Ch. 7: Bacterial and Viral Genetic System
gene mapping with transformation
**The bacteria takes up DNA from the environment through crossing over. the rate of cotransformation is inversely proportional to the distances between genes, because genes that are physically close together on a chromosome are more likely to be present on on the same DNA fragment and be recombined together SO the CLOSER the genes, the more often co-transformed
F+ x F- Conjugation (F factor transfer)
-F+ plasmid contains genes required for sex pilus and conjugation. Bacteria without this plasmid are termed F-. A cell containing the F factor produces sex pili, one which makes contact with a receptor on an F- cell and pulls the two cells together. DNA is transferred from the F+ cell to the F- cell *****Conjugation can ONLY take place between a cell that possesses the F factor and a cell that lacks the F factor. If the ENTIRE F factor is transferred to recipient F- cell, that cell becomes an F+ cell
Hfr conjugation
-donor HFr cell has F factor integrated into its chromosome -donor genes are transferred to recipient cell----In conjugation between Hfr and F- cells, the integrated F factor is nicked and the end of the nicked strand moves into the F- cell. BUT in an Hfr cell, the F factor is integrated, the chromosome follows the F factor into the recipient cell. -a complete copy of the F factor is usually not transferred---the F- cell ALMOST NEVER becomes F+ or Hfr because the F factor is nicked so the recipient cell does NOT receive the entire F factor. For this to happen, the entire donor chromosome must be transferred. THIS RARELY HAPPENS because most conjugated cells break apart before entire chromosome can transfer. -gene transfer can be clockwise or counterclockwise
Transduction in bacteria
...type of bacterial recombination (create genetic diversity) in which there is an exchange of bacterial DNA through bacteriophages - viruses that infect bacteria. GENETIC MODIFICATION due to virus **THE closer the genes, the more often they co-transduce.
What are 2 ways to analyze bacteria?
1. Liquid culture 2. Solid media
Mechanism of bacterial transformation
1. One strand of the DNA fragment enters the cell, the other is hydrolyzed 2. The single stranded fragment pairs with the bacterial chromosome and recombination takes place 3. The remainder of the single stranded DNA fragment is degraded 4. When the cell replicated and divides, one of the resulting cells is transformed and the other is not.
3 ways bacteria exchange DNA
1. Transformation 2. Transduction 3. Conjugation ***Each type of genetic transfer consists of a ONE WAY movement of genetic info to the recipient cell.
lytic cycle steps
1. Virus (Phage) attaches to host cell and inserts genetic material into cell 2. Viral genetic material takes over host cell 3. Host cell makes viral parts and assembles them to make viruses. 4. Host cell burst and releases viruses and the cycle continues with other cells.
lysogenic cycle steps
1. Virus (phage DNA) attaches to host cell and inserts genetic material into cell. 2. Genetic material is copied into the host cell's own chromosome. (repressors produced which gives cell immunity from phage infection) 3. Every time the cell divides the viral genes divide too (acts as another bacterial cell. NOW daughter cell has phage DNA but inactive) 4. A trigger from the environment could causes the viral genes to begin a different cycle
2 life cycles of bacteriophages
1. lytic cycle 2. lysogenic cycle
If 1 million F- bacteria received an F factor through conjugation with F+ bacteria, how many of these would be expected to become Hfr bacteria?
100
Types of genetic recombination in bacteria
3 types of horizontal gene transfer
Bacteria grows in Solid medium
A growth medium is suspended in gelatin-like agar Add a dilute solution of bacteria to petri plate Spread bacterial solution evenly with glass rod (plating) After incubation for 1 to 2 days, bacteria multiply, forming visible colonies
Prophage
A phage genome that has been inserted into a specific site on the bacterial chromosome. Integrated phage with potential to become an activ phage
F factor (fertility factor)
A plasmid containing genes for plasmid replication and conjugation pilus formation (controls mating and gene exchange) Contains a number of genes that regulate its transfer into a bacterial cell, replication, and insertion into the bacterial chromosome
How are chromosomal genes transferred?
A small number of the F+ cells become Hfr Cells F+ becomes Hfr 1. Host chromosome is F+ 2. Crossing over takes place between F factor and chromosome 3. F factor integrated into host cell ***Becomes episome: segment of Dna can be represented as a plasmid or integrated into the host genome. (what happens to small amount of F factor)
Virus
A tiny, nonliving particle that invades and then reproduces inside a living cell. Made up of nucleic acid surrounded by a protein coat (Can be ssDNA or ds DNA; ssRNA or dsRNA)
Genes transfer unidirectionally
A= Donor B= recipient So Donor= F+= fertility factor present Recipient= F-= NO fertility factor
Sex Pilus (Bacteria)
Appendage made from the donor male (+) bacterial cell connects to the recipient female (-), allowing for the formation of the cytoplasmic bridge and transfer of genetic material.
