BIOL253: Genetics L11 – Bacterial Genetics

Bacterial Genetics – Transduction & Transposition

Specialised transduction mediated by temperate bacteriophages e.g., bacteriophage l

Generalised transduction (discussed L10) – map order

of genes when within around 100Kbp of each other
around the entire chromosome.

Specialised transduction – cannot be used to map genes around the entire chromosome – just in specific
regions.
• Mediated be temperate bacteriophages. (virulent in generalised)
• Temperate bacteriophages – can adopt 2 different lifestyles: lytic lifecycle or lysogenic lifecycle.
• Genome in the head – infects bacteria.
• Bacteriophage lambda – within the bacteriophage head the DNA is linear. As soon as injected into
bacterium – it circularises.
• Then dependent on levels of gene expression of genes that are present on the bacteriophage – there
are two alternatives.
• Lytic cycle – produce many copies of the bacteriophage, ultimately produce lysozyme to release
bacteriophage.
• Lysogenic cycle – injected bacteriophage genome is inserted into genome of bacteria – can stay there
and maintained in bacterial chromosome (in replication). Occasionally there are circumstances where
the bacteriophage chromosome when sitting in E.coli chromosome is called a prophage. Occasionally
circumstances cause it to excise (come out of) bacterial chromosome and then enter the lytic cycle.
Circumstances include, DNA damage.
• release of lambda genome form prophage into bacteriophage into lytic cycle – happens as part of the

SOS response.

Bacteriophage l can adopt alternative life cycles

 Therefore can do specialised transduction.
Linear chromosome first circularized in bacteria by annealing of
complementary ends and ligation
- 12 nucleotide overhang at each end of linear genome. 5’
protruding ssDNA, when introduce dinto host bacterium,
sticky ends base pair (complementary base pairing).
Circularises DNA.
Two possible fates for circularised l:
(1) Lytic pathway – circularised l DNA nick introduced into one strand and helicase then displaces the 5’ end.
The 3’ end initiates synthesis by rolling-circle mechanism (using info on intact ssDNA) to produce a
concatamer of linear DNA (long DNA molecule consisting of multiple end to end copies of the lambda
chromosome). Second strand is produced within the same bacterium. Circle can be used to make several
copies of the linear DNA.
Concatamer cut at cos sequence by Ter
enzyme to produce l genome sized
BIOL253: Genetics L11 – Bacterial Genetics
molecules (includes the sticky ends) – packaged into phage particles. (condense down to genome size units to
pack into bacteriophage head)

(2) Lysogenic pathway

• Circular l DNA integrates into bacterial genome at a specific site – site specific recombination
• Recombination occurs between bacterial attB site (attachment site for the DNA in the bacterial
chromosome) and attP (site in phage chromosome) in the lambda DNA
• Both attP and attB contain a central spacer region, O, that is flanked by different integrase binding sites
(B, B’ and C, C’) (integrase enzyme that coded for by bacteriophage – required to insert phage
chromosome into bacterial chromosome)
• Site-specific recombination occurs within sequence ‘O’ – the core sequence common to both att sites.
To integrate the prophage chromosome.
End up with prophage, flanked by p’ and c’ sequences, O sequence and B.
Specific mechanism by which phage chromosome integrates into bacterial chromosome – by site specific
recombination occurring at the specific site between attP and attB sequences. When it sits in the chromosome,
rather than having attP and attB. Now have attL, attR (left and right) - composites of the phage and the
bacterium attB and attP sites.

• Integrated prophage NORMALLY excised from chromosome by recombination between core O

sequences of attL (B O C’) and attR (C O B’)- release of normal lambda phage gene which can go into
the lytic cycle. Generating intact attP and attB site.
• On rare occasions prophage is excised from chromosome aberrantly – some prophage DNA remains in
bacterial chromosome, excised bacteriophage lacks some prophage genome (d defective), but carries
bacterial DNA from region flanking integration site.
• attR site in bacteria rather than attB, and attL site in released phage. Phage release is a lambda d gal+
phage chromosome. Gal+ as taken gal+ gene with it, d stands for defective, although released
bacteriophage and this itself can be packaged into bacteriophage head, as all of the genes required for
that are in the one cell. The bacteriophage can infect another cell, if it does this no longer has all of the
proteins in a new cell to synthesize the bacteriophage head and tail, cant act as proper bacteriophage.
• Results in mixed phage lysate – containing wt l phage and ld gal+ phage. (small proportion will be
genes that were sitting outside where the prophage was integrated.

ld gal+ phage mediates specialized transduction

• Defective phage cannot integrate at attB site (lacks attP – have attL instead).
• Defective phage cannot replicate and enter lytic cycle.
• Defective phage integrates by recombination with homologous chromosomal DNA.
• Specialised transducing phage transfers gal+ gene into recipient bacteria
• Released lambda d has now become gal-, cant really do anything as it only has par of the lambda
genome, as part was left behind in the previous bacterial chromosome.
• Recombined into recipient chromosome – converts gal- to gal+
• Specialised transduction limited to transducing genes close to att site. Only can map genes that are
located close to the site of attB withing the bacterial chromosome.
BIOL253: Genetics L11 – Bacterial Genetics
• Some modifications to force the lambda to integrate at sites other than attB, but is limited to what it can
do.
• Generalized transduction is more useful than specialised. Because generalized can map anywere
within the bacterial genome.

