E.coli systems and recombination: Determinants of diversity: Overall aims ML

1
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.
• 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.

2
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

3
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
4
R100 as an example of the Cassette model
mer amp sul str kan cm
5
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
6
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

7
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

8
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

9
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 NONE

38
5
Tn 1721 5.0 tetr and
INTEGRON system 38
5
10
Structure of IS10 and composite Tn10 as an example
Active in transposition
Defective
tetR
IR-L
IR-R
Host
Tn10
Transposase
11
Structure of Tn3 as an example
5bp duplication 5bp duplication
IR-R
tnpA
tnpR
bla
IR-L
12
Transposition Mechanisms
CONSERVATIVE VS REPLICATIVE Independent of
RecA
Donor
CONSERVATIVE TRANSPOSITION
REPLICATIVE TRANSPOSITION
13
Tn3 Transposition is replicative
Tn3
Transposase cut
Replication
5bpTarget cut
14
Tn3 Transposition is replicative cont..
Resolution site analogous to cer
Donor Intact Transposed element replicated
15
IS10 (Tn10) transposition is conservative
Donor DNA lost / degraded
IS10

Transposition complete
16
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
17
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
18
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)
19
Repressor regulation Tn3
IR-R
tnpA
tnpR
bla
IR-L
20
Antisense RNA and methylation IS10R fromTn10
180 base overlap from Pout causes 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)
21
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
22
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
23
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
24
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
25
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