Module 5: Bacterial Genetics (Gene Transfer) Flashcards
Horizontal Gene Transfer
The movement of DNA between microbes
AKA. Lateral Gene Transfer
What is vertical gene transfer?
The INHERITANCE of genes from a direct ancestor
What are the main mechanisms of horizontal gene transfer?
1) Transformation
2) Conjugation
3) Transduction
4) Transposition
No matter the method of horizontal gene transfer, what is a necessary process for the transfer to occur successfully?
Some form of RECOMBINATION occurs to incorporate foreign DNA into the bacterial genome
DNA that enters a cell will be degraded and not maintained if…
it does not have a way of replicating
(not linked to usable origin of replication in host cell)
Chromosomes + plasmids vs DNA fragments in cells
–> What is the difference between what happens to them, why?
Chromosomes + Plasmids = Have origin/s of replication –> Replicate on their own; are maintained within cells
DNA Fragments = NO origin of replication –> Gets degraded and is NOT maintained within cell
What must DNA fragments “do” to maintain within a cell?
MUST incorporate into host genome to get replicated and therefore maintain presence in cell
Incorporation of DNA fragments into genome occurs via
Recombination
Recombination is a “spin off” of _____________.
It requires breaking and __________ DNA strands.
Recombination is a “spin off” of DNA repair mechanisms.
It requires breaking and rejoining DNA strands.
What are the main forms of recombination?
Homologous and NON-homologous
Homologous Recombination (define)
Process of 2 DNA segments with IDENTICAL or very SIMILAR DNA sequences pair up and exchange or replace some portion of their DNA
(Process of DNA exchange/replacement between 2 DNA segments that have some level of sequence homology)
Homologous Recomb. AKA =
CROSSOVER
RecBCD
An enzyme complex (RecB+ C + D) that functions in homologous recombination to:
1) UNWIND DNA
2) CLEAVE DNA
What type of enzyme complex is RecBCD?
Helicase-Nuclease
Helicase = unwinds, Nuclease = clips
What is responsible for initiating homologous recombination?
RecBCD activity on DNA
(forming the single stranded knick in the DNA)
RecA
Protein (ssDNA BP) that catalyzes DNA strand exchange (and invasion) in homologous recombination
What catalyzes holiday junction formation?
RecA
Catalyzes strand invasion + exchange leading to holiday junc. forming
Homologous Recombination: Process
1) RecBCD binds to foreign DNA fragment and begins to unwind the DNA
2) RecBCD encounters chi site while unwinding
3) Upon recognition of the chi site, RecBCD cuts ONE of the strands of the DNA at this site, producing a segment of SINGLE stranded DNA (on one side of the cut; the other side remains base paired)
4) RecA is recruited to the single stranded segment of the DNA and binds to it
5) RecA bound to the single strand scans chromosomal DNA for a homologous sequence (to the single stranded segment)
6) Once a homologous sequence is found, RecA invades the homologous region of the chromosome with the single strand it is bound to, annealing to the complementary chromosomal strand
–> Effectively, displaces a strand of the chromosomal DNA, making a single stranded segment of chromosomal DNA
7) Displaced chromosomal strand basepairs to the free bases on the foreign DNA fragment
8) Holiday Junction forms (crossed structure)
9) Holiday structure is cleaved in 2 spots (one cut made in each non-recombinant strand; “outer” strands that have not been cut in this entire process)
10) The free strands created from holiday junction cleavage are ligated to form 2 recombinant dsDNA molecules
What is invasion in terms of RecA function?
Strand bound to RecA is brought to a homologous segment of chromosomal DNA and aligned with the complementary strand in the homologous segment
–> The strand then base pairs to this complementary strand, kicking out the chromosomal strand it was already base paired with
Holliday Junction
4-way structure of DNA formed by exchange of strands between homologous DNA duplexes (dsDNA)
Where is the holliday junction cleaved?
Cleaved at ONE spot on BOTH non-recombinant strands (the strands that have remained in their original form thus far)
–> Cut in TWO places
What determines the TYPES of molecules that are produced from homologous recombination?
