Genetic Adaptation II

Microbial Physiology
Module 3
 Topics
• Topic 4: Transposable Elements
• Topic 5: Exchange of Genetic Material Between
Organisms
• Topic 5a: Protection Against Foreign DNA
 Aims and Objectives
• By the end of this module you should…
– be able to distinguish between IS and Tn elements
– understand the importance of transposable elements to
the evolution and adaptation of microorganisms
– have a detailed understanding of the three main
mechanisms of DNA transfer and be able to distinguish
between them
– know how bacteria protect themselves against the
introduction of foreign DNA
 Last Time…
• Look at…
– How mutations arise
– Macrolesions
• Insertions, deletions, inversions, duplications
– Microlesions
• Frame-shift mutations
• Base substitutions
– Transitions
– Transversions
• Point mutations
– Silent
– Missense
– Nonsense
 Last Time…
• Mutations can allow organisms to acquire new
functions over many generations, under suitable
conditions (selective pressures)
 This time…
• Look at how organisms can acquire genetic
material from other sources
– Transposable elements
– Other bacteria
• Transformation
• Transduction
– Generalised
– Specialised
• Conjugation
 Recombination
• Central to all the mechanisms of DNA transfer via
transposable elements, transduction,
transformation and conjugation
• Recombination can be…
– homologous or non-homologous
• non-homologous is also called site-specific
– reciprocal or non-reciprocal
 Homologous Recombination
• Occurs between DNA sequences that are the same
or very similar
• Results in a reciprocal exchange of genetic
material between the two sources
• Best example: crossing over that occurs between
pairs of chromosomes
• Bacterial example: Gene mapping using
conjugation of Hfr with F- cells
 Site-specific Recombination
• Exchange of material in sequences that show little
or no sequence homology
• Exchange is non-reciprocal
– DNA transfer only occurs one way
• Can only happen at specific sites in the genome
• Example: Prophage formation in bacteriophage λ
 Topic 4: Transposable Elements
• Elements within the bacterial genome
(chromosome and accessory genetic elements) that
are capable of translocating to new locations
– “Jumping genes”
– Transfer generally occurs through non-homologous
recombination
– Some leave a copy in the original location
– Some move without duplicating the sequence
• Two types of transposable genetic elements
– Insertion sequences (IS)
– Transposons (Tn)
 Insertion Sequences
• Simplest transposable elements
• Small
• Don’t code for structural proteins
• Code for transposase
– Enzyme responsible for facilitating transposition
• Functions of the transposase
– Helps create the insertion site
– Joins the IS ends to the target site
– Fills all the gaps created by insertion
Structural Features of IS

Inverted terminal repeats

IS gene(s)

Target site duplication

(direct repeats)
 Mechanism of Transposition
Transposase

TAGGGCAAA AAACGGGAT
ATCCCGTTT IS TTTGCCCTA

Inverted terminal repeats

ACCGTCGGCATCA
TGGCAGCCGTAGT

Target site
 Mechanism of Transposition
Transposase

TAGGGCAAA AAACGGGAT
ATCCCGTTT IS TTTGCCCTA

Transposase
ACCGTCGGCATCA
TGGCAGCCGTAGT

DNA is cleaved at target site

 Mechanism of Transposition

TAGGGCAAA AAACGGGAT
ATCCCGTTT IS TTTGCCCTA

Transposase

ACCGTCGGCATCA CA
TG TGGCAGCCGTAGT
 Mechanism of Transposition

ACCGTCGGCATCA TAGGGCAAA AAACGGGAT CA

TG ATCCCGTTT IS TTTGCCCTA TGGCAGCCGTAGT
 Mechanism of Transposition

ACCGTCGGCATCA TAGGGCAAA AAACGGGAT ACCGTCGGCATCA

TGGCAGCCGTAGT ATCCCGTTT IS TTTGCCCTA TGGCAGCCGTAGT

Transposase Transposase
 Mechanism of Transposition

Target site duplication

(direct repeats)

Inverted terminal repeats

ACCGTCGGCATCA TAGGGCAAA AAACGGGAT ACCGTCGGCATCA

TGGCAGCCGTAGT ATCCCGTTT IS TTTGCCCTA TGGCAGCCGTAGT
 IS General Features
• Inverted terminal repeats
– Inverted repeats (generally)
– 9 to 40 bp
• Target site duplications
– Direct repeats
– 2 to 13 bp
IS Size (bp) Target site Terminal Repeats
duplication
IS1 768 9 23, inverted

IS2 1 327 5 32, inverted

IS3 1 258 3-4 32, inverted

 Functions of IS
• Chromosomes may contain multiple copies of IS
elements
– E. coli chromosome has 6-10 copies of IS1
• May also be present on plasmids
– F plasmid has IS2 and IS3
– When also present on chromosome…
• Homologous recombination between like Is’
• Integration of F plasmid into chromosome
– Hfr
• Higher frequency recombinant
• Promotes genetic exchange (including transposons)
 Transposons (Tn)
• Also called composite transposons
• More complex than IS
– involve genes other than those required for
transposition
– larger
• Formed by the proximity of two ISs on a
chromosome
– sequence between the ISs is captured an transposed
along with the IS elements
 Tn9