Define autotroph and phototroph
Autotroph needs supplements to grow; grow in complete media Phototroph are the wild type; DO NOT need supplements so grow on minimal media
auxotrophs vs prototrophs
Auxotrophs: require supplement to grow Phototrophs: Wild type; grow on minimal media (MM) ***SO MM determines if you have auxotrophs
What happens to an F- cell when it mates with an F+ cell?
BOTH become F+ through conjugation
Mapping bacterial genes via transduction
Co transduction: Only genes located close together on the bacterial chromosome will be transferred together. ***Bacterial genes can be mapped with the use of generalized transduction.
Conjugation vs Transformation vs Transduction
Conjugation: -Cytoplasmic bridge forms -DNA replicates and transfers from one cell to another -Transferred DNA replicates Transformation: -Naked DNa is taken up by recipient cell INVOLVES FOREIGN DNA Transduction: -A virus attaches to a bacterial cell -...Injects its DNA -...And replicates taking up bacterial DNA. The bacterial cell lyses. -The virus infects a NEW bacterium -...Carrying bacterial DNA with it. ALL 3 PROCESSES have the same last 2 steps: -A crossover in the bacterium or recipient leads to... -The creation of a RECOMBINANT chromosome
horizontal gene transfer types
Conjugation: DNA transferred from a donor to a recipient cell via a direct connection Transduction: the movement of genes into a bacterial recipient cell via a phage vector Transformation: free DNA in the extracellular environment taken up by bacterial cells
F+ bacterium
Contains a plasmid with only the F factor Contains a plasmid with the gene for genetic transfer **Include a sex pilus, which allows it to conjugate its plasmid to F- bacteria
F- bacterium
Does not contain the F factor sequences. Recipient of DNA during bacterial conjugation
Are the transfer of genes unidirectional or bidirectional?
Donor vs. recipient Are genes from strain A passed to strain B? And are strain b genes passed to strain A?
F' bacterium
F factor is EXCISED from the chromosome of an Hfr strain and reverts to F+ state. In the process, the F factor (referred to as F") often brings several adjoining genes with it. Transfer of F' to an F- cells results in a partially diploid cell called a merozygote. Contains a plasmid with the F factor and some genomic DNA
What is F'?
F factor is excised from Hfr cell so the F factor and chromosomal DNA go from one circle to 2 circles
Conjugations and cell types present after conjugation
F+ X F- -End up with 2 F+ cells (F- cell becomes F+) Hfr X F- -End up with 1 Hfr cell and one F- (no change) F' X F- -End up with 2 F' cells (F- cell becomes F')
F factor characterisitics
F+= F factor present as separate circular plasmid; ACTS as donor F-: F factor is absent. Acts as recipient Hfr: F factor is present, integrated into bacterial chromosome; Acts as HIGH FREQUENCY donor F'= F factor PRESENT as separate circular plasmid; Donor carrying some bacterial genes
Bacterial fertility factor types
F- bacterium: DOES NOT contain F factor sequences; RECIPIENT of DNA during bacterial conjugation F+: Contains plasmid with ONLY the F Factor F': contains plasmid with F factor and some genomic DNA Hfr: Contains F factor but NOT in separate plasmid (1 cell)
What happens to an F- cell when it mates with an Hfr cell?
F- does NOT become an F+ cell BUT it can become prototype if Hfr donates F factor.
F prime plasmid
F-plasmid that has integrated into and exited from chromosome Contains a copy of the F factor with some chromosomal genes.
What happens after the nicking of Hfr bacterium?
Following the nick in the Hfr bacterium's chromosome, the free 5' end (leading strand) of the donor moves through the transfer pore into the F- recipient bacterium. As a result, those genes that follow the free 5' end of the nicked DNA strand are more likely to be transferred than those near the 3' end of the nick which would only enter the F- bacterium if the entire chromosome is transferred, which happens only rarely.
Why can you use streptomycin to select for F- recombinant bacteria if the first gene entering the F- bacteria can potentially convert the streptomycin resistant F- strain into a streptomycin sensitive bacterium?
