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MOLECULAR
MICROBIOLOGY

Due to selection pressure and the mutations that arise in these situations, variation is created –
Darwinian evolution
...
As the generations go on more and more mutations occur and reproduce to
fill the gap left by the unfavoured
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Through adaptation these animals (considered extremophiles) have
gained adaptations leading to troglomorphism (adaptation to cave living)
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Some of these blind fish can still sense light via the use of light
sensing cells in the brain such as the Mexican tetra, others have developed electroreception,
chemoreceptors and a form of echolocation which uses the suction of water to sense objects
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Gene Gain events
For successful gene gain to occur there has to be a selection pressure for those genes to be useful
...

A transposon (jumping genes which can move between chromosome, phage and plasmid In the
absence of a complementary sequence in the host DNA) is a linear segment of DNA which can
contain resistance to Antibiotics
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coli infected patient in
1964 and contains:
OriT – Origin of transcription
tra genes – Plasmid transfer via a T4SS – pilus – contains almost a dozen genes
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Tn10 – tetracycline resistance gene
Plasmid is 94
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pneumoniae now the most common
cause of nosocomial (health system acquired infections) respiratory infections with no vaccine in the
health care system
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7kbp
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coli p0157
An accessary plasmid 92kbp long
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KatP
espP – Proteolytic
stcE
ehxA – D = Haemolysis

Yersinia pestis
3 pathogenic species of Yersinia carry the plasmid virulence plasmid pYV 69
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Y
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enterocolitica
Y
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15 species in total which, 12 with-out plasmids are completely not dangerous
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Evolution will compose of both loss and gain of genes
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pseudotuberculosis strain which produced a biofilm
VirA, B, C and G are all shaped by the subtypes of this plasmid and encode for secretion of
intracellular components
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Y
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pFRA
encodes Antigen fraction 1 as well as
phospholipase D an enzyme used in endocytosis,
cytoskeleton rearrangement and exocytosis
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coli EHEC such as O157 and
O104
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ipaA - binds with vinculin (membrane bound protein) to interact
with F-actin and induce depolymerisation
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B is also able
to cause apoptosis in macrophages
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There will be two of these IS1 or 2 sequences on either side
of a single gene such as a toxin or res genes (multiple in the more complex IS)
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More complex IS are called transposons and carry a number of genes such as on the R100 plasmid
which contains two transposons
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The RTF (resistance transfer factor) which encodes the
transcriptional part of the plasmid is left on its own
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Intergrons – found in chromosomes
Salmonella enterica ssp
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In0 (the intergrating sequence containing an DNA intergrase, intl1)
P – promoter
attl site – genes are intergrated here, can be dozens or just one
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cholera and contained virulence genes and res genes and had a
length of 130kbp and 170 cassettes although the term has fell out of use since there is a huge variety
in length
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This PI is
the difference between
O157 and K12 which is
harmless
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During
an infection there is an
increases chance of
Horizontal Gene Transfer (HGT)
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Transposable elements – Prophage DNA with transposable element can more into the genome
(second infection from taking DNA via temperate phage method)
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Gene gain examples
SPI-1 the salmonella pathogenicity Island for survival allows the modulation of the host cell functions

to suit its needs
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It can effect Cytokine expression in favour of the organism
Makes the cell think that the SCV is an essential organelle, the cell then moves the SCV into the
centre of the cell and feeds it
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There are 2 speculated gene gain events in the B
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cereus and the second to separate it completely from all of the closet
known organisms
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Another gene gain in the same stage is the internalin which allows the crossing of host barriers and
survival in phagocytic and non-phagocytic cells, this is a gene was is homologous with one found in
Salmonella enterica ssp
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Typhi
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Stage 2 gains includes prophage (which may have introduced some of the genes at this stage)
proteins and the Anthracis toxin lethal factor, protection Antigen (Ag) and calmodulin-sensitive
adenylate cyclase
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The
genes gained by E
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Those factors which are next to
the gene they aid the transcription (neighbouring regulators of are believed to be gained through
HGT, whereas global regulators evolve vertically with the gamma-Proteobacteria group
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coli’s
TF families has been
that they arose via
genetic duplication
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Although
slim there is a chance
these independently acting TF’s may have evolved and ended up back in the same species via
allotropic gene divergence
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coli
K12
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An example of genes believed to have developed in that way are xapR (regulator) and XapA which
encodes for the catabolism of xanthosine (Price, 2008)
Gene loss is the change in a gene which causes it to become inactive or to be deleted
...

