Download Friends Foes Bacterial Friends and Foes

Biotechnology and
Bacterial
Friends and
Foes
Examples of how research is
helping in the battle against
disease-causing bacteria, and
offering new opportunities to
use beneficial bacteria in
agricultural, food, pharmaceutical
and other industries.
Suitable for post-16 studies
Includes
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Bacteria that make medicines
Bacteria that cause disease
Bacteria and the environment
Lactic acid bacteria
Bacteria that make insecticides
Bacteria as sources of enzymes and
food additives
Bacteria as factories
Bacteria as sensors
For some people the word “bacteria”conjures up a picture of
disease and decay. It’s true that bacteria cause illnesses
including,for example, tuberculosis, food-poisoning,
whooping cough and cholera,and they can cause serious
infections in burns and other wounds.But it is also true that
bacteria are crucial to life on Earth. For example, their role in
decomposition is vital to the recycling of nutrients which
would otherwise be trapped and so ‘lost’in the bodies of
dead animals and plants.Some bacteria ‘fix’atmospheric
nitrogen,ultimately making it available to other organisms,
including plants and animals.
Some bacteria have been used for thousands of years to make
fermented dairy products such as cheese and yoghurt. In the
past fifty years, bacteria have been an important source of
antibiotics,contributing to longer life expectancy and quality
of life.Today, bacteria are used as mini ‘factories’to make
medicines and other high value natural products.
Bacteria can be both a problem and an asset. For example,
some strains of Pseudomonas may be useful for cleaning up
the environment,or making new medicines,whereas other
strains can cause disease. One strain of E. coli is a serious food
poisoning organism while another is used to make life-saving
medicine. Erwinia bacteria produce antibiotics, but they also
attack plants.
Keeping up with bacterial evolution
Bacteria can divide frequently. One human
generation of about 25 years is equivalent to
100,000 bacterial generations. Bacteria evolve
rapidly because each contains a single copy of
genes (haploid).They can readily take up
additional small loops of DNA (plasmids)
containing genes that confer resistance to
antibiotics.The widespread use of antibiotics has
led to some disease-causing organisms becoming
resistant, not only to a single antibiotic but to
many, leading to the re-emergence of some
diseases thought to have been conquered, e.g.
tuberculosis. Other conditions can no longer be
cured easily. Methicillin Resistant Staphylococcus
aureus (MRSA) can be a problem in hospitals.We
need to find new antibiotics and to develop better
ways of using them responsibly so that this
problem does not recur in future.
Studying bacteria in the laboratory
Studying bacteria in the laboratory can be difficult. Bacteria g row
by division.Each type has optimum growth conditions.Usually
bacteria are cultured in a nutrient medium,either a broth or
solidified with agar in a Petri dish where the growth can be seen
in the form of colonies.Certain chemicals or antibiotics may be
added to make the medium selective, for example, in order to
isolate one type of bacteria from a mixed culture. Bacterial g rowth
is also affected by temperature, with pathogens multiplying fastest
at blood heat (37 oC) and cold loving organisms growing best at
low temperatures.pH and oxygen availability are also important.
Some bacteria can grow only when oxygen is available; others can
only multiply in its absence.
Some bacteria stop growing at relatively low population densities,
and so on a Petri dish the colonies may stop growing before they
are large enough to be seen.Some bacteria cannot be cultured by
traditional methods at all.
Microbiologists are increasingly turning to molecular biology to
study bacteria.The sequencing of genomes of known species is
providing a mass of fascinating data (see page 12). Many thousands
of bacteria living on Earth have yet to be discovered.DNA analysis
of, for example, marine sediments is r evealing a whole range of
previously unknown species.
Some bacteria can appear to be dead, but can recover and start
growing when placed in a different environment. For example, the
food poisoning organism E .c o l i 0157:H7 (see page 2) behaves
differently in the presence and absence of oxygen.Proper cooking
kills these bacteria. But if they are inadequately heated they can
survive and recover.They recover better when grown in the absence
of oxygen that in its presence. So, if inadequately cooked products
were tested for E. coli using aerobic (oxygen-rich) counting
techniques, far fewer bacteria would be found, because they recover
more slowly under these conditions than if the test were conducted
under anaerobic conditions where E.coli would have recovered faster
and started to multiply.This could result in false estimates of how
effective the cooking had been in killing the organisms.
Another point to remember is that when they are growing on a
rich growth medium in the laborator y, bacteria may not need to
carry out all the metabolic functions they perform in their natural
habitat.Some of their genes may be ‘switched off ’ for example as
they grow under highly f avourable conditions.
