Chapter 9
Lecture Outline
Gene Transfer, Mutations,
and Genome Evolution
Covered in this Lecture
9.1 The mosaic nature of genomes
 9.2 Gene transfer: Transformation,
conjugation, transduction
 9.3 Recombination
 9.4 Mutations
 9.5 DNA repair
 9.6 Mobile gene elements

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The Mosaic Nature of Genomes


DNA sequence is not static
Over the millennia microbes have undergone extensive
gene loss and gain



Horizontal gene transfer
Recombination events
Mutations





New functions useful in particular situations



Single bases
Large deletions
Large insertions of sequence
Transferred from other species
Pathogenicity islands
Fitness islands
Maintained via interaction with environment

Survival determined by having appropriate genes for the specific
environment
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Gene Transfer

Transformation
 Uptake

of naked DNA
Conjugation
 Uptake
of DNA from cell to cell with direct
contact

Transduction
 Transfer
of DNA via virus
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Transformation: History

Griffith: demonstrated the phenomena of
transformation in 1928

Avery, MacLeod, and MacCarty: proved that
DNA is the transforming
element in 1944
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Gene Transfer: Transformation

Uptake of DNA directly from surrounding medium




Natural transformation is found in many species




Competent cells
Gram +: Streptococcus, Bacillus
Gram-: Haemophilus, Neisseria
Translocasome takes up DNA


DNA used as nutrient
For repair
Adjustment to the environment
Located in envelope
Competence induced in different species

Gram+ cells secrete signal


Species specific competence factor
Gram- cells do not produce competence factor


Always competent
Stress induces competence

Starvation
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Artificial Transformation
In the laboratory
 Alter membrane to allow DNA to pass
 Chemical

 CaCl2

Electroporation
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Gene Transfer: Conjugation
Sex pilus connecting 2 E. coli cells.

Requires cell to cell contact




“Bacterial sex”
In gram- bacteria: sex pilus
In gram+ bacteria: non-pilus
attachment proteins
Requires transferable plasmid


Contains all the genes for
pilus/attachment information and
DNA export (ex.: F factor)
If plasmid incorporates into host
chromosome it may also transfer
host genes
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The Conjugation Process
1. Sex pilus from the F+ donor cell attaches to
receptors on recipient cell.
Donor (F+) Cell
Recipient (F-) Cell
2. Contraction of the pilus draws the two cells
together and forms a conjugation bridge.
3. The F factor is nicked at oriT and the 5’ end
begins transfer through the bridge.
4. The strand remaining in the
donor is replicated by rolling circle
with Pol III.
5. Once in the recipient the
transferred strand circularizes and
replicates.
6. The recipient has been converted to a donor.
F+ Cell
F+ Cell
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Gene Transfer: Conjugation

Episome


Plasmid that can exist in extrachromosomal and integrated form
Hfr


High frequency recombination
F factor integrates on bacterial chromosome



Bacterial strain is called Hfr strain
More cells are capable to transfer chromosomal DNA
Bacterial
chromosome
Tries to transfer entire chromosome




F factor
Requires 100 minutes for E. coli (44 kbp/min)
Transfers genes in order
Can determine order of genes on chromosome by interrupted mating
F (F prime) factor


Integrated F factor is excised along with some chromosomal DNA
F plasmid contains extra genes


In addition to genes for pilus, transfer
Transfers extra genes to recipient
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lac genes
oriT
Flac
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Conjugation across Species

Relatively frequently
across species
 Ex.:
from E. coli to
Salmonella

Some bacteria can cross
biological domain
 From
Agrobacterium
tumefaciens to plants


Tumor inducing plasmid
Crown gall disease
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Gene Transfer: Transduction


Bacteriophage inject DNA into cell
After replication and capsid production package
DNA into viral capsid
 Viral
DNA
 Sometimes accidentially package bacterial host DNA

Transfer DNA to new host
 Viral
DNA
 Host DNA


Generalized transduction
Specialized transduction
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Generalized Transduction




During lytic life cycle host
DNA is degraded
Can be packaged into
capsid
Can replace entire virus
genome
Transducing particle is
generated

Defective: can infect a new
host cell but not be
replicated
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Specialized Transduction



During lysogenic life cycle
host DNA is accidentally
excised when prophage
is reactivated
New virus genome
contains host genes lying
adjacent to the phage
insertion site
Can be packaged into
capsid along with viral
DNA

Infective virion: can infect a
new host cell and will be
able to replicate
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Defense against Transferred DNA

Bacteria cut entering DNA to
pieces


Cut at specific restriction sites
with restriction enzymes
Bacteria add methyl groups to
DNA

Prevents restriction at those
sites
 Added as cell replicates
chromosome

Entering DNA is destroyed

Unless it comes from a similar
species


And has methyl groups
protecting DNA
Prevention of promiscuous
DNA transfer
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EcoRI
restriction/
modification
site
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Recombination


New DNA enters via transformation, conjugation,
transduction
Fate of DNA




Independent existence as plasmid
Degradation
Integration into host chromosome through recombination
Recombination is the process by which DNA sequences
can be exchanged between DNA molecules



Replaces variable-sized section of DNA
DNA repair
Enhancement of fitness
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Generalized Recombination