Transformation as a method for gene transfer

Transformation = uptake of ‘naked’ DNA
- Doesn’t require plasmids or bacteriophage.
Third mechanism by which genes can be transferred between
different bacteria.

Transformed DNA exchanged into (recombined) recipient genome.

Cotransformation frequency can be used to map bacterial gene

order. If two genes close – then more likely to be on same bit of
DNA.
Co-transformation efficient up to 20Kbp. Limit of mapping genes.

Gross level mapping = conjugation.

Medium level mapping – within 100Kbp = use transduction.

Cotransformation – map genes within 10Kbp of each other.

More frequently co-transformed if closer together.

Transposable elements (transposons) and transposition

• Transposable elements (aka transposons) are specifically defined pieces of DNA that can move around
the genome and insert at target sites by transposition present in both prokaryotes and eukaryotes.
• Transposons are found in all organisms and can comprise large parts of genomes. (Particularly for
eukaryotes/plants)
• Transposons move to different places in the genome, or move between chromosomal DNA and
extrachromosomal elements e.g. plasmids within the same cell.
• Transposable elements - a major source of mutations
– insertion of transposon in a gene will knock-out its
function. E.g. can end up in promoter region,
interrupting expression.
• Transposon insertion can affect genes, gene
regulation, chromatin structure (affects gene
regulation), genome stability and evolution.
• No independent form of the element.

Transposons can move by excision and integration or by replication

• Transposition does not require sequence homology for insertions.
• Insertions result in target-site duplications - small sequences at the insertion site that become
duplicated due to DNA repair mechanisms.
• The amount of duplication is characteristic of the particular transposon.
Whenever a transposon moves (can move in 2 possible ways), one of the features of where it ends up in the
genome, there becomes a duplication of sequence either side of where
transposon inserts.
Excision and integration – cut out and inserted into target DNA. Short
sequence, length of which is characteristic of specific transposons, ends
up becoming either side of insertion of transposon.
BIOL253: Genetics L11 – Bacterial Genetics
Replicative transposition – transposons remains in donor site, but copy introduced at target site. Same features
seen – small target site duplications.
Always flanked by short target site duplications. Target sites duplication lengths are specific to different types
of transposons.

Three main types of transposon

• DNA only-cut and paste elements have terminal inverted repeats (TIR) (at end of transposon, there are
repeat sequences that are inverted) that are recognized by the encoded transposase. (no RNA involved
in the transposition stage)
• Autonomous transposons encode the proteins needed to move the DNA –(have TIR and a gene that
encodes a transposase – protein required to remove a transposon around in a cell) Non-autonomous
transposons rely on proteins made by an autonomous element (rely on autonomous transposons in the
same cell to move around as no transposases present in the non-autonomous transposons. Can use
another transposons transposase to move around).
• DNA –only cut and paste transposons are widespread in bacteria and eukaryotes.
• DNA-only transposons are the only type of transposon found in bacteria.

DNA-only transposons are in virtually all organisms examined to date

• DNA-only transposons always contain an element bordered by
short inverted repeats that are recognized by element-encoded
transposase.
• (a) Bacterial elements carry other genes such as pathogenicity
factors and genes for antibiotic resistance (this has contributed
to spread of antibiotic resistance in bacteria).
- Insertion sequences – have terminal inverted repeated, but the
only other thing that they encode is the transposase.
- Composite transposons (e.g. Tn5, Tn10) consist of a pair of
insertion sequences typically flanking a set of gens that encode
antibiotic resistance.
- Non-composite transposon e.g., Tn7. No insertion sequences at
each end. Have inverted repeats at each end. And have several
genes between the inverted repeats.
• (b) Eukaryotic DNA-only elements usually only contain the
transposase. Typically inverted repeats and a gene encoding a
transposase.

Three main types of transposon:

(NOT present in prokaryotes)
b) Long terminal repeat (LTR) elements are retroviruses, or similar to retroviruses. They encode several
proteins including a reverse transcriptase. First producing an mRNA copy of the transposon, reverse
transcribed to DNA that then moves around the genome.

c) Non-LTR elements can be autonomous (encode genes to allow them to move around) or non-autonomous
(rely on proteins produced by other transposons to move around), and encode proteins with a range of
activities
BIOL253: Genetics L11 – Bacterial Genetics
Long Interspersed Elements (LINE) encode proteins that mediate their own transposition (these are common in
the human genome and are autonomous)
Short Interspersed Elements (SINE) do not encode proteins for their movement, and so rely on those from
LINEs. (non-autonomous)

45% of human genome is derived from transposable elements.

