Transferencia del material Transferencia del material genético.genético.

Conjugación, transformación y Conjugación, transformación y transducción.transducción.

Mapeo genéticoMapeo genético

Mutations in BacteriaMutations in Bacteria

• Mutations arise in bacterial populationsMutations arise in bacterial populations– InducedInduced– SpontaneousSpontaneous

• Rare mutations are expressedRare mutations are expressed– Bacteria are haploidBacteria are haploid– Rapid growth rateRapid growth rate

• Selective advantage enriches for mutantsSelective advantage enriches for mutants• Gene transfer occurs in bacteriaGene transfer occurs in bacteria

Gene Mapping in Bacteria and Gene Mapping in Bacteria and BacteriophagesBacteriophages

Mapping bacteria, 3 different methodsMapping bacteria, 3 different methods:: ConjugationConjugation

TransformationTransformation

TransductionTransduction

Bacteriophage mappingBacteriophage mapping::

Bacteriophage gene mappingBacteriophage gene mapping

Cis-transCis-trans complementation test complementation test

Bacteria transfer (or receive) genetic Bacteria transfer (or receive) genetic material 3 different waysmaterial 3 different ways

Transfer Transfer alwaysalways is is unidirectionalunidirectional, and , and no complete diploidno complete diploid stage forms. stage forms.

1.1. ConjugationConjugation

2.2. TransformationTransformation

3.3. TransductionTransduction

Mating types in bacteriaMating types in bacteria

– DonorDonor• F factor (Fertility factor)F factor (Fertility factor)

– F (sex) pilusF (sex) pilus

– RecipientRecipient• Lacks an F factorLacks an F factor

Donor

Recipient

General Features of General Features of Gene Transfer in BacteriaGene Transfer in Bacteria

• UnidirectionalUnidirectional– Donor to recipientDonor to recipient

• Donor does not give an entire Donor does not give an entire chromosomechromosome– MerozygotesMerozygotes

• Gene transfer can occur between Gene transfer can occur between species species

ConjugationConjugation

1.1. Discovered by Discovered by Joshua LederbergJoshua Lederberg and and Edward Edward TatumTatum in 1946. in 1946.

2.2. Unidirectional transfer of genetic material Unidirectional transfer of genetic material between donor and recipient cells by direct between donor and recipient cells by direct contact.contact.

3.3. Segment (rarely all) of the donor’s chromosome Segment (rarely all) of the donor’s chromosome recombines with the homologous recipient recombines with the homologous recipient chromosome.chromosome.

4.4. Recipients containing donor DNA are called Recipients containing donor DNA are called transconjugantstransconjugants..

Lederberg & Tatum Lederberg & Tatum (1946) Experiment (1946) Experiment

demonstrating demonstrating recombination in recombination in E. E.

coli.coli.

• Recombination Recombination of 2 of 2 complimentary complimentary auxotrophs auxotrophs gives rise to a gives rise to a strain that can strain that can synthesize all synthesize all nutrients.nutrients.

Bernard Davis experiment demonstrated that Bernard Davis experiment demonstrated that physical contact is required for bacterial physical contact is required for bacterial

recombination.recombination.

E. coliE. coli conjugation conjugation

Conjugation-transfer of the Conjugation-transfer of the sex factor Fsex factor F

1.1. William Hayes (1953) demonstrated that William Hayes (1953) demonstrated that genetic exchange in E. coli occurs in only one genetic exchange in E. coli occurs in only one direction.direction.

2.2. Genetic transfer is mediated by sex factor F.Genetic transfer is mediated by sex factor F.

3.3. Donor is F+ and recipient is F-.Donor is F+ and recipient is F-.

4.4. F is a self-replicating, circular DNA plasmid F is a self-replicating, circular DNA plasmid (1/40 the size of the main chromosome).(1/40 the size of the main chromosome).