Considerable stretch of DNA homology
Involves RecA and intermediate ssDNA
Cross over sites appear as crosses
 Holliday

junction
Deinococcus radiodurans uses RecA
homologue for radiation induced DNA repair
 Has
multiple copies of each of its 2 chromosomes
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Site-Specific Recombination
Exganche between DNAs with little overall
sequence homology
 Does not require RecA
 Requires insertion sequences

– 20 bp sequence recognizable by
restriction enzymes is required
 10

Intergrases mediate site-specific
recombination
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Recombination
Animation: Recombination
Click box to launch animation
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Mutation

Heritable permanent change in DNA sequence





Harmful, beneficial, or neutral
Change in genotype
May result in a change in phenotype
Change is base sequence and failure to repair
Point mutations

Change in one base pair



Transition: purine  purine,
pyrimidine  pyrimidine
Transversion: purine  pyrimidine
Larger mutations

Insertion


Deletion


addition of 1 or > nucleotides
subtraction of 1 or > nucleotides
Inversion

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flipping a fragment
of the DNA
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(Missense)
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Silent Mutations


Most mutations are silent
No effect on organism
Normal
 Mutations
in regions between genes
 Mutations that change 3rd base of a codon
 Mutations that change 1 amino acid into a similar one

Protein still retains normal function
 Mutations

that change a protein that is not needed
In current environment of organism
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Types of Mutations

Silent
 No

CGA  CGC
effect on organism
Missense mutation
 Change
one codon to
another

CGA  CAA
Arginine  Glutamine
Nonsense mutation
 Change

Arginine  Arginine
a codon to Stop
CGA  TGA
Arginine  STOP
Frameshift mutation
 Insert
or delete a single base
 Changes bases read by
AAA CGA CCC  AAA CTG ACC C
Lysine Arginine Proline  Lysine Leucine Threonine
ribosome
 Alters all codons downstream
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of mutation
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Mutagens

Increase the rate of mutations
 Number of mutations formed per cell doubling
 Spontaneous mutation rate is 106 to 108 per
generation

Increase of mutation frequency
 Number
of mutant cells per population (ratio of
mutant cells/ total number of cells)

Most mutagens are carcinogens
 Cancer
results from multiple
mutations
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Sectored Colonies reflect Mutation
Mutant yeasts
accumulate red
compound
Normal
Lac- bacteria mutate
and revert
to Lac+.
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Measurement of Mutagens: Ames Test


Reversion test
Uses bacterial strain auxotrophic for histidine
 Has
mutation in hisG gene
 Cannot grow unless histidine is supplied


Expose bacteria to the mutagen
Mutagen causes reversion
 Changes

mutation to normal form
Normally rare mutation
 More
colonies = stronger mutagen
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Classical and Modified Ames Test
Classical

Modified
Bacteria are plated on medium
w/o his and a disk with mutagen is
added

Bacteria are incubated with
mutagen and liver extract and
then plated onto medium w/o his
Mutagen on
paper disk
Revertant
colonies
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DNA Repair

Methyl mismatch repair

Parent strand is methylated
 Newer strand is less methylated and more likely to
contain errors

Nucleotide excision repair


Clips out patches of ssDNA
Base excision repair

Excision of structurally altered bases without cleaving
the phosphodiester backbone resulting in an apurinic
and apyrimidic site (AP site)
 AP nucleases follow up

Recombinational repair


Error-Proof
Intact strand is used to replace a homologous
damaged strand
SOS response


Coordinated response to extensive damage
Set of genes and repair mechanisms activated
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Error-Prone
29
Mobile Genetic Elements

Chromosomes sequence is not fixed
 Transposable
elements insert into chromosome
Found in all species
 Retroviruses (HIV) insert DNA in mammals

 Can

Nonreplicative transposition
 Can

jump from one site to another
copy itself to a new site
Replicative transposition
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Transposable Elements


DNA sequences
Are not autonomous


Insertion sequences (IS)




Cannot exist outside of a larger DNA molecule
500 – 1500 bp
5’-AATCGAT……….ATCGATT-3’
Transposase gene
Flanked by short inverted repeat sequences
Transposons (Tn)


More complex than IS
Carry additional genes

Antibiotic resistance or catabolic genes
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Basic Transposition and Origin of
Target Site Duplication
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Non-Replicative and Replicative
Transposition
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Mobile Genetic Elements
Animation: Transposition
Click box to launch animation
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Concept Quiz
Gene transfer by means of a bacteriophage
is called:
Transformation
b. Conjugation
c. Transduction
a.
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Concept Quiz
In an Ames Test, a very strong mutagen will
cause the appearance of:
Many colonies over the plate, with a
clear space close to the disk of mutagen.
b. A few colonies throughout the plate up
to the edge of the disk.
c. Many colonies covering the whole plate,
extending up to the disk.
a.
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Concept Quiz
Even though transposons disable genes into
which they jump, transposons aren’t
immediately detrimental for a cell because:
Transposons speed evolution through
lateral transfer.
b. Transposons are unmethylated, and
are destroyed by the recipient cell.
c. Transposons bring antibiotic genes into
the cell.
a.
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