Mutations are stable, heritable changes in the sequence of bases in DNA.
Point mutations are the most common type, involving alteration of single pairs of nucleotides.
Mutations can also involve the addition or deletion of nucleotide pairs.
Larger mutations, such as insertions, deletions, inversions, duplication, and translocations of nucleotide sequences, are less common.
Mutations can occur spontaneously or be induced by external factors.
Spontaneous Mutations
Spontaneous mutations occur naturally.
The rate of spontaneous mutations is low due to the proofreading capability of DNA polymerase.
Insertions and Deletions
Insertions and deletions can occur due to slippage during DNA replication.
(a) Slippage leading to an insertion:
Slippage in the new strand can cause an extra nucleotide to be added, resulting in an insertion mutation.
(b) Slippage leading to a deletion:
Slippage in the parental strand can cause a nucleotide to be skipped during replication, resulting in a deletion mutation.
Induced Mutations
Base analogs:
These are structurally similar to normal bases (e.g., 5-bromouracil, an analog of thymine).
Mistakes occur when they are incorporated into the growing polynucleotide chain.
DNA modifying agents:
These alter a base, causing it to mispair (e.g., methyl nitrosoguanidine).
Intercalating agents:
These distort DNA to induce single nucleotide pair insertions and deletions (e.g., acridine orange).
Mutagens
Chemical Mutagens:
5-Bromouracil: Base analog
2-Aminopurine: Base analog
Ethyl methanesulfonate: Alkylating agent
Hydroxylamine: Hydroxylates cytosine
Nitrogen mustard: Alkylating agent
Nitrous oxide: Deaminates bases
Proflavin: Intercalating agent
Acridine orange: Intercalating agent
Physical Mutagens:
UV light: Promotes pyrimidine dimer formation
X-rays: Causes base deletions, single-strand nicks, cross-linking, and chromosomal breaks
Chemical Mutagens: Induced mutations
Chemicals induce mutations at a higher rate than found spontaneously.
Nitrous acid (HNO2) causes oxidative deamination of bases:
Adenine is converted to hypoxanthine.
Cytosine is converted to uracil.
Guanine is converted to xanthine.
Physical Mutagens: Induced mutations
Ultraviolet light can cause a higher than expected mutation rate in DNA by forming thymine dimers.
Effects of Mutations
Wild type: Most prevalent form of a gene.
Forward mutation: Wild type converts to mutant form (wild type → mutant form).
Reversion mutation: Mutant phenotype converts to wild type phenotype (mutant phenotype → wild type phenotype).
Suppressor mutation
Mutations in Protein Coding Genes
Point mutations in protein-coding genes can affect protein structure in various ways.
Mutations are named according to their effect on the encoded protein.
The most common types are silent, missense, nonsense, and frameshift mutations.
The Genetic Code
The genetic code consists of codons that specify amino acids.
The table shows the relationship between codons and amino acids.
Some codons specify stop signals.
Point Mutations
Silent mutation:
Changes the base sequence of a codon but does not change the encoded amino acid.
Missense mutation:
A single base substitution that changes a codon for one amino acid into a codon for another amino acid.
Nonsense mutation:
Converts a sense codon to a stop codon.
Frameshift mutation:
Results from insertion or deletion of one or two base pairs in the coding region of the gene.
Missense Mutations
A missense mutation results in the replacement of one amino acid with another in the protein.
Nonsense Mutations
A nonsense mutation results in a stop codon, leading to a truncated protein.
Frameshift Mutations
A frameshift mutation results from the insertion or deletion of one or more base pairs, altering the reading frame and leading to a completely different amino acid sequence downstream of the mutation.
Other Types of Mutations
Conditional mutations:
Expressed only under certain environmental conditions.
Auxotrophic mutant:
Unable to make an essential macromolecule such as an amino acid or nucleotide.
Has a conditional phenotype and can survive if the nutrient is provided.
Prototroph:
The wild-type strain from which an auxotroph arose; it does not require the nutrient.
Other Types of Mutations
Mutations in regulatory sequences:
Changes to promoter regions can affect transcription.
Mutations in tRNA and rRNA genes:
Protein synthesis is disrupted.
The Birth of Bacterial Genetics
Bacteria are ideal candidates for genetic research because they have one chromosome, making mutation detection easy.
Early studies used nutritional mutants (auxotrophs) to study one gene based on its inability to utilize (or produce) a particular nutrient.
