2. Genetic Basis of Variation in Bacteria
NB:
Antibiotic resistance is one phenotype of genetic
transfer between bacteria and that the same
principles allow other genes like pathogenicity and
virulence factors to spread.
Bacteria change their DNA very easily and very
readily.
Aim: understanding how this occurs and the
consequence it has on the changing variety of
bacteria and bacterial pathogenicity.
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3. Genetic Basis of Variation in Bacteria
1. Vertical Inheritance of mutations
Bacteria multiply exponentially.
The generation time varies:
20 min. in perfect conditions
hours in a real infection.
Growing exponentially means one cell can turn into millions of
bacteria.
Daughters are identical to the parent – this is a clonal population,
all are genetically identical.
A clone is represented on an agar plate by a single bacterial colony.
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4. Genetic Basis of Variation in Bacteria
NB: But DNA changes.
This affects the properties of the bacteria and
creates a subclone within the population.
Mutations occur at a low frequency, 1 in a million
cells will have a mutation in any gene.
Because bacteria grow so rapidly, this is actually a
significant number.
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5. Genetic Basis of Variation in Bacteria
Mutation Outcomes:
1) Deleterious: blocking or disrupting a gene causes a
disadvantage (lethal, slow growth).
This population dies out by being taken over by wild type
(normal) bacteria.
2) Beneficial: mutation has added an advantageous
function to the cell, like antibiotic resistance.
Under the appropriate conditions, this advantageous mutation
will be selected for and will overtake the other populations of
bacteria.
3) Random/Spontaneous: no obvious effect on
phenotype, silent mutations.
These can accumulate and the sum can then lead to
change in gene function
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6. Genetic Basis of Variation in Bacteria
Two kinds of physical mutations (occur at the same low
spontaneous rate)
1) Point mutations: change of a single nucleotide
2) DNA rearrangements: shuffling of the genetic
information
insertions, deletions, inversions, or changes in
structure (several thousand nucleotides)
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7. 2. Horizontal inheritance.
DNA can be transferred from one bacteria to another and
assuming stable inheritance
this acquisition of genetic material will form a new subclone
population.
1) Transformation – results from the release and uptake of naked
DNA (e.g from lysed cells).
New DNA is incorporated into the chromosome.
This is the most inefficient form of transfer since the DNA
is open to the damaging environment, and requires a high
density of bacteria.
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8. Recombination refers to changes in genetic
information
Homologous
recombination
involves replacement of
DNA sequence with a
similar Sequence
Bacteria may also
acquire additional DNA
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10. Mechanism of Bacterial Transformation
Natural transformation
is limited to particular
species
Transformation requires
specialized proteins in the
recipient cells for
competence
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12. 2. Horizontal inheritance.
2) Transduction – bacterial genes are transferred in virus
particles.
Bacteriophages package DNA and inject DNA into other
bacteria.
More efficient because of protection of the DNA in a safe
protein coat.
However, the amount of DNA is limited by the capsid size.
Furthermore, phage can only infect bacteria expressing the
correct receptor, so there is a tropism to the transfer of DNA.
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13. Transduction
Bacteriophage (phage) are viruses of bacteria - can be
either lytic or temperate
i. Lytic - always lyse (kill) host bacterial cell
ii. Temperate - can stably infect and coexist
within bacterial cell (lysogeny) until a lytic phase is
induced
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14. Life Cycle of a Bacteriophage (Bacteriophage Lambda)
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15. Lysogeny
i. The phage genome during lysogeny is called
the prophage, and the bacterial cell is called a lysogen
ii. If the phage genome encodes an observable
function, the lysogen will be altered in its phenotype –
lysogenic conversion (e.g., diphtheria toxin in
Corynebacterium diphtheriae)
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17. Specialized transduction
i. Some prophages integrate into the bacterial genome at
a specific location
ii. When a prophage is induced to lytic phase, it may
drag along a piece of the bacterial genome next to the
integration site and move that bacterial sequence into the
new recipient host cell, changing the recipient's genome
iii. Not very important medically since only selected
genes can be transferred
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19. Generalized transduction
i. When a phage lyses the host bacterial cell, it normally
packages phage genome into the capsid
ii. Sometimes the capsid is accidently filled with random
pieces of bacterial genome, possibly including plasmids
iii. When the capsid injects the host genes into a new
recipient, the new gene can recombine into the recipient
genome and cause a change
iv. Virulence and antibiotic resistance genes can be moved
by generalized transduction
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22. 2. Horizontal inheritance.
