M5 - Prokaryotic Genetics Flashcards
Why do we study bacterial genetics?
To understand molecular biology, human health, bacterial ecology, and to apply knowledge in biotechnology.
What is bacterial genetics?
The study of how heritable information is transferred in bacteria through chromosomes, plasmids, transposons, and phages.
Why are bacteria good model organisms?
They are haploid, reproduce asexually, have short generation times, grow easily in defined media, and are easy to genetically manipulate.
What is the structure of the bacterial genome?
Most bacteria have a single circular, double-stranded DNA chromosome, although some, like Borrelia burgdorferi, have a linear chromosome.
What is binary fission?
A form of asexual reproduction where the bacterial cell elongates, replicates its DNA, and divides into two identical daughter cells.
What is a biosynthetic auxotroph?
A mutant strain that cannot synthesize a specific compound (e.g., an amino acid) and requires it from the environment for growth.
What is a catabolic auxotroph?
A mutant strain that cannot break down a specific carbon source, limiting its ability to use that substrate for growth.
What are housekeeping genes?
Genes essential for bacterial survival, involved in critical functions like DNA replication, transcription, translation, and cell division.
What are temperature-sensitive (ts) mutants?
Mutants that function normally at a permissive temperature but show defects at a restrictive temperature due to protein misfolding.
How are genes named in bacterial genetics?
Using three lowercase italicized letters for the pathway and a capital letter for the specific gene (e.g., leuB for leucine biosynthesis).
How are phenotypes denoted in bacterial genetics?
Using the same three-letter code as the gene but capitalized and non-italicized (e.g., Leu⁻ indicates a strain that requires leucine).
What is a conditional lethal mutant?
A mutation that is lethal under certain conditions but allows survival under permissive conditions (e.g., specific temperatures).
What does an auxotroph for histidine (His⁻) require to grow?
It needs histidine supplied in its growth medium.
What are suppressor mutations (sup)?
Mutations that allow translation to continue past a stop codon, compensating for nonsense mutations.
What does Δ (delta) symbolize in bacterial genetics?
A gene deletion, meaning a specific gene or sequence has been removed (e.g., leuA Δ indicates a deletion of the leuA gene).
What is Lamarckian evolution?
A theory suggesting that changes in organisms are directed by the environment, with traits acquired during life passed on to offspring.
How does Darwinian evolution differ from Lamarckian evolution?
In Darwinian evolution, changes occur spontaneously through random mutations, and natural selection favors the survival of the fittest.
What was the main conclusion of the Luria-Delbrück experiment (1943)?
Mutations occur randomly before exposure to selective agents, supporting the Darwinian model of evolution in bacteria.
How did the Luria-Delbrück experiment test bacterial resistance?
E. coli cultures were grown, then exposed to T1 phage; large variations in resistant colonies across plates suggested pre-existing mutations.
What is the significance of the Luria-Delbrück experiment for bacterial evolution?
It demonstrated that bacterial resistance results from random mutations rather than directed changes due to environmental factors.
What did the Newcombe experiment (1949) show about bacterial resistance?
Re-spreading bacteria allowed pre-existing resistant cells to be evenly distributed, confirming that resistance mutations were present before exposure.
What technique was introduced by Lederberg and Lederberg (1952)?
Replica plating, a method to detect bacterial mutants by transferring cells from a master plate to selective media.
What was the conclusion of the Lederberg and Lederberg experiment?
Resistant bacteria existed before exposure to the selective agent, and their consistent position on plates supported random mutation theory.
How do antibiotics drive bacterial evolution?
Antibiotics select for pre-existing mutations in bacteria, allowing resistant strains to survive and proliferate.
What is Lysenkoism, and how did it challenge evolutionary theory?
A politically driven rejection of Darwinian evolution in Soviet science, incorrectly promoting Lamarckian ideas of directed inheritance.
Why were prokaryotes initially thought to be exceptions to Darwinian evolution?
Early scientists believed prokaryotes did not follow typical evolutionary patterns due to a lack of supporting data at the time.
What is meant by “selection of the fittest” in bacterial evolution?
It refers to the process where bacteria with beneficial mutations survive environmental pressures, such as antibiotics, and pass on these traits.
How does the environment influence bacterial evolution?
While mutations are random, environmental pressures like antibiotics select for advantageous mutations, shaping the bacterial population.
What is the significance of T1 phage in bacterial evolution studies?
T1 phage infects and kills E. coli, allowing scientists to study bacterial resistance mechanisms through selective pressure.
