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E.coli systems and recombination: Determinants of
diversity: Overall aims ML
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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.
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.
©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast.
Plasmid Evolution and Role of mobile DNA Elements
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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
©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast.
Cassette model for Plasmid evolution.
 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
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
R100
tra genes
F
Tn10 found on R100
©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast.
R100 as an example of the Cassette model
IS1
Tn3 on R1
mer
amp
sul
str
kan
cm
Tn4
tra
IS2
Tn2571
Tn10
Tn903 on R6
IS1
IS10
IS10
©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast.
Resistance
Determinants
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
PO
E
T
K
MUTATION
TRANSCRIPTION BLOCKED.
NO ENZYME EXPRESSION
©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast.
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
Melt and self anneal
dgalpolar mutant
+
dgal+
Melt an anneal
©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast.
Classes of transposable DNA in bacteria
 Many elements discovered since first ones
 There are four basic types
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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
©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast.
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
©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast.
Properties of some transposable DNA elements
 TYPE I Insertion sequences and their composite transposons
shown in handout. See IS FINDER WWW SITE http://wwwis.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
Tn 1
5.0
ampr
38
5
Tn 3

5.0
5.0
ampr
NONE
38
38
5
5
Tn 1721
5.0
tetr and INTEGRON system
38
5
©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast.
Inverted repeats
Target dup’
Structure of IS10 and composite Tn10 as an example
Defective
IS10-L
Active in transposition
IS10-R
9.3Kb
tetR
1057 bps
Tn10
IR-L
IR-R
9bp duplication
9bp duplication
Transposase
©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast.
Host
Structure of Tn3 as an example
Resolution site
5bp duplication
IR-L
5bp duplication
tnpA
Transposase
©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast.
tnpR
Resolvase/
repressor
bla
-lactamase
IR-R
Transposition Mechanisms
CONSERVATIVE VS REPLICATIVE
Independent of RecA
TRANSPOSON
Target sequence
Donor
+
CONSERVATIVE
TRANSPOSITION
REPLICATIVE
TRANSPOSITION
RESOLUTION
+
+
Donor may be degraded
©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast.
Tn3 Transposition is replicative
Tn3
Ligation
Transposase cut
Replication
5bpTarget
cut
©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast.
Tn3 Transposition is replicative cont……..
Resolution site
analogous to cer
Resolution by
TnpR
©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast.
+
Donor Intact
+
Transposed element
replicated
IS10 (Tn10) transposition is conservative
IS10
Double strand
cuts
9bpTarget
cut
Donor DNA lost / degraded
+
Repair of 9bp gap
Transposition complete
©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast.
Demonstration of IS10 conservative transposition
IS10 constructed into  phage int-, replication deficient: needs permissive host
lacZ- insert
lacZ+ insert
OR
Melt, mix then reanneal
Plate on tet/Xgal plates for transposants
Package into  phage heads.
Infect recA, lac deletion, non-permissive
host cells
Some sectored
colonies
But
10% sectored and
still segregating
Therefore transposition must be conservative
90% blue or white
©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast.
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
©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast.
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)
©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast.
Repressor regulation: Tn3
Resolution site analogous to cer
IR-L
tnpA
Transposase
©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast.
tnpR
Resolvase/
repressor
bla
-lactamase
IR-R
Antisense RNA and methylation: IS10R fromTn10
180 base overlap from Pout causes
multicopy inhibition
Tn10
IR-L
Pout
IR-R
Host
Pin
Transposition x10 higher in dam mutants
Tn10
IR-L
No expression when methylated
only after replication and hemimethylation
IR-R
GATC
CTAG
Host
In Pin region
Combination leads to ONLY 0.25 molecules (1 per 4 cells)
of transposase (measured using cat gene fusions)
©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast.
Transcriptional frameshift control: IS1
IS1 768 bps: Complex internally.
Occasionally a transcriptional frameshift to give fused
insA/insB protein and full transposase
IR-L
insA
insB
No full transposase
Transposase
©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast.
IR-R
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
©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast.
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
Recombinase
3’conserved
5’conserved
7 bps core sites
in variable region
New gene acquired
©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast.
Target DNA
Overview of gene spread
The relative role of transposons vs other recombinational
and mutational events.
A SPECTRUM of activities leads to variation
Plasmid
transfer
Homologous
recombination
*10-1
10-2
Integron action
Point
mutation
Transposition
10-3
10-4
High frequency
Low diversity
* As frequency per cell per generation
©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast.
10-5
10-6
10-7
10-8
Low frequency
High diversity
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
©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast.