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Microbiology 1
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BIO1004
Microbiology Fundamentals

Robert Hooke (1635-1703)
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Organisms too small to see by eye couldn’t be identified or studied until there was a
microscope
Hooke used a microscope to demonstrate the presence of minute organisms – fungi

Antony van Leeuwenhoek (1632-1723)
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Developed a solar light microscope
Was able to see and draw bacteria for the first time

Edward Jenner (1749-1823)
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Carried out successful vaccination against smallpox
Cowpox gave similar pustules to smallpox but wasn’t a fatal disease and people with cow pox
did not get small pox
Injected a boy with pus from cow pox and once he recovered he infected him with small pox
– he did not become ill

Louis Pasteur (1822-1895)
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Provided evidence against belief of spontaneous generation
Showed small organisms could be isolated from air
This was done by sterilising media with heat and sterilising air that entered the media, as it
cooled he prevented microbial growth
Showed that yeast could grow without air – fermentation
Developed the process of pasteurisation – initially to preserve wine

Robert Koch (1843-1910)
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Identified specific agents of disease
Initially with anthrax
Formulated Koch’s postulates

Alexander Fleming (1881-1955)
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Believed natural compounds were safe for injection to help prevent infection
He identified that lysozyme, an enzyme in nasal mucus, inhibited the growth of
Staphylococcus aureus
He found first the antibiotic penicillin produced by the mould Penicillium notatum

Prokaryotic Cells
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No membrane bound nucleus
Generally, no membrane bound internal organelles

Eukaryotic Cells
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Contain membrane bound nuclei
Also have membrane bound internal organelles

Multicellular: an organism made up of two or more cells EG fungi

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Unicellular: a single celled organism EG bacteria, archaea, protozoa
Growing Microorganisms
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Grown in a liquid or solid medium EG agar
The medium must contain all of the nutrients needed for the microorganism to grow
It must be sterilised before adding the sample
This then needs to be incubated at an appropriate temperature and gaseous environment

On Solid Media
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Streak Plate
o A cotton bud containing the microorganism is swiped across the agar plate
o Used for different sections
Spread Plate
o A small volume of liquid culture is pipetted onto the surface of the agar plate
o A sterile spreader is used to spread the culture across the plate as evenly as possible
Pour Plate
o Either a small volume of liquid culture is added to cooled molten agar, and after
mixing, the agar is poured into a petri dish and allowed to solidify
o The liquid culture could be put into an empty petri dish and cooled molten agar is
poured over it and mixed

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Microbial Growth

Single celled organisms divide by binary fission (doubling)
This gives an exponential increase
A culture will grow exponentially when it has a ready
supply of its nutrients

The Microbial Growth Curve

Time
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When growing bacteria in flasks or test tubes,
you will not get exponential growth all the time
 Lag: the organism is adapting to its new
environment, the enzymes are being
synthesised, cells are increasing in size but not
dividing
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Log: cells are undergoing exponential growth
with constant growth rate and mean generation time
...
)
They attach, divide and increase in
number with many also producing extra
cellular
polysaccharide
material
forming a protective matrix
They may excrete molecules that
attract other microbes, which will
increase the size & complexity of the biofilm
Some parts of the biofilm will break off and spread

Counting in Liquid Media
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Using a counting chamber
A glass slide with an engraved precise grid of known area is flooded with a dilution of culture,
this is covered with a cover slip
The depth between the grid and coverslip is measured
The number of cells in several squares is counted, and then the average no
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of cells
per ml of culture

Counting on Solid Media
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Use either a pour or spread plate technique for colonies to be seen
The original sample must be serially diluted to ensure a good number of clearly separated
colonies can be counted

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From the serial dilutions, 0
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01
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0-11
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4-8
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 Moist heat: autoclaving using pressurised steam
o Pasteurisation
 Temperatures below 100C
 The aim is to kill pathogens and reduce the number of other organisms
 Doesn’t kill spores
Antimicrobial Chemicals
o Used to kill or inhibit microbial growth
o Organic compounds: phenol, formaldehyde
o Halogens: chlorine, iodine
o Oxidising agents: hydrogen peroxide, ozone
o Disinfectants kill microorganisms but also potentially damage the host tissue
o Antiseptics are less strong disinfectants which don’t damage host tissue

