Plasmids and Bacteriophages
Plasmids: autonomously replicating extrac
hromosomal DNA molecules present mostl
y in the bacterial cells.
Bacteriophages (phages): bacterial viruses.
Plasmids General properties of plasmids:
dsDNA, mostly circular
Size: 3 kb-150 kb
Copy number: low, intermediate, or high
Host range: narrow v.s. broad
Stability
Incompatibility
Transfer: self-transmissible;mobilizable, nonconjugative;nonconjugative, nonmobilizable
Features of selected plasmids of E. coli
Plasmid Size(kb)
Copy number
Conjugative Other phenotype
ColE1 6.6 10–20 No Colicin productionand immunity
F 95 1–2 Yes E. coli sex factor
R100 89 1–2 Yes Antibiotic-resistance genes
P1 90 1–2 No Plasmid form is prophage; produces viral particles
R6K 40 10–20 Yes Antibiotic-resistance genes
Plasmid replication:
Mode of replication
Cairns intermediate (θ or butterfly form)
Rolling circle
Requirements for host enzymes
Control of copy numberNegative control
Inhibitor-target mechanism
Iteron-binding mechanism
Replication of ColE1
Rop
Evolution of pUC18/19
ColE1
pBR322
pUC18/19
oriV
oriT
rop
Amplification
For ColE1-related plasmids, like pBR322
By chloramphenicol treatment
Copy number increases up to 1,000X
MechanismsReplication of host chromosomal DNA decreases, while the machinery for plasmid replication is more stable.
Rop concentration decreases, and that way blocks the negative control of plasmid copy number.
Plasmid R1 control circuit for replication
Iteron-binding mechanism
Stability of plasmids
Partitioning
Lethal segregation
Resolution of plasmid multimers(e.g., Xer and cer of ColE1)
Centromere-like function
Regions involved in partitioning of plasmids
The binding of ParA and ParB to parS serves as a unit for interaction with the host components involved in segregation.
parB-mediated post-segregational killing of plasmid free cells of R1 plasmid
h: hok (host killing) mRNA; s: sok (suppression of killing) mRNA.
Targets of poisons: cell membrane, gyrase, DnaB, unknown
Antitoxins:mRNA (type I)Protein (type II)
Incompatibility
Incompatibility groups: IncA-IncZ
Factors determining host range
Stable mating pair formation and mobilization
Restriction enzymes in the recipient
Replication defect
F plasmid
Cell contact via the sex pilus
DNA mobilization and transfer
DNA replication while transferring
Surface exclusion:
The F+ strains do not usually serve as the recipient
F plasmid
F’ plasmid
Hfr strain
Integration of F plasmid into host chromosome
Aberrant excision of F plasmid from host chromosome
Integration of F’ plasmid
Excision of F plasmid from host chromosome
E. coli Hfr strains
Transfer of chromosomal DNA from Hfr into a F- recipient
Time of entry
Hfr x F-
Hfr: str-s; a+, b+, c+, d+, e+
F- : str-r; a-, b-, c-, d-, e-
Tran
scon
juga
nts
Construction of the genetic map of E. coli by conjugation
PhagesBacterial viruses;
replicate only within a metabolizing bacterial cell.
Structures
Coat
Nucleic acid (ds- or ss-DNA or RNA; linear or circular)
Lytic (virulent) phages
Lysogenic (temperate) phages
Life cycle of phages
Multiplicity of infection (moi)Poisson’s law P(n)=mne-m/n!
