e coli systems and recombination determinants of diversity overall aims ml
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E.coli systems and recombination: Determinants of diversity: Overall aims ML. Nine/ten lectures with Key topics. Homologous recombination and DNA repair Role of methylation and repair. Role of Plasmids; control of replication, transfer and stability.

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e coli systems and recombination determinants of diversity overall aims ml
E.coli systems and recombination: Determinants of diversity: Overall aims ML
  • Nine/ten lectures with Key topics.
  • Homologous recombinationand DNA repair
  • Role of methylation and repair.
  • Role of Plasmids; control of replication, transfer and stability.
  • Illegitimate recombination: transposons and integrons
  • Regulation of DNA transposition.
  • You should:
    • Have a basic grounding for further reading and other systems covered in the course (e.g pathogens).
    • Be able to critically read key papers in the area.
    • Critically assess the development of ideas to date.
plasmid evolution and role of mobile dna elements
Plasmid Evolution and Role of mobile DNA Elements
  • Plasmid structure and evolution: Cassette model
  • Discovery of transposable elements in bacteria
  • Classes of transposable element
  • Distribution of these elements
  • Mechanisms of transposition
  • Negative control of transposition
  • Target site specificity and immunity
  • Integron mechanism for acquisition of genes
  • Overview of gene spread via plasmid / transposon vectors
  • You should be able to discuss the RELATIVE role of moveable or transposable DNA elements and the host factors controlling them in the evolution of diverse microbial genomes
cassette model for plasmid evolution
R100

F

tra genes

Tn10 found on R100

Cassette model for Plasmid evolution.

Many antibiotic resistance plasmids

such as R1, R6 and R100 are closely related to F- plasmids in the Enterobacteriaceae

e.g. F and R100 from Shigella flexneri

  • Many different types of plasmid
  • Three basic units / regions
    • 1.Transfer / 2. Replication / 3. Determinant
  • Antibiotic resistance plasmids
  • Phages replicate as plasmids
  • Catabolic plasmids e.g. Pseudomonas spp and Rhodococcus spp
  • Most are closed circular
  • More large linear mega plasmids / second chromosomes discovered e.g. Borrelia,Streptomyces and Rhodococcus spp
r100 as an example of the cassette model
Resistance

Determinants

IS1

Tn3 on R1

Tn4

tra

Tn2571

IS2

Tn10

Tn903 on R6

IS1

IS10

IS10

R100 as an example of the Cassette model

mer

amp

sul

str

kan

cm

discovery of transposable dna elements in bacteria
P O

E

T

K

MUTATION

TRANSCRIPTION BLOCKED.

NO ENZYME EXPRESSION

Discovery of Transposable DNA elements in bacteria
  • First noted in 1967 in E.coli as cause of polar mutations in;
    • gal operon (Saedler)
    • lac operon (Shapiro)
    • High frequency of spontaneous reversion to gal or lac +
  • Hedges and Jacob (1974) demonstrated 1st Transposon Tn1 (Tn3 related): Ampr in plasmid RP4

gal operon on defective lambda phage ; dgal

discovery of transposable dna elements in bacteria6
Melt and self anneal

dgal-

polar mutant

dgal+

+

Melt an anneal

Discovery of Transposable DNA elements in bacteria
  • DNA of dgal phage analysed by density gradient centrifugation and by homology annealing and EM sizing
  • Inserts detected as approx’ 800 bps or 1500 bps
  • Responsible for the POLAR effect on gene expression
  • Looping indicated that there were inverted repeats at the ends
  • Named Insertion Sequences IS1 and IS2
classes of transposable dna in bacteria
Classes of transposable DNA in bacteria
  • Many elements discovered since first ones
  • There are four basic types
    • The Insertion sequences and their composite elements TYPE I
    • The Tn3 family of elements TYPE II
    • The transposing bacteriophages (e.g. mu - not covered here) TYPE III
    • The conjugative transposons (e.g. Tn916 carrying tet resistance around a range of host cells in Enterococcus and other bacteria). Large family found in these Gram positive bacteria with broad host range. Carry Integration / excision determinants and plasmid transfer genes. INTEGRATE - EXCISE -TRANSFER ON PLASMID (not covered in detail here).
  • Many features in common but with exceptions
    • MUST have precise end recognition EITHER use terminal inverted repeat sequences OR in some cases integrate at specific sequences to produce a consensus sequence for end recognition
    • Often generate duplications at target sites
classes of insertion sequences in bacteria
Classes of Insertion sequences in bacteria
  • 19 families based on combinations of the following criteria:
  • 1) similarities in genetic organisation (arrangement of open reading frames)
  • 2) marked identities or similarities in their Transposases (common domains or motifs); DDE Motif conserved
  • 3) similar features of their ends (terminal IRs)
  • 4) fate of the nucleotide sequence of their target sites (generation of a direct target duplication of determined length).
  • IS DATABASE is best reference source
  • http://www-is.biotoul.fr
properties of some transposable dna elements
Properties of some transposable DNA elements
  • TYPE I Insertion sequences and their composite transposons shown in handout. See IS FINDER WWW SITE http://www-is.biotoul.fr/is.html. Indicates size, duplications and inverted repeats
    • Composite elements flanked by IS elements
    • Multiple copies in different bacteria WIDELY DISTRIBUTED
  • TYPE II The Tn3 like elements.
    • Many ANTIBIOTIC RESISTANCE DETERMINANTS

Type Kbps Marker Inverted repeats Target dup’