Cell Membranes
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It is the boundary of a cell
They have separate internal aqueous, polar environment from external aqueous, polar
environment
They consist of a lipid which is non polar, so acts as a barrier
The membrane lipids have associated polar regions
Arranged in a phospholipid bilayer so the polar regions face towards the interior and exterior
of the cell and the non-polar region in between

Phospholipid Bilayer
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Made up of fatty acids linked to a glycerol molecule by an ester bond
The fatty acids with non-polar hydrocarbon chains aren’t branched
There is one highly polar group including a phosphate group
Found in bacteria and eukaryote

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Cell Walls
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Cell walls provide rigid structure to prevent eukaryotic cells from bursting
There is a higher concentration of solutes inside the cell than outside meaning that water will
move into the cell
This causes the cell to swell, and without the cell wall the cell would burst

Peptidoglycan
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Peptide chains linking polysaccharide
A polysaccharide is a long chain with the repetition of
two units
Murein – muramic acid and protein

Teichoic Acids
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They’re derivatives of polyalcohol’s – glycerol
and polyribotol
They have a terminal –OH link to phosphate
groups that ionise to give negative charges
Some sugars or alanine may attach to the nonterminal –OH groups
Teichoic acids link to the NAM in peptidoglycan
Can also link to lipids in the membrane

Outer Membrane
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Present in some bacteria
It looks like a standard lipid bilayer but:
o The inner half contains a lipoprotein which links to
peptidoglycan, called LPP, and phospholipids
o The outer half contains lipopolysaccharides
(LPS)
o The LPS is composed of lipid A and long
polysaccharide chains extending to the outside of
the cell
o Lipid A has 4 fatty acid chains linked to
a glucosamine phosphate complex
o Polysaccharide links to one of the
glucosamine
o They have important antigenic properties and
may be toxic

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Microbial Diversity 1

Gram Positive and Gram Negative Cell Walls
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Bacteria can be divided into two groups on the basis of a stain called the gram stain
The difference depends on the structure of the cell wall
Gram Positive:
o Thick layer of peptidoglycan
o Teichoic acids
o Some surface proteins

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Gram Negative:
o Thin layer of peptidoglycan
o Outer membrane contains lipopolysaccharides
o Porin transport protein in the outer membrane
Gram Negative
Gram Positive

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Bacteria (Proteobacteria/Eubacteria)
 Morphological characteristics of bacteria
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Colony Morphology
 This is whether the colony is glistening or smooth
 Feathered edges or smooth edges
 Raised or flat
 Stringing or solid
 General colour characteristics
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Cell Wall Characteristics
 Cell wall biochemistry and structure has been one of the main discriminative
characteristics between different groups of bacteria
 The gram stain works because of the differences in cell wall properties
between the Gram positive (+) and the gram negative (-) species
 Sensitivity to some antibiotics (Penicillin) is related to cell wall properties
 Unique bacterial cell walls components:
 Peptidoglycan (Murein): unique to all bacteria, forms a thin sheet in
which the 2 sugar derivatives are connected by peptide cross links
...
This forms the main wall layer in Gram positive
species
 Lipopolysaccharide layer (LPS): this forms an additional outer lipid
bilayer to the walls of Gram positive bacteria, the space in between is
known as the periplasm
 Teichoic acids: acidic polysaccharides are unique to Gram positive
bacteria and give their walls an overall negative charger

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Gram negative species also often have an additional surrounding
layer of capsular polysaccharides