m: moi; n: no. absorbed phage
PlaqueInfective centerBurst size
Infe
ctiv
e c
ente
rs (
frac
t io
n o
f yi
eld
)
Min. after infection
One step growth curve of phage
Factors contributing to host specificity:
Receptors
Ability of bacterial RNA pol to recognize phage promoters
Host restriction and modification
Properties of a phage-infected bacterial culture
Properties of several phage typesPhage Host DNA;
RNAForm Lysoginize Mode of
release
X174 E DNA ss, circ - Lysis
M13, fd, f1 E DNA ss, circ - Extrusion
Mu E DNA ds, lin + Lysis
T7 E DNA ds, lin + Lysis
E DNA ds, lin + Lysis
P1 E DNA ds, lin + Lysis
SPO1 B DNA ds, lin + Lysis
T2, T4, T6 E DNA ds, lin + Lysis
MS2, Qb, f2 E RNA ss, lin - Lysis
CTX VC DNA ss, cir + Extrusion
Lytic cycle
Temporal control of SPO1 transcription
Regulatory cascade in lytic cycle
Control of phage life cycle
Lytic cycle
Early gene expression
Expression of late genes Expression of repressor
Lysogenic cycle
Genetic map of phage
PRPL
PR’
cos
Temporal control of transcription during lytic infection by phage
tL1 tR1
tR3
Without N
A
tR2
With NA
Gene expression regulation by antitermination
(nutL)
N protein is an RNA-binding protein (via an Arg-rich domain), recognizing a stem loop formed at the nut sites.
Host proteins are involved in antitermination.N causes antitermination at both -dependent and -independent
terminators by restricting the pause time at the terminator.
Antitermination by N protein
Q binds to the qut site, which overlaps PR’, alters the RNA
pol in a way that it resumes transcription and ignores the t
erminator, continuing on into the late genes.
Late gene expression
Antitermination by Q protein
DNA replication and maturationModes of replication:
and rolling circle
Cutting and packaging of DNA (38-51 kb)
terminase (Ter system)
two cos sites
Lysogenic cycleGeneral properties
Turbid plagues
Immunity
Induction
Mechanism of immunity
CI repressor; OR and OL
Homoimmune and heteroimmune
cI -; vir
Site-specific recombination
gal
bio
Prophage integration
att sites (attP and attB)
Integrase (Int)
IHF (host factor)
Prophage exision
att sites (attL and attR)
Integrase
Exisionase (Xis)
IHF
Synthesis of cI: promoters PRE and PRM
CII acts on PRE, PI and Panti-Q
CIII protects CII from degradation by HflA
cyL and cyR mutations prevent establishment of lysogeny in cis
PRE
OL and OR contains three repressor-binding sites
AT-rich spacers allow DNA twist more readily which enhances the affinity of the operator for repressor
Blocks access of RNA pol to the promoter
The lytic cascade requires Cro (the repressor for lytic infection)
Fate of a infection: lysis or lysogeny
CI:
OR1 > OR2 > OR3
OL1 > OL2 > OL3
Cro:
OR3 > OR2 = OR1,
OL3 > OL2 > OL1
The key to the fate is CII
CII is degraded by host protease, and stabilized by CIII
Induction of prophage
TransductionSpecialized transduction (e.g. transduction)Generalized transduction (e.g. P1 transduction)
Methods to determine plasmid copy number
Quantification of gene products
→Activity of enzymes→Fluorescence (GFP)→Rate of segregation
Quantification of nucleic acids
Without separation/isolation of plasmid DNA
→Dot blot→Sequence-specific assay/ILA→PCR
With separation/isolation of plasmid DNA
→HPLC→Density gradient centrifugation→AGE and densitometry→AGE and image analyzer→AGE and Southern blot→PCR and TGGE→CGE→FIA/FIP
Friehs K. 2004. Adv Biochem Engin/Biotechnol. 86: 47-82
Methods to avoid segregational plasmid instability
Friehs K. 2004. Adv Biochem Engin/Biotechnol. 86: 47-82
Adding an antibiotic to the medium
Complementation of chromosomal mutations
Post segregational killing of plasmid free cells
Influences of plasmid size and form
Active plasmid partitioning
High copy number and plasmid distribution
Lowering the difference in specific growth rates by the internal factors
Influences of cultivation conditions (pH, O2, phosphate, etc.)
Integration into the chromosome
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