2 factors:
1) The TYPE (linear or circular) of DNA starting molecules
2) The NUMBER of crossovers that occur (1 or 2 free strands invading?)
What is the result of homologous recombination for:
Single cross-over between 2 circular molecules
= ONE circular recombinant molecule (that contains all the DNA of BOTH starting molecules)
–> No exchange; it’s like a summation
What is the result of homologous recombination for:
Double cross-over between 2 circular molecules
= TWO recombinant circular molecules (that have exchanged a segment of DNA)
–> Reciprocal exchange!
What is the result of homologous recombination for:
Double cross-over between one circular and one linear molecule
= one circular recombinant molecule AND one linear recombinant molecule (that have exchanged a segment of DNA)
–> Reciprocal exchange!
Double Crossovers
TWO sites of recombination between two DNA molecules
–> Yields 2 molecules in which there is reciprocal exchange (DNA swap!)
Single Crossovers
ONE site of recombination between two DNA molecules
–> Yields ONE molecule in which there is only DNA integration (NO exchange!) = the product molecule has the DNA of BOTH starting molecules!
Single crossovers are unlikely to occur between ____ and ________
because…
Unlikely to occur between linear and circular molecules
–> Because this would result in an unstable linear molecule
Non-Homologous Recombination
Process of two DNA segments, with LITTLE to NO sequence homology, to exchange or replace some portion of their DNA
Non-homologous recombination takes place in…
ALL life forms!
What DNA transfer components does non-homologous recombination usually involve?
1) Transposable elements
2) Certain viruses
What are examples (2) of non-homologous recombination?
1) The lysogenic integration of temperate bacteriophage genome into host cell
2) Transposition (insertion of a transposon into DNA)
Temperate Bacteriophage
A bacteriophage which integrates its DNA into host cell genome
(Upon infection, has a lysogenic life cycle rather than a lytic cycle)
Lysogenic Integration involves what form of recombination?
Site-Specific Integration
= Non-homologous integration at a specific site/DNA sequence
–> In lysogenic integration, insertion of viral DNA into host cell DNA sequence occurs non-homologously at specific points
What enzyme is needed for lysogenic integration
A virally encoded INTEGRASE enzyme
What are the uses/implications of recombination
(1 general, 3 for lab)?
1) EVOLUTION: Naturally occurring process that is a major contributor to evolution through producing genetic variability
Lab Uses:
2) Make defined mutations
3) Replace segments of genome with other segments (genetic engineering)
4) Disrupt genes –> Make KO mutations
Knockout Mutations
Recombination that disrupts a gene rendering it dys/non-functional
(usually by inserting DNA into the middle of a gene)
What insights do KO mutations provide?
Can help in research to determine the functions of a gene by analyzing what processes are inhibited/altered upon removing the function of that gene
What are the two different types of KO mutation that can form?
1) Single crossover KO mutation
2) Double crossover KO mutation
Generation of a single crossover KO mutation employs what kind of vector with what characteristics?
SUICIDE PLASMID VECTOR
With:
1) Internal fragment of the gene to you want to disrupt (to provide homology for the crossover)
2) Selectable marker
3) Origin of replication for the CLONING cell (the cell that produces the vector but NOT for the cell the mutation is being induced in)
What type of recombination is used to make KO mutations?
Homologous recombination
Uses crossovers (think of the pictures)
What is a suicide plasmid?
A plasmid vector that CANT replicate in a target cell as it LACKS an origin of replication for this cell
What type of integration occurs in a single crossover mutation with a suicide vector?
CO-INTEGRATION
–> suicide vector gets linearized and then the ENTIRE thing is inserted into the gene you want to knock out
If this is the suicide vector used, what will the resulting single crossover KO mutation be?
What type of integration occurs in a double crossover mutation?
RECIPROCAL EXCHANGE (but the reciprocal given to the suicide vector is ultimately degraded)
Generation of a double crossover KO mutation employs what kind of vector with what characteristics?