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(they can cause problems in the food processing industry)
 The location of spores within cells varies – terminal or central etc
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Conjugation
 Bacterial conjugation occurs via pili
 There is a donor cell containing a conjugative plasmid and a recipient cell
without
 Only the donor cells have pili
The Archaea (Archaebacteria)
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Originally thought to be the most primitive form of ‘Monera’, and in text books published over
20 years ago they are typically shown to be at the very base of the tree of life
Many species are extremophiles, allowing them to grow in environments of high salinity and
extreme temperatures and pressure
These conditions were equated to those that existed in the early stages of evolution of life on
this planet
Morphologically and cytological Archaea species do not look significantly different from true
bacteria
They most likely share more biochemical characteristics in common with eukaryotes than with
true bacteria
They were presumable the ancestral cell form which gave rise to the eukaryote cell
BACTERIA
Cell walls contain peptidoglycan (murein)
Contain ester-linked lipids
Have only one RNA polymerase
No histones
Sensitive to streptomycin, kanomycin and
chloramphenicol

Ribosomes not sensitive to diphtheria toxin

ARCHAEA
No peptidoglycan (murein) in cell walls
Contain ether-linked lipids
Have several RNA polymerases
Contain histone proteins
Not sensitive to streptomycin, kanomycin and
chloramphenicol
Ribosomes sensitive to diphtheria toxin

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Halophilic genera: bacteria that are able to survive in high salt concentrations, EG
Halobacterium
Hyperthermophilic genera: bacteria that can survive very hot temperatures, EG Ferroglobus,
Pyrococcus
Methane-producing genera: bacteria that can survive high methane concentrations, EG
Mathanococcus
Black smoker microbes: microbes that can survive high temperature and pressure, red cells
from 3500m deep

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Microbial Diversity 2

Viruses
 Discovery
o In 1886 the Russian Ivanovsky noted the agent which caused the mosaic mottling
disease of tobacco passed through filters which retained bacteria
o In 1886 the Dutchman Beijerinck proposed that this agent was a new type of organism
– a virus
o IN 1935 the American Stanley crystallized the virus agent and showed that it was
composed of a protein and a nucleic acid
 The viruses do not fit into the normal tree of life scheme
 They can be thought of as pieces of rogue DNA and RNA surrounded by coat proteins
 The viral nucleic acid integrates itself into the host cell and causes the cell to replicate the viral
DNA and coat proteins which self-assemble into new virus particles
 Viruses are very small, around 20-300nm
 They’re able to infect: bacteria, animals (AIDS, flu etc
...
They aren’t coated in proteins and are important plant pathogens
Prions: in their extracellular form they are entirely protein, they’re infectious and can cause
crapie in sheep
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Chytriomycota
 Do not usually have nucleate hyphae but globose thalli with anucleate feeding
rhizoids
 Posyeriorly uniflagellate zoospores
 They play a key role in the breakdown of plant fibre in the rumen
 It was recently discovered to be lethal parasites of tropical frogs, frogs die by
dehydration
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Glomeromycota
 These are wide-spread on roots of herbaceous plants (the earliest known
fossil fungi) and share nutrients with the plant
 They form large globose thalli which produce fine thread like hyphae which
produce branches arbuscles that invade root cells
 There is no known sexual stage and are unable to be maintained in laboratory
culture
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Basidiomycota pistol fungi
 These are the most highly evolved fungi; they have septate hyphae with clamp
connections which ensure dikaryotic distribution of nuclei in hyphal
compartments
 The spores produce on pistol-like basidia bearing 4 basidiospores
 These include typical mushrooms and toadstools but also important plant
pathogenic rusts and smuts

Protozoa
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Flagellates: have flagella, many flagellate protists are important pathogens of man EG gut
parasites, malaria
Amoebae: have locomotory extensions of the cytoplasm enclosed by the cell membrane,
called pseudopodia
Ciliates: EG Paramecium ingest food by phagocytosis, they’re covered in short, stiff cilia

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Plants
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Algae: (single celled plants that contain chloroplasts)
o There are red & green algae so allow for different pigments to absorb different light
wavelengths

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Microbial Biotechnology

What is Biotechnology?
 Using organisms to make useful products
 The application of organisms or their cellular components to specific processes
 This can include organisms manipulated buy genetic engineering
 EG insulin, antibiotics, bread, cheese, washing powder enzymes, Botox, biosensors & sewage
treatment etc
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g
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grow, there will be spoilage