Screening
Screening involves looking for changes in phenotype.
It use media that grows parental strain
It is an inefficient process
Mutations, Mutants, and Strains
Changes in genes are often visible by changes in phenotype or growth patterns.
Selection
Selection involves using a growth medium that will inhibit microbes lacking the desired gene(s).
Antibiotic selection is commonly used.
Replica Plating
Treatment of E. coli cells with a mutagen.
Inoculate a plate containing complete growth medium and incubate.
Both wild-type and mutant survivors form colonies.
Replica plating using a velvet surface to transfer colonies to new plates.
Replica plates with complete medium and medium minus lysine are used to identify lysine auxotrophs.
Patch plating
Pick colonies from master plate with sterile toothpick.
Sequentially inoculate gridded test plates with each picked colony; incubate.
Compare growth on test plates.
Defined growth medium
Complex growth medium
No growth
Carcinogenicity Testing
Based on the observation that most carcinogens are also mutagens.
Tests for mutagenicity are used as a screen for carcinogenic potential.
Ames test (Bruce Ames, 1970’s):
Does the agent increase the rate of mutation?
A quick, cheap method using bacteria instead of animals.
Uses Salmonella enterica serovar Typhimurium (histidine auxotroph) and looks for reversion.
If reversion rate in presence of suspected carcinogen > reversion rate in absence of suspected carcinogen, then the agent is a mutagen and may be a carcinogen.
Ames Test for Mutagenicity
Culture of Salmonella histidine auxotrophs plated on minimal medium plus a small amount of histidine.
Incubation at 37°C.
Spontaneous revertants appear.
Minimal medium with test mutagen and a small amount of histidine is used to detect revertants induced by the mutagen.
Repair of Mutations
Complex mechanisms exist for repairing DNA.
These mechanisms include:
DNA pol 3’ to 5’ exonuclease proofreading activity
Mismatch repair systems
Photolyase
Alkyltransferase
Proofreading
Correction of errors in base pairing made during replication.
Errors corrected by DNA polymerase 3’ to 5’ exonuclease proofreading activity.
Excision Repair
Corrects damage that causes distortions in the double helix.
Three types of repair systems are known:
Mismatch repair
Nucleotide excision
Base excision repair
Mismatch Repair
Mismatch correction enzyme scans newly synthesized DNA for mismatched pairs.
Mismatched pairs are removed and replaced by DNA polymerase and DNA ligase.
Enzymes used: MutS, MutH, MutL, DNA ligase.
Methyl-Directed Mismatch Repair in E. coli
The MutS protein slides along the DNA until it finds a mismatch.
MutL binds MutS, and the MutS/MutL complex binds to MutH, which is already bound to a hemimethylated sequence.
The complex recruits helicase and exonuclease to remove the mismatched region.
DNA polymerase and ligase fill the gap.
Nucleotide Excision Repair in E. coli
The UvrAB complex tracks along the DNA in search of damaged DNA.
After damage is detected, UvrA is released, and UvrC binds.
UvrC makes cuts on both sides of the thymine dimer.
UvrD is a helicase that removes the damaged region. UvrB and UvrC are also released.
DNA polymerase fills in the gap, and DNA ligase joins the fragments.
Base Excision Repair
Damaged single bases are recognized and removed from the DNA molecule.
Uses DNA glycosylase.
Example: removing uracil (deamination of cytosine).
Direct Repair
Photoreactivation:
Used to directly repair thymine dimers by splitting them using visible light.
Thymines are separated by a photochemical reaction catalyzed by photolyase.
Direct repair of alkylated bases:
Catalyzed by alkyltransferase or methylguanine methyltransferase.
Repairs DNA with damage in both strands or a gap opposite a lesion.
Involves recombination with an undamaged molecule (another copy of the chromosome is often available in rapidly dividing cells).
RecA protein catalyzes recombination events.
Requires an undamaged DNA strand, DNA polymerase, and free single-strand (overhang) ends.
Recombinational Repair
By recombination, a region in strand A is swapped for the same region in strand C.
The gap that is now in strand A is filled in by DNA polymerase and DNA ligase, using strand B as a template.
The gap has been repaired, but the thymine dimer remains and will be removed by another repair system.
The SOS Response - 1
Inducible repair system.
A global control network.
Used to repair excessive damage that halts replication, leaving many gaps.