Suicide plasmid vector with:
1) Entire gene of interest BUT modified to have a selectable marker WITHIN it
2) Counterselectable marker
3) Origin of replication for the CLONING cell (the cell that produces the vector but NOT for the cell the mutation is being induced in)
Counterselectable Marker
A gene that makes cells SENSITIVE to a compound (kills) to select for cells WITHOUT the marker
Cells w/ counterselec. marker DIE + cells w/o it LIVE
Process of Secondary crossover KO mutation generation:
1) Homologous recombination between suicide plasmid and gene of interest (reciprocal exchange)
2) Isolating Recombined Cells: Plate cells in medium containing the selectable marker compound = kills all cells that did NOT successfully get plasmid integration (ie the cells that did not get resistance to the selective compound)
3)Isolating ONLY the SECONDARY crossovers:
Counterselective marker test -
–> cells that underwent a singular crossover event will still have the counterselectable marker = will die in presence of this compound
–> Cells that underwent a double crossover event will NOT have the counterselectable marker (will have gotten lost to the degradation of the suicide vector)
What are the products of secondary crossover KO mutation generation?
1) Recombined Chromosomal DNA w/ Inhibited gene of interest (due to selectable marker inserted into middle of it)
2) Suicide plasmid with the reciprocal exchange from the DNA of interest –> GETS DEGRADED so the cell loses it!
What is an example of a counterselective marker?
SacB gene
SacB gene
Gene that produces levansucrase
Levansucrase
Enzyme that hydrolyzes sucrose to produce units of fructose (which then polymerize themselves into LEVAN)
Why is the SacB gene a counterselective marker?
Because SacB produces levansucrase which in the presence of sucrose will produce a lot of LEVAN which is toxic to cells! = kills cells!
What is the role of selectable and counterselectable markers in secondary crossovers?
Selectable Markers = To isolate cells that successfully underwent integration (isolate cells that underwent SOME crossover to gain the resistance to the selective compound)
Counterselectable Markers = To isolate cells that specifically underwent a SECONDARY crossover (causing LOSS of the sensitivity to the counterselective compound)
Transformation
Introduction of extracellular DNA directly into an organism
(DNA uptake)
For transformation to occur, cells must be…
COMPETENT
Competent Cells
Cells that possess the capability to uptake DNA from surroundings
What are the two types of cell competency?
1) Naturally competent
2) MADE competent
Naturally Competent vs MADE competent
Naturally Competent= Bacteria with specialized cell surface receptors and transport system for DNA uptake
–> contain cellular machinery for DNA uptake
MADE Competent = Bacteria that LACK cellular machinery for DNA uptake! Instead, competence is ARTIFICIALLY induced
Transformation process by NATURALLY competent cells
1) Extracellular dsDNA binds to receptor proteins in the CELL WALL
–> Binding = dsDNA is passed through cell wall into the periplasm
2A) ONE strand of the dsDNA is degraded in the periplasm
2B) ONE strand of the dsDNA is pulled through a PORE in the plasma membrane by DNA TRANSLOCASE to enter the cytoplasm
3) Single-strand DNA Binding Proteins (ssDNA BPs) and RecA bind to the ssDNA in the cytoplasm
What happens to transformed DNA in a cell (2 potential cases)?
1) can go and get integrated into genome
2) Can be used as a nutrient source
How are cells made to be competent?
Two main methods:
1) Cation treatment (mainly Ca2+) = makes membrane temporarily more permeable to larger molecules
2) Electroporation = application of an electrical current to generate temporary holes in cell membrane to allow for DNA uptake
What is the competency of E.coli
NOT naturally competent!
Conjugation
Transfer of DNA from one cell to another via DIRECT CELL-CELL CONTACT
What are main characteristics of DNA transfer that occurs by conjugation (3):
(Essentially, conjugation can result in the transfer of what kind DNA with what features?)
1) Can transfer LARGE segments of DNA
2) can transfer DNA between organisms of different domains
3) Can transfer virulence factors (spreading pathogenicity)
Fertility Factor F
AKA F plasmid
–> A plasmid that contains the genes needed for the generation of conjugal structures
What is absolutely necessary for conjugation?
