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What are the 3 mechanisms of transfer of DNA into bacterial cell?
- 1. transformation
- 2. transduction
- 3. conjugation
- such processes are of great biomedical importance as they are the basis for virulence & drug resistance gene transfer among bacteria
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competent bacteria
- bacteria with the ability to import DNA
- aka they have the ability to make specific proteins that import DNA into the cell
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What can limit the ability of DNA from one species to be acquired by a different species?
restriction enzymes --> cleave foreign DNA that enters the cell
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How may bacteria be induced to take up DNA from the environment?
by changing their cell envelope structure
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Transformation
- introduction of free DNA from the environment into bacteria
- confers a special property to transformation: if you add DNAse, DNA will be consumed & no transformation will occur
- (DNAse = nucelase that destroys DNA)
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Transduction (2)
- the transfer of DNA from one bacterium to another by a bacteriophage vector
- generalized transduction: when a transducing virus packages random fragments of host DNA
- specialized transduction: when viruses pick up genes that lie near the site of prophage integration
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Stages of Generalized Transduction
- 1. Adsorption/Attachment
- 2. Injection of Bacterial DNA
- 3. Transduction of Bacterial DNA
- able to interact with new host chromosome via homologous recombination
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Conjugation
- allows the direct transfer of DNA from one bacterium to another
- two cells, the donor cell and the recipient cell, join via sex pilus, a special attachment structure, & a single strand of DNA moves from the donor to the recipient cell
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What is the one thing that conjugation requires that neither transformation nor transduction require?
- conjugation requires cell to cell contact
- donor cells physically attach to recipient cells
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Recombination
- the generation of new versions of DNA from precursor DNA molecules by enzymes that can break and ligate DNA
- is a COVALENT association (bond-wise)
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conjugative plasmids
- plasmids that carry special conjugation genes
- donor DNA that's transferred may be plasmid or portions of chromosomal DNA
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Steps of Conjugation
- 1. donor bacterial cell makes sex pilus using genes found on it's chromosome's “transferable element” or a “plasmid” it has
- 2. donor’s sex pilus attaches to the recipient cell --> brings the donor and recipient cells together
- 3. a single-stranded copy of the plasmid (transferable element) is transferred into the recipient cell
- 4. the single-stranded copy of plasmid in the recipient cell circularizes & replication completes the complementary strand --> new sex pilus can be made
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Where are the genes that enable a cell to make a special sex pilus?
the genes may be found on the transferable element in the bacterial chromosome or its extrachromosomal plasmid
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What happens if the recipient cell gets only a piece of the chromosome that's transferred?
- it's integrated into the recipient cell’s chromosome by homologous recombination
- if the recipient gets the entire plasmid, a new donor cell is created
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RecA
- enzyme that aligns homologous DNA regions & functions in the recombination process
- cuts & pastes incoming DNA into a bacterial chromosome
- a gene might be immediately selected for & integrated because it can confer antibiotic resistance or utilize nutrient in environment (i.e. sugar)
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What are all the different ways bacteria can undergo genetic changes?
- 1. mutations
- 2. acquisition of new DNA (transformation, transduction, conjugation)
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What are the DNA elements in bacterial cells?
- Chromosome
- Plasmids
- Transposons
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plasmid
- non-essential, extra-chromosomal, self-replicating DNA (replicon)
- usually circular
- non-essential = not required for normal cellular survival
- can carry genes necessary for pathogenesis (eg. anthrax)
- can confer drug resistances upon their hosts
- can be spread among bacteria or other species
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replicon
- any molecule of DNA that can be autonomously replicated (capable of self-replicating)
- eg. the chromosome, ALL plasmids & some autonomous viruses
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origin of replication (oriR)
- special site in/on plasmids where replication initiates
- is a specific, short DNA sequence DIFFERENT from the chromosomal origin of replication
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rep protein
encoded by plasmid itself binds to oriR & recruits host DNA pol to initiate replication
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Is plasmid segregation highly regulated?
- YES, plasmid segregation is highly regulated to ensure that each daughter cell receives at least one copy of the plasmid
- plasmids DO NOT partition randomly to the daughter cells
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What controls the plasmid copy number?
plasmid genes
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incompatible plasmids
- if you try to introduce into a bacterium a plasmid that has the SAME oriR-rep system as a plasmid that was ALREADY PRESENT in the bacterium, only ONE of the two plasmids will be maintained
- plasmids of the same family type are incompatible within the same cell
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What are 3 intrinsic properties of plasmids?
