1. Plasmids and Bacteriophages
Plasmids: autonomously replicating extrachromosomal DNA molecules present mostly in the bacterial cells.
Bacteriophages (phages): bacterial viruses.

2. 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

3. Features of selected plasmids of E. coli
Size (kb)
Copy number
Conjugative
Other phenotype
ColE1
6.6
10–20 No
Colicin production and 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

4. Plasmid replication: Mode of replication
Cairns intermediate (θ or butterfly form)
Rolling circle
Requirements for host enzymes

5. Control of copy number Negative control Inhibitor-target mechanism
Iteron-binding mechanism

6. Replication of ColE1
Rop

7. Evolution of pUC18/19
ColE1
pBR322
pUC18/19
oriV
oriT
rop

10. Amplification Mechanisms For ColE1-related plasmids, like pBR322
By chloramphenicol treatment
Copy number increases up to 1,000X
Mechanisms
Replication 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.

11. Plasmid R1 control circuit for replication

12. Iteron-binding mechanism

13. Stability of plasmids
Partitioning
Lethal segregation
Resolution of plasmid multimers
(e.g., Xer and cer of ColE1)

14. Regions involved in partitioning of plasmids
Centromere-like function
The binding of ParA and ParB to parS serves as a unit for interaction with the host components involved in segregation.

15. parB-mediated post-segregational killing of plasmid free cells of R1 plasmid
Targets of poisons: cell membrane, gyrase, DnaB, unknown
Antitoxins:
mRNA (type I)
Protein (type II)
h: hok (host killing) mRNA; s: sok (suppression of killing) mRNA.

16. Incompatibility
Incompatibility groups: IncA-IncZ

17. Factors determining host range
Stable mating pair formation and mobilization
Restriction enzymes in the recipient
Replication defect

18. F plasmid

19. Surface exclusion: 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

20. F plasmid Hfr strain F’ plasmid
Excision of F plasmid from host chromosome
Integration of F plasmid into host chromosome
Hfr strain
Aberrant excision of F plasmid from host chromosome
Integration of F’ plasmid
F’ plasmid

21. E. coli Hfr strains

22. Transfer of chromosomal DNA from Hfr into a F- recipient

23. Hfr x F- Time of entry Hfr: str-s; a+, b+, c+, d+, e+
F- : str-r; a-, b-, c-, d-, e-
Transconjugants
Time of entry

24. Construction of the genetic map of E. coli by conjugation

25. Phages Structures Bacterial viruses;
replicate only within a metabolizing bacterial cell.
Structures
Coat
Nucleic acid (ds- or ss-DNA or RNA; linear or circular)

26. Life cycle of phages Lytic (virulent) phages
Lysogenic (temperate) phages

27. Properties of a phage-infected bacterial culture
Multiplicity of infection (moi)
Poisson’s law
P(n)=mne-m/n!
m: moi;
n: no. absorbed phage
Plaque
Infective center
Burst size
One step growth curve of phage
Infective centers (fraction of yield)
Factors contributing to host specificity:
Receptors
Ability of bacterial RNA pol to recognize phage promoters
Host restriction and modification
Min. after infection

28. Properties of several phage types
Host
Form
Lysoginize
DNA; RNA
Mode of release
fX174 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 l 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
CTXf VC
DNA
ss, cir +
Extrusion

29. Lytic cycle

30. Regulatory cascade in lytic cycle
Temporal control of SPO1 transcription

31. Control of phage l life cycle
Lytic cycle
Lysogenic cycle
Early gene expression
Expression of late genes
Expression of repressor

32. Genetic map of phage l PR PL
PR’

33. cos

34. Temporal control of transcription during lytic infection by phage l
tL1
tR1
tR3
Without N A
tR2
With N
Temporal control of transcription during lytic infection by phage l

35. Gene expression regulation by antitermination
(nutL)

36. Antitermination by N protein
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 r-dependent and r-independent terminators by restricting the pause time at the terminator.

37. Antitermination by Q protein
Q binds to the qut site, which overlaps PR’, alters the RNA pol in a way that it resumes transcription and ignores the terminator, continuing on into the late genes.
Late gene expression

38. DNA replication and maturation
Modes of replication:
q and rolling circle
Cutting and packaging of DNA (38-51 kb)
terminase (Ter system)
two cos sites

39. Lysogenic cycle General properties Turbid plagues
Immunity
Induction
Mechanism of immunity
CI repressor; OR and OL
Homoimmune and heteroimmune
lcI -; lvir

40. att sites (attP and attB) Integrase (Int) IHF (host factor)
Prophage integration
att sites (attP and attB)
Integrase (Int)
IHF (host factor)
Prophage exision
att sites (attL and attR)
Integrase
Exisionase (Xis)
IHF
Site-specific recombination
gal
bio

41. Synthesis of cI: promoters PRE and PRM PRE
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

42. OL and OR contains three repressor-binding sites
Blocks access of RNA pol to the promoter
AT-rich spacers allow DNA twist more readily which enhances the affinity of the operator for repressor

43. The lytic cascade requires Cro (the repressor for lytic infection)

44. Fate of a l 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

45. Induction of l prophage

46. Transduction Specialized transduction (e.g. l transduction)
Generalized transduction (e.g. P1 transduction)

48. Methods to determine plasmid copy number
Quantification of gene products
→Activity of enzymes
→Fluorescence (GFP)
→Rate of segregation
Quantification of nucleic acids
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
Without separation/isolation of plasmid DNA
→Dot blot
→Sequence-specific assay/ILA
→PCR
Friehs K Adv Biochem Engin/Biotechnol. 86: 47-82

49. Methods to avoid segregational plasmid instability
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
Friehs K Adv Biochem Engin/Biotechnol. 86: 47-82