Bread Production
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This depends on the growth of yeast (Saccharomyces cerevisae)
Amylase enzymes in the flour break down the starch in the flour to glucose
The yeast then metabolises this glucose to carbon dioxide and water aerobically (and
produces ethanol anaerobically)
The production of the carbon dioxide is what causes the dough to rise (leavening)
Baking makes ethanol volatile and sets the bread with the holes in it
Adding other yeasts and bacteria can produce specific flavours

Beer Production
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This depends on the growth of yeasts
Cereals (barley) are allowed to germinate (malting) which produces enzyme that degrade
starch
Malted cereals are then mashed with warm water, the enzymes degrade the starch to maltose
and glucose
A clear liquid called wort is removed and boiled with the hops
After cooling, the yeast is added to ferment any sugars into alcohol
Bottom fermentation:
o Saccharomyces carlsbergensis
o The yeast will settle at the bottom of the fermentation vat
o This produces lager
o Fermentation occurs at 6-12°C in 8-14 days and is then stored at around -1°C before
packaging
Top fermentation:
o Saccharomyces cerevisiae
o The yeast does not settle
o Produces beer
o The fermentation occurs at 14-23°C in 5-7 days and is stored at 4-8°C

Food Poisoning
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The consumption of food can cause illnesses
Several potential causes can be:
o Microorganisms: bacteria, fungi, viruses
o Allergies
o A chemical naturally in the food, or due to contamination
o Parasites
Gastroenteritis is a common, but not the only, symptom of food poisoning, symptoms include:
o Abdominal pain
o Diarrhoea

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o Vomiting
o Fever
Intoxications
o Caused by a toxin being released by bacteria growing on food
o Ingestion of live bacteria is not required
o The toxins are exotoxins
o Enterotoxins
 These cause the release of water and sodium ions to pass from the
enterocytes (cells lining the gut) into the gut lumen, leading to diarrhoea
 These may also stimulate vomit receptors in the gut which send impulses to
the vomit centre in the brain
 The toxin will remain in the gut
 Staphylococcus aureus
 There is an incubation period of 30minutes to 6 hours
 Causes nausea, vomiting, diarrhoea and abdominal pain
 Can last from a few hours to 3 days
 There needs to be 5x106 organisms/gram of food or 1 ng of the
toxin/gram of food
 Contaminates via the skin, nose throat
 Common from cooked meat, dairy products & canned foods
o Neurotoxins
 These pass through the lining of the gut and then enter the blood stream
 They are then transported to all regions on the body
 They bind to nerve endings in tissues causing paralysis

o

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Campylobacter
 C
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coli are the species that cause most infections
 They are a major cause of food poisoning
 Symptoms are visible within 2-5 days and lasts the same amount of time, but
can linger for weeks
 They produce flu-like symptoms, abdominal pain, nausea
 The animal gut acts as the reservoir EG pigs, cattle, sheep etc
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coli 0157:H7 are the most important causes of food poisoning
 There is an incubation period of 3-4 days
 Causes abdominal pain, diarrhoea with bleeding, internal bleeding
 Has a duration of about 2-9 days

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There is a significantly high death rate in children and the elderly
10-100 organisms are infective
Its natural habitat is in the gut of cattle and so can be sourced from
undercooked minced beef and raw milk

Infections
o Live cells need to be ingested but the organism doesn’t grow or multiply in the gut
o These live cells are ingested and attach to cells lining the gut and multiply, they may
penetrate through the gut wall

