gal or bio lociphage defective λdgal requires helper phage
67 TransductionRestricted transduction only genes adjacent to insertion site phage λ - gal or bio lociphage defective λdgal requires helper phage
68 Fates of DNAIncorporation of genes transformation, conjugation, transduction
69 Fates of DNAIncorporation of genes transformation, conjugation, transductionPersist and replicate - clone of cells with the DNA self-replicating plasmids
70 Fates of DNAIncorporation of genes transformation, conjugation, transductionPersist and replicate - clone of cells with the DNA self-replicating plasmidsPersist but not replicate abortive transduction
71 Fates of DNAIncorporation of genes transformation, conjugation, transductionPersist but not replicate abortive transductionPersist and replicate - clone of cells with the DNA self-replicating plasmidsDegradation or host restriction
75 Host restrictionrestriction endonucleases cut at specific nucleotide sequences – 6-8 base pairs most generate ‘sticky ends’host DNA protected by methylation-G-A-A-T-T-C- -C-T-T-A-A-G-
76 Genetic engineering. Tools:. vectors - plasmids, bacteriophage Genetic engineering Tools: vectors - plasmids, bacteriophage restriction enzymesProducts proteins - see Table 9.2 in text resistant plants
77 ConjugationDonorDefinition: Gene transfer from a donor to a recipient by direct physical contact between cellsMating types in bacteriaDonorF factor (Fertility factor)F (sex) pilusRecipientRecipientLacks an F factor
78 Physiological States of F Factor Autonomous (F+)Characteristics of F+ x F- crossesF- becomes F+ while F+ remains F+Low transfer of donor chromosomal genesF+
79 Physiological States of F Factor Integrated (Hfr)Characteristics of Hfr x F- crossesF- rarely becomes Hfr while Hfr remains HfrHigh transfer of certain donor chromosomal genesF+Hfr
80 Physiological States of F Factor Autonomous with donor genes (F’)Characteristics of F’ x F- crossesF- becomes F’ while F’ remains F’High transfer of donor genes on F’ and low transfer of other donor chromosomal genesHfrF’
81 Mechanism of F+ x F- Crosses Pair formationConjugation bridgeF+F-DNA transferOrigin of transferRolling circle replication
82 Mechanism of Hfr x F- Crosses Pair formationConjugation bridgeHfrF-DNA transferOrigin of transferRolling circle replicationHomologous recombination
83 Mechanism of F’ x F- Crosses Pair formationConjugation bridgeF’F-DNA transferOrigin of transferRolling circle replication
84 Conjugation Significance Gram - bacteria Gram + bacteria Antibiotic resistanceRapid spreadGram + bacteriaProduction of adhesive material by donor cells
85 Transposable Genetic Elements Definition: Segments of DNA that are able to move from one location to anotherProperties“Random” movementNot capable of self replication (not a replicon)Transposition mediated by site-specific recombinationTransposaseTransposition may be accompanied by duplication
86 Types of Transposable Genetic Elements Insertion sequences (IS)Definition: Elements that carry no other genes except those involved in transpositionNomenclature - IS1StructureImportanceMutationPlasmid insertionPhase variationTransposaseABCDEFGGFEDCBA
87 Phase Variation in Salmonella H Antigens H1 geneH2 geneISH1 flagellaH2 flagella
88 Types of Transposable Genetic Elements Transposons (Tn)Definition: Elements that carry other genes except those involved in transpositionNomenclature - Tn10StructureComposite TnsISResistance Gene(s)ImportanceAntibiotic resistance
89 The insertion of the IS-element is accompanied by a duplication of a short sequence around the insertion site.A proposed mechanism known as the cut and paste model for the occurence of this effect is illustrated opposite. A staggered single-strand cut is made in the target sequence followed by an insertion of the respective IS-elements. The cuts are subsequently sealed and as a result the target sequence is duplicated.
90 PlasmidsDefinition: Extrachromosomal genetic elements that are capable of autonomous replication (replicon)Episome - a plasmid that can integrate into the chromosome
91 Classification of Plasmids Transfer propertiesConjugativeNonconjugativePhenotypic effectsFertilityBacteriocinogenic plasmidResistance plasmid (R factors)
92 Some Examples of Plasmid Encoded Genes Antibiotic resistance genes (enzymes that modify or degrade antibiotics) plasmids with these genes are generally called R factorsHeavy metal resistance (enzymes that detoxify metals by redox reactions)Growth on unusual substrates (enzymes for hydrocarbon degradation, etc.)Restriction/modification enzymes (protect DNA, degrade unprotected DNA)Bacteriocins (proteins toxic to other bacteria lacking the same plasmid)ToxinsProteins that mediate plasmid transfer to uninfected strains (i.e. the tra genes)Genes which provide the cell with a pathogenic feature: such as the ability invade cells (Yersinia pestis) or produce toxins (E coli 0:157) plasmids of this type are often called virulence plasmids.