For linear DNA to recombine with the bacterial chromosome, a double crossover is required. Streptomyc indictates that the crossovers eliminate the strs allele from the donor DNA without recombination.
Types of Transduction in bacteria
Generalized: any gene is transferred Specialized: ONLY A FEW genes are transferred.
Bacterial Conjugation
Genetic material passes directly from one bacterium to another. 2 bacteria lie close together and a connection forms between them. A plasmid or part of the bacterial chromosome passes from one cell (the donor) to the other (recipient) After conjugation, crossing over may take place between homologous sequences in the transferred DNA and the chromosome of the recipient cell. In conjugation, DNA transferred ONLY from a donor to a recipient cell with NO reciprocal exchange of genetic material
Which are essential to map the bacterial chromosome in a single interrupted mating experiment?
Individual pairs of Hfr and F- bacteria must begin conjugation nearly simultaneously. One must be able to remove aliquots of the conjugating bacteria and stop the mating at different time points. One must be able to select against Hfr cells following conjugation. One must start with Hfr bacteria from the same strain (having the same site and orientation of F factor integration).
Which of the following can be accomplished by an interrupted mating experiment between Hfr and F- bacteria?
Interrupted mating experiments can determine gene order on the Hfr bacterial chromosome.
If lac+ Hfr bacteria mated with a lac- F- bacteria in an interrupted mating experiment, the F- bacteria will only gain the ability to metabolize lactose if a copy of the lac+ gene recombines with the F- bacteria's chromosome. Why is recombination into the bacterial chromosome necessary?
Linear DNA is unstable in bacterial cells and unless the lac+ gene is recombined into the bacterial chromosome, it will be destroyed.
How to analyze bacteria
Liquid Culture or solid medium Solid medium--> where bacteria from liquid is spread onto an agar plate **Often clones form where there are clusters of bacteria that derive from a single cell and are Genetically identical
Mapping bacterial genes with interrupted conjugation
Mapping using Hfr X F- Interrupted mating experiment: 1. Hfr with factor (a+b+c+) mates with F- cell (a-b-c-). 2. After 5 minutes, the mixture is put in blender to break up conjugation 3. Cells are put in different media; Found that after the 5 minutes, c+ gene was transferred 4. After 10 minutes, b gene was transferred 5. After 20 minutes, a gene was transferred. Map units= minutes **Supply different supplements and determine which gene is transerred _____c___5_____b____10___a After 5 minutes, c transferred. After 10, b transferred. and after 20, a was transferred ****The F factor integrates at different positions and orientations, what is important is the distance in time units BETWEEN THE GENES
Hfr x F-
One Hfr cell and one F- (no change) Hfr has F factor so that cell is donor. Segment of F factor enters F- cell through conjugation bridge. HIGH rate of recombination. Crossing over takes place within the Hfr chromosome. VERY LOW (unlikely) transfer of entire F factor SO F- Remains F- When the F factor excises from the bacterial chromosome it may carry some bacterial genes with it CONJUGATION= bacteria together
Streptomycin experiment
PHASE 1 Strain A (a-b-c+) was treated with streptomycin which allowed for no cell division. Then the strep was washed out. Strain B (a+b+c-) was left untreated BOTH strains were mixed in a complete media and placed on a plate. Growth was seen. WHICH direction did the DNA transfer? Any growth on the plate had to come from strain b because it was untreated SO Genes transferred from Strain A to strain B PHASE 2 Strain A was left untreated. Strain B was treated with streptomycin and then the strep was washed out. SO strain B could not divide. Combined both strains in complete media and plated on MM and NO growth was found. (NO phototrophs) FOUND that since strain B did not transfer genes to strain A, GENE transfers UNIDIRECTIONAL
lytic and lysogenic cycle
RACE between 2 cycles -If phage DNA excised out of cell, then it goes into lytic cycle (CALLED a temperate phage)
Auxotroph experiment
Strain A (a-, b-, c+) and Strain B (A+ B+ C-) were grown in a complete media and then plated on minimal media NO GROWTH occurred because it required supplement C Well Ledenburg and Tatum mixed strain A and strain B in a complete media and plated both on minimal media. term-45 FOUND that a small amount of colonies grew on the plate. In order for growth to occur, the colonies had to be phototrophs (a+ b+ c+) GENETIC exchange occurs****
What best describes the most common fate of an F- bacterium after conjugation with an Hfr bacterium?
The F- bacterium remains F- but might also undergo a phenotypic change resulting from recombination.
Which gene is closest to the leading side of the F factor origin of transfer?
The gene with the SMALLEST time.