Conjugation-transfer of the Conjugation-transfer of the sex factor Fsex factor F

5.5. F plasmid contains an origin sequence (O), F plasmid contains an origin sequence (O), which initiates DNA transfer. Also contains which initiates DNA transfer. Also contains genes for hair-like cell surface (F-pili or sex-genes for hair-like cell surface (F-pili or sex-pili), which aid in contact between cells.pili), which aid in contact between cells.

6.6. No conjugation can occur between cells of the No conjugation can occur between cells of the same mating type.same mating type.

7.7. Conjugation begins when the F plasmid is Conjugation begins when the F plasmid is nicked at the origin, and a single strand is nicked at the origin, and a single strand is transferred using the rolling circle mechanism.transferred using the rolling circle mechanism.

8.8. When transfer is complete, both cells are F+ When transfer is complete, both cells are F+ double-stranded.double-stranded.

Transfer of the Transfer of the FF factor factor

Conjugation of high-Conjugation of high-frequency recombinant frequency recombinant

strainsstrains1.1. No chromosomal DNA is transferred by standard No chromosomal DNA is transferred by standard

sex factor F.sex factor F.2.2. Transfer of chromosome DNA is facilitated by Transfer of chromosome DNA is facilitated by

special strains of F+ integrated into the bacteria special strains of F+ integrated into the bacteria chromosome by crossing over.chromosome by crossing over.

3.3. Hfr strains = high frequency recombination Hfr strains = high frequency recombination strains.strains.

4.4. Discovered by William Hayes and Luca Cavalli-Discovered by William Hayes and Luca Cavalli-Sforza.Sforza.

5.5. Hfr strains replicate F factor as part of their main Hfr strains replicate F factor as part of their main chromosome.chromosome.

Conjugation of high-Conjugation of high-frequency recombinant frequency recombinant

strainsstrains5.5. Conjugation in Hfr strains begins when F+ is Conjugation in Hfr strains begins when F+ is

nicked at the origin, and F+ and bacteria nicked at the origin, and F+ and bacteria chromosomal DNA are transferred using the chromosomal DNA are transferred using the rolling circle mechanism.rolling circle mechanism.

6.6. Complete F+ sequence (or complete Complete F+ sequence (or complete chromosomal DNA) is rarely transferred chromosomal DNA) is rarely transferred (1/10,000) because bacteria separate randomly (1/10,000) because bacteria separate randomly before DNA synthesis completes. before DNA synthesis completes.

7.7. Recombinants are produced by crossover of the Recombinants are produced by crossover of the recipient chromosome and donor DNA containing recipient chromosome and donor DNA containing F+.F+.

Transfer of the Transfer of the HfrHfr FF++ factor factor

Excision of the Excision of the FF++ factor factor also occurs also occurs spontaneously at low spontaneously at low frequency.frequency.

1.1. Begin with Begin with HfrHfr cell cell containing containing FF++..

2.2. Small section of host Small section of host chromosome also may chromosome also may be excised, creating an be excised, creating an F’F’ plasmid. plasmid.

3.3. F’ F’ plasmid is named for plasmid is named for the gene it carries, e.g., the gene it carries, e.g., F’ (lac)F’ (lac)

Using conjugation to map bacterial Using conjugation to map bacterial genesgenes

1.1. Begin with appropriate Begin with appropriate HfrHfr strains selected from strains selected from FF++ x x FF-- crosses and perform an crosses and perform an interrupted interrupted mating experimentmating experiment..

2.2. HfrHHfrH thr+ leu+ azithr+ leu+ aziRR ton tonRR lac+ gal+ lac+ gal+ strstrRR

FF-- thr leu azithr leu aziSS ton tonss lac lac gal gal strstrSS

3.3. Mix 2 cell types in medium at 37°C.Mix 2 cell types in medium at 37°C.

Using conjugation to map bacterial Using conjugation to map bacterial genesgenes

4.4. Remove at experimental time points and agitate Remove at experimental time points and agitate to separate conjugating pairs.to separate conjugating pairs.

5.5. Analyze recombinants with selective media.Analyze recombinants with selective media.

6.6. Order in which genes are transferred reflects Order in which genes are transferred reflects linear sequence on chromosomes and linear sequence on chromosomes and time in time in mediamedia..

