Microbial Genetics / Molecular Biology Flashcards
(32 cards)
Bacterial Chromosome vs. Plasmid
Chromosome
- Primary DNA molecule in the cell
- Located in the nucleoid
- Large, circular DNA (millions of base pairs -> Mbp)
- One copy per cell
- Carries essential genes for cell survival and function
- Replicates during binary fission
Plasmid*
- Extra-chromosomal, accessory DNA molecule
- Found in the cytoplasm
- Small, circular DNA (thousands of base pairs -> Kbp)
- Multiple copies and types per cell
- Carries non-essential genes (e.g., antibiotic resistance, virulence factors)
- Controls its own replication
- May not divide evenly during binary fission
- Can be lost, degraded, or incorporated into the chromosome. There is a “burden” to carrying a plasmid and may be better for bacteria to dump plasmid
Mutation Rates – Viruses vs. cell
Bacteria and Eukaryotes
- Mutation rate: ~1 in 250,000,000 bases
- Use high-fidelity DNA polymerases with proofreading activity
Viruses
- Mutation rate: ~1 in 250,000 bases (on average)
- RNA viruses have especially high mutation rates
- Use viral RNA-dependent RNA polymerases, which are more error-prone
- Many lack proofreading, but not all
- DNA viruses vary:
- Some use host polymerases → lower mutation rate
- Others encode their own → higher mutation rate
Summary:
- RNA viruses mutate faster due to error-prone, self-encoded polymerases
- DNA viruses can resemble host mutation rates if they rely on host enzymes
Mutation – Overview and Types
- an alteration in the DNA (or RNA) sequence
- effect depends on:
- type of nucleotide change
- location in the genome
- impact on resulting protein
- outcomes can be deleterious, neutral (null), or beneficial
types of mutation:
- point mutation (base substitution)
- silent mutation → no change in amino acid
- missense mutation → changes one amino acid
- nonsense mutation → codon becomes stop codon
- sense mutation → stop codon becomes an amino acid codon
- insertion or deletion
- frameshift mutation → alters the reading frame, changing all downstream amino acids
note:
- in viruses, mutations can occur in RNA
- RNA viruses mutate faster due to use of error-prone viral RNA polymerases
Gene Transfer – Vertical vs. Horizontal
vertical gene transfer
- Transfer of DNA from parent to offspring
- Occurs in both sexual and asexual reproduction
- In eukaryotes, involves sexual reproduction (meiosis + fertilization) -> genetic diversity
- In bacteria, occurs through asexual reproduction (binary fission)
- Ensures genetic continuity between generations
horizontal gene transfer
- Transfer of DNA between individuals of the same generation
- Common in bacteria and archaea, rare in eukaryotes
- Involves transfer between unrelated cells, not through reproduction
- Increases genetic diversity and adaptability
- Mechanisms: transformation, transduction, conjugation
Bacterial Transformation
- Definition: Uptake of naked DNA from the environment into a bacterial cell
- Mechanism of horizontal gene transfer
natural transformation
- Requires competence: the physiological ability to take up DNA -> competence pilus.
- Only some species are naturally competent -> AKA have competence pilus
- Use a competence pilus to bind and import environmental DNA
- Imported DNA may recombine with the chromosome or exist as a plasmid
artificial transformation
- Involves lab manipulation to force uptake of DNA (e.g., plasmids)
- Methods include:
- Heat shock (opens pores in membrane)
- Electroporation (electric pulse to disrupt membrane)
- Used in recombinant DNA technology and genetic engineering
- Does not occur naturally; bypasses normal physiological barriers
note:
- Transformation differs from transduction (virus-mediated) and conjugation (cell-to-cell contact)
Generalized Transduction
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Phage infection
- A lytic bacteriophage infects a bacterium and injects its DNA
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Host DNA degradation
- Viral enzymes degrade the host chromosome into fragments
-
Mistake in assembly
- During the synthesis/assembly phase, a random fragment of bacterial DNA is mistakenly packaged into a phage capsid
- Creates a defective phage (transducing particle) that lacks viral genes
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Injection into new host
- Transducing phage injects donor bacterial DNA into another bacterial cell
- No productive infection occurs (no viral replication)
-
Genetic recombination
- Injected DNA may undergo homologous recombination with the recipient genome
- Introduces new traits to the recipient bacterium
note:
- DNA transferred is random
- Enables horizontal gene transfer of any gene from donor
Transduced DNA must recombine with the chromosome to be stably inherited It does not form or act as a plasmid, unless — by rare chance — it contains an origin of replication and remains extrachromosomal (very uncommon)
Specialized Transduction
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Lysogenic phage infection
- A bacteriophage infects a bacterium and integrates its genome into the host chromosome
- The integrated phage DNA is called a prophage
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Prophage excision (induction)