There may be a base pair substitution which can lead to silent mutations, nonsense mutations (an
Incomplete protein), missense mutation (a faulty protein)

Insertions and deletions
in where a bp is either inserted into or deleted from the DNA strands during replication
...

Gene loss can be advantageous as the less genes the less energy must be required to maintain those
genes

examples
The loss of the 150kb PBP3 gene from Burkholderia pseudomallei led to the rise of ceftazidime
resistant strains (ceftazidime is the common drug of choice to treat the disease melioidosis as it is
usually intrinsically resistant to many beta-lactams) believed to have been caused by random
genomic recombination whilst the host is being treated with ceftazidime
...
The loss of this gene has dramatically
decreased the growth speed and subsequent colony morphology of this organism but it also means
it is surviving stage 1 of a stage 3 treatment to eradicate it (Torok 2012)
...
This organism shows lineage (ST403) specific mutations which separate it
from the other lineages in the species
...
A series of mutations throughout the genome also introduce stop codons which are not found
in non-st403 complexes
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Lactobacillus helveticus DPV4571 (an organism used in the dairy industry) has 65 – 75% conserved
genome compaired to various commensal species of lactobacillus
...
Many of these pseudogenes, associated with loss of function
mutations, are actually transposase enzyme genes belonging to IS elements
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Callanan, 2007
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The promoting regions consists of a -35 sequence and a prinbow bow at -10, this consensus
sequence is recognised by the sigma factor of the RNA molecule, the sigma factor attracts the RNA
polymerase core enzyme and begins transcription as the sigma factor dissociates
...

This pathway consists of enzymes which produce Arginine from citrulline, such as Argininosuccinate
synthetase (ASS) and Argininosuccinate lyase (ASL)
...

Arginine is an amino acid used in the production of various proteins and maintain the cell
membrane
...
So when there is a high concentration of arginine in the
cell it binds to the repressor which in turn binds to the operon of the genes which encode for the
enzymes which would synthesise more
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Negative regulation – Induction
Lactose, in the presence of lactose and therefore allolactose the repressor will dissociate from the
operon and allow RNA polymerase to transcribe the genes involved in the further conversion of
lactose to allolactose
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GFP (green fluorescent protein) is a protein, found on the GFP plasmid, used widely in molecular
biology as a reporter gene
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When in the presence arabinose the genes for both arabinose metabolism
and the production of GFP are activated
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Activator protein mechanisms

Some operons need the use of a activator for the
RNA poly to begin transcribing
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Supercoiled DNA
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Bacteria have a global control system to
control multiple operon, this can be up to 70% of the bacterial genes
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For example
Glucose and then lactose as glucose is much easier to metabolism than the disaccharide lactose
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Activator protein is called cyclic AMP receptor protein (CRP) also known as Catabolite Activator
Protein (CAP)
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Mostly found on genes for metabolism
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This enables DNA to bend and

show previously hidden operons to RNA polymerase, then the gene is expressed
...

In the absence of glucose, adenylate cyclase is activated, produced cAMP from ATP
High levels of cAMP bind to CRP which (unwinds the DNA) binds to the lac operon and starts the
transcription of lactose
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Stringent response – where access to amino acids is lost and the cell must work to produce them its
self
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Heat Shock Response
Induces by heat or other stress factors that result in protein unfolding, depends on an alternative
sigma factor called RpoH
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coli) prevent aggregation of newly synthesized proteins and stabilises unfolded
proteins (stops them binding to each other and wrongly)
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coli catalyse the correct folding of misfolded proteins
...

These all al stable at higher temperature and self-regulate by binding to their own consensus sites to
promote transcription
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Sigma factors
Many different Sigma factors
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Specific Sigma factor SigmaL, increases
...