Turns on numerous repair mechanisms (e.g., Uvr excision repair).
Cell division is blocked.
SOS Response - 2
RecA protein initiates recombination repair.
RecA protein also acts as a protease, destroying LexA repressor protein and thereby increasing the production of excision repair enzymes.
Last-ditch effort after about 40 min - DNA polymerases IV and V synthesize unrepaired DNA using incorrect bases to increase the likelihood that some cells will survive.
SOS Response - 3
DNA damage activates RecA.
Active RecA activates LexA protease activity; LexA is degraded.
LexA causes partial repression of recA.
Error-prone DNA polymerase IV.
UvrA protein: Error-free DNA repair.
UmuCD proteins: Error-prone DNA repair.
Transfer of Genetic Information
Vertical Gene Transfer
Transfer of genetic material from parents to progeny.
Important when mutations are passed between generations.
Particularly important in Eukaryotic sexual recombination
Horizontal Gene Transfer
Transfer of genetic material between cells that do not share an ancestor–descendant relationship.
Stable recombinant has characteristics of donor and recipient.
Important in the evolution of many bacterial and archaeal species (e.g., expansion of metabolic diversity, increased virulence, emergence of diverse populations)
HGT Mechanisms
Three main mechanisms of HGT have evolved in bacteria to create recombinants:
conjugation
transformation
transduction
HGT Mechanisms-An Overview
Integration
Uptake
Transformation
RecA proteins
Pore
Transduction
Conjugation
'Non-canonical'
Pilus
Vesicle
Phage
Nanotubes
GTAS
PhageDNA
ICES
Plasmid
DNA Fragments
RecA mediated
RecA independent
Extrachromosomal
1 RecA-mediated recombination, includes preprocessing by RecA proteins
2 RecA-independent recombination: for example, illegitimate recombination from microhomologies or non-homologous end joining
3 Site-specific recombination via integrases, recombinases or transposases
Fate of Donor DNA after HGT
Integration of donor DNA leading to a population of stable recombinants.
Donor DNA self-replicates.
Donor DNA cannot self-replicate.
Host restriction leading to no stable recombinants.
Recombination at the Molecular Level
Two types of genetic recombination can occur:
homologous recombination (reciprocal and nonreciprocal)
site-specific recombination
Reciprocal Homologous Recombination
Most common.
Involves a reciprocal exchange between DNA molecules with the same or similar nucleotide sequence.
RecA proteins carry out.
Double-strand break model:
Most widely accepted.
Involves DNA breakage, crossing over, and reunion.
Both DNA strands get new material.
Reciprocal Homologous Recombination Proteins
RecBCD:
Recognizes double-stranded breaks and then generates single-stranded regions at the break site that are involved in strand invasion.
Single-strand binding protein:
Prevents excessive strand degradation by RecBCD.
RecA:
Promotes strand invasion and displacement of complementary strand to generate D loop.
RecG:
Helps form Holliday junctions and promotes branch migration.
RuvABC:
Endonuclease that binds Holliday junctions, promotes branch migration, and cuts strands in the Holliday junction to separate chromosomes.
Double Stranded Break Model of Homologous Recombination
Double-stranded break
Strand degradation via RecBCD occurs at the double-stranded break site to yield single-stranded ends.
RecA promotes strand invasion and D-loop formation.
Gap repair synthesis fills in the vacant region.
Branch migration and resolution can produce recombinant or nonrecombinant chromosomes.
Nonreciprocal Homologous Recombination
Incorporation of a single strand of DNA into a chromosome, forming a stretch of heteroduplex DNA.
Proposed to occur during bacterial transformation (e.g., Griffith experiment).
Site-Specific Recombination
Recombination occurs at specific target sites in DNA.
There is only a small region of homology between inserted genetic material and the host chromosome.
It is catalyzed by recombinases.
Important in the insertion of viral genomes or some plasmids into host chromosomes.
Transposable Elements (TE)
Segments of DNA that move about the genome in a process called transposition.
Can be integrated into different sites in the chromosome.
Are sometimes called “jumping genes.”
Include insertion sequences (IS) and transposons (Tn).
Discovered in the 1940s by Barbara McClintock, who won the Nobel Prize in 1983.
TE- Insertion Sequences (IS)
Short sequences of DNA (700 – 1,600 bp).
Contain only the gene for the enzyme transposase with inverted repeats (15 to 25 bp) at each end.
