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3 Vectors of Escherichia coli 3.1 Plasmid vectors 3.2 Phage vectors 3.3 Cosmids - Fosmids 3.4 Phagemids 3.5 Vectors allowing integration of genes into the E. coli chromosome 3.1 Plasmid Vectors 3.1.1 Isolation of Plasmids Native Plasmid DNA Can be Present in Three Different Topological Forms and in Oligomers Topological forms: ƒ Circular covalently closed = CCC form ƒ Open circular = OC form ƒ Linear form (there are linear plasmids in some bacterial species) Oligomeric forms: ƒ Monomer ƒ Dimer ƒ Trimer ƒ etc. The Three Different Topolocial Forms of Plasmids CCC = SC OC linear Supercoiling Supercoils are introduced by topoisomerases Relaxed and Superhelical Plasmids Gel Electrophoresis of Native Plasmid DNA sc = supercoiled Formation and Resolution of a ColE1 Dimer The CCC Form of Plasmid DNA Consists of Different Topoisomers Topoisomers are distinguished by the number of superhelical turns Topoisomers can be separated by using chloroquine Analysis of the Distribution of Topoisomers = OC form Natural topoisomers Plus topoisomerase I Collection of Superhelical DNA from CsCl Gradients Containing Ethidium Bromide Bouyant density: Protein: 1.3 g/cm3 DNA: 1.5 g/cm3 RNA: 1.7 g/cm3 Fast methods of plasmid isolation: 1. Alkali lysis method 2. Boiling method Principle of both methods: Linear and OC form DNA cannot renature after strand separation 3.1.2 Plasmid Biology Classification of plasmids: 1. Conjugative – mobilizable 2. Incompatibility groups 3. Copy-number 4. Host-range Electron Micrograph of Two Conjugating E. coli Cells Donor Recipient Conjugation in Gram-Negative Bacteria Needs Three Essential Components 1. The transferosome: Transmembrane multiprotein complex = pilus 2. The relaxosome: Protein complex binding at the oriT and responsible for a singlestrand cleavage at the nic site mob genes) 3. The coupling protein: Connecting the two entities The Relaxase Exerts Four Different Functions 1. DNA-binding protein with sequence specificity for the oriT (origin of Transfer) 2. Endonuclease recognizing the nic site within the oriT 3. Pilot protein: covalently linked to the 5' end of the single-stranded DNA 4. DNA ligase Transfer of Plasmid DNA by Conjugation Plasmid Transfer by Conjugation Mobilizable Plasmids 1. Lack the genetic information coding for the transferosome 2. Need a conjugative plasmid coding for the transferosome Mobilization of a plasmid can occur by one of two mechanisms: Donation: Needs Tra functions (transferosome) provided by a conjugative plasmid Conduction: Needs a mobile element Donation E. coli / ColE1 ⇒ E. coli / F, ColE1 no transfer ⇒ transfer of ColE1 independent of F Mobilization of an E. coli Vector by Donation oriT Conduction Conjugative plasmid with A B Plasmid without oriT mobile element A B Cointegrate = DNA molecule with two different ori 's Donation versus Conduction Donation: A conjugative plasmid codes for the transferosome (pilus) which allows a mobilizable plasmid containing its own oriT and coding for its relaxosome to be transferrred from the donor to the recipient cell Conduction: A conjugative plasmid codes for at least one mobile element which can form an unstable cointegrate structure during the transposition process, and this cointegrate can be transferred to a recipient cell The F Factor IS = insertions element Tn = transposon Integration of the F factor into the E. coli chromosome occurs by one of two mechanisms: 1. Homologous recombination using an IS element present on both the F factor and the E. coli chromosome 2. Illegitimate recombination Integration of the F Factor by Homologous Recombination Localization of IS Elements on the E. coli K-12 Chromosome Illegitimate Recombination Using either IS2 or IS3, the F factor can integrate at any site within the E. coli chromosome thereby forming a cointegrate These cointegrates are rather stable Incompatibility Observation: A plasmid is not welcome when it enters a cell that already harbours a plasmid of the same anchestry Entering and resident plasmids segregate from each other over succeeding generations to form single-plasmid stable lines Phenomenon = Incompatibility Incompatibility Definition: The inability of two plasmids to stably coexist within the same cell in the absence of selective pressure The molecular basis of incompatibility: 1. Due primarily to interference between replication control functions (high copy number plasmids) 2. Due to identical partition loci (low copy number plasmids) Mechanism One Interference between copy number control functions: A few plasmid copies are chosen arbitrarily and replicated several times till the total copy number has doubled Mutual Inhibition by Replication