Protects nucleic acid from adverse temperatures by wrapping around the DNA
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Transition from Log to stationary phase often
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It is the central regulator which switches on genes required for survival in these conditions
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Regina Hengay found that stationary phase will start much earlier than previously thought so there
are still enough nutrients to survive on
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As (p)ppGpp increases in response to starvation it:
-Promotes an increases RpoS transcription
-Promotes increased RpoS translation
-Inhibits degradation
-improved activity of RpoS
Over 60 genes are transcribed once RpoS has been translated into a protein some are involved in the
cell wall maintenance
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This change results in changes in DNA supercoiling at the promoters, this isn’t under the effect of
gyrase and isomerase
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coli
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These effect DNA topology at the promoter and therefore effect transcription
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Two-Component Systems (TCS)
Involved the use of two proteins
1, Sensor Kinase proteins
- A transmembrane protein
- Autophosphorylates His residues (histidine kinases) in response to a signal
2, Response regulator protein
- DNA binding protein
- Phosphorylated by the active sensor kinase
- Activated regulator can then regulate transcription
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Examples
EnvZ-OmpR
Osmolarity of the environment
controls the expression of OmpC or
OmpF porins
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OmpF is a large pore which is
expressed in low osmotic pressure
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PhoP/PhoQ
Low Mg2+ influx from PhoQ (sensor) phosphorylates PhoP Which then upregulates MgtA, a mg2+
influx proteins
...

PhoP (p) (in S
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It is
required for survival of oxidative burst in the host macrophages
Activation of the pmrD (PmrA/B) causes further LPS changes to infer resistance to polymyxin B and
the upregulation of its self
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Salmonella
-Preference to M (Microfold) cells which are used in the innate immune system in mucosal surfaces
of the intestines at Ag samplers (which are then taken to Ag Presenting cells)
...

-Once the ruffling has collapsed and the organism has been taken into the cell in a phagosome,
without any survival mechanisms any organism will be destroyed by oxidative burst with in 30
minutes, but the salmonellae can induce the formation of a spacious phagosome which acts to dilute
the oxidative species, the increase of antimicrobials in the phagosome also activate the PhoP/PhoQ
TCS and upregulate certain genes involved in pathogenicity such as effector proteins which will stop
the fusion of lysosomes to the phagosome (creating a phagolysosome) and becoming a Salmonella
Containing Vacuole (SCV) this is done by the use of the T3SS-2 effector proteins
...
The change in membrane bound markers on the surface of the
phagosome such as the deregulation of Rab5 and upregulation of LAMPS causes the phagosome to
be moved towards the cell in a juxtanuclear position near the MTOC (Microtubule Organising
Complex)
...

-As the number of Salmonellae increase the SCV will stretch and form microtubules called Sif
tubules
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Quorum Sensing
Bacterial communication is essential for colonisation, although they are mainly free living, it
would be pointless to try and start a colony of their own
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AHL is Acyl Homoserine
Lactone an Autoinducer
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(Luxi – the AHL synthase
LuxR is bound to by the AHL to
act as a promoter and allow the
transcription of genes which are
useful when the organisms are at
a sufficiently high cell density)
Quorum sensing is used in
specialised organs of some
complex organisms such as the Hawaian bobtail squid which is nocturnal (using the
organism Vibrio fisheri)
...

Vibrio Harvei is free living, V
...

Group behaviours include:: Symbiosis, Virulence, Competence, Conjugation, Antibiotic
Production, Motility, Sporulation and Biofilm production, fermentation, bacteria –complex
organism interations
Pathogenesis
The body has various numbers of barriers
to infection by any organism
...
coli binds to DAF
receptor) and receptors on the host cell
...


Phase variation
Phase variation is a method for dealing with rapidly varying environments without requiring random
mutation employed by various types of bacteria, including Salmonella species
...
Although it has been most commonly studied in the context of immune evasion, it is
observed in many other areas as well
...
Phase variation contributes to virulence by
generating heterogeneity; certain environmental or host pressures select those bacteria that express
the best adapted phenotype