F Plasmid
What are the key features of an F plasmid (4)?
1) Origin of replication
2) Tra Region (containing genes needed to begin and direct conjugation)
3) OriT (origin of transfer)
4) IS elements and TN1000 transposon
Tra Region
A region of the F plasmid that contains >30 different Tra genes which encode for proteins needed to form conjugal structures
OriT
Origin of transfer = where single stranded nick occurs in conjugation to begin DNA transfer
Conjugation occurs in ___________ direction, from _______ to ___________
Conjugation occurs in ONE direction, from F+ (donor) cell to F- (recipient) cell
F+ vs F- cells
F+ = Donor cell; HAS F-plasmid
F- = Recipient cell; does NOT have F plasmid
Conjugation turns a ________ into a ___________
Conjugation turns an F- cell into an F+ cell
General conjugation process:
1) Tra genes in donor cell initiate sex pilli formation
2) Sex pilli expand and contract looking for a recipient cell
3) Sex pilli find and bind to recipient cell and pull the recipient closer to the donor
4) Complex of tra-encoded proteins forms the MATING BRIDGE, physically connecting the two cells
5) Endonuclease associated with the mating bridge nicks ONE strand of the F plasmid at the origin of transfer
6) The free single strand of the F plasmid begins to move into the recipient cell while one end of it remains bound to the mating bridge
7) DNA replication occurs in both cells to reform the dsDNA of the F-plasmid
8) Once transfer is done, the bound end of the transferred single strand of F plasmid dissociates from the mating bridge and the F-plasmid recircularizes in the recipient cell
9) Cells break apart == TWO F+ cells!
An entire F plasmid is transferred to recipient ONLY if…
mating bridge stays “open” long enough
What causes conjugation with chromosomal involvement?
The integration of F plasmid into the F+ chromosome!
and the subsequent initiation of conjugation by the F plasmid while it is IN the integrated state!
How does F plasmid integrate into host chromosome?
Via a SINGLE crossover event!
In conjugation with chromosomal involvement, OriT of F plasmid always points…
AWAY from the first gene that is transferred to the recipient cell
Hfr Strain
“High Frequency of Recombination” cells
= cells with an integrated F plasmid!
Called Hfr b/c they are very efficient at transfer of chrom. genes
Conjugation between Hfr and F- cell results in_________
but does NOT result in ____________-
Conjugation between Hfr and F- cell results in transfer of chromosomal genes
but does NOT result in conversion of F- to F+
Why does Hfr + F-+ NOT convert F+ to F-?
Because when F plasmid is in integrated form and conjugation is initiated by cleavage of OriT, half of the F plasmid goes to one end of the free single strand while the other half goes to the other end
–> Due to the extensive size of the chromosome, it is rare for the ENTIRE integrated chromosome to transfer (because mating bridge does not stay open that long)
…as such the second half of the F plasmid never gets transferred! (because it’s at the end of the integrated chromosome structure!)
In conjugation of Hfr and F- cell, what would need to occur in order for F- to convert to F+?
The ENTIRE chromosome of donor cell would need to transfer to the recipient in order for BOTH halves of the F plasmid to transfer = full F plasmid in recipient
In conjugation with chromosomal involvement:
Genes ______ to the origin of transfer have a GREATER rate of transfer to recipient cell
CLOSER
In conjugation with chromosomal involvement:
> mating time = …
> # of genes transferred!
Conjugation with chromosomal involvement allows us to ________ which provides key insights in chromosomes!
MAP the locations of genes in a chromosome!
(because genes will get transferred “in order”)
What happens if an integrated F plasmid excises wrong?
F plasmid excises chromosomal genes with it!
= Host cell loses genes from its chromosome and F plasmid gains genes
F’ Plasmid
= F plasmid that looped out of (excised) integrated state improperly (and now carries some amount of chromosomal genes)
What is special about the gene transfer of F’ strains?
