Plasmids and Bacteriophages

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 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

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. Rop Replication of ColE1

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

10. Amplification 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. 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 Integration of F plasmid into host chromosome Excision of F plasmid from 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- 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 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 Form Lysoginize Phage Host 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. PR’ PL PR Genetic map of phage l

33. cos

34. tR1 tL1 Without N tR2 With N A tR3 A 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. 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. 2004. 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. 2004. Adv Biochem Engin/Biotechnol. 86: 47-82