Bacterial Conjugation notes PDF

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lecture notes on bacterial conjugation methods to supp...

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Bacterial Conjugation Horizontal Gene Transfer -

Refers to the ability or process by which pieces of DNA are transferred from one cell to another cell not through generational processes (not through single cell dividing, but rather the DNA ‘hops’)

Three different kinds 1. Transformation  A bacterium will shed its DNA into the extracellular space, or you can artificially lyse a cell open and take its DNA which can be taken up through specialised machinery to allow DNA to move into a new cell 2. Transduction  DNA in a cell can be packaged into phage’s which can then be released from cells or lysed out of cells and then infect new cells to introduce that DNA 3. Conjugation  DNA is directly transferred from one cell to another through specialised machinery  The original donor doesn’t need to die or be lysed so both donor and recipient can end up with the DNA  If the machinery is transferred along with the DNA the recipient can itself become a donor allowing for exponential spread of conjugative elements in populations of bacterial cells First description of conjugation in 1946

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Took 2 different strains of E. coli 1. Y-10 which carries metabolic deficiencies so can’t synthesise threonine, leucine and thiamine 2. Y-24 which is defective in biotin, phenylalanine, cysteine

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Each strain was grown independently and then plated onto minimal media and you didn’t get any colonies without supplementing these deficiencies If you mix the strains together and then plate them onto minimal media you get a bunch of colonies growing The genes present in one or the other strain were able to complement the deficiencies of the other strain through DNA transfer from one type of cell to the other

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Conjugation -

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Occurs in both bacteria and archaea (between bacterial cells individually and archaeal cells individually) Conjugative elements can be both plasmids or integrative elements  Plasmids can autonomously replicate themselves or might require insertion into the chromosome in order to replicate  Integrative elements can sometimes bring along parts of the chromosome that they are inserted into Transfer requires direct cell-cell contact between a Donor and Recipient- if two different bacteria don’t come into physical contact DNA cannot be exchanged Machinery that enables this process to occur is a specialized secretion system (Type IV Secretion System; T4SS) Different conjugative systems have different host-range- different systems are able to conjugate into different types of cells as well as the element being delivered can persist in different types of cells

Steps of conjugation 1. Pili formation  Pili are extracellular structures like long filaments that extend out of the cell  Bacteriophage MS-2 only infects F+ E. coli  Pilus structure only observed on F+ cells  The pili can extend out of the cell and bind to a potential recipient cell

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Pilus biogenesis Type IV secretion system (T4SS) which is the structure of a pilus

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Multicomplex

F pilus extension and retraction  Can visualise the process refer to lecture 13:40  Fluorescent R17 bacteriophage (labelled with green dye) bind to the F pilus  Bacteria expressing dsRed (red fluorescent protein) F pili mediate cell-cell interaction  Pili extend, attach to recipient cell  Pili retract, pulling cells into close contact RP4 pili  Has a wide host range so can go into many different types of bacteria  Called P-like pili or P-plili  Only about 0.2-1 micrometer long  Rigid- easily broken off  Probably don’t “capture” targets

2. Mating pair formation  End goal is to have two different cells that form close interactions with each other F pilus  when you form the mating pair its hypothesised that the pilus that forms is a form of a channel that allows things to translocate across it  Has a hole that has the right hydrophobicity that allows things to flow through it

RP4 mating pair formation (MPF)  Donor and recipient form close contact with each other  By electron microscopy you can see contacts forming and type of strong density forming a bridge/link between the two cells  Not enough information to see if there are any transfers forming

3. Relaxosome assembly  Relaxosome is comprised of an enzyme called relaxase + oriT (a DNA sequence that is recognised by the relaxase) + accessory proteins (different conjugative systems use different accessory proteins, they bind to the relaxase and help bind it to the right spot on the oriT  Relaxase is the only essential component that is always present; other accessory proteins vary from system to system 1. Relaxase binds to the oriT 2. Nicks the DNA (cuts one strand) and starts unwinding it 3. Accessory proteins bind to stabilize this complex/bend the DNA  Coupling protein then bridges the relaxosome with the T4SS (secretion machinery/membrane component)  Relaxosome and coupling protein are not needed for mating pair formation Relaxosome structure

4. DNA transfer  T4SS switches mode from “pilus biogenesis” to “Substrate transfer”

5. Complementary strand synthesis

 DNA becomes doubled stranded again Integrative conjugative elements  ICE lack of replication origin – must integrate into the chromosome to replicate  To conjugate, ICEs will excise from the chromosome to form circular DNA (technically not a “plasmid” though) Pieces of the chromosome can be transferred from one cell to another, an example of this are Hfr strains of E. coli - E. coli strains that have the F plasmid integrated  F- bacteria lack the F plasmid completely (recipients)  F+ bacteria carry the F plasmid independent of the chromosome  Hfr (High Frequency of Recombination) bacteria have the F plasmid integrated into

Chromosome mapping  Before rapid genome sequencing, Hfr strains were used to identify the position of genes on the chromosome  The farther away a particular gene is from the hfr insertion the less efficient the process will be for the DNA to be transferred  If you have a particular strain where the donor has particular markers present and then look for how frequently the markers are transferred, you can get an estimation for how close the makers are to the F insertion  Hence the closer a gene is to the integrated F, the more rapidly that gene will be conjugated along with the F  Also, the closer a gene is to the integrated F, the more frequently that gene will be transferred

6. Donor and recipient separate Fertility inhibition - Plasmids are selfish: they don’t want other plasmids to spread - Donor bacteria with multiple conjugative plasmids often only able to transfer a subset of these plasmids - Another form of “plasmid incompatibility” - Conjugative elements encode mechanisms that supress the transfer of other conjugative elements  Interfere with transcription  Prevent pilus formation  Block coupling protein  Other mechanisms