What is the scientific method’s role in understanding bacterial evolution?
It involves making observations, testing hypotheses through experiments, and refining conclusions based on consistent evidence.
Why are bacterial mutants important in scientific research?
They help us understand biochemistry, cell biology, disease control, and have applications in biotechnology.
What is a spontaneous mutation?
A mutation that arises naturally due to replication errors, tautomerization, or DNA damage without exposure to external mutagens.
What are mutagens?
Chemical or physical agents that increase mutation rates by damaging DNA, such as nitrous acid, UV light, or alkylating agents.
What is a point mutation?
A mutation affecting a single base pair in DNA, which can be a substitution, deletion, or insertion.
What is a frameshift mutation?
A mutation caused by insertions or deletions that shift the reading frame, leading to altered or nonfunctional proteins.
What is a missense mutation?
A substitution that changes a codon, leading to the incorporation of a different amino acid into the protein.
What is a nonsense mutation?
A mutation that converts a codon into a stop codon, prematurely terminating protein synthesis.
What is the Ames test?
A biological assay using Salmonella typhimurium to identify mutagenic chemicals, often linked to carcinogenicity.
What is a revertant mutation?
A mutation that restores the original DNA sequence and phenotype.
What is suppressor mutation?
A secondary mutation that compensates for the effects of a primary mutation, restoring the normal phenotype.
What are intercalating agents, and how do they affect DNA?
Flat molecules (e.g., ethidium bromide) that insert between DNA base pairs, distorting the helix and causing frameshift mutations.
What is a temperature-sensitive (ts) mutant?
A mutant that functions normally at permissive temperatures but shows defects at restrictive temperatures.
How can replica plating help identify mutants?
It transfers bacterial colonies from a master plate to selective media, helping identify mutants that cannot grow under specific conditions.
What is cross-feeding in bacterial mutants?
When two mutant strains support each other’s growth by exchanging essential metabolites that each strain cannot produce on its own.
What are large-scale mutations?
Mutations that affect large DNA segments, including deletions, inversions, tandem repeats, or transposon insertions, which can disrupt gene function.
What is an operon?
An operon is a group of genes under the control of a single promoter, allowing them to be regulated and transcribed together.
What are the three main genes in the lac operon, and what do they encode?
lacZ: β-galactosidase (breaks down lactose)
lacY: β-galactoside permease (transports lactose into the cell)
lacA: Galactoside acetyltransferase (possibly detoxifies harmful compounds)
What is the default state of the lac operon?
The lac operon is normally repressed (OFF) to prevent energy waste when lactose is absent.
What molecule acts as the inducer of the lac operon?
Allolactose, a derivative of lactose, binds the repressor to deactivate it and initiate transcription.
How does the lac operon respond when glucose is present?
Glucose inhibits cAMP production, preventing CAP from enhancing transcription, a process known as catabolite repression.
What is diauxic growth?
A two-phase growth pattern where bacteria first consume glucose and then switch to lactose once glucose is depleted.
What is the role of the lacI gene?
It encodes the lac repressor protein, which binds the operator region to block transcription in the absence of lactose.
What is the role of cAMP in the lac operon?
cAMP binds to the catabolite activator protein (CAP), enhancing the binding of RNA polymerase to the promoter, increasing transcription.
How does glucose inhibit lac operon activation?
Glucose suppresses adenylate cyclase activity, lowering cAMP levels and preventing CAP activation.
What is IPTG, and how is it used in biotechnology?
IPTG is a non-metabolizable lactose analogue that induces the lac operon without being broken down, commonly used in lab experiments.
Why is the lac operon significant in biotechnology?
Its promoter is widely used to control the expression of genes, allowing scientists to produce proteins like insulin.
How does β-galactosidase enable lactose metabolism?
It breaks lactose into glucose and galactose and also forms allolactose to induce the lac operon.
What happens in a lacZ⁻ mutant?
The bacteria cannot metabolize lactose due to the absence of β-galactosidase activity.
What is catabolite repression?
A regulatory mechanism where the presence of glucose inhibits the transcription of other carbon metabolism genes, including the lac operon.
Why don’t bacteria express the lac operon when glucose is present?
To conserve energy, as glucose is a preferred carbon source, and the machinery to process lactose is unnecessary until glucose is depleted.
Write 15 question and answer cards based off the attached lecture, making sure that someone revising from the cards would know the contents of the lecture off by heart by revising from the cards.
The transforming principle—non-pathogenic bacteria can acquire genetic material from heat-killed pathogenic bacteria and become virulent.
What is bacterial transformation?