7.7. Frequency of recombinants declines as donor Frequency of recombinants declines as donor gene enters recipient later.gene enters recipient later.

Interrupted Interrupted mating mating

experimentexperiment

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

Generating a map Generating a map for all of for all of E. coliE. coli

1.1. Location and orientation Location and orientation of the of the HfrHfr FF++ in the in the circular chromosome circular chromosome varies from strain to varies from strain to strain.strain.

2.2. Overlap in transfer Overlap in transfer maps from different maps from different strains allow generation strains allow generation of a complete of a complete chromosomal map.chromosomal map.

Circular Circular genetic map of genetic map of

E. coliE. coli

Total map units = Total map units = 100 minutes100 minutes

~time required for ~time required for E. coliE. coli chromosome to chromosome to replicate at replicate at 37°C. 37°C.

SignificanceSignificance

• Gram - bacteriaGram - bacteria– Antibiotic resistanceAntibiotic resistance– Rapid spreadRapid spread

• Gram + bacteriaGram + bacteria– Production of adhesive material by Production of adhesive material by

donor cells donor cells

TransformationTransformation

• Unidirectional transfer of extracellular DNA Unidirectional transfer of extracellular DNA into cells, resulting in a phenotypic change in into cells, resulting in a phenotypic change in the recipient.the recipient.

• First discovered by Frederick Griffith (1928).First discovered by Frederick Griffith (1928).• DNA from a donor bacteria is extracted and DNA from a donor bacteria is extracted and

purified, broken into fragments, and added to purified, broken into fragments, and added to a recipient strain.a recipient strain.

• Donor and recipient have different Donor and recipient have different phenotypes and genotypes.phenotypes and genotypes.

• If recombination occurs, new recombinant If recombination occurs, new recombinant phenotypes appear.phenotypes appear.

More about transformationMore 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 Bacteria such as Bacillus subtilis take up DNA naturally.naturally.

• Other strains are engineered (i.e., competent Other strains are engineered (i.e., competent cells).cells).

• Competent cells are electroporated or treated Competent cells are electroporated or treated chemically to induce chemically to induce E. coliE. coli to take up to take up extracellular DNA.extracellular DNA.

Bacteria known to be Bacteria known to be capable of transformationcapable of transformation

• Natural transformationNatural transformation– Gram positive bacteriaGram positive bacteria

• Streptococcus pneumoniae, S. sanguis, B. Subtilis, B. Streptococcus pneumoniae, S. sanguis, B. Subtilis, B. Cereus, B. StearothermophilusCereus, B. Stearothermophilus

– Gram negative bacteriaGram negative bacteria• Neisseria gnonorrheae, Acinetobacter calcoaceticus, Neisseria gnonorrheae, Acinetobacter calcoaceticus,

Moraxella osloensis, M. urethansMoraxella osloensis, M. urethans• Psychrobacter sp., Azotobacter agilis, Haemophilus Psychrobacter sp., Azotobacter agilis, Haemophilus

influenzae, H. Parainfluenzae, Pseudomonas stutzeriinfluenzae, H. Parainfluenzae, Pseudomonas stutzeri

• Artificial transformationArtificial transformation• Escherichia coli, Salmonella thyphimurium, Escherichia coli, Salmonella thyphimurium,

Pseudomonas aeruginosasPseudomonas aeruginosas y muchas otras. y muchas otras.

TransformationTransformation

– RecombinationRecombination• Legitimate, Legitimate,

homologous or homologous or general general

• recA, recB and recC recA, recB and recC genesgenes

• SignificanceSignificance– Phase variation in Phase variation in NeiseseriaNeiseseria– Recombinant DNA technologyRecombinant DNA technology

• StepsSteps– Uptake of DNAUptake of DNA

• Gram +Gram +• Gram -Gram -

Heteroduplex DNA

Transformation of Transformation of Bacillus Bacillus subtilissubtilis

Hanahan and Bloom, 1996, Chapter 132, Hanahan and Bloom, 1996, Chapter 132, Escherichia coliEscherichia coli and and SalmonellaSalmonella, ASM Press, ASM Press

