Microbial Genetics Flashcards
RecA
Protein required for homologous recombination
lateral gene transfer
gene transfer between unrelated cells
-Due to lateral gene transfer organisms with similar genes may not have evolved from a common ancestor
vertical gene transfer
Gene transfer from mother to daughter
transformation
gene transfer by taking up soluble DNA from the environment
What is transduction?
Gene transfer by means of bacteriophages
Generalized Transduction Process
- Bacteriophage injects its genome into a host bacterium
- The phage genome directs the bacterial cell to synthesize new
phage genomes and new phage coats. Phage genomes are
packaged into phage heads to make mature phage particles. At
an extremely low frequency, there is a mistake in phage
packaging, and bacterial genes (rather than phage genes) can
be packaged in phage heads, producing “transducing phage.” - Mature bacteriophage particles are released from the host cell
and can now infect another bacterial cell. - A recipient bacterial cell infected by a transducing phage
acquires genes that originated from the first host bacterium.
These genes will be inherited by progeny cells only if the new
genes are integrated into the recipient’s genome by
homologous recombination.
Generalized transduction
Any of the hosts bacterium’s genes can be transferred
Specialized Transduction
only certain genes can be transferred
-genes that are neighboring to the attB site will be transferred
Conjugation
Gene transfer that requires cell-cell contact. Includes R plasmid, F plasmid, F’ plasmid, and Hfr transfer
Mechanism of Transformation
- Naked DNA fragments from disintegrated cells in the area of a potential recipient cell. This cell must be of the correct genus and be in a state of competence, a proper physiological condition, to permit entry of the DNA fragment.
- Entry of naked DNA into competent cell
3.Recombination
-Some DNA fragments replace host cell DNA. The resultant recombinant cell is said to have genetically transformed and will now express the foreign genes it has received and pass them on to all its progeny.
-DNA that has not recombines is broken down by enzymes
natural vs artificial transformation
-Natural: is fairly rare
*Requires naturally competent cells, which can be found in dense
cultures
*The DNA source for natural transformation is thought to be from dead
or lysed cells, although this is controversial.
-Artificial: In the laboratory
*chemically competent cells: Ca +2 ions make E.coli competent to take
up DNA
*electroporation: DNA entry through transient holes in cell membranes
made by a pulse of high voltage
*a fundamental technique in recombinant DNA technology
what is competence?
Cells that can take up DNA from the environment
Virulent Phage
Always replicate using the lytic cycle
Temperate Phage
can replicate using the lytic and cycle or lysogenic cycle
Lytic cycle
1.adsorption
2. penetration
3. synthesis
4. assembly
5. lysis
Lysogenic Cycle
During penetration prophage is integrated
2. prophage is excised—≥ assembly
F plasmid
-Rolling circle transformation
-a single strand of the F plasmid enters the F- plasmid
What happens during Hfr conjugation?
-Genes immediately following the oriT are transferred to a recipient cell first
-The amount of DNA transferred depends on the amount of time the conjugation lasts
Hfr strain
-Integration of the F plasmid at an insertion sequence into the E.coli chromosome results in a Hfr strain that can mediate transfer of chromosomal genes into a recipient F- cell
What is F’ conjugation?
-imprecise excision of F plasmid from the chromosome of a Hfr cell
-the genes on the F’ plasmid are transferred to a F- cell during conjugation.
-only genes that contain F plasmid are transferred
R plasmid
R plasmid: (resistance plasmid) contains resistance (R) genes that confer resistance to multiple antibiotics and to heavy metals that would ordinarily kill bacteria.
-R plasmids are transferred quickly from bacterium to bacterium, often via conjugation, so many bacteria can acquire antibiotic resistance quickly.
-many contain transposons, which also contain antibiotic resistance genes
-transfer of resistance plasmids can occur btwn closely related genera such as Escherichia, Klebsiella, Salmonella, Serratia, Shigella, & Yerisinia
virulence Plasmid
carry instructions for structures, enzymes, or toxins that enable a bacterium to become pathogenic
oriT
Marks the region on F that enters the recipient cell first
tra genes
in gram-negative bacteria, conjugation requires cell-cell contact facilitated by sex pili. this requires the “tra” (transfer) genes, which includes genes encoding proteins in the sex pili.
Example of mutations
*auxotrophic – causes a defect in a biosynthesis pathway, so the
mutant cell requires exogenous building blocks for growth
*temperature-sensitive – causes loss of a particular function at high
or low temperature
*reversion – (also called back mutation) changes a mutated
sequence back to the original wild-type sequence
mutation
Any change in a cells DNA sequence
mutagen
Any agent that causes a mutation(physical or chemical)
genetic screen
-used to discover genes in a process you’re interested in
Process:
- Determine the phenotype of the mutant you’re looking for
• how will you distinguish your mutant from all of the other cells? - Make a pool of mutants
• want to have a collection of thousands of different mutants
• want to have 1 mutation per cell
• want mutants in ALL the different genes in the genome
represented (E. coli has 4500 genes, so if screening with E. coli,
want at least 4500 different mutants) - Let mutants grow up in “permissive” conditions
• lets virtually ALL the mutants grow - Replica and grow in “restrictive” conditions to identify
mutants with the desired phenotype
• ONLY mutants with the desired phenotype will NOT grow - Identify which gene contains the mutation
transposon
↝ transposon: a mobile, genetic sequence, flanked by terminal inverted repeats, that contains genes for transposition (the transposase); often contains resistance (R) genes
-transposons can insert randomly in DNA, causing mutations and conferring antibiotic resistance.