Direct repeats of IS1

768 bp 768 bp

Tn9 IS1 camR IS1

23 bp IS1
terminal inverted repeats
 Tn5

Inverted repeats of LS50

1533 bp 1533 bp

IS50L kanR bleR strR IS50R

9 bp terminal inverted repeats

 Regulation of Tn5 transposition
• Different IS elements at each end
– IS50L and IS50R
• IS50R produces 2 proteins
– Transposase
– Repressor
• translated from start site within the transposase gene
• more abundant than the transposase
Regulation of Tn5 transposition
IS50L kanR bleR strR IS50R
Transposase

Repressor

Phage DNA Phage DNA

containing Tn5 containing Tn5

Tn5 Tn5

Repressor Repressor
> Transposase: >>> Transposase: Tn5
Tn5 occasionally Tn5 seldom
transposes transposes

Genomic DNA Genomic DNA

without Tn5 containing Tn5
 Tn3 Elements
• Larger than IS elements
• Have inverted terminal repeats
• Produce target site duplication
• Carry genes other than other required for
transposition
– Not flanked by other IS elements
• Transposition is a two-step process
– cointegration
– resolution
 Tn3 Structure
38 bp inverted terminal repeats

tnpA tnpR bla

Transposase
Resolvase /
Repressor
B-lactamase
Tn3 Transposition: Cointegration
Tn3

Donor Recipient
Transposase
Plasmid Plasmid

Cointegrate
Tn3 Transposition: Resolution

Cointegrate Resolvase

Tn3 Tn3

Donor Recipient
Plasmid Plasmid
 Conservative and Replicative
Transposition
• Conservative transposition
– transposon is excised from one location and inserted
into another
– no duplication of transposon DNA
– eg. Tn5
• Replicative transposition
– transposon DNA is duplicated during the transposition
process
– source transposon remains in its original location
– eg. TnA transposon such as Tn3
 Important Note re Transposition
• Transposition IS (essentially) a recombination
event
– Generally does not involve homologous sequences
• i.e. is non-homologous
– Does not use the general recombination system
– Happens at specific DNA sequences
• Site-specific recombination
• transposase used instead of RecA
 Properties of IS and Tn elements
Name Size Direct Terminal Repeats Genes
(bp) Repeats Carried
IS1 768 9 bp 23 bp, inverted None

IS2 1327 5 bp 32 bp, inverted None

IS3 1400 3-4 bp 32 bp, inverted None

Tn3 4957 5 bp 38 bp, inverted ampR

Tn5 5700 9 bp 1400 bp (IS50), inverted kanR, bleR, strR

Tn9 2500 9 bp 768 (IS1), direct camR

Tn10 9300 9 bp 1400 bp (IS10), inverted tetR

 Significance of Transposable
Elements
• Medical significance
– transmission of antibiotic resistance genes
– passage of genes from chromosome to plasmid
• from plasmid to other cells
• cells need not be the same strain
– process occurs in pathogenic strains of
• Staphylococcus, Enterococcus, Neisseria, Shigella
and Salmonella
– bacteria can acquire resistance to multiple antibiotics
• MDR strains
– multiple drug resistant
 Phase Variation
• Phenomenon observed in Salmonella
• Associated with flagella formation
• Flagella are usually composed of a single protein
– flagellin
• Phase variation results in flagella proteins being
one of two different types
• Which protein is produced depends on the
orientation of an invertible DNA segment
 Topic 5: Exchange of Genetic
Material between Organisms
• Mutations are essential for the generation of
microbial diversity
• The ability to test combinations of mutations
under selective pressure accelerates the
evolutionary process
• Process accelerated by the ability of bacteria to
acquire DNA from other sources
• Three mechanisms
– Transformation
– Transduction
– Conjugation
 Transformation
• The uptake of naked DNA from the environment
into a recipient cell
– free DNA is usually the result of lysis of the donor cell
• Only competent cells can be transformed
– secrete competency factors which induce 8-10 proteins
required for transformation
• Competency depends on
– growth stage
– best if dividing at maximal rate
– competency can be increased by cations and
temperature
 Mechanism of Transformation in
Streptococcus
Naked DNA

Membrane-Associated Nuclease
DNA binding protein

Chromosome
Mechanism of Transformation in
Streptococcus
DNA binds to the
binding protein

Nuclease nicks and

degrades on strand
Mechanism of Transformation in
Streptococcus
Nucleotides
released
Mechanism of Transformation in
Streptococcus
 Mechanism of Transformation in
Streptococcus
Integration of single
stranded DNA into
chromosome
 Mechanism of Transformation in
Streptococcus