Exclusion -

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In addition to fertility inhibition this is another process that blocks conjugation It is wasteful to try to conjugate into cells that already have the conjugative element Conjugative elements have exclusion factors Two types:  Surface exclusion- prevents mating pair formation  Entry exclusion- prevents DNA transfer  These names are often used interchangeably Different plasmids have different types of exclusion: F has both types, RP4 only has one

Applications of Conjugation -

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Conjugation is a mechanism for transferring DNA from one cell to another Genetic engineering- delivering DNA into cells  Bacteria  Plants Efficient (unlike transformation) General (broad host range, unlike viruses/phage)- can go into a large number of cells Easy to remove the delivery vehicle  Antibiotic can kill off donor bacteria

RP4 and plasmid mobilization -

RP4 can be integrated into chromosomes, one of the work horse strains that people use is sm10 - Plasmids that carry oriT can be mobilized - Useful for bacteria that are not naturally competent  Clone genes into plasmid carrying oriT  Transform into E. coli donor strain  Mate with another non-competent organism - Conjugation can be scaled up… much more efficient than transformation… necessary for rare events (transposition, allelic exchange)  Can mate 1010 cells (or more) Conjugation to introduce plasmids into non-competent species

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E. coli transformation efficiency is pretty high (with excess DNA is roughly 10-2 to 10-3) Pseudomonas aeruginosa transformation efficiency is more than 1000 lower than E. coli  Transformation can also be stressful to P. aeruginosa can induce mutations

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Conjugative efficiency between E. coli and P.aeruginosa is roughly 10-3 to 10-4

Allele exchange -

homologous recombination Rate is variable between 10-3 and 10-7  Best case scenario for every 1000 cells that carry a plasmid, one will successfully recombine  Normal transformation is too inefficient  Conjugation used instead  Suicide vector can only be replicated in donor strain

Agrobacterium tumefaciens Ti plasmid

Binary Artificial Ti Plasmids

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Helper plasmid doesn’t get transferred itself- it sits inside the bacteria, but it encodes the type IV secretion system machinery that enables the shuttle vector to be delivered into the target recipient Plant selection marker to see if the piece of DNA has been delivered MCS to insert any chunk of DNA

Bacterial conjugation definition -

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Way by which a bacterial cell transfers genetic material to another bacterial cell The genetic material that is transferred through bacterial conjugation is a small plasmid, known as F-plasmid (F is for fertility factor) -the F plasmid carries genetic information different from that which is already present in the chromosome of the bacterial cell The F plasmid can replicate in the cytoplasm separately from the bacterial chromosome A cell that already has a copy of the F plasmid = F+ cell = donor cell and the recipient is FThe transfer of the F-plasmid takes place through horizontal connection by which the donor cell and the recipient cell directly contact each other to form a bridge between the two through which the genetic material is transferred Where the F-plasmid is a donor cell is integrated in the cell’s genome (i.e. in the chromosome) a part of the chromosomal DNA may also be transferred to the recipient cell together with the F-plasmid

Steps: 1. The F+ cell (donor) produces the pilus- a structure that projects out of the cell and begins contact with the recipient cell 2. The pilus enables direct contact between the donor and the recipient cells 3. The F-plasmid is a double-stranded DNA molecule forming a circular structure and the enzyme relaxase (or relaxosome when it forms a complex with other proteins)

nicks one of the two DNA strands of the F-plasmid and this strand (also called Tstrand) is transferred to the recipient cell 4. The donor cell and the recipient cell, both containing ssDNA, replicate this DNA and thus end up forming a dsF-plasmid identical to the original F-plasmid and given that the F-plasmid contains information to synthesize pili and other proteins, the old recipient cell is now a donor cell with the F-plasmid and the ability to form pili so both cells are now donors

DNA transfer - To avoid the F-plasmid being transferred to an already F+ cell- the donor cell contains info to detect and avoid cells that already have one - F-plasmid contains:  two main loci (tra and trb)  an origin of replication (OriV)  origin of transfer (OriT) - tra locus contains the genetic information to enable the donor cell to be attached to a recipient cell: the genes in the tra locus code for proteins to form the pili (hence is the pilin gene) in order to start the cell-cell contact and other proteins to get attached to the F- cell and to start the transfer of the F-plasmid - trb locus contains DNA that code for other proteins (e.g. some involved in creating a channel through which the DNA is transferred from the F+ to the F- cell) - the OriV is the site at which replication of the DNA occurs - the OriT is the site at which the enzyme relaxase nicks the DNA strand of the Fplasmid (Although the DNA that is transferred in bacterial conjugation is that present in the Fplasmid, when the donor cell has integrated the F-plasmid into its own chromosomal DNA, bacterial conjugation can result in the transfer of the F-plasmid and of chromosomal DNA.

When this is the case, a longer contact between the donor and the recipient cells results in a larger amount of chromosomal DNA being transferred.) Extra: F-plasmids contain genetic information that is not present in the bacterial chromosomes, although they can be integrated in the chromosomes. Moreover, F-plasmids have the genetic information to enable the production of pili so that the F+ cell can get attached to a new recipient (F–) cell. Surface exclusion protein: mechanism by which F plasmids prevent the redundant entry of additional F plasmids into the host cell during exponential growth- conferred only by trbK and only when gene dosage is high or when trbJ is also present in cis or in trans Fertility inhibition: F-like plasmids consist of an antisense RNA protein which act together to prevent the translation of TraJ, the positive regulator of the transfer operon...