Chemical competenceChemical competence• In some bacteria, includingIn some bacteria, including E. col E. coli, treatment of cells with i, treatment of cells with

divalent cations at low temperature, facilitates the uptake of divalent cations at low temperature, facilitates the uptake of plasmid DNA into the cell (linear DNA can be taken up, but is plasmid DNA into the cell (linear DNA can be taken up, but is shredded by cytoplasmic DNases before it can do anything)shredded by cytoplasmic DNases before it can do anything)

• Remains unclear how this worksRemains unclear how this works

Uptake channels made Uptake channels made of polyP, PHB, and Caof polyP, PHB, and Ca

High field strengths result in very transient holes in the cellular High field strengths result in very transient holes in the cellular envelopeenvelope

Under the appropriate conditions, DNA leaks in and DNA leaks out.Under the appropriate conditions, DNA leaks in and DNA leaks out.

A high concentration of plasmid outside results in a rapid influx of A high concentration of plasmid outside results in a rapid influx of plasmids into the cell.plasmids into the cell.

Electroporation Electroporation cuvettecuvette

Cells go hereCells go hereHigh voltageHigh voltageshockshock

ElectroporationElectroporation

Transformation efficiency

Saturating cells (# of transformants/g of DNA)

106-109/g of pBR322app. 1011 plasmids/g pBR322can also be analyzed as % of cells that receive plasmid

Saturating DNA

% of DNA molecules that successfully transform cells

How well has your How well has your transformation worked?transformation worked?

Protocol Sat. cells Sat. DNA

Chemical 1% 12%

Electro 10% 90%

Dubnau. 1999. Ann. Rev. Microbiol. 53:217Dubnau. 1999. Ann. Rev. Microbiol. 53:217

Natural Natural transformation in transformation in Gram positives Gram positives

Examples: Examples: Streptococcus pneumoniaeStreptococcus pneumoniaeBacillus subtilisBacillus subtilis

• no base specificityno base specificity• limited # of uptake sites limited # of uptake sites

(30-75)(30-75)• nicked internallynicked internally• complement is degraded complement is degraded

during transportduring transport• recombines in recipientrecombines in recipient

Dubnau. 1999. Ann. Rev. Microbiol. 53:217Dubnau. 1999. Ann. Rev. Microbiol. 53:217

Natural Natural transformation in transformation in Gram negatives Gram negatives

Examples: Examples: Haemophilus influenzaeHaemophilus influenzaeNeisseriae gonorrhoeae Neisseriae gonorrhoeae

• sequence specific – sequence specific – uptake sequencesuptake sequences

• 4-8 sites/cell4-8 sites/cell• no cell bound intermediateno cell bound intermediate• import of ds DNA to import of ds DNA to

periplasmperiplasm• complement is degraded complement is degraded

during transport into during transport into cytoplasmcytoplasm

• recombines in recipientrecombines in recipient

Dubnau. 1999. Ann. Rev. Microbiol. 53:217

the reverse of a conjugal transfer system- some components similar to Tra functions

Gram positive uptake Gram positive uptake machinerymachinery

-dedicated machinery for the transport of DNA into the cell

-dedicated machinery for the transport of DNA into the cell-dedicated machinery for the transport of DNA into the cell- must cross periplasm and outer membrane - must cross periplasm and outer membrane

Dubnau. 1999. Ann. Rev. Microbiol. 53:217

Gram-negative uptake Gram-negative uptake machinarymachinary

Energy for driving the Energy for driving the process?process?

• Intracellular ATP Intracellular ATP hydrolysishydrolysis

• pH gradient – PMF?pH gradient – PMF?

• Complement degradationComplement degradation

Function for natural Function for natural transformationtransformation

• NutritionNutrition

• DNA repairDNA repair

• Genetic diversificationGenetic diversification

Diferencias entre los sistemas de transformación natural codificados porStreptococcus pneumoniae y Haemophilus influenzae.

