MICR-3050 Chapter Notes - Chapter 11: Ampicillin, Nucleoid, Wild Type
30 Apr 2018
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Department
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Professor
The Nucleoid
Nucleoid: location of chromosome and assoc. proteins
Has dsDNA
○
•
Irregularly shaped
Not membrane bound
○
•
Plasmids
Extra- chromosomal DNA
Like a BONUS!
○
•
Replicate independently of chromosome
Contain nonessential genes
○
•
Can give bacteria an advantage
BUT if the plasmid is not used, the cell will lose it
If a bacteria has a plasmid that codes for ampicillin resistence
but it grows on erythomycin plates, it will lose the resistence
for ampicillin because it doesn’t need it anymore
§
○
•
DNA
Four nucleotide or bases
A=T
○
C=G
○
•
DNA as the genetic material
Griffith (1928)
○
Avery, Macleod and McCarthy (1994)
○
Hershey and Chase (1952)
○
•
DNA structure
Watson and Crick (1953)
○
•
Terms to know
Gene: Dna segment that codes for a polypeptide, rRNA or tRNA
Ex. hisC gene => HisC protein
○
•
Genotype: set of genes an organism possesses
Nucleotide sequence
○
•
Phenotype: set of observable characteristics
Ex. His+ or His-
○
•
Wild type strain: isolated from nature
•
Mutation: stable, heritable change in sequence
Alter genotype
○
May or may not have effect on phenotype of org.
○
•
Expression of Mutations
Wild type: most prevalent form of a gene
•
Forward mutations
Wild type to mutant form
○
Ex. Prototroph to auxotroph (nutritional mutation)
○
•
Reverse mutations
Mutant to wild type
○
Ex. Auxotroph to prototroph
○
•
Revertant
Same site
○
Second site
Suppressor mutations
§
○
•
Detection and Isolation of Mutants
Screening: detecting mutants via observation
•
Selectable mutation: bacteria receive some type of advantage when they
have this mutation
Ex. Drug resistance
○
•
Selection: place organism under conditions where the growth of those w
favorable genotype will thrive
•
Replica Plating process
Pick and transfer colonies to fresh media
One complete medium a.
One selective medium b.
1.
Incubate the plates and examine
Results:
Complete medium: all colonies will grow i.
Selective medium: mutants will not grow ii.
a.
2.
Gene transfer in Bacteria
Fate of donor DNA in horizontal gene transfer
Integration of donor DNA1.
Donor DNA self replicates (ex. Plasmid)2.
Donor DNA cannot self replicate 3.
Host restriction 4.
Genetic Recombination
Genetic recombination: one or more nucleic acid molecules rearrange or
combine to produce new nucleotide sequence
•
•Homologous recombination: involves a reciprocal exchange between a
pair of DNA molecules w same nucleotide sequence
○DNA strand breakage and reunion leads to crossover
Homologous Recombination
•DNA strand is nicked
•SSB protein and Rec A protein complex formed
•Recipient DNA invaded
•Crossover leads to exchange
○Ligated to form 2 recomb DNA molecules
Process of homologous recombination
1. Endonuclease nicks Donor DNA
2. Binding of SSB protein to one end of the nicked DNA
3. The nicked strand bound with SSB protein invades the Recipient DNA
with the help with RecA protein
4. A criss-cross strand exchange develops
5. Result: get recombination horizontally and vertically
Recombinant Detection
•Tryptophan negative cells put on agar lacking tryptophan = no growth
•Tryptophan negative cells put on tryptophan agar = growth of
recombinant colonies
Transformation
•Transformation: Uptake of naked DNA by a competent cell
○Followed by incorp of DNA into recipient cell's genome
•Natural and artificial
Process of transformation
1. Binding DNA
2. Uptake of single stranded DNA
3. RecA-mediated homologous recombination
Transduction
•Transduction: transfer of DNA from one cell to another by a
bacteriophage
•Two modes:
○Generalized transduction: DNA from any portion of the host
genome is packaged inside the virion
§general
○Specialized transduction: DNA from specific region of host
chromosome is integrated directly into the virus genome
§Specific
Generalized Transduction
•Occurs during lytic cycle
•During viral assembly, fragments of host DNA mistakenly packaged into
phage head
○Defective virus
○Note: lytic cycle just happens meaning there is not specific
packaging of a genome
Specialized Transduction
•Carried out by temperate phages that have established lysogeny
•Occur when prophage is incorrectly removed
Bacterial conjugation
Process of conjugation
1. Pilus retracts and forms bridge between F+ cell and F- cell
Cell pairs are stabilized
F plasmid is nicked in one strand a.