The process by which bacteria take up extracellular (naked) DNA from their environment, incorporating it into their genome.
What is competence in bacterial transformation?
The ability of a bacterial cell to take up external DNA, which can occur naturally or be induced artificially (e.g., in E. coli for biotech purposes).
Name two naturally competent bacteria.
Streptococcus pneumoniae and Bacillus subtilis.
What is horizontal gene transfer (HGT)?
The transfer of genetic material between organisms that are not parent and offspring, increasing genetic diversity.
Why does competence often occur near the stationary phase of bacterial growth?
It is linked to quorum sensing and nutrient limitation, allowing bacteria to acquire genetic material for survival.
What role does the ComX pheromone play in Bacillus subtilis competence?
ComX concentration increases with cell density and binds to ComP, triggering gene expression changes that induce competence.
How do bacteria recognize DNA from the same species during transformation?
Through specific recognition sequences found more frequently in their own DNA (e.g., 5’ AAGTGCGGTCA 3’ in Haemophilus influenzae).
What is transduction in bacteria?
A process where bacteriophages (viruses) transfer DNA from one bacterial cell to another.
What is generalized transduction?
Random bacterial DNA fragments are mistakenly packaged into phage particles and transferred to new host cells during infection.
What is specialized transduction?
Specific bacterial genes near prophage integration sites are transferred due to improper excision of lysogenic phage DNA.
What is a lysogen?
A bacterial cell carrying a prophage (phage DNA integrated into the bacterial genome), which can be passed on during cell division.
How does the lytic cycle of a phage differ from the lysogenic cycle?
The lytic cycle leads to immediate phage replication and host cell lysis, while the lysogenic cycle integrates phage DNA into the host genome, remaining dormant until triggered.
What is the restriction-modification system in bacteria?
A defense mechanism where bacterial enzymes (restriction enzymes) cut invading phage DNA, while methylation protects the host’s own DNA.
How is phage therapy being reconsidered in modern medicine?
As an alternative to antibiotics, particularly for treating antibiotic-resistant bacterial infections.
What is bacterial conjugation?
A process of horizontal gene transfer where genetic material, usually plasmids, moves from a donor to a recipient via direct cell-to-cell contact.
What was the significance of the Lederberg and Tatum (1946) experiment?
It demonstrated genetic recombination in bacteria by showing that mixing two mutant strains could produce wild-type progeny.
How did the Davies (1950) experiment confirm the need for cell-to-cell contact in conjugation?
Using a semipermeable filter, he showed that physical separation prevented gene transfer, ruling out cross-feeding, transformation, and transduction.
What are plasmids?
Small, circular double-stranded DNA molecules that replicate independently of chromosomal DNA and often carry useful genes like antibiotic resistance.
What are conjugative plasmids?
Plasmids that encode the genes necessary for their own transfer, such as tra genes responsible for pilus formation and DNA transfer.
What is the F plasmid?
A fertility plasmid in E. coli that facilitates DNA transfer during conjugation and can integrate into the bacterial genome.
What is the function of the F pilus?
It forms a physical bridge between the donor (F⁺) and recipient (F⁻) cells, enabling DNA transfer.
How is DNA transferred during conjugation?
The plasmid is nicked at the origin of transfer, unrolled as single-stranded DNA, and replicated in both donor and recipient cells using rolling circle replication.
What is a High-Frequency Recombination (HFr) strain?
A bacterial strain where the F plasmid has integrated into the chromosome, allowing partial transfer of chromosomal genes during conjugation.
Why don’t HFr strains convert F⁻ recipients to F⁺?
The entire F plasmid is rarely transferred because the mating pair usually separates before the full transfer is complete.
What is an episome?
A plasmid capable of integrating into the host genome, such as the F plasmid in E. coli.
What is an F’ plasmid?
A plasmid that excises imprecisely from the chromosome, carrying some chromosomal genes along with the original plasmid genes.
What is a merodiploid cell?
A cell that is haploid overall but temporarily carries two copies of certain genes—one on the chromosome and one on the F’ plasmid.
What is time-of-entry mapping in conjugation?
A technique to determine gene order and distance on the bacterial chromosome by measuring the time it takes for different genes to transfer during conjugation.
What is the biological significance of bacterial conjugation?
It promotes genetic diversity, spreads antibiotic resistance, and can transfer virulence factors, contributing to bacterial adaptation and evolution.
Why might scientists want to clone or express a gene?
To determine its nucleotide sequence, analyze control sequences, identify mutations, study protein structure and function, produce large amounts of protein, and create tagged versions for purification.