Propiedad Streptococcus Haemophilus

Factores de competenciadesencadenan la competencia

Sí No

Forma en que el DNA entra enla célula

Hebra sencilla Hebra doble

Fuente de DNA que puedeentrar a la célula

Cualquiera Sólo homóloga

Forma del DNA unido a l asuperficie celular

Hebra doble Hebra doble

Estado físico del DNA dent rode la célula

Unido a proteínas Contenido en eltransformasoma

Diferencias entre los sistemas de transformación natural codificados porStreptococcus pneumoniae y Haemophilus influenzae.

Propiedad Streptococcus Haemophilus

Factores de competenciadesencadenan la competencia

Sí No

Forma en que el DNA entra enla célula

Hebra sencilla Hebra doble

Fuente de DNA que puedeentrar a la célula

Cualquiera Sólo homóloga

Forma del DNA unido a l asuperficie celular

Hebra doble Hebra doble

Estado físico del DNA dent rode la célula

Unido a proteínas Contenido en eltransformasoma

Mapping using transformationMapping using transformation

Recombination frequencies are used to infer Recombination frequencies are used to infer gene order.gene order.

p+p+q+ o+q+ o+ xx p q o p q o

1.1. If p+ and q+ frequently cotransform, order is If p+ and q+ frequently cotransform, order is p-q-o.p-q-o.

2.2. If p+ and o+ frequently cotransform, order is If p+ and o+ frequently cotransform, order is p-o-q.p-o-q.

TransductionTransduction

1.1. Bacteriophages (bacterial viruses) transfer genes Bacteriophages (bacterial viruses) transfer genes to bacteria (e.g., T2, T4, T5, T6, T7, and to bacteria (e.g., T2, T4, T5, T6, T7, and ).).

1.1. Generalized transductionGeneralized transduction transfers any gene. transfers any gene.

1.1. Specialized transductionSpecialized transduction transfers specific transfers specific genes.genes.

2.2. Phages typically carry small amounts of DNA, Phages typically carry small amounts of DNA, ~1% of the host chromosome.~1% of the host chromosome.

3.3. Viral DNA undergoes recombination with Viral DNA undergoes recombination with homologous host chromosome DNA.homologous host chromosome DNA.

TransductionTransduction

• Genetic exchange mediated Genetic exchange mediated by bacterial viruses by bacterial viruses (bacteriophage)(bacteriophage)

• Two basic types of bacterial Two basic types of bacterial virusesviruses

• Lytic viruses – infect Lytic viruses – infect cells, multiply cells, multiply

rapidly, rapidly, lyse cellslyse cells• Lysogenic viruses – infect Lysogenic viruses – infect

cells, can integrate cells, can integrate into into genome and go genome and go dormant dormant (a prophage)(a prophage)

• - at some point, can - at some point, can excise, multiply and excise, multiply and

lyse lyse cells.cells.

Phage Composition and Phage Composition and StructureStructure

• CompositionComposition– Nucleic acidNucleic acid

• Genome sizeGenome size• Modified basesModified bases

– ProteinProtein• ProtectionProtection• InfectionInfection

• Structure (TStructure (T44))

– SizeSize– Head or capsidHead or capsid– TailTail

Tail

Tail Fibers

Base Plate

Head/Capsid

Contractile Sheath

Infection of Host Cells by PhagesInfection of Host Cells by Phages

• AdsorptionAdsorption– LPS for T4LPS for T4

• Irreversible attachmentIrreversible attachment• Sheath ContractionSheath Contraction• Nucleic acid injectionNucleic acid injection• DNA uptakeDNA uptake

Types of BacteriophageTypes of Bacteriophage

• Lytic or virulent – Phage that multiply within the host Lytic or virulent – Phage that multiply within the host cell, lyse the cell and release progeny phage (cell, lyse the cell and release progeny phage (e.g.e.g. T4)T4)

• Lysogenic or temperate phage: Phage that can Lysogenic or temperate phage: Phage that can either multiply via the lytic cycle or enter a quiescent either multiply via the lytic cycle or enter a quiescent state in the bacterial cell. (state in the bacterial cell. (e.g.,e.g., ))– Expression of most phage genes repressed Expression of most phage genes repressed – ProphageProphage– LysogenLysogen