3) Conjugation –involves cell to cell contact.
Two cells come into contact, a pore is formed and
DNA is transferred from one to the other.
Very efficient and rapid and is able to transfer
large amounts of DNA.
This is the most prevalent form of DNA transfer.
NB: CONJUGATION IS PROMISCUOUS.
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23. Conjugation - Plasmid transfer
Plasmids are circular DNA molecules replicated
independently of the bacterial chromosome
Plasmids encode proteins that allow for their transfer to cells
without the plasmid
Plasmid transfer is accompanied by “rolling circle” replication
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24. Conjugation - Formation of an Hfr cell
Recombination between the plasmid and the chromosome
leads to integration of the plasmid into the chromosome
Or is that integration of the chromosome into the plasmid?
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25. Conjugation - Transfer of chromosomal genes
The plasmid begins rolling circle replication and transfer into
the recipient
This time, the chromosomal DNA of the Hfr is dragged along
The transferred chromosomal DNA may undergo
homologous recombination into the recipient chromosome
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26. 2. Horizontal inheritance.
The DNA has to be stabilized in bacteria via two ways:
1) Genetic recombination – the incoming DNA is
inserted into the chromosome and replicates within the
bacteria’s own genome and is passed into the daughter’s
cells.
2) Plasmid – the incoming DNA forms a plasmid,
accessory genetic elements that replicate outside of the
chromosome that have their own replication signals,
independent of the chromosome. i.e.“minichromosome”.
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27. Gene transfer is extremely efficient.
Example of how horizontal gene transfer has
real world consequence:
Vancomycin requires 5 genes to be altered
for resistance– this took 30 years to generate.
In the few years since resistance has
developed, there has been 30 fold increase in
resistance to vancomycin.
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28. 2. Horizontal inheritance.
Why so efficient?
Remember properties of bacterial cell
1) Single chromosome
Can be double stranded linear or circular.
2) Bacteria are haploid. One copy of each gene.
3) Replication time is short, bacterial are small
evolution is rapid
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29. DNA makes RNA makes PROTEIN and this can all
be mutated
This is a gene.
There is a start codon and
A stop codon.
There is a promoter for the binding of RNA
polymerase to transcribe the DNA
RNA is taken to ribosome to make protein,
which reads the RNA in codons
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30. Point Mutations
Mutations which affect codons:
1) Missense: One nucleotide change can alter
the amino acid of that codon.
2) Nonsense: creates a truncated protein by
inserting a stop codon early
3) Frameshift: insertion or deletion of one
nucleotide, causes an out of frame shift reading
by the ribosome usually result in a truncation.
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31. Gene expression can be altered as well.
Mutations can occur outside the coding sequence
E.g in ribosome binding sites,
promoters,
repressor binding site,
transcription activator binding sites.
Mutations can:
Increase or decrease levels of protein expression
or gene transcription depending on where the
mutation occurs.
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32. How do nucleotide changes occur (physically)?
1) DNA polymerase is extremely accurate.
Only 1 in a billion misreadings occurs.
Genes are a thousand nucleotides, thus about 1 in a
million genes will have a change in it.
But there are billions of bacteria mutations
can then accumulate relatively rapidly.
2) Mutagens (chemicals) can change a base from one to
another.
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33. 3) If DNA is damaged very heavily?
A system called SOS response corrects DNA damage.
It also induces the expression of a number of compensatory
genes
One of is a proofreading protein which lowers the fidelity
of DNA polymerase.