- Under stress (e.g., UV light), the prophage is induced to exit the host genome and enter the lytic cycle
-
Mistake in induction
- During excision, the phage accidentally takes adjacent bacterial genes with it
- A specific, non-random region of the host DNA is packaged along with viral DNA
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Phage assembly
- New phage particles are assembled containing both viral DNA and specific bacterial genes
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Infection of a new host
- The defective phage infects another bacterial cell and injects its hybrid DNA
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Integration or recombination
- The bacterial genes may recombine with the new host genome
- May result in lysogenic conversion, where the recipient gains new traits (e.g., toxin genes)
note:
- DNA transferred is non-random and located near the prophage insertion site
- Occurs only in lysogenic phages
- Can influence pathogenicity -> Specialized transduction by temperate phages is a major mechanism by which bacteria acquire toxin genes and virulence factors
Conjugation
\Transfer of DNA from one bacterium (F⁺ donor) to another (F⁻ recipient) through direct contact
Steps of conjugation:
1. F⁺ donor contains an F plasmid that encodes a conjugation pilus
2. The pilus brings the F⁺ and F⁻ cells into contact (but does not transfer DNA itself)
3. A copy of the F plasmid is replicated and transferred to the F⁻ cell via a mating bridge (not the pilus)
4. After transfer, both cells are F⁺ — each carries a full F plasmid
Additional notes:
- Transfer occurs during rolling-circle replication
- Major mechanism for spreading antibiotic resistance genes
- Can lead to a rapid increase in F⁺ cells in a population
- Only plasmid DNA is transferred in standard conjugation, not chromosomal DNA
Operons
- A group of functionally related structural genes controlled by a single promoter and transcribed as one polycistronic mRNA → one mRNA, multiple ORFs
- Genes in an operon are turned on or off together
- Common in prokaryotes, especially for genes in metabolic pathways
- Two major types:
- Inducible operons (e.g., lac operon) → usually off, turned on by inducer
- Repressible operons (e.g., trp operon) → usually on, turned off by corepressor
Operon Components:
- Regulatory gene (not part of the operon): Encodes a regulatory protein such as a repressor, activator, or both
- Regulatory region of the operon = promoter + operator (+ activator-binding site if present)*
- Promoter: Site where RNA polymerase binds to begin transcription
- Operator: Site where a repressor may bind to block transcription
- Activator-binding site: DNA sequence (often upstream of promoter) where an activator binds to enhance transcription
- Structural genes: Encode proteins (often enzymes) that carry out a shared function
Regulatory Elements:
- Repressor: Binds to the operator to block RNA polymerase
- Activator: Binds to the activator-binding site to enhance RNA polymerase binding
- Inducer: Small molecule that binds to a repressor or activator to alter its function
- Example: Allolactose inactivates the lac repressor (inducing transcription)
Note:
- One operon → one long mRNA → multiple proteins translated independently
- Regulation allows coordinated expression and energy-efficient gene control
Inducible vs. Repressible Operons
inducible operon
- typically controls catabolic pathways (breakdown of nutrients)
- off by default – transcription blocked by an active repressor
- turned on when the substrate is present. Most often the substrate is the inducer (lac operon)
- repressor is active by default → binds operator and blocks transcription
- inducer binds to repressor, inactivating it → transcription proceeds
- example: lac operon (responds to lactose availability)
repressible operon
- typically controls anabolic pathways (biosynthesis of essential molecules)
- on by default – transcription proceeds unless turned off
- turned off when product accumulates and is no longer needed
- repressor is inactive by default → cannot block transcription alone
- co-repressor binds to repressor, activating it → blocks transcription. Often co-repressor is the end product of the regulated pathway
- example: trp operon (shuts off tryptophan synthesis when tryptophan is abundant)
note:
- both systems use negative control via repressor proteins
- allow bacteria to adapt gene expression to environmental conditions
Lac Operon
- lac operon is an inducible operon used to metabolize lactose in E. coli
- controls production of enzymes for lactose breakdown
- normally off by default because the repressor is active and blocks transcription
absence of lactose (no inducer):
- repressor binds operator, blocking RNA polymerase
- structural genes (lacZ, lacY, lacA) are not transcribed
- conserves energy by not producing enzymes when lactose is unavailable
presence of lactose (inducer present):
- lactose acts as the inducer
- binds the repressor → changes its shape (allosteric inhibition)
- repressor cannot bind operator → RNA polymerase binds promoter
- transcription of structural genes occurs → enzymes produced for lactose metabolism
Trp Operon
- trp operon is a repressible operon that controls anabolic synthesis of tryptophan
- normally on by default so enzymes for tryptophan biosynthesis are made