What are the key steps for cloning a gene into E. coli?
Restriction digest, ligation of the gene into a plasmid, transformation into E. coli, and selection of successfully transformed cells.
What are restriction enzymes, and how do they work?
Enzymes that recognize specific DNA sequences and cut the DNA at or near those sites, facilitating gene insertion.
How does DNA ligase function in gene cloning?
It joins DNA fragments by forming phosphodiester bonds, allowing seamless insertion of DNA into plasmids.
Why is complementary DNA (cDNA) used when cloning eukaryotic genes into bacteria?
Bacteria cannot remove introns from eukaryotic genes, so cDNA (which lacks introns) is used for proper expression.
How is cDNA synthesized from mRNA?
Reverse transcriptase converts mRNA into single-stranded DNA, followed by DNA polymerase to make it double-stranded for cloning.
What is a plasmid, and what are its key features?
A circular DNA vector containing an origin of replication, selectable markers, and multiple cloning sites for gene insertion.
What is bacterial transformation?
The process by which bacteria take up foreign DNA from their environment, often facilitated by chemical treatment or electroporation.
How does blue-white screening identify successful clones?
White colonies indicate successful insertion (disruption of lacZ’), while blue colonies have non-disrupted lacZ’ and lack inserts.
How does electroporation work in bacterial transformation?
An electric pulse creates temporary pores in the bacterial membrane, allowing DNA to enter the cell.
What is an expression vector?
A plasmid engineered with bacterial promoters, ribosome-binding sites, and terminators to ensure proper transcription and translation of cloned genes.
What is a His-tag, and why is it useful in protein purification?
A sequence of six histidines added to proteins, allowing purification through binding to nickel columns.
What are fusion proteins, and why are they important?
Proteins made by combining two genes, enabling researchers to study protein functions, localizations, and interactions.
What role does GFP play in molecular biology?
Green Fluorescent Protein (GFP) is used to visualize gene expression, protein localization, and molecular interactions within cells.
How do restriction enzymes recognize specific DNA sequences?
They identify palindromic sequences and cut symmetrically within or near these sites, producing sticky or blunt ends for cloning.
What is recombination in bacterial genetics?
Recombination breaks and joins DNA to form new genetic combinations, facilitating rapid evolution and enabling gene complementation.
What is the difference between homologous and non-homologous recombination?
Homologous recombination: Requires sequence similarity (e.g., swapping alleles of the same gene).
Non-homologous recombination: Joins DNA fragments without sequence similarity, often during double-stranded break repair.
What are the main mechanisms of horizontal gene transfer in bacteria?
Transformation, transduction, conjugation, and conjugative transposon transfer.
What enzymes are crucial for homologous recombination in E. coli?
RecA (catalyzes DNA strand exchange) and RecBCD (initiates DNA nicks and unwinds DNA for recombination).
What is a Holliday junction?
A cross-shaped DNA structure formed during homologous recombination, allowing the exchange of genetic material between DNA strands.
What role does RuvC play in recombination?
RuvC is a nuclease that resolves Holliday junctions by cleaving the DNA to complete recombination.
What is non-homologous recombination used for in biotechnology?
Techniques like CRISPR-Cas9 introduce double-stranded DNA breaks at specific sites, enabling gene editing without the need for sequence similarity.
What are insertion sequences (IS elements)?
Small DNA segments (~1000 bp) that move within the genome via transposition, catalyzed by transposase, without carrying additional genes.
How can insertion sequences affect bacterial genes?
They can disrupt genes if inserted into coding regions, which may result in gene knockouts or lethal mutations.
What are transposons, and how do they differ from insertion sequences?
Transposons are larger DNA elements that carry additional genes (e.g., antibiotic resistance) and sometimes tra genes for conjugation.
What is transposition?
The movement of transposable elements (insertion sequences or transposons) from one genomic location to another.
What are the two types of transposition mechanisms?
Conservative transposition: The transposon is cut out and inserted elsewhere.
Replicative transposition: A copy of the transposon is made and inserted, while the original remains in place.
How can transposons contribute to antibiotic resistance?
They can carry resistance genes and spread them through bacterial populations via horizontal gene transfer.
What is the biological significance of recombination in bacteria?
It generates genetic diversity, repairs DNA damage, and facilitates adaptation through horizontal gene transfer.
What is the function of the RecBCD complex in homologous recombination?
It initiates DNA strand nicking, unwinds DNA, and prepares single-stranded DNA for RecA-mediated recombination.