2.
3. One strand of plasmid moves from F+ cell to F- cell and simultaneously
the other strand in the F+ cell is replicated
4. Synthesis of the complementary strand inside the F- cell begins
5. Cells separate as DNA transfer completes
Transposable elements
•Transposition: segments of DNA that move around the genome
○Can integrate at target sites in chromosome
○Have transposase gene
○Bound by inverted terminal repeats
•Insertion sequences (IS): only carry the gene for transposase
○Participate in transposition
○Short segment
•Transposons (Tn): contain genes other than those used for transposition
○Larger than IS
○Can consist of insertion sequences
Insertion of Transposable element into the chromosome
1. The transposable element drops into the target DNA sequence.
a. The gene could duplicate itself
Two mechanisms of transposition
Conservative transposition
•Transposon is excised from donor and transposition into target DNA
where target sequence is located
Replicative transposition
•Transposon replicates in original host cell
Chapter 11 Bacterial Genetics
Monday, April 16, 2018
1:08 PM
The Nucleoid
Nucleoid: location of chromosome and assoc. proteins
Has dsDNA
○
•
Irregularly shaped
Not membrane bound
○
•
Plasmids
Extra- chromosomal DNA
Like a BONUS!
○
•
Replicate independently of chromosome
Contain nonessential genes
○
•
Can give bacteria an advantage
BUT if the plasmid is not used, the cell will lose it
If a bacteria has a plasmid that codes for ampicillin resistence
but it grows on erythomycin plates, it will lose the resistence
for ampicillin because it doesn’t need it anymore
§
○
•
DNA
Four nucleotide or bases
A=T
○
C=G
○
•
DNA as the genetic material
Griffith (1928)
○
Avery, Macleod and McCarthy (1994)
○
Hershey and Chase (1952)
○
•
DNA structure
Watson and Crick (1953)
○
•
Terms to know
Gene: Dna segment that codes for a polypeptide, rRNA or tRNA
Ex. hisC gene => HisC protein
○
•
Genotype: set of genes an organism possesses
Nucleotide sequence
○
•
Phenotype: set of observable characteristics
Ex. His+ or His-
○
•
Wild type strain: isolated from nature
•
Mutation: stable, heritable change in sequence
Alter genotype
○
May or may not have effect on phenotype of org.
○
•
Expression of Mutations
Wild type: most prevalent form of a gene
•
Forward mutations
Wild type to mutant form
○
Ex. Prototroph to auxotroph (nutritional mutation)
○
•
Reverse mutations
Mutant to wild type
○
Ex. Auxotroph to prototroph
○
•
Revertant
Same site
○
Second site
Suppressor mutations
§
○
•
Detection and Isolation of Mutants
Screening: detecting mutants via observation
•
Selectable mutation: bacteria receive some type of advantage when they
have this mutation
Ex. Drug resistance
○
•
Selection: place organism under conditions where the growth of those w
favorable genotype will thrive
•
Replica Plating process
Pick and transfer colonies to fresh media
One complete medium
a.
One selective medium
b.
1.
Incubate the plates and examine
Results:
Complete medium: all colonies will grow
i.
Selective medium: mutants will not grow
ii.
a.
2.
Gene transfer in Bacteria
Fate of donor DNA in horizontal gene transfer
Integration of donor DNA
1.
Donor DNA self replicates (ex. Plasmid)2.