- expression is turned off when tryptophan is abundant
absence of tryptophan (no co-repressor):
- repressor is inactive → cannot bind operator
- RNA polymerase binds promoter → transcribes structural genes (e.g., trpE, trpD, trpC, trpB, trpA)
- tryptophan is synthesized
presence of tryptophan (co-repressor present):
- tryptophan binds to repressor, changing its shape
- active repressor binds operator → blocks RNA polymerase
- transcription is halted → no unnecessary tryptophan made
note:
- trp operon is regulated by negative feedback
- this conserves resources by shutting down biosynthesis when product accumulates
antigenic drift / antigenic shift
- influenza A has a segmented -ssRNA genome (8 segments), allowing for rapid genetic variation
- two major mechanisms of viral change: antigenic drift and antigenic shift
- both affect surface glycoproteins: hemagglutinin (HA) and neuraminidase (NA) → critical for immune recognition
antigenic drift
- caused by random point mutations in the RNA during replication. This causes slight changes in the spike proteins hemagglutinin and neuraminidase.
- due to lack of proofreading by viral RNA polymerase
- leads to small-scale changes in glycoprotein structure (especially HA/NA)
- can alter epitopes and help virus evade adaptive immunity
- causes seasonal flu epidemics
- occurs frequently — every 1–3 years
- responsible for yearly flu vaccine updates
antigenic shift
- caused by reassortmentof RNA segments when two different influenza viruses infect the same host cell
- produces novel combinations of glycoproteins
- leads to major, sudden change → creation of a new viral subtype
- population may have no pre-existing immunity
- can result in pandemics
- occurs less frequently — every 10–20 years
- requires segmented genome and coinfection in a shared host species (e.g., pigs, birds)
summary
- drift = minor, frequent mutations → epidemic flu
- shift = major, rare reassortment → pandemic flu
note:
- these mechanisms explain why influenza evolves so quickly, making long-term vaccines difficult
antigenic drift is common to RNA viruses, Antigenic shift is restricted to influenza A. Influenza B has a segmented genome but much more restricted host range → doesn’t undergo shift
Antigenic Shift in Influenza A vs B
- Influenza A: Undergoes antigenic shift via reassortment of RNA segments from different strains, often across species (e.g., human + avian)
-
Influenza B: Also has a segmented genome and can undergo reassortment between human strains, but:
- Has a narrow host range (only humans)
- Shows limited genetic diversity
- Reassortment events are rare and mild → do not cause pandemics
- Conclusion: Antigenic shift is functionally significant only in Influenza A
2009 H1N1 Pandemic
- the 2009 H1N1 influenza pandemic was caused by a novel influenza A virus
- formed through antigenic shift: reassortment of RNA segments from multiple influenza viruses infecting the same pig host (mixing vessel)
contributing viral strains:
- Eurasian pig strain
- Classic H1N1 pig strain
- North American human strain (H3N2)
- Avian strain
process:
- co-infection in pigs allowed RNA segments from different strains to mix
- resulted in a new H1N1 virus with unique combinations of genes from swine, human, and avian origins
- people had little to no pre-existing immunity to this strain
outcome:
- the reassorted virus spread rapidly → caused global pandemic in 2009
- known as “swine flu”
- demonstrated how antigenic shift creates pandemic potential
note:
- pigs can be infected by both avian and human influenza viruses
- this cross-species susceptibility makes them a key reservoir for genetic reassortment
Biotechnology
- Use of living organisms or their biological components to produce a product or perform a function
Examples:
- Bread, beer, wine – fermentation by yeast
- Cheese – uses molds, bacteria, or animal-derived enzymes
- Insulin – produced by genetically modified yeast containing recombinant DNA
Key distinctions:
- Biotechnology ≠ genetic engineering
- Biotechnology includes any biological process, traditional or modern
- Genetic engineering is a subset of biotechnology that specifically involves modifying genetic material
Summary:
- Biotechnology does not require genetic engineering, but
- Genetic engineering always uses biotechnology
Genetic engineering
- The intentional modification of an organism’s genome to achieve a desired trait or function
- Involves recombinant DNA: combining DNA from different sources into a single molecule
Types of recombinant DNA:
- Cisgenic recombinant DNA
- DNA comes from the same or closely related species*
- Example: blight-resistant potatoes
- Transgenic recombinant DNA
- DNA comes from a different, unrelated species
- Example: Bt toxin–producing Brinjal (eggplant)
Key notes:
- Recombinant DNA can also occur naturally, but genetic engineering uses lab techniques to direct it
Recombinant DNA
DNA made by combining genetic material from different sources. Most often engineered in the lab, but natural mechanisms also produce recombinant DNA
Cisgenic recombinant DNA: Novel DNA sourced from closely related / same species -> Blight resistant potatoes
transgenic recombinant DNA: Novel DNA sourced from unrelated species -> BT toxin producing Brinjal.