Salmonella use this technique to switch between different types of the protein flagellin
...
Once an adaptive response has been mounted
against one type of flagellin, or if a previous encounter has left the adaptive immune system ready
to deal with one type of flagellin, switching types renders previously high affinity antibodies, TCRs
and BCRs ineffective against the flagella
...
When inverted the gene will be switched
off and be unable to be read
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coli this occurs with the gene for pilin, S
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FimA – E
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Insertion and excision
If excision is precise and the original DNA
sequence is restored
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Phase variation mediated by transposition
targets specific DNA sequences
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When
excised the IS element becomes
circularised and the gene switched on
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gonorrhoea) and the silent genes (PilS, not being used) can recombine into the active pilin gene
(PilE) and produce a new phenotype
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coli
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fetus surface proteins where the promoter can shift between copies of the gene and allow the
transcription of a different gene
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Listeria
internalised into cell via InlA of InlB (Internalin, which are also adhesins) pathways
A relies on the interaction of A and surface protein E-Cadherin (only expressed on epithelial cells)
which activates catenin (a protein which interacts with actin) and Rho GTPase to rearrage the
cytoskeleton
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Once in the cell the organism will produce listeriolysin to rupture it
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The organism will replicate in the cytosol of the host cell and eventually use actA to accumulate factin behind the cell and force it through the cell membrane into an adjacent cell (where it will now
have two membranes which are then “popped” by listeriolysin
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as well as effector molecules
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Required direct contact, once bound the plug on the end of the T3SS is release
...
These effects can create pores in the host cell so that the needle
can enter the cell wall and inject effector proteins
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Overall there is a very varied effect on
the host cell
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EPEC and EHEC form pedestals to push the organism into the intestinal lumen for better acquisition
of nutrients
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coli cell surface
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Once phosphorylated Tir which is also a Receptor Tyrosine Kinase (RTK) activates condensation
(cofilin) and polymerization (profilin) of actin filaments under the bacterial cell to form a pedestal
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DPT
Diphtheria, Pertussis, Tetanus vaccine
The disease is characterised by the formation of a pseudomembrane (layer of dead/dying cells) in
the throat, and death caused by a mixture of suffocation and myocardial tissue destruction
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Colonises the
throat and tonsils and produces a potent AB toxin (which halts host cell protein synthesis)
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The AB toxin is made up of A, B and a translocation segment
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Once in the endosome, pH
changes and changes the shape of the T domain which can now insert its self into the endosomal
membrane
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Whooping cough
Acute highly infectious respiratory disease usually seen in children under 5, recurrent violent
coughing lasting upto 6 weeks – patients make whooping sound trying to inhale
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Bordetella pertussis is a g-ve rod which spread via airborne transmission
...
Very complex pathogen which carries at least 4 toxins
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The pertussis toxin is required for full virulence, AB5 toxin
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This disrupts cellular functions and causes ciliary stasis
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Anthrax
High potential to be used in bioweapons
...

Death via inhaled anthrax is due to the growth of the organism in the lymphatic system and causing
it to drain into the lungs
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Toxin is made up of Lethal factor (LF), protective Ag and Edema (swelling) factor (EF)
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Although a three
component toxin it still follows the AB toxin paradigm A being enzymatic and B being the
transporting segment
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Each toxin is secreted individually from the organism
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Once endocytosed, the pH increase
causes the PA-63kDa to convert into a cation selective channel
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EF acts as a calcium2+ and
calmodulin dependent
Adenylate Cyclase that greatly
increases the amount of cAMP
in the cell
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LF also aids the evasion of the immune system by killing of macrophages
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This stops the MAPKK from binding to their substrates which would normally end with the
transcription of anti-apoptotic genes
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If it
is the gene should be mutated or
not expressed to explain the
avirulence
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Introducing the gene to an
avirulent strain should induce
virulence
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The gene can be disrupted by cutting the foreign DNA and a
vector with the same restriction enzyme
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Suicide plasmids
Plasmids which require the host cell to in a
certain condition:
pKNG101 and pERFORM, only surviv in E
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pRK and p-GM only replicate at 30oC and
cannot exist as plasmids at 42
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This gene encodes for an enzyme called levans sucrose which converts sucrose to sucrose
...
Growth in the presence of sucrose means the plasmid cannot be
maintained to ensure survival
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Mainly using X-rays on a growth of organisms the methods have
since moved onto Error-prone PCR where the product is cloned into a vector and then expressed in
an organism such as E
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Mini Transposons are jumping genes derived from transposons (typically TN5 and 10) which have
been rearranged and optimised to a specific use
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coli cat gene which encodes for Chloramphenicol Acetyl-Transferase and a
origin of transcription OriR6K-gamma
...
This crease genomic
DNA with the mini transposon intergrated
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To identify the location of the transposon then the Genomic DNA must be isolated and digested,
self-ligated and transformed into E
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