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Disease in Humans

Disease can be caused by many different types of microbe: bacteria (meningitis), viruses (HIV, Ebola),
protozoa (malaria) and fungi
Meningitis & Septicaemia
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Different symptoms of the same disease
They can kill within hours of the symptoms appearing
May be caused by bacteria or viruses
Bacterial meningitis is more severe, and several types can cause it
Before 1990s Haemophilus influenzae type b was most important
Now Neisseria meningitidis is the major bacterial cause (meningococcal disease) with
Streptococcus pneumoniae (pneumococcal meningitis) next
Causes & Symptoms of Meningitis:
o Bacteria cross from the blood to the tissues around the brain and lining of the spinal
chord
o Toxins cause inflammation of the tissues
o This leads to headaches, stiff necks and drowsiness
o Eventually ends in a coma which can be potentially fatal
Causes & Symptoms of Septicaemia:
o Bacteria transfer to the bloodstream, multiply and produce toxins
o A fever develops
o Toxins damage the capillary walls so that blood is able to escape under the skin,
causing a rash
o Circulatory system is reduced at the periphery to conserve the blood, this results in
cold hands and feet & rapid breathing
o This can result in shock, heart failure or multiple organ failure
Neisseria meningitidis infection
o It is a gram negative non-motile coccus
o They often occur in pairs and may have a capsule
o Infection can lead to meningitis or septicaemia
o Usually produces symptoms of both
o Referred to as a meningococcal disease
o Can kill within hours of the symptoms appearing
o Accounts for more than half of meningitis case in the UK
o Fatal for about 1 in 10 cases
o Survivors may be left with serious disability, EG deafness or brain damage
o Its normal environment is in the nose, throat or upper respiratory tract without
causing disease

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It is transmitted through coughing, sneezing & intimate kissing
Cannot survive long outside of the body
Vaccination:
 A vaccination was produced against Meningitis C; it has been offered to those
under 18 since 1999
 The vaccine is not live but based on a cell polysaccharide

HIV: Human Immunodeficiency Virus
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Ebola

It is a single-stranded RNA retrovirus in an enveloped, icosahedral capsid structure
72 glycoproteins spikes stick out from the envelope
This infects cells with the CD4 protein on their surface
The most common are T-helper cells and macrophages
which are involved in the normal immune response
The HIV envelope fuses with the cell membrane and the
capsid containing the RNA enters the cell and breaks
down
Associated with the RNA are preformed
viral
proteins
including
reverse
transcriptase (RT) and integrase
The RT catalyses the conversion of single
stranded RNA into double stranded DNA
Integrase insert the DNA into the host cell
genome
The integrated cDNA can stay dormant (a
provirus), replicating slowly, for long
periods of time
After an initial bout of illness following
infection there may be little evidence of
disease for many years
After induction the provirus can initiate
mRNA production and the synthesis of viral
proteins
New viral particles are assembled and leave the cell by budding
The new virus particles infect other non-infected CD4 cells
Eventually this will reduce the number of active cells in the immune system
Which eventually leads to AIDS
The number of CD4 cells depletes from 600k-100mil/ml to less than 200k/ml
When the number of CD4 cells depletes this low, opportunistic infections appear usually
leading to death – diseases that are not normally seen in individuals with normal immune
function
Pneumonia is the most frequent infection
The virus will be present in the blood, macrophages and in CD4 cells
The transmission requires transfer of the virus, usually through bodily fluids EG blood, semen
& breast milk

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Is a member of the virus family Filoviridae
(it is a filovirus)
Contains single stranded RNA of a long
filamentous shape which can branch
Can be up to 14k nm long and have an
average diameter of 80nm
It has a nucleocapsid surrounded by a
helical capsid
Surrounded by a lipoprotein layer derived
from the host cell
Has many spikes on the surface
The genome codes for 7 proteins:
o Polymerase L plus VP35: this replicates viral RNA in the cytoplasm
o Nucleoprotein: encapsulates the RNA
o VP30: a transcription factor
o Glycoprotein: thought to be responsible for attachment for host cells
o Viral proteins VP24, matrix VP40 and GP associate with the cell membrane
After the RNA is coated by the nucleoprotein it buds out through the cell membrane gaining
an outer lipoprotein coat from the host cell

Zoonotic disease
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A disease that can be transmitted between animals and man
These diseases can be transmitted through direct contact of the eyes, mouth & skin
openings with body tissues from infected individuals

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They produce initially flu like symptoms followed by vomiting, bloody diarrhoea
Haemorrhagic fever also occurs causes haemorrhages due to the damage of endothelial
cells lining blood vessels

Malaria
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Caused by the protozoans Plasmodium vivax and P
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Has a temperature of 39°C