Brock Biology of Microorganisms, vol. 9, Chapter 8Brock Biology of Microorganisms, vol. 9, Chapter 8

Bacteriophage have a range of morphologies Bacteriophage have a range of morphologies from simple filaments to large complex from simple filaments to large complex

structuresstructures

• 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

Smithsonian (Oct 2000)

Attach to specific receptors on the surface of their host Attach to specific receptors on the surface of their host bacteriabacteria

T4 bacteriophage on the surface of an T4 bacteriophage on the surface of an E. coli cellE. coli cell

Transfer their nucleic acid Transfer their nucleic acid into the host cellinto the host cell

Life cycle Life cycle of phage of phage

Generalized Generalized transduction transduction of E. coli by of E. coli by phage P1phage P1

Transduction mapping is similar to Transduction mapping is similar to transformation mappingtransformation mapping

Gene order is determined by frequency of Gene order is determined by frequency of recombinants.recombinants.

If recombination rate is high, genes are If recombination rate is high, genes are close together.close together.

If recombination rate is low, genes are far If recombination rate is low, genes are far apart.apart.

Mapping genes of bacteriophagesMapping genes of bacteriophages1.1. Infect bacteria with phages of different genotypes using two-, three-, Infect bacteria with phages of different genotypes using two-, three-,

or four-gene crosses or four-gene crosses crossovercrossover..

2.2. Count recombinant phage phenotypes by determining differences in Count recombinant phage phenotypes by determining differences in cleared areas (no bacteria growth) on a cleared areas (no bacteria growth) on a bacterial lawnbacterial lawn..

3.3. Different phage genes induce different types of clearing (Different phage genes induce different types of clearing (small/largesmall/large clearings with clearings with fuzzy/distinctfuzzy/distinct borders). borders).

Fine structure gene-mapping of Fine structure gene-mapping of bacteriophagesbacteriophages

Same principles of Same principles of intergenic mappingintergenic mapping also can also can be used to map mutation sites within the be used to map mutation sites within the same gene, same gene, intragenic mappingintragenic mapping..

1.1. First evidence that the gene is sub-divisible First evidence that the gene is sub-divisible came from C. P. Oliver ‘s (~1940) work on came from C. P. Oliver ‘s (~1940) work on DrosophilaDrosophila..

2.2. Seymour Benzer’s (1950-60s) study of the Seymour Benzer’s (1950-60s) study of the rIIrII region of bacteriophage T4.region of bacteriophage T4.

Seymour Benzer’s (1950-60s) study Seymour Benzer’s (1950-60s) study of the of the rIIrII region of T4 region of T4

1.1. Studied 60 independently isolated Studied 60 independently isolated rIIrII mutants mutants crossed in all possible combinations.crossed in all possible combinations.

2.2. Began with two types of traits: Began with two types of traits: plaque plaque morphologymorphology and and host range property.host range property.

1.1. Growth in permissive host Growth in permissive host E. coli BE. coli B; all four ; all four phage types grow.phage types grow.

1.1. Growth in non-permissive host Growth in non-permissive host E. coli K12(E. coli K12());; rare rare r+ r+ recombinants grow (rare because the recombinants grow (rare because the mutations are close to each other and mutations are close to each other and crossover is infrequent).crossover is infrequent).

Seymour Benzer’s (1950-60s) study Seymour Benzer’s (1950-60s) study of the of the rIIrII region of T4 region of T4

3.3. Benzer also studied 3000 Benzer also studied 3000 rIIrII mutants showing mutants showing nucleotide deletionsnucleotide deletions at different levels of at different levels of subdivision (subdivision (nested analysesnested analyses).).

4.4. Was able to map to T4 to level equivalent to 3 Was able to map to T4 to level equivalent to 3 bp.bp.

5.5. Ultimately determined that the Ultimately determined that the rIIrII region is sub- region is sub-divisible into >300 mutable sites by series of divisible into >300 mutable sites by series of nested analyses and comparisons.nested analyses and comparisons.