Its only function is to recognize and insert at complementary places in the chromosome.
Moves by simple (cut-and-paste) transposition.
TE- Composite Transposons
Have the transposase gene plus other genes (i.e., antibiotic resistance).
Usually consist of two IS elements on each side of a gene with inverted repeats.
Move by simple (cut and paste) transposition.
TE- Replicative Transposons
Transposons with additional genes.
Move by making copies of themselves.
Contain a transposase, a resolvase gene, and inverted repeats on each end.
Simple Transposition Mechanism
Transposase recognizes the inverted repeats and cleaves at both ends of the transposable element, releasing it from its original site.
Transposase carries the TE to a new site and cleaves the target DNA at staggered sites.
The TE is inserted into the target site.
DNA gap is filled.
Replicative Transposition Mechanism
Transposase makes cuts, and the strands are exchanged and ligated, forming a cointegrate.
Gaps are filled in by DNA polymerase and sealed by DNA ligase.
Resolvase catalyzes recombination between the two elements, resolving them into two separate structures, each with a copy of the transposable element.
Effects Caused by Transposable Elements
Can insert within a gene to cause a mutation (mutagenic effect).
Can carry stop codons or termination sequences to block transcription or translation.
May carry promoters and activate genes near insertion points (regulatory mutations) - On/Off switch.
Can participate in plasmid fusion, insertion of plasmids into chromosomes, and in plasmid evolution.
Role of Transposons in Plasmid Evolution
Plasmids have several different transposon target sites, so transposons frequently move between plasmids.
Concerned with the transfer of antibiotic resistance genes between plasmids and between plasmids and chromosomes.
Bacterial Plasmids
Small, autonomously replicating DNA molecules.
Can exist independently or, as episomes, integrate reversibly into the host chromosome.
Can transfer copies of themselves to other bacteria during conjugation (e.g., conjugative plasmids -F plasmid).
Can expand their hosts’s ecological niches (e.g., degradation of toxic compounds, virulence, antibiotic resistance, new metabolic pathways).
Plasmid Prevalence in Bacteria and Archaea
Plasmids are widespread in bacteria and archaea, contributing to their genetic diversity and adaptability.
Bacterial Conjugation
Involves direct cell-to-cell contact mediated by the F pilus and unidirectional DNA transfer from donor to recipient.
This process depends on direct cell to cell contact.
Bacterial Conjugation
J. Lederberg and E. Tatum demonstrated the transfer of genes between bacteria that depends on
direct cell to cell contact mediated by the F pilus
unidirectional DNA transfer from donor to recipient (can be distantly related)
The F Plasmid
F factors contain the information for the formation of a sex pilus.
attaches F+ cell to F- cell for DNA transfer during bacterial conjugation
F factors have insertion sequences (IS) that assist in plasmid integration.
The F Plasmid
The F plasmid carries genes needed for conjugation.
Encodes pilin protein
Encodes proteins that are components of a type IV secretion system
region of F factor with genes needed for conjugation
Encodes coupling protein
Encodes relaxase
F Plasmid Integration
The F factor can integrate into the bacterial chromosome via insertion sequences (IS).
This converts the F+ cell into an Hfr cell.
The Mechanism of Conjugation
The F plasmid carries gene to form sex pilus, a “bridge” between two cells.
The F plasmid can be copied using the rolling circle method and sent across the bridge into the recipient cell.
This can turn an F– cell into an F+ cell, capable of conjugating with another F– cell.
F+ x F- Mating
A copy of the F factor is transferred to the recipient and does not integrate into the host chromosome.
Donor genes usually are not transferred.
F factor codes for sex pilus.
Type IV secretion system.
makes contact between cells that DNA moves across
Conjugation in E. coli
Sex pili pull the cells together, mating bridge is formed, and single-strand nick is made at the oriT.
One strand of the F plasmid is transferred into the recipient, and replication occurs in both donor and recipient cells to result in double-stranded DNA molecules.
Completion of F-plasmid transfer results in two F+ bacteria.
F Factor-Mediated Conjugation
Sex pilus makes contact with F- recipient cell.
Sex pilus contracts, bringing cells together.
Type IV secretion system is constructed and joins two cells.
Relaxosome makes a cut at the origin of transfer and begins to separate one DNA strand. The intact strand is replicated by the rolling-circle mechanism.
Accessory proteins of the relaxosome are released.
