Microbio Plasmids & Transposons (7/8/9)

• 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

• bacteria with the ability to import DNA
• aka they have the ability to make specific proteins that import DNA into the cell

restriction enzymes --> cleave foreign DNA that enters the cell

by changing their cell envelope structure

• 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)

• 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

• 1. Adsorption/Attachment
• 2. Injection of Bacterial DNA
• 3. Transduction of Bacterial DNA
• able to interact with new host chromosome via homologous recombination

• 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
• 

• conjugation requires cell to cell contact
• donor cells physically attach to recipient cells

• 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)

• plasmids that carry special conjugation genes
• donor DNA that's transferred may be plasmid or portions of chromosomal DNA

• 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

the genes may be found on the transferable element in the bacterial chromosome or its extrachromosomal plasmid

• 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

• 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)

• 1. mutations
• 2. acquisition of new DNA (transformation, transduction, conjugation)

• Chromosome
• Plasmids
• Transposons

• 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

• any molecule of DNA that can be autonomously replicated (capable of self-replicating)
• eg. the chromosome, ALL plasmids & some autonomous viruses

• special site in/on plasmids where replication initiates
• is a specific, short DNA sequence DIFFERENT from the chromosomal origin of replication

encoded by plasmid itself binds to oriR & recruits host DNA pol to initiate replication

• 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
• 

plasmid genes

• 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

• 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

a plasmid which contains an oriR sequence, a rep gene, & something like the tra operon, a series of genes that code for transfer proteins

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

sequence of DNA found in an F plasmid's tra operon where transfer replication initiates

• 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

• 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

• plasmids that carry drug resistance genes rendering the host cell resistant to certain antibiotics
• can be transferable

• 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"

• 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
• 

• 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)

• 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)

• 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

• 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

• 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]

• 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

• 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

because the repressor is nonfunctional - long ago an insertion mutation occurred in the repressor gene

• 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
• 

• 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

• 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

• 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
• 

• 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

• 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

transfer of either plasmid occurs at LOW frequency

• 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

• RP4
• not only does it transfer to almost ALL bacterial species, it can even transfer DNA to yeast cells and to plants

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

• 1. Insertion Sequences (IS) [700-1500 bp]
• 2. Simple or Noncomposite Transposons
• 3. Composite Transposons
• 4. Itegrating Conjugal Elements (ICE)

• 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

short (<100 bp), inverted repeat sequences at their 5' & 3' ends

• 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

• 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
• 

• a mobile genetic element containing additional genes unrelated to transposition (eg. a gene encoding drug resistance, toxins, conjugative properties, etc.)
• 

• 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

• 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

• 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'

resolves the cointegrate: a transposition intermediate

• 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

• 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
• 

• resolvase (TnpR in Tn3): resolves quickly because it's dedicated to this specific function
• RecA: resolves more slowly

• 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

combines properties (genes) of transposons, temperate phages, AND conjugal elements all in the same unit

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

• 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