Heteroduplex
chromosome
Mechanism of Transformation in
Streptococcus

Replication
Successful Transformation

DNA degraded
by recipient:
Unsuccessful
Transformation

DNA incorporated
into recipient
genome:
Successful
Transformation
 Transformation Conclusion
• Transformed plasmid DNA need not recombine to
be maintained by the cell
Natural Transformation
Gram Positive Streptococcus pneumoniae
Bacillus subtilus
Bacillus cereus
Gram Negative Neisseria gonorrheae
Haemophilus influenzae
Pseudomonas stuzeri
Artificial Transformation
Escherichia coli
Salmonella Typhimurium
Pseudomonas aeruginosa
 Transduction
• Transfer of DNA from a donor to a recipient via a
bacteriophage (bacterial virus)
• Uses errors in the life cycle of the bacteriophage
to transfer the DNA
• Two classes of transduction
– generalised
– specialised
Bacteriophage Lifecycles: Lytic
Bacteriophage Lifecycles: Lysogeny
 Generalised Transduction
• Replication cycle of virulent bacteriophage has 3
phases
– invasion
– replication
– release
• During infection…
– host DNA is fragmented
– occasionally packaged instead of phage DNA
• defective phage
• can be any DNA fragment
• Defective phage can infect
– can’t replicate or induce lysis
 Generalised Transduction Mechanism
Viral
Virus DNA
infects cell

Host DNA
Infection

Viral DNA
enters cell
Degradation of Host DNA

Fragmentation
of host DNA
by viral
enzymes
Replication

Virus coat protein

 Assembly
Wild-type Transducing
phage phage
Lysis
 Infection by Transducing Phage
Donor DNA
Virus
infects cell

Host DNA
Infection

Donor DNA
enters cell
 Fate 1: Degradation

X Unsuccessful gene transfer X

Donor DNA is
degraded by
host
enzymes
 Fate 2: Not Replicated

X Abortive transduction X

Donor DNA is
stable but does
not integrate into
the chromosome
 Fate 3: Integration

9 Stable gene transfer 9

Donor DNA replace

homologous region in
recipient chromosome
 Specialised Transduction
• Utilises the lysogenic cycle of some bacteriophage
– eg. bacteriophage λ
• Phage DNA is propagated along with the cell’s
chromosome
• Integration into the chromosome is a site-specific
recombinational event
• DNA transfer is the result of errors in the
bacteriophage lifecycle
• Only DNA around the site of insertion can be
transferred
 Prophage
Prophage: Integration of
phage DNA into host
chromosome at a specific site

Host
chromosome
Induction
Prophage
excision error
incorporates
some host
DNA with the
phage DNA
 Initiation of the Lytic Cycle

Prophage
Replication
Assembly
Specialised transducing phage
Lysis
 Infection by Transducing Phage
Donor DNA
Virus
infects cell

Host DNA
Infection
Fate 1: Recombination

Recombination
between homologous
sequences
 Fate 1: Recombination
9 Bacterial chromosome contains
donor DNA
Fate 2: Integration

Normal
bacteriophage
Fate 2: Integration
Fate 2: Integration
Prophage DNA

Donor DNA

Recipient DNA

Two copies of genes

around the insertion
point
 Conjugation
• Sex in bacteria
• Direct transfer of DNA from one cell to another
– across a protein bridge
• pilus
– rolling-circle replication
• Requires an F-plasmid
– transfer of plasmid DNA from F+ to F- cell
– when an episome…
• can get Hfr
• transfer of chromosomal DNA
 Bacterial Mating
F+ cell F- cell
Pilus Forms and Replication
F+ cell F- cell

Rolling
Circle
Replication
Plasmid Transferred
F+ cell F+ cell
 Disrupt Mating
F+ cell F+ cell
Higher Frequency Recombinants
Hfr cell F- cell
Rolling-Circle Replication
Hfr cell F- cell

Rolling
Circle
Replication
Transfer Continues…
Hfr cell F- cell
 Disrupt Mating
Donor Recipient

Recombination
between
homologous
sequences
Successful Recombinant
Donor Recipient
Distinguishing between Mechanisms
of DNA Transfer

Transfer Cell-Cell contact Sensitive to

Process required? Dnase?
Transformation No Yes

Transduction No No

Conjugation Yes No
Topic 5a: Protection Against Foreign
DNA
• Repair mechanisms protect against mutation
• Mechanism exists to protect cells against foreign
DNA
• Restriction endonucleases
– cleave DNA at specific sequences
• eg. EcoRI from Escherichia coli RY13 cuts at the
sequence GAATTC

5’…GAATTC…3’
3’…CTTAAG…5’
 Restriction Enzymes and the
Bacterial Immune System
• Any DNA sequence that matches the recognition
sequence of the enzyme will be cleaved
– eg. EcoR I cuts λ DNA 5 times
• What protects the cell from it’s own REs?
– Methylation
– Specific sequences that would be recognised by the
host RE are methylated
– RE can’t digest methylated DNA
– bacterial immune system
 Suggested Reading and Learning
Exercises
• Atlas: Chapter 7, Sections 7-2 to 7-4
• Learning Exercises
– Rolling-circle replication
– Bacteriophage Mu
– Module 3 question sheet
 Next Week…

Module 4

Physiological
Adaptation