Bacterial DNA-only transposons:

1. Insertion sequences
Just have a transposase between a pair of inverted repeats.
Code just for enzyme needed for transposition, flanked by short inverted terminal repeats which are essential
for transposition. Transposase binds to short inverted terminal repeats as part of the process of transposition.
Bacterial DNA cut and paste elements that encode their own transposition functions are called insertion
sequences (IS elements)
Insertion sequence – codes for enzyme needed for transposition, flanked by short inverted terminal repeats

Bacterial DNA-only transposons:

2. Composite transposons
• In composite transposons a pair of IS (insertion
sequences) elements flank another gene for
antibiotic resistance.
• IS elements may be in same (Tn9) or inverted
orientation (Tn10, Tn5)
• Composite transposons often carry genes for drug
resistance
• Functional IS element (IS10) can transpose alone
- IS10 elements are in opposite directions.
• Composite transposon flanked by pair of IS
elements can transpose
Important for spreading antibiotic resistance.
Can transpose either as whole transposon or transpose with just one of the insertion sequences.

Bacterial DNA-only transposons:

3. Non-composite transposons – TnA family
• Large, ~5 kb transposons, not dependent on IS-type
elements. Have inverted repeats.
• Independent units, genes for transposition as well as
genes encoding drug resistance
• e.g. Tn3, Tn1000 (gd)
• Terminal inverted repeats, generate 5 bp direct repeat at
target site.
BIOL253: Genetics L11 – Bacterial Genetics

Mechanism of transposition
• Most DNA-only transposons move by cut-and-paste mechanisms (a)
• Duplication is a result of a small amount of repair that is required at the site at which the
transposon inserts. Some move by nick and paste (these ones end up with a copy in
donor and target)
• In cut-and-paste, the element excises completely and inserts into the target, using a small
amount of replication to repair the join sites
• In bacteria only, some DNA-only transposons move by nick-and-paste (b)
• In nick-and-paste, the transposon remains attached to the donor DNA and is joined to the
target (forming a cointegrate which is eventually resolved into two molecules, each
containing a transposon
• In bacteria a few transposons can move by both mechanisms.

Transposition at the molecular level

• DNA cut-and-paste transposition occurs in transpososome structures
• Terminal inverted repeat DNA recognized by transposase (1)
• Transposases oligomerize, bringing transposon ends together (2) activating
transposon cleavage from the background DNA (3)
• Only when interaction between both transposases at either end of a
transposon, that we get cutting. Transposase bound at the right – cuts a the left
end and vice versa. Allows transposon to be released from its donor site.
• Transposase/cleaved transposon complex binds to target DNA, and the 3′ OH
ends attack target DNA and join at staggered positions, staggered cut in target.
Staggered cut (characteristic of a specific transposase) with 5 nucleotide
overhang, transposase cuts target DNA and joins 3’ end of the release
transposon to where the cuts have been introduced. (4)
• Single-stranded DNAs are filled by host repair (5)
Staggered cuts result in duplication at same sequence at either end of transposon.
If transposon leaves cleanly – leave footprint of where is has been as short repeat
sequence where it has been left.

Transposons in episomes/plasmids

F factor has insertion sequences.

These insertion sequences within the F factor that recombine with the same transposons in the bacterial
chromosome that is required to generate Hfr strains.

Hfr strains are generated by recombination between transposons on F plasmid and

bacterial chromosome
Recombination between IS sequences on chromosome and plasmids can result in
plasmids being integrated into host chromosome. Plasmid integration is reversible

• Bacteria – well-defined genetic systems, easy to grow

• Early studies focused on nutritional mutants (auxotrophs)
• Genetic material can be transferred between bacteria by:
- Conjugation – F plasmid, Hfr strains, F’ strains
- Transformation – naked DNA uptake
- Transduction
• Generalised - lytic bacteriophages (e.g. P1vir)
• Specialised – temperate bacteriophages (e.g. l)
BIOL253: Genetics L11 – Bacterial Genetics
• Gene map order can be determined by:
• Conjugation (scale of complete chromosome)
• Cotransduction frequency (scale ~100 kbp)
• Cotransformation frequency (scale ~20 kbp)
• Bacterial chromosomes/plasmids carry DNA-only transposons: insertion sequences, composite and
non-composite transposons.
• Recombination between plasmid-borne transposons and chromosomal transposons results in
integration of plasmid into bacterial chromosome.