Bacteria at the industrial scale
To make products from bacteria on a large scale, the organisms
must be g rown in very large vessels (called fermenters or
bioreactors).It is important to provide the best possible conditions
for bacterial g rowth by:
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optimising the pH and temperature;
providing nutrients;
removing end products and any inhibitors which might slow
bacterial metabolism.
Scaling-up processes from the laboratory to an industrial scale can
bring problems such as a build up of heat,and inhibited transfer of
gases.Because bacteria can evolve rapidly, it is important that
cultures used in industrial processes are carefully checked and
monitored to ensure that the product is always identical.
Bacteria that make medicines
Antibiotics are natural compounds made by bacteria and other
microorganisms.They are part of the natural defence mechanisms
of these microorganisms against others in their environment.They
can be extracted and used to control harmful bacteria in humans
and other animals.The world’s first antibiotic, penicillin, was
discovered in the 1920s and isolated from the mould Penicillium
notatum. In 1944,a second antibiotic called streptomycin was
isolated from a bacterium called Streptomyces griseus. Since then,
Streptomyces bacteria have been a major source of antibiotics,
including tetracycline and erythromycin.
Streptomyces bacteria live rather like fungi:they grow in the soil as
branching threads which bear chains of reproductive spores.Each
species or strain of Streptomyces makes its own customised set of
antibiotics which it uses to fight off other bacteria.So far only
about 1% of these natural antibiotics have been worth developing
as medicines. But scientists are optimistic that new information
about the Streptomyces genome (see page 12) will enable them to
design and develop new antibiotics,for example, by ‘mixing and
matching’ products from different Streptomyces strains.
In 1984, researchers at the John Innes Centre made the world’s
first hybrid antibiotic by mixing clusters of genes from two
different strains of Streptomyces. Although this particular compound
is not useful in medicine, it shows that in principle new antibiotics
can be made in this way.
Several antibiotics,including erythromycin,are polyketides.These
are long chain molecules that are made in a way similar to fatty
acid synthesis in animal cells, but in a complex series of cycles at
which different chemical modifications are made to the backbone.
Caption: colonies of Streptomyces bacteria. Around the edges of
the colonies are droplets containing antibiotics.
Picture courtesy: Professor K. Chater FRS
The enzymes responsible (polyketide synthases) are huge and very
complicated because they have multiple activities. For example, the
ability to make the basic erythromycin structure is coded for by
just three genes, but involves about forty different catalytic
activities.Scientists at the University of Cambridge have shown
that they can make hybrid enzymes by combining genes for
polyketide synthases from different organisms.This offers a way of
making novel polyketides that might be useful as antibiotics.
In the soil, Streptomyces bacteria produce only very small amounts
of antibiotic.When grown industrially in fermenters they can be
persuaded to make much larger amounts.Scientists are identifying
the genes that regulate the level of antibiotic production so that
even greater production efficiency can be obtained.
Streptomyces bacteria are a source of
many useful natural products
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Caption: three strains of Streptomyces. One makes a blue
antibiotic, another a brown antibiotic; the third which makes a
purple antibiotic is a hybrid made by combining genes from the
other two strains.
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antibiotics including tetracycline, streptomycin and
erythromycin which work by inhibiting protein
synthesis at bacterial ribosomes
an anti-fungal agent, nystatin
an anti-worm agent, avermectin, used to combat the
tropical disease river blindness
an anti-tumour drug, daunorubicin
FK-506, used in transplant surgery to suppress
rejection of the new organ by the immune system.
Picture courtesy: Professor Sir David Hopwood FRS.
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Bacteria that cause disease
E. coli 0157:H7
Escherichia coli is a Gram negative bacterium that li ves in the guts of
humans and other animals.Most strains are harmless: indeed
scientists sometimes use safe strains of E.coli bacteria like E.coli
K12 as ‘factories’to make compounds (see also page 10).But one
strain,known as E.coli 0157:H7,is a serious food poisoning
organism.This strain was responsible for a major incident in
Scotland in 1996 which resulted in the deaths of 20 people.
E.coli 0157:H7 can live in the gut of healthy cattle and may get
onto meat when the animals are slaughtered.It can also get into
milk,and can be found on vegetables which have been fertilised
with contaminated cow manure.The organism is killed if food is
cooked thoroughly.
E.coli 0157:H7 can cause a rare but serious condition known as
haemolytic uraemic syndrome in susceptible people.The cause of the
problem is a toxin produced by the E .c o l i.This toxin is made up of
two different types of protein subunits,A and B.There are five Bs and
one A in a toxin.The B units form a ring and bind to carbohydrate
molecules on the surface of kidney cells, enabling the toxic A subunit
to enter the cells.Once inside, the A subunit can switch off protein
synthesis inside the kidney cells.This causes serious kidney damage.