F' x F- conjugation
Two F' cells (F- cell becomes F') During conjugation, the F factor with the gene is transferred to the F- cell. AND produces a partial diploid with two copies of the gene. (merozygote)
F+ x F- mating
Two F- cells (F- cell becomes F+) Low rate of recombination High transfer of F factor.
Bernard Davis U-tube experiment
U-tube was divided into 2 components by a filter with fine pores. Filter allowed liquid medium to pass from one side of tube to the other but the pores were toos mall for bacteria to pass 2 auxotrophic strains of bacteria were placed on opposite sides of the filter, and suction was applied to ends of U tube, which caused the medium to flow back and forth between 2 compartments Even though the bacteria was incubated for hours, the bacteria plated on minimal medium did NOT grow--> NO genetic exchange between strains Conclusion: Genetic exchange requires direct contact between bacterial cells.
Transfer of genes is...
Unidirectional
Lederberg and Tatum experiment
Used two triple auxotrophic e. coli strains (Y10 and Y24) with different nutrient requirements and established three separate bacterial cultures growing in complete medium. Autotrophic bacterial strain Y10 cannot synthesize Threonine, Leucine, or vitamin thiamine. Strain Y24 cannot synthesize Biotin, Phenylalanine, or Cysteine.So neither auxotrophic strain can grow on minimal medium. Moved culture 1 (Y10) into a minimal medium, and it did not grow. Moved culture 2 (Y24) into a minimal medium, and it did not grow. Moved culture 3 (which was a combination of Y10 and Y24) into a minimal medium, and phototrophic cells grew. ****They concluded that the strains transferred genetic information between themselves such that the two autotrophic strains "filled in" the genetic gaps and created a phototropic strain. (Genetic exchange and recombination took place between the mutant strains)
temperate phage
able to enter lysogenic cycle
Auxotroph
an organism that lacks the ability to synthesize essential molecules and will grow only on medium supplemented with those essential molecules
Hfr bacteria
bacteria that produce a high frequency of recombinants for chromosomal genes in mating experiments because their chromosomes contain an integrated F plasmid Behave as donor---special class of F+ cells Hfr strain donates genetic information to F-cell, but recipient DOES NOT become F+
How does an F+ cell become an Hfr cell?
by integrating the F plasmid (F factor) into the chromosome
Plaque of bacteria
clear patches of lysed cells on a lawn (continuous layer) of bacteria that occur after several rounds of phage production. Each plaque derived from single phage of bacteria that multiplied and lysed many cells (Clear areas because of lysing of cells) FULL of phage that are GENETICALLY the SAME (clones)
Bacteria grows in Liquid medium
components are dissolved in water and sterilized Bacteria grown in test tubes that contain sterile liquid medium or "broth" Bacteria grow and divide--Few bacteria added to broth tube and grow and divide until all of nutrients are used up OR--more commonly--until the concentration of their waste products becomes toxic
How transformation really occurs
exogenous DNA is taken from environment during transformation 1. Small pieces of extracellular DNA (ex: from lysed bacterial cell) taken up by living bacterial cell Leads to stable genetic change in recipient cell. (Single strand enter bacterial cell and causes recombination) ***Competent cells take up foreign DNA 2. Plasmid DNA uptake--NOT incorporated into the chromosome of the recipient. Heteroduplex is created: 50% transformed and 50% non transformed
Complete media
has all necessary compounds to grow
Lysogeny
persistence of a prophage without phage replication and destruction of bacterial cell
replication of plasmids
replication in a plasmid begins at the origin of replication, the ORI site. (Allows plasmid to replicate independently of the bacterial chromosome) Strands separate and replication takes place in BOTH directions Eventually producing 2 circular DNA molecules
Plasmid
small, extra chromsomal circular autonomously replicated DNA Carry genes that are not essential to bacterial function but may play important role in the life cycle and growth of their bacterial hosts. Responsible for the spread of antibiotic resistance among bacteria
F factor integration
take place at different sites and orientations F factor can start out with different locations and orientations
Bacterial Transformation
takes place when a bacterium takes up DNA from the medium in which it is growing. After transformation, recombination may take place between the introduced genes and those of the bacterial chromosome. free DNA in the extracellular environment taken up by bacteria
Bacterial Transduction
takes place when bacterial viruses (bacteriophages or phages) carry DNA from one bacterium to another. Inside the bacterium, the newly introduced DNA may undergo recombination with the bacterial chromosome. movement of genes into a bacterial recipient cell via a phage vector