Control Systems in a Two-Plasmid Strain Ap Selective pressure Tc Mechanism Two Based on the partition(ing) control mechanism This mechanism ensures that both daughter cells receive one copy of the plasmid What is the basis of partition incompatibility ? Partition(ing) is ƒ an active process ƒ functionally equivalent to mitosis ƒ used by low-copy-number plasmids to ensure regular distribution of their copies to the daughter cells Low-Copy-Number Plasmids Possess a Specific Partition Module The three elements: ƒ A cis-acting centromere-like site consisting of an array of sequence repeats = parC (sopC) ƒ parA (sopA) codes for an ATPase ƒ parB (sopB) codes for a centromere-binding protein The Plasmid Partition Mechanism J-Y Bouet (2007) Mol. Microbiol. 65: 1405 The sequence of events: 1. ParB binds to parC to form a partition complex on each plasmid 2. ParA specifically interacts with ParB in the partition complex 3. This interaction prompts ParA to form metastable assemblies that push or drag plasmid copies towards each pole The Mixed Pairing Model J-Y Bouet (2007) Mol. Microbiol. 65: 1405 How to find out whether two different plasmids belong to the same or to different incompatibility groups ? 1. Transform both plasmids (carry different marker genes!) into the same cell and grow strain under double selective pressure 2. Grow strain for 50-100 generations in the absence of selective pressure 3. Check for the presence of both plasmids How do plasmids ensure that they do not get lost from the bacteria ? Three mechanisms have been described leading to the survival of plasmids in bacterial cells The Three Plasmid Stability Systems 1. Stable partitioning system: Consists of three determinants: ParA: ATPase; binds to ParB ParB: binds to the centromere-like parS sequence The nucleoprotein complexes assemble in the cell at separate locations with respect to the division plane The Three Plasmid Stability Systems 2. Multimer resolution system: A resolvase acts on the plasmid res site to resolve plasmid multimers 3. Post-segregational killing: Toxin-antitoxin mechanism killing plasmidfree cells Copy number = Number of plasmid copies per chromosome Low-copy number: 1-2 (mini-F, pSC101) Medium-copy number: 2-10 (pACYC177/184) High-copy number: 20-50 (pBR322) Very high-copy number: ~100 (pUC plasmids) Host-range narrow – wide host range All Vector Systems Depend on Naturally Occurring Plasmids Many naturally occurring plasmids do not code for an antibiotic resistance marker = cryptic plasmids 3.1.3 Plasmid Vectors ƒ ColE1 family ƒ p15A family ƒ pSC101 family ƒ F-factor family ƒ R6K family ƒ RP4 family 3.1.3.1 ColE1-Derived Vectors The Naturally Occurring Plasmid ColE1 bacteriocin Bacteriocin = Plasmid-encoded protein able to kill bacteria of the same or closely related species; neutralized by the immunity protein The First Plasmid Vector pBR322 (High-Copy-Number) Present in 39-55 copies per cell, depending on the growth rate of the cell F Bolivar (1977) Gene 2: 75 The Very High-Copy-Number Plasmids pUC18/19 500-700 copies per cell at 37°C C YanishPerron (1985) Gene 33: 103 Chromosomal Mutations Altering the Copy Number of ColE1-Type Plasmids pcnB: plasmid copy number B Codes for an poly(A) polymerase Involved in polyadenylation of RNA I (antisense RNA of RNA II) J Lopilato (1986) Mol. Gen. Genet. 205: 285 L Lle (1993) Mol. Microbiol. 9: 1131 rpoC: RNA polymerase C Codes for the β' subunit G1161R and a 42-amino-acid deletion reduce the copy number J Ederth (2002) Mol. Gen. Genet. 267: 587 3.1.3.2 p15A-Derived Vectors The Medium-Copy-Number Vectors pACYC177 and pACYC184 ACY Chang (1978) J. Bacteriol. 134: 1141 3.1.3.3 pSC101-Derived Vectors The Low-Copy-Number Plasmid pSC101 SN Cohen (1973) PNAS 70: 1293 The pSC101-Derivative Vector pHSG415 T Hashimoto-Gotoh (1981) Gene 16: 227 pSC101 Vectors with Elevated Copy Numbers Copy number: 27 - ~240 J Peterson (2008) Plasmid 59: 193 3.1.3.4 F-Factor Derived Vectors The F Factor The Mini-F Factor pDF41 = BAC Bacterial Artificial Chromosome M Kahn (1979) Methods Enzymol. 68: 268 The BAC Vector pBeloBAC11 loxP: permits introduction of additional DNA segments via Cre-mediated recombination cosN: linearization by λ terminase VJ Kim (1996) Genomics 34: 213 Cloning with BAC A BAC vector can stably maintain DNA fragments >300 kb F' factors H Shizuya (1992) PNAS 89: 8794 Stepwise Assembly of a Large Contiguous DNA Fragment by Recombination A B C C A B C D D E E Stepwise Assembly of a Large Contiguous DNA Fragment by Recombination needed for sequencespecific recombination growth at high temperature 3.1.3.5 R6K-Derived Vectors The Naturally Occurring Plasmid R6K The R6K-Derived Vector pRK353 M Kahn (1979) Methods Enzymol. 68: 286