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

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

Benzer’s deletion Benzer’s deletion analysis of the rII analysis of the rII

region of T4region of T4

No recombinants can be No recombinants can be produced if mutant produced if mutant strain lacks the region strain lacks the region containing the mutation.containing the mutation.

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

Benzer’s composite map of the Benzer’s composite map of the rIIrII region indicating >300 mutable region indicating >300 mutable sites on two different genes. sites on two different genes.

Small squares indicate point mutations mapping to a given site.Small squares indicate point mutations mapping to a given site.

Seymour Benzer’s Seymour Benzer’s cis-transcis-trans complementation testcomplementation test

1.1. Used to determine the number of functional Used to determine the number of functional units (genes) defined by a given set of units (genes) defined by a given set of mutations, and whether two mutations occur mutations, and whether two mutations occur on the same unit or different units.on the same unit or different units.

2.2. If two mutants carrying a mutation of If two mutants carrying a mutation of different genes combine to create a wild type different genes combine to create a wild type function, two mutations compliment.function, two mutations compliment.

3.3. If two mutants carrying a mutation of the If two mutants carrying a mutation of the same gene create a mutant phenotype, same gene create a mutant phenotype, mutations do not compliment.mutations do not compliment.

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

Example of complementation in Example of complementation in DrosophilaDrosophila

Transposable Genetic ElementsTransposable Genetic Elements

• Definition: Segments of DNA that are able to Definition: Segments of DNA that are able to move from one location to anothermove from one location to another

• PropertiesProperties– ““Random” movementRandom” movement– Not capable of self replicationNot capable of self replication– Transposition mediated by site-specific recombinationTransposition mediated by site-specific recombination

• TransposaseTransposase– Transposition may be accompanied by duplicationTransposition may be accompanied by duplication

TransposaseABCDEFG GFEDCBA

Types of Transposable Types of Transposable Genetic ElementsGenetic Elements

• Insertion sequences (IS)Insertion sequences (IS)– Definition: Elements that carry no other genes Definition: Elements that carry no other genes

except those involved in transpositionexcept those involved in transposition– Nomenclature - IS1Nomenclature - IS1– Structure Structure – ImportanceImportance– MutationMutation– Plasmid insertionPlasmid insertion– Phase variationPhase variation

ISISH1 geneH1 gene H2 geneH2 gene

H1 H1 flagellaflagella

H2 H2 flagellaflagella

Phase Variation in Phase Variation in Salmonella H AntigensSalmonella H Antigens

Types of Transposable Genetic Types of Transposable Genetic ElementsElements

• Transposons (Tn)Transposons (Tn)– Definition: Elements that carry other genes Definition: Elements that carry other genes

except those involved in transpositionexcept those involved in transposition– Nomenclature - Tn10Nomenclature - Tn10– StructureStructure

• Composite TnsComposite Tns

– Importance

• Antibiotic resistance

IS ISResistance Gene(s)

IS ISResistance Gene(s)

PlasmidsPlasmids

• Definition: Extrachromosomal genetic Definition: Extrachromosomal genetic elements that are capable of elements that are capable of autonomous replication (replicon)autonomous replication (replicon)

• Episome - a plasmid that can integrate Episome - a plasmid that can integrate into the chromosomeinto the chromosome

Classification Classification ofof Plasmids Plasmids

• Transfer propertiesTransfer properties– ConjugativeConjugative– NonconjugativeNonconjugative

• Phenotypic effectsPhenotypic effects– FertilityFertility– Bacteriocinogenic plasmidBacteriocinogenic plasmid– Resistance plasmid (R factors)Resistance plasmid (R factors)

Structure of R FactorsStructure of R Factors

• RTFRTF– Conjugative Conjugative

plasmidplasmid– Transfer genesTransfer genes Tn 9

Tn

21

Tn 10

Tn 8

RTF

R determinant

• R determinantR determinant– Resistance genesResistance genes– TransposonsTransposons