Donor DNA cannot self replicate 3.
Host restriction 4.
Genetic Recombination
Genetic recombination: one or more nucleic acid molecules rearrange or
combine to produce new nucleotide sequence
•
•Homologous recombination: involves a reciprocal exchange between a
pair of DNA molecules w same nucleotide sequence
○DNA strand breakage and reunion leads to crossover
Homologous Recombination
•DNA strand is nicked
•SSB protein and Rec A protein complex formed
•Recipient DNA invaded
•Crossover leads to exchange
○Ligated to form 2 recomb DNA molecules
Process of homologous recombination
1. Endonuclease nicks Donor DNA
2. Binding of SSB protein to one end of the nicked DNA
3. The nicked strand bound with SSB protein invades the Recipient DNA
with the help with RecA protein
4. A criss-cross strand exchange develops
5. Result: get recombination horizontally and vertically
Recombinant Detection
•Tryptophan negative cells put on agar lacking tryptophan = no growth
•Tryptophan negative cells put on tryptophan agar = growth of
recombinant colonies
Transformation
•Transformation: Uptake of naked DNA by a competent cell
○Followed by incorp of DNA into recipient cell's genome
•Natural and artificial
Process of transformation
1. Binding DNA
2. Uptake of single stranded DNA
3. RecA-mediated homologous recombination
Transduction
•Transduction: transfer of DNA from one cell to another by a
bacteriophage
•Two modes:
○Generalized transduction: DNA from any portion of the host
genome is packaged inside the virion
§general
○Specialized transduction: DNA from specific region of host
chromosome is integrated directly into the virus genome
§Specific
Generalized Transduction
•Occurs during lytic cycle
•During viral assembly, fragments of host DNA mistakenly packaged into
phage head
○Defective virus
○Note: lytic cycle just happens meaning there is not specific
packaging of a genome
Specialized Transduction
•Carried out by temperate phages that have established lysogeny
•Occur when prophage is incorrectly removed
Bacterial conjugation
Process of conjugation
1. Pilus retracts and forms bridge between F+ cell and F- cell
Cell pairs are stabilized
F plasmid is nicked in one strand a.
2.
3. One strand of plasmid moves from F+ cell to F- cell and simultaneously
the other strand in the F+ cell is replicated
4. Synthesis of the complementary strand inside the F- cell begins
5. Cells separate as DNA transfer completes
Transposable elements
•Transposition: segments of DNA that move around the genome
○Can integrate at target sites in chromosome
○Have transposase gene
○Bound by inverted terminal repeats
•Insertion sequences (IS): only carry the gene for transposase
○Participate in transposition
○Short segment
•Transposons (Tn): contain genes other than those used for transposition
○Larger than IS
○Can consist of insertion sequences
Insertion of Transposable element into the chromosome
1. The transposable element drops into the target DNA sequence.
a. The gene could duplicate itself
Two mechanisms of transposition
Conservative transposition
•Transposon is excised from donor and transposition into target DNA
where target sequence is located
Replicative transposition
•Transposon replicates in original host cell
Chapter 11 Bacterial Genetics
Monday, April 16, 2018 1:08 PM
Document Summary
But if the plasmid is not used, the cell will lose it. If a bacteria has a plasmid that codes for ampicillin resistence but it grows on erythomycin plates, it will lose the resistence for ampicillin because it doesn"t need it anymore. Gene: dna segment that codes for a polypeptide, rrna or trna. May or may not have effect on phenotype of org. Wild type: most prevalent form of a gene. Selectable mutation: bacteria receive some type of advantage when they have this mutation. Selection: place organism under conditions where the growth of those w favorable genotype will thrive. Pick and transfer colonies to fresh media a. b. Fate of donor dna in horizontal gene transfer. Genetic recombination: one or more nucleic acid molecules rearrange or combine to produce new nucleotide sequence: homologous recombination: involves a reciprocal exchange between a pair of dna molecules w same nucleotide sequence. Dna strand breakage and reunion leads to crossover.