Tn 1 5.0 ampr 38 5

Tn 3 5.0 ampr 38 5

 5.0 NONE38 5

Tn 1721 5.0 tetr and INTEGRON system38 5

structure of is10 and composite tn10 as an example
IS10-R

IS10-L

9.3Kb

1057 bps

9bp duplication 9bp duplication

Structure of IS10 and composite Tn10 as an example

Active in transposition

Defective

tetR

IR-L

IR-R

Host

Tn10

Transposase

structure of tn3 as an example
Resolution site

Resolvase/

repressor

Transposase

-lactamase

Structure of Tn3 as an example

5bp duplication 5bp duplication

tnpA

tnpR

bla

IR-R

IR-L

transposition mechanisms
TRANSPOSON

Target sequence

+

RESOLUTION

+

+

Donor may be degraded

Transposition Mechanisms

CONSERVATIVE VS REPLICATIVE

Independent of RecA

Donor

CONSERVATIVE

TRANSPOSITION

REPLICATIVE

TRANSPOSITION

tn3 transposition is replicative
LigationTn3 Transposition is replicative

Tn3

Transposase cut

Replication

5bpTarget

cut

tn3 transposition is replicative cont
+

Resolution by

TnpR

Tn3 Transposition is replicative cont……..

Resolution site

analogous to cer

Donor Intact

+

Transposed element

replicated

is10 tn10 transposition is conservative
Double strand

cuts

9bpTarget

cut

Repair of 9bp gap

IS10 (Tn10) transposition is conservative

Donor DNA lost / degraded

IS10

+

Transposition complete

demonstration of is10 conservative transposition
Melt, mix then reanneal

Package into  phage heads.

Infect recA, lac deletion, non-permissive

host cells

Some sectored

colonies

But

10% sectored and

still segregating

90% blue or white

Demonstration of IS10 conservative transposition

IS10 constructed into  phage int-, replication deficient: needs permissive host

lacZ- insert

lacZ+ insert

OR

Plate on tet/Xgal plates for transposants

Therefore transposition must be conservative

transposition demonstrated in vitro
Transposition demonstrated in vitro

IS10 transposase makes double stranded cuts

And can form circles via single stranded ligation

Only Mg+ needed in reaction

In vitro transposition shown using  vectors

Rates of about 1 in 106 shown following

packaging and infection of host cells

Host factors such as;

Hu protein

Integration host factor(Ihf)

and supercoiled DNA needed

negative control of transposition
Negative control of transposition

All transposons appear to be under negative regulation

This brings transposition recombinational frequencies

down to around 10-3 to 10-6

In E. coli the growth temperature greatly affects many

transposition events.

Higher frequencies at lower temperatures (below 37oC)

Especially IS1 and Tn3. Basis not known.

Negative control due to:

A. Repressor molecule Tn3 (earlier)

B. Antisense RNA (Tn10)

C. Methylation (Tn10 and many IS elements)

D. Transcriptional frameshift (IS1 specifically)

repressor regulation tn3
Resolution site analogous to cer

Resolvase/

repressor

Transposase

-lactamase

Repressor regulation: Tn3

tnpA

tnpR

bla

IR-R

IR-L

antisense rna and methylation is10r fromtn10
Pout

IR-L

IR-R

Host

Tn10

Pin

IR-L

IR-R

Host

Tn10

GATC

CTAG

In Pin region

Antisense RNA and methylation: IS10R fromTn10

180 base overlap from Poutcauses

multicopy inhibition

Transposition x10 higher in dam mutants

No expression when methylated

only after replication and hemimethylation

Combination leads to ONLY 0.25 molecules (1 per 4 cells)

of transposase (measured using cat gene fusions)

transcriptional frameshift control is1
IR-L

IR-R

Transposase

Transcriptional frameshift control: IS1

IS1 768 bps: Complex internally.

Occasionally a transcriptional frameshift to give fused

insA/insB protein and full transposase

insB

insA

No full transposase

target site specificity and immunity
Target site specificity and “immunity”

Many relatively NON specific in target preference

Often NO common features

Tn5 and IS1 prefer hot spot AT rich DNA

Tn7 has specific target

Tn10 shows some preference for a consensus

NGCTNAGCN but not clear cut.

“IMMUNITY” shown by Type II elements (Tn3)

Low probability of second transposition in a plasmid

E. coli chromosome shows strong “immunity”

Basis is not known

integron mechanism for acquisition of genes
Recombinase

3’conserved

5’conserved

7 bps core sites

in variable region

Target DNA

Integron mechanism for acquisition of genes

Discovered in some Tn3 like elements such as Tn21

They are found WITHIN these elements

They explain the acquisition of new genes/markers

New gene acquired

overview of gene spread
Overview of gene spread

The relative role of transposons vs other recombinational

and mutational events.

A SPECTRUM of activities leads to variation

Plasmid

transfer

Integron action

Homologous

recombination

Point

mutation

Transposition

*10-1 10-2 10-3 10-4 10-5 10-6 10-7 10-8

Low frequency

High diversity

High frequency

Low diversity

* As frequency per cell per generation

the end for now
The END for NOW

The force that through the green fuse drives the flower

Drives my green age; that blasts the roots of trees

Is my destroyer.

And I am dumb to tell the crooked rose

My youth is bent by the same wintry fever

The force that drives the water through the rocks

Drives my red blood; that dries the mouthing streams

Turns mine to wax.

And I am dumb to mouth unto my veins

How at the mountain spring the same mouth sucks

Dylan Thomas 1914 - 1953

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