Insulin example:
1. Isolate human insulin gene from a somatic cell
2. Insert it into a bacterial plasmid → forms recombinant DNA
3. Introduce recombinant plasmid into a bacterial host -> TRANSFORMATION
4. Bacteria replicate, producing human insulin
5. Insulin is harvested from bacterial cultures
Key concept:
- The bacteria are transgenic because they express DNA from another species (human)
- Common example of genetic engineering used in medicine
CRISPR
Clustered Regularly Interspaced Short Palindromic Repeats
- Definition: A DNA-based adaptive immune system found in bacteria and archaea that defends against viral infections
- Structure:
- Repeats: Short palindromic sequences
- Spacers: Unique sequences between repeats, derived from viral DNA
- Acquisition:
- When a bacterium survives a viral infection, it captures a fragment of the viral DNA
- This fragment is inserted into the CRISPR array as a new spacer, near the leader sequence
- This process is mediated by Cas proteins (e.g., Cas1 and Cas2)
- Immunity function:
- CRISPR array is transcribed into a long RNA → processed into crRNAs
- crRNAs guide Cas nucleases (e.g., Cas9) to matching viral DNA
- Cas proteins then cleave the foreign DNA, preventing infection
- Key point: CRISPR provides heritable, sequence-specific immunity based on past viral exposures
Define Gene Editing
Using enzymes to cut or alter specific target DNA sequences, including within a gene.
CRISPR-Cas9 Gene Editing
- Definition: A gene-editing tool adapted from the bacterial CRISPR-Cas immune system, used to make precise changes in eukaryotic genomes
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Components:
- Cas9: A programmable endonuclease that introduces a double-stranded break (DSB) in DNA. Uses gRNA to locate target DNA sequence. Cuts DNA.
- Guide RNA (gRNA): A synthetic RNA molecule that is complementary to target DNA sequence.
- Together, the gRNA guides Cas9 to the target site by base-pairing with the complementary DNA sequence
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Mechanism:
- gRNA binds to a specific DNA sequence next to a PAM (Protospacer Adjacent Motif)
- Cas9 makes a DSB at the target site
- The cell repairs the break via:
- Non-homologous end joining (NHEJ) → may cause insertions/deletions (gene disruption)
- Homology-directed repair (HDR) → allows insertion of desired DNA if a repair template is provided
- Applications: Gene knockout, correction of mutations, gene insertion, research, and therapeutics
Bt Brinjal
- Definition: Genetically modified eggplant engineered to produce Bt toxin (from Bacillus thuringiensis)
- Purpose: Confers resistance to fruit and shoot borer (Leucinodes orbonalis)
- Gene inserted: Often cry1Ac
- Status: Commercially grown in Bangladesh; not approved in India and some other countries
- Benefit: Reduces need for chemical insecticides
Bt Toxin
- Definition: Insecticidal protein produced by Bacillus thuringiensis
- Target: Specific insect larvae (e.g., Lepidoptera, Diptera, Coleoptera)
-
Mechanism:
- Ingested by insect
- Binds to gut receptors
- Creates pores in gut lining → cell lysis
- Causes death by gut disruption
- Use: Expressed in GM crops (Bt corn, Bt cotton, Bt brinjal) for biological pest control
Blight-Resistant Potatoes
- Definition: Genetically modified or selectively bred potatoes that resist late blight caused by the fungus-like pathogen Phytophthora infestans
- Gene source: Resistance (R) genes often derived from wild potato species (e.g., Solanum bulbocastanum)
- Mechanism: R genes detect pathogen effectors and activate immune response to stop infection
- Purpose: Prevent crop loss, reduce dependence on fungicides, and improve yield stability
- Example: GM varieties like Innate® potatoes (also have reduced bruising and acrylamide formation)