Badly damage DNA causes intrinsic hypermutagenesis.
This might be evolutionarily advantageous
Since a bacteria which finds itself in toxic conditions
can undergo massive DNA change and perhaps gain the
ability to cope with that damage and survive.
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34. Gross DNA Rearrangements:
Majority of these changes are caused by transposable elements.
These are segments of DNA that have the ability to move from
one location in the chromosome to another.
In the process of moving, they can generate changes in DNA
structure.
These changes are deletions, inversions, formation of circles,
translocation or mobilization of other genes.
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35. Transposons - “Jumping genes”
First described for eukaryotes by Barbara McClintock
Simplest are insertion sequences
Complex transposons have contributed to evolution of R plasmids
with genes for multiple antibiotic resistances
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37. Insertion sequences (IS)
Insertion sequences (IS) are the generic transposable element.
They are present in large quantities in all bacterial chromosomes
(the number is variable).
It is a defined sequence of DNA (700-3000 bp long)
Has flanking inverted repeats and
Has one or two genes that encodes a transposase
– a protein involved in movement of this element from one location
to another.
•ORF encodes the transposase
•Inverted repeats are identical and
of variable length
•Different IS exist (IS1, IS2,
IS50….)
•Function unknown?
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38. Insertion sequences (IS)
The gene is expressed from an internal promoter.
Transposase is a recombination enzyme that recognizes the IR
and cuts the junction between the IR and normal DNA.
It can cut one strand at each end and ligate the single
stranded nicks to other locations in the chromosome.
Or it can make a double strand cut and excise the element
and move it into another location.
They move at a low frequency, the same frequency as point
mutations – so 1/million to 1/100 million will get a mutation in
any gene
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39. 2 Types of transposition mechanisms
I. Replicative transposition:
When the IS element copies itself and then the new
copy inserts elsewhere in the chromosome.
II. Conservative transposition (non replicative/cut-and paste):
When the IS element excises itself completely and
jumps to another place in the chromosome.
It ligates the ends of the excision.
This process is either precise, or imprecise leaving or
taking single nucleotides from the site.
This has the potential for frameshift mutations
at the point of transposition.
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41. What are the consequences??
When a IS element jumps,
i. it can totally destroy that gene’s function.
ii. can have other consequences if that gene product
effects other genes
(e.g. jumping into a repressor of an operon will
cause the operon to be transcribed more because
of disruption of the repressor).
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42. What are the consequences??
IS elements can directly alter gene expression.
They have their own promoters that not only point inwards for
their gene products, but outwards as well for nearby (quiescent)
genes.
Thus they can insert their strong promoters upstream near
important genes (like b -lactamase gene).
It’s a portable promoter.
IS elements can insert in just about any sequence, for the most
part random (occasionally some specificity).
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43. What are the consequences??
If the target of the IS is between genes c and d, there are two
outcomes to this scenario
1) Inversion of the sequence, this could be significant if the
arrangement of genes affects expression, (e.. the c gene promoter
upregulates b gene expression once b gene is rearranged
downstream of the c gene).
2) Deletion of the DNA is the other outcome, with the
deleted piece forming an extrachromosomal circle.
The consequence of this circle is that it carries a
transposable element and can then target other locations
in the chromosome, or it can interact with a plasmid
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45. Composite transposons.
These are when a chromosomal gene(s) is flanked by IS
elements on both sides, allowing the transposition of that
gene(s) to other parts of the genome.
This arrangement is demonstrated in Figure 8 as the result of
IS-mediated intramolecular inversion.
Horizontal transfer of that gene will increase since the gene
will more easily incorporate into plasmids or be packaged into
phage.
This could be dangerous if the gene encodes antibiotic resistance or
virulence.]
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46. Genome Organization
Replication
The Genome of Escherichia coli
Genomes of eurkaryotes
are usually composed of
multiple linear chromosomes
Genomes of prokaryotes
are often single circular
chromosomes
Prokaryotes are
monoploid
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