plasmids and bacteriophages plasmids: autonomously replicating extrachromosomal dna molecules...

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