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Has a very large aqueous environment (5-10L in a sheep & 40-100L in cattle)
Food enters the rumen before and other part of the gastro-intestinal tract
Why is low flow rate important?
o It gives the microbes sufficient time to reproduce, this must be done before the food
is washed out of the gut
Why are pH & temperature important?
o Microbes have an optimal pH and temperature, which produces the best growth rate
o They are unable to maintain themselves in conditions very different from their
surroundings
What do the microbes provide for the ruminant?
o Nutrients
o They ferment sugars & polysaccharides including cellulose & hemicellulose to provide
energy for their own growth & waste products, the conditions in the gut are anaerobic
and so organic products are produced EG short chain (volatile) fatty acids
(SCFAs/VFAs, gases)
o The ruminant then absorbs VFAs and uses them as sources of energy and for glucose
synthesis
Protein entering the rumen is broken down by the microbes to give amino acids and ammonia
The amino acids are used to build new microbial cells, these pass into the small intestine
where they are digested and the amino acids are absorbed by the ruminant

Both Organisms have to Compromise
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Bacteria
o The bacteria have to grow anaerobically
 Limits the type of organisms
 Limits the energy productions per unit of carbohydrate (3-4 ATP v 38 ATP)
 Limits the rate of growth, and they can potentially be washed out
Ruminant
o Uses volatile fatty acids for energy production rather than glucose from starch (less
efficient)
o It must synthesise all glucose that it will require (EG for milk or for a baby)
o A lower quality protein in microbial cells compared to some components of diet (not
compared to poor forage)
o This makes them susceptible to metabolic diseases

Rumen Bacteria
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There are around 1010-1012 bacteria/ml of rumen fluid
There is a mixed population of anaerobes and facultative anaerobes
Both gram positive and gram negative bacteria
All shapes of bacteria: robs, cocci, spirals, ovals & unique shapes
Some common ones: Lactobacillus, Streptococcus & Escherichia
Some are unique to the ruminant’s gut: Selenomanos, Prevotella, Ruminococcus
They are grouped according to the substrate they utilise i
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cellulolytic, amylolytic, proteolytic
etc
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Mycorrhizal Associations
 Fungi which grown in association with roots – to mutual benefit of both partners
 Fungi gains sugars and the host plant shows improved efficiency of nutrient uptake –
particularly phosphorus
 Nearly every plant has a mycorrhizal fungus which are divided into a number of groups
 They play key roles in improving agriculture production and bioremediation of polluted
soils
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Arbuscular/Endomycorrhiza
 Glomeromycota
o These have the longest fossil history of any fungi, appearing in the roots of the
earliest rooted land plants
o Mainly associated with herbaceous spp
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Rhizobia are attracted to the root
 The plant releases compounds called flavonoids that attracts the rhizobia
bacteria (chemotaxis)
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Root hairs curls and the rhizobia enter via invagination process
 There interaction between the bacterium and causes the root hairs to bend,
this leads to the development of an invaginated thread which facilitates the
invasion of rhizobia into the interior root tissues
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An infection thread forms allowing invasion of bacteria into the root cortex
 The bacterial cells now rapidly divide forming a new cell type – bacteroids –
which accumulate in the modified host cells
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The cells
containing the bacteroids continue to divide, forming the characteristic pink spherical
nodules on the roots
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o Fruits: fail to ripen, premature fall or in situ rotting
o Stems: cankers or lesions, if vascular tissue becomes blocked, shoots will wilt or lose
leaves
o Roots: galls, dieback, typically leads to wilting and rapid plant death
Organisms that cause plant disease:
o Viruses
 Many types of plant viruses e
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tobacco mosaic, often vectored by insects EG
aphids
 Many cause mottling or other changes and blister-like lesions
 Normally causes a loss of yield rather than causing death
 Difficult to control
 Most have small single stranded RNA genomes which only encode for 3 or 4
proteins: a replicase, a coat protein and a movement protein to allow cell to
cell movement
o Fungi
 The most numerous and important plant pathogens
 Necrotroph: kill tissue and live off decaying remains EG apple rot

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Facultative biotrophs: infect and survive on living tissue, easily
cultured
Obligate biotrophs: only infect living cells, difficult to culture and
usually produce highly specialised infection structures

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