- 1. replication: they control their own (oriR & rep)
- 2. segregation: they themselves make sure one copy is given to a daughter bacterium
- 3. how many of themselves should be present in each bacterium
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transferable/conjugative plasmid
a plasmid which contains an oriR sequence, a rep gene, & something like the tra operon, a series of genes that code for transfer proteins
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tra operon
a series of genes that code for transfer proteins that allow the host bacterial cell to act as a donor of genetic material by conjugation
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oriT (origin of transfer)
sequence of DNA found in an F plasmid's tra operon where transfer replication initiates
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Non-Transferable Plasmids
- plasmids that aren't self-transferable, aka they do not contain an oriT sequence or encode the tra proteins that mediate conjugation
- they could also lack both an oriT sequence & the tra operon
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Mobilizable Plasmids
- a plasmid which itself lacks a tra operon BUT has an oriT sequence recognizable by a separate transferable plasmid's Tra proteins
- if these two exist in the same cell the non-transferable plasmid may be mobilized by the transferable plasmid & passed to a recipient cell
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R factors/plasmids
- plasmids that carry drug resistance genes rendering the host cell resistant to certain antibiotics
- can be transferable
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transposable elements
- units of DNA that can move, within the cell, from one site to another on the same DNA molecule or from one DNA molecule to another
- often carry within them drug resistance genes
- "jumping genes"
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bacterial gene exchange
- 1. transDUCtion: DNA transfer between donor and recipient is mediated by a virus
- 2. transFORmation: donor DNA in solution is taken up by recipient cells
- 3. Conjugation: direct contact between a donor and recipient cell occurs via F plasmid for DNA transfer to occur
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Steps of Conjugation
- 1. donor cell contain genes for sex pili synthesis encoded in a plasmid (F plasmid) & therefore expresses sex pili on its surface
- 2. an extended sex pilus makes initial contact with a recipient cell
- 3. the pilus is retracted bringing the two cells in contact w/ each other
- 4. a copy of the F plasmid is nicked at oriT
- 5. F plasmid DNA is transferred as a linear single strand into recipient bacterium
- 6. the newly transferred F plasmid is circularized & converted to double-stranded DNA in recipient cell while the F plasmid of donor cell is converted back to dsDNA
- (non-conjugal plasmids WILL NOT transfer)
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Will a non-conjugative plasmid with an oriT site in the donor cell transfer if there's a conjugative plasmid being transferred?
- Yes, it can be transferred individually or by using the proteins made by the conjugative plasmid
- (if the OriR is the same between 2 plasmids, only ONE will surivive, NOT OriT)
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oriT (origin of transfer)
- sequence on the F plasmid nicked by an endonuclease (encoded in the tra operon)
- cutting the plasmid DNA at oriT is necessary to initiate transfer of the plasmid to the recipient cell
- the first segment of DNA to be transferred to a recipient cell is adjacent to the nick
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tra operon
- region of the F plasmid which encodes genes involved in conjugation
- said genes encode many proteins involved in the synthesis of the sex pili & DNA transfer proteins, including the endonuclease that nicks the DNA at oriT
- other proteins encoded by the tra operon include pilin, the major component of the pilus, the proteins of the structural base of the pilus, pilus assembly proteins, & proteins that modify the surface of the donor cell
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surface exclusion
- the phenomenon that conjugative plasmids encode proteins which modify the bacterial cell surface of the cell in which they reside
- these modifications BLOCK the ability of the sex pilus of a second cell carrying a related plasmid to attach to the cell surface
- this prevents a cell that carries a conjugative plasmid from inheriting additional, related conjugative plasmids [rendering the original one useless]
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tra repressor gene
- inactivates the synthesis of traJ (positive regulator of tra operon)
- found in the R factor - a drug resistance plasmid similar to the F factor plasmid
- most conjugative plasmids regulate their fertility
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Why are tra repressor genes seen in most conjugative plasmids?
- because the pili plasmids encode can easily be bound by phages, resulting in the bacterium's death
- it is advantageous to be able to control pili expression
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Why is the tra operon of F expressed constitutively?
because the repressor is nonfunctional - long ago an insertion mutation occurred in the repressor gene
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What are some regions of homology shared between the F factor & the bacterial chromosome (+ other plasmids)?
- IS3 gene (2 copies)
- IS2 gene (1 copy)
- Tn1000 (1 copy of transposon)
- *in the presence of RecA, these regions of homology can serve as RECOMBINATION SITES
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How can a transferable (conjugative) plasmids mediate the transfer of chromosomal genes?
- chromosomal genes may only be transferred if the conjugative plasmid becomes INTEGRATED into the bacterial chromosome
- this is a rare event (occurs in 1 out of every 106 cells) BUT in a culture of a billion cells there would be about 1000 cells in which independent integrations would occur
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Where in the bacterial genome can transferable plasmids integrate?
- transferable plasmids can integrate at a variety of sites (unlike temperate phages like lambda which integrate at a specific site in the chromosome)
- therefore a population of cells carrying a transferable plasmid is heterogeneous: some cells contain free plasmid DNA, some have a plasmid integrated in their chromosome, and those cells will have the plasmid integrated in different sites of their chromosome
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Hfr Strains
- a new strain of bacteria that have an F factor integrated into a chromosome - it can transfer genes proximal to the integrated conjugative plasmid at high frequency
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F' factor
- an F factor that has excised itself from a bacterial Hfr chromosome taking with it a piece of bacterial DNA
- Type I excision: F factor has some bacterial DNA & is itself incompletely excised
- Type II excision: F factor is completely excised & takes with it some bacterial DNA
- these excisions are an important mechanism by which plasmids acquire additional genes, notably genes for pathogenesis factors which they can then pass on to future generations and to other cells
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R100
- an R factor (a plasmid) that conveys drug resistance against antibiotics chloramphenicol, streptomycin, tetracycline, & sulfonamide to bacteria such as Shigella dysenteriae (cause dysentery)
- contains a tra operon, so is a transfer plasmid
- fertility is repressed in established cultures but not in cells that have NEWLY acquired R100 because the R100 sex pilus is s receptor for many phages
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What happens if a cell contains both R100 (R factor) and an F factor?