The DNA/relaxase complex is recognized by the coupling factor and transferred to the secretion system.
The secretion system pumps the DNA/relaxase complex into the recipient cell.
As the DNA enters, the F-factor DNA is replicated to become double stranded.
Rolling Circle Replication
The displaced strand is > 1 unit length in the F+ cell.
The displaced strand is almost 1 unit length in the original location.
Complementary strand synthesis occurs to create a dsDNA
The Type IV Secretion System
Encoded by the F factor.
Hfr x F- Conjugation
Donor Hfr (High Frequency of Recombination) cell has F factor integrated into its chromosome.
Leads to donor genes transfer to recipient cell (mobilization of donor chromosome).
A complete copy of the F factor is usually not transferred.
Gene transfer can be clockwise or counterclockwise.
Hfr cells & Hfr x F- Conjugation
Insertion of the F factor into the chromosome creates an Hfr cell.
Sex pilus of Hfr cell makes contact with F- cell and contracts to pull Hfr and F- cells together. Type IV secretion system connects cells.
Transfer of Hfr chromosome occurs via rolling-circle replication.
Recombination occurs between donor DNA and recipient DNA.
Recombination and DNA transfer
Hfr strains transfer genes in a specific order and at a specific rate.
The order of transfer can be used to map the location of genes on the bacterial chromosome.
F’ Plasmid
Results when the F factor is aberrantly excised from the host chromosome.
Excision product contains host genes.
Some F factor genes may be retained in the chromosome.
F’ plasmid conjugation mediates transfer of host genes to the recipient cell.
Presence can lead to some gene duplicates.
F' Conjugation
Involves deintegration of the F factor, including part of the bacterial chromosome.
Cells are connected by a type IV secretion system.
The F' plasmid begins replication and transfer.
The F' plasmid is replicated and transferred.
Bacterial Transformation
F. Griffith demonstrated transformation.
Uptake of naked DNA by a competent cell followed by incorporation of the DNA into the recipient cell’s genome.
Bacterial Transformation
Fragments of DNA are taken up by the bacteria
Integration occurs by nonreciprocal DNA recombination leading to a stable transformation
Unsuccessful transformation leads to degradation.
Plasmids can also be taken up for stable transformation.
Transformation Mechanism
DNA from the donor cell binds to DNA receptor proteins on the recipient cell.
One strand of the DNA bound to receptor proteins is pulled by the DNA translocase through the channel formed by pore proteins while the other strand is degraded. The remaining single DNA strand is bound by single-stranded DNA-binding protein and RecA.
Introduced DNA is integrated into the chromosome by recombination.
Fate of transformation
DNA Uptake leads to expression
Transduction
The transfer of bacterial genes by viruses.
Viruses (bacteriophages) can carry out the lytic cycle in which the host cell is destroyed, or the viral DNA can integrate into the host genome, becoming a latent prophage (lysogenic cycle).
Lytic and Lysogenic Cycles of Temperate Phage
Phage DNA can integrate into the host chromosome.
Exposure to stress leads to excision from host chromosome.
Phage DNA leads to synthesis of many new phages.
Cell lyses and releases the new phages. leads to Cell lyses
Generalized Transduction
Any part of the bacterial genome can be transferred.
Occurs during the lytic cycle of virulent phage.
During viral assembly, fragments of host DNA are mistakenly packaged into the phage head (transducing particles).
Host DNA fragments cannot replicate but can recombine with the new host DNA.
Generalized Transduction in Bacteria
Phage infects bacterial cell.
Host DNA is hydrolyzed into pieces, and phage DNA and proteins are made.
Phages assemble; occasionally, a phage carries a piece of the host cell chromosome.
Transducing phage injects its DNA into a new recipient cell.
The transduced DNA is recombined into the chromosome of the recipient cell.
Specialized Transduction
Carried out only by temperate phages that have established lysogeny.
Only a specific portion of the bacterial genome is transferred.
Occurs when the prophage is incorrectly excised.
Specialized Transduction by a Temperate Bacteriophage
Prophage induction leads to a lysogenized cell with prophage.
Rare deintegration that includes some bacterial genes occurs.
Replication of defective virus DNA with incorporated host genes occurs.
Assembly and release of transducing phage particles.
Infection of recipient cell.
Crossover to integrate bacterial genes.
Integration as prophage
Bacterial chromosome containing both virus and donor DNA