How is gene exchange in eukaryotes different from gene exchange in
prokaryotes? What mechanisms are commonly used for gene exchange in
eukaryotes? in prokaryotes?
-Eukaryotes reproduce sexually while prokaryotes reproduce asexually
-during prokaryotic gene exchange only a small of DNA is transferred
-This small amount of DNA is only propagated (e.g.,
replicated and handed down to progeny cells) if it
integrates into a chromosome or plasmid or if it is a
plasmid itself.
-eukaryotes are diploids vs prokaryotes are haploids
-in eukaryotes gene exchange only occurs through gamete fusion
-gene exchange in prokaryotes through transduction, transformation, and conjugation
Why is gene exchange important?
-Bacterial genetic exchange has implication for the evolution of phenotypes that are either beneficial to humans, such as biodegradation of toxic xenobiotic chemicals, or that are detrimental, such as the evolution of pathogenesis and the spread of antibiotic resistance.
If a plasmid enters a
bacterial cell, what are its possible fates?
Doesn’t get degraded
If a linear piece of foreign DNA
enters a bacterial cell, what are its possible fates?
-Integration through double recombination which requires the recA protein
- or degradation if DNA is not part of a replicon by nucleases in the cytoplasm
What needs to happen or be present before genes on a foreign piece of DNA
can be inherited by progeny bacterial cells?
-Transferred DNA must be integrated into a chromosome or be a plasmid itself
How is lateral gene transfer different from vertical gene transfer? Why does
lateral gene transfer make the study of bacterial evolutionary relationships
difficult?
Vertical gene transfer is the transfer form mother to daughter while lateral gene transfer is the transfer of genes between unrelated cells. Lateral gene transfer makes it so related genes don’t evolve from a common ancestor.
How are transformation, transduction, and conjugation all different from each
other? Consider the mode of transfer and the types of bacterial genes that can
be transferred
⤷ Transformation: gene transfer by taking up soluble DNA from the environment
-only competent cells can take up DNA from envm, thus participating in transformation
⤷ Transduction: gene transfer by means of bacteriophages. Can be generalized or specialized.
-generalized: any of hosts bacterium; occurs due to error in phage replication during Assembly stage
-specialized: only certain genes adjacent to the integration site (attB)–usually bio & gal genes
⤷ Conjugation: gene transfer that requires cell-cell contact. Includes R plasmid, F plasmid, F’ plasmid, & Hfr transfer
-Hfr conjugation: genes immediately following oriT. amount transferred depends on how long conjugation lasts
-F’ conjugation: the imprecise excision of the F plasmid of an Hfr chromosome. So, it carries only the genes around it.
-Larger amount of DNA can be transferred through conjugation compared to transformation and transduction
Describe how Griffith performed his experiment and how the data led to the
conclusion.
Conclusion: something from the dead S cells had “transformed” the
R cells, making them smooth and virulent
How is generalized transduction different from specialized transduction? Consider
what “mistake” occurs in each process, which type of phage can perform each,
which genes can be transferred, and how the transferred genes can be inherited
by progeny cells.
⤑ Generalized transduction: lytic phage infects bacterium, leading to the cleavage of bacterial DNA and synthesis of viral proteins. parts of bacterial chromosomal DNA may become packaged in viral capsid. phage infects another bacterium, transferring these genes. All genes can be transferred
⤑ Specialized transduction: lysogenic phage infect bacterium; viral DNA incorporated into bacterial chromosomes. when phage DNA is excised, flanking bacterial genes may be excised w/ it. DNA is packaged into phage viral capsid and can infect another bacterium. Only some genes can be transferred
How is F plasmid conjugation different from Hfr and F’ plasmid conjugation?
Consider the genes that can be transferred and how the transferred genes can
be inherited by progeny cells.
Why are genetic screens useful? What is the purpose of EACH step in a genetic
screen? What are advantages of using transposons in genetic screens?
-Used for identifying genes in a process you are interested in
➟Step 1: determine the phenotype of the mutant you’re looking for. This will help you distinguish your mutant from all the other cells
➟Step2: make a pool of mutants.
-want to have a collection of thousands of different mutant
-want to have 1 mutation per cell
-want mutants in ALL the different genes in the genome represented (E coli has 4500 genes = 4500 diff mutants)
➟Step 3: let mutants grow up in “permissive” conditions–let’s virtually ALL the mutants grow
-permissive conditions: environmental conditions that allow conditional lethals to live
-conditional lethals: any mutant whose viability depends on the conditions of growth–grows normally in permissive conditions, but does not grow in restrictive conditions
➟Step 4: replica and grow in “restrictive” conditions to identify mutant w/ the desired phenotype–only mutants w/ the desired phenotype will NOT grow
Step 5: identify which gene contains the mutation
Which of the following methods of gene transfer REQUIRE homologous recombination for the transferred genes to be inherited by progeny? Why?
*transformation with linear DNA
*transformation with plasmid DNA
*generalized transduction
*F plasmid conjugation
*Hfr conjugation
*R plasmid conjugation
*transformation w/ linear DNA
*generalized transduction
*Hfr conjugation
- Why is CRISPR-Cas important for prokaryotes? Briefly explain how CRISPR-
Cas works in prokaryotes
Protects prokaryotes from repeat viral infections
- Prokaryotes keep a record of encountered viral DNA, so the DNA is cleaved if encountered again