They transfer a limited # of chromosomal genes BUT at a very high frequency
Conjugation between F’ cell and F- results in…
TWO F’ cells
WE can use conjugation to (2):
1) Create merodiploids (bacterial cells w/ 2 copies of SOME genes; one copy in chrom., one in F’)
2) Transfer other plasmids (aside from the F plasmid, that are genetically modified to have an OriT)
Merodiploids
PARTIALLY diploid cells
–> = diploid for one or a few genes
1 copy in chrom., 1 copy in F’ plasmid
How can conjugation create Merodiploids?
by addition of a second copy of a gene by an F’ plasmid!
1 copy of gene will exist in chromosome and second copy of gene will exist in the F’ plasmid!
How can conjugation be used to transfer plasmids OTHER than F palsmid?
We can engineer other plasmids to contain an OriT AND tra genes!
Triparental Conjugation
Tra genes are provided by a helper plasmid to create conjugal structures and then the actual plasmid of interest contains OriT to get transferred!
(F plasmid of helper strain trasmitted into actual donor strain of interest THEN donor strain is able to conjugate with wanted recipient strain by the tra genes from the helper plasmid and the OriT of the desired plasmid)
Mobilizer Cell and Helper Plasmid
Mobilizer cell = Has the helper plasmid
Helper plasmid = Plasmid that gets transferred to donor cell to act as a source of tra genes to produce conjugal structures in donor cell (for triparental conjugation)!
Triparental Conjugation Process:
1) Mobilizer cell transfers helper plasmid to donor cell
2) Donor cell develops conjugal structures from the helper plasmid tra genes
3) Donor cell conjugates to recipient cell and transfers plasmid of interest!
Transposition
Movement of DNA via mobile genetic elements (transposable elements)
What are transposable elements?
Mobile genetic elements
(“jumping genes”)
–> DNA sequences that can move to different locations within a genome
What are two of the main types of transposable elements?
1) Insertion sequences (IS)
2) Transposons (Tn)
Insertion Sequences
DNA sequences which encode proteins needed for transposition
Transposons
Transposable elements that encode proteins needed for transposition AND contain other genes!
What is the difference between insertion sequences and transposons?
BOTH encode for proteins needed for transposition
BUT transposons contain other genes in their sequences as well!
Transposable elements mediate genomic ________ by doing what (2)?
Mediate genomic rearrangements by moving:
1) Within and 2) Between genomes
Transposition is the method by which some genes can…
some genes can move from one part of the genome to another
Transposable elements are very useful for generating _______ within a lab
GENERATING MUTATIONS
Why are transposable elements good for generating mutations?
Because they often INSERT RANDOMLY into genome and almost always disrupt the function of the gene they insert into
Process of Transposon Mutagenesis:
1) Donor cell with suicide plasmid that contains a transposon passes suicide plasmid to recipient cell
2) Suicide plasmid degrades in the recipient cell = releases carried transposon
3) Transposon inserts itself into recipient chromosome
How does the “other gene containing” quality of transposons impact their use in mutagenesis?
Transposons can carry antibiotic resistance genes = allows for selection of cells that were successfully transposed!
Transduction
Transfer of DNA from one cell to another via altered bacteriophage
Transduction occurs through the use of?
Transducing Particles
(“odd viruses”)
Transducing Particles
Phages with accidentally packaged fragment of host cell DNA
(= phage is missing some of its viral genome as a result!)
Transducing particles can transfer___________ without actually __________
They can transfer mistakenly packaged host cell DNA into another cell without actually infecting the cell (don’t produce more viral particles upon DNA entry into cell)!
Process of Transduction:
1) Regular phage infects a host cell
2) Phage DNA replicates
3) Phage enzymes fragment the host cell genome
4) Phage packaging occurs = frenzy in which host DNA accidentally gets packaged into phage
5) Phage production which leads to lysis of cell
6) Phages and accidentally made transducing particles are released from cell
7) Transducing particles and phages go off to bind and infect new host cells
8) Transducing particle binds to host cell and inject the non-viral DNA = No infection occurs BUT new DNA has been added to the cell
9) DNA undergoes recombination with chromosome to produce a transductant cell
Transductant
Bacterial cell that contains DNA obtained through transduction!
What type of recombination occurs in transduction?
Homologous