transfer of either plasmid occurs at LOW frequency
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R factor
- plasmids which encode resistance to at least one antibiotic
- they can be conjugative plasmids or have no conjugative capability at all
- some R factors have very limited host ranges (can only be maintained in one or few related species), while others have very broad host ranges
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Which R factor is the most 'promiscuous'/transferable?
- RP4
- not only does it transfer to almost ALL bacterial species, it can even transfer DNA to yeast cells and to plants
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Which R factor genes generally contain antibiotic and chemical resistances?
genes that are parts of transposable elements: DNA sequences that can move as a unit to new sites on the same DNA molecule OR to other DNA molecules within the same cell
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What are the 4 classes of transposable elements?
- 1. Insertion Sequences (IS) [700-1500 bp]
- 2. Simple or Noncomposite Transposons
- 3. Composite Transposons
- 4. Itegrating Conjugal Elements (ICE)
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Insertion Sequences (IS)
- mobile, short (~0.7-1.8 kb) DNA elements that ONLY encode a single protein, transposase, involved in their transposition
- they are found ubiquitously (in the F factor, bacterial chromosome, R factors, phage genomes…)
- can transpose between any DNA molecule present in the SAME cell, aka their transposable properties are limited to only functioning intracellularly
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What do all IS elements tend to have at their ends?
short (<100 bp), inverted repeat sequences at their 5' & 3' ends
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Why are chromosomes not riddled extensively with transposable elements?
- despite the fact that an IS can hop randomly, transposable elements have evolved so that the transposase protein both mediates transposition AND represses its own transcription
- too much transposition would inactivate genes & be deleterious
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What happens at the target site of transposition where the transposon or IS element is incorporated into the piece of DNA?
- after the target site is cut & the transposon or IS element is ligated in, DNA polymerase fills in gaps that resulted from cutting of the target site, effectively DUPLICATING it
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Transposon
- a mobile genetic element containing additional genes unrelated to transposition (eg. a gene encoding drug resistance, toxins, conjugative properties, etc.)
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Composite Transposons
- typically consists of a drug resistance gene sandwiched between two matching IS sequences
- the whole transposon can either "hop" as a unit or the ends can transpose independently
- two matching IS sequences can transpose themselves & everything in between
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composite vs. simple transposon
- composite: a drug resistance gene between by two matching IS sequences
- simple: a single drug resistance gene, a transposase gene, & a resolvase gene between short terminal repeats
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Simple (Noncomposite) Transposons
- are structurally similar to IS elements, but carry a SINGLE drug resistance gene, a transposase gene, & a resolvase gene between the short terminal repeats (STRs)
- simple transposons MUST transpose as a unit
- unlike how composite transposons, there is no independent IS sequence at either end of a simple transposon that can 'hop'
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resolvase
resolves the cointegrate: a transposition intermediate
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Tn3
- classic examples of a simple transposon
- contains an It has an AMP resistance gene between the short terminal repeats
- its transposase functions are provided by two genes
- doesn't use a cut & paste mechanism like ISs use; instead it inserts itself into new genetic material via replicative transposition
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replicative transposition
- the mechanism by which transposons move between & within genetic material
- 1. trasposon is copied/duplicated
- 2. form a cointegrate: intermediate consisting of the donor and receptor DNA sequences in a characteristic configuration ('theta')
- 3. one copy of transposon remains where it existed originally & the copy is transferred
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What proteins are capable of resolving the cointegrate transposition intermediate resulting in the original genetic material and the new, slightly altered genetic material?
- resolvase (TnpR in Tn3): resolves quickly because it's dedicated to this specific function
- RecA: resolves more slowly
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What is another way a nonconjugal plasmid can be transferred from a donor to a recipient cell?
- by forming a cointegrate with a transfer factor & the complex is transferred in it's cointegrate form
- when resolved in recipient cell, nonconjugal plasmid has now been TRANSFERRED
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Integrating Conjugal Elements (ICE)
combines properties (genes) of transposons, temperate phages, AND conjugal elements all in the same unit
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STX element
an integrating conjugal element from Vibrio cholera that integrates at a specific site in the V.cholerae genome, promotes DNA transfer to recipient cells, & contains a transposon that includes a gene for drug resistance
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Conservative (Cut & Paste) Transposition
- some transposons are transmitted by a cut-and-paste mechanism where the transposon is actually excised from the site at which it resides and is inserted into a new site
- there is NO duplication of target site
- NO cointegrate formation
- the transposon LEAVES the donor site & MOVES to the target site
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