Scientists at the University of Leeds are working to find new drugs
which will block the interaction between the B subunits and the
carbohydrate molecules on the cells.Their aim is to identify which
bits of the B subunits and carbohydrate recognise each other, and
then to make replicas of the carbohydrate portion.These replicas
will act like decoys:binding to the toxin molecules and blocking
them from reaching the kidney cells.
Caption: a section taken through a potato tuber infected with
Erwinia carotovora. The bacteria were inoculated into the tuber and
incubated for three days. The extent of the rot has been highlighted
by staining the tuber section with iodine.
Pictures courtesy: Professor G. Salmond.
Erwinia carotovora
Erwinia carotovora is a Gram negative bacterium, subspecies of
which are responsible for two economically important diseases of
plants in the UK:soft rot in stored potatoes and black leg or stem
rot in seed potatoes.
The bacterium produces and secretes a cocktail of enzymes pectinases,cellulases and proteases - which breakdown the cell
walls of plant tissue.
Scientists at the University of Cambridge are investigating how E.
carotovora produces and secretes these enzymes.They are comparing
mutant forms of E.carotovora with the normal bacteria.Some
mutants produce less of the harmful enzymes than normal and
secrete them less effectively. Others produce the enzymes
efficiently but cannot secrete them.Some can secrete only the
proteases but not the other enzymes.Eventually, it may be possible
to block the secretory pathw ays and prevent the bacteria from
causing disease.
Friend as well as foe
Caption: the structure of a simple carbapenem antibiotic is shown (inset).
This photograph also displays an agar plate showing growth of a bacterial
‘lawn’ of Escherichia coli across the surface of the plate. This bacterium is
sensitive to the carbapenem antibiotic made by the Erwinia carotovora
(growing in a patch in the middle of the plate). As the Erwinia strain has
grown, it has made the antibiotic and this has diffused out to kill the sensitive
bacteria (shown as a zone of inhibition of growth around the Erwinia growth.)
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Erwinia bacteria make natural antibiotics called
carbapenems. Scientists at the Universities of
Cambridge and Nottingham have studied how the
bacteria make these antibiotics. They have identified
and isolated the genes that govern production of
these antibiotics. The genes have been transferred
into E. coli to enable analysis of antibiotic production.
In principle, it should now be possible to make new
semi-synthetic antibiotics by taking precursor
molecules and modifying them chemically.
Campylobacter
In 1998 there were 58,000 reported cases of Campylobacter food
poisoning in the UK.But there may well have been a hundred
times this number of actual cases,because most people do not
report the illness.The organism responsible for this food poisoning
is Campylobacter jejuni. Until the 1970s, it was not thought to pose
a health risk to humans,although it was known to be present
harmlessly in the guts of healthy poultry and other animals
including cattle, and to be found sometimes in un-chlorinated
water such as ponds and streams.
C. jejuni has become the principal food-borne pathogen in
countries such as the UK and the USA, despite the fact that it is
relatively fragile and susceptible to drying, heating and disinfectants.
Proper cooking of food and pasteurisation of milk kill C. jejuni.
Poorly cooked food (e.g. inadequately barbecued chicken) and raw
milk are probably the major sources of infection.The infectious
dose is thought to be small, perhaps a few hundred cells.
Most cases of infection do not require antibiotics.If treatment is
needed, erythromycin (see also page 1) is one of the antibiotics
sometimes used.
Nobody knows precisely how C. jejuni causes food poisoning.
There is evidence that it produces a toxin,which may cause
diarrhoea, and that the organism is able to invade the cells which
line the human gut.
Scientists at the Sanger Centre near Cambridge have recently
sequenced the genome of C. jejuni which they estimate contains
about 1700 genes. Eventually they hope to know what each gene
does.If they can find genes that code for proteins which enable
the organism to g row inside the human gut,then they might be
able to design drugs to knock out the activity of those proteins.
Similarly, if they can find genes that help the organism to cope
with changes in its environment,such as changes in temperature,
pH and salt concentration,they might be able to block these and
stop the growth of C. jejuni in foods.
Scientists can compare genes found in C. jejuni with those found
in other species of bacteria.If a gene in C. jejuni is very similar to
one from another species,it may well have a very similar function.
Understanding the role of one gene provides clues about very
similar genes.This approach has led scientists to believe that some
of the C. jejuni genes are involved in ‘camouflaging’ the organism,
so that it escapes detection by the host’s immune system.But about
one third of the genes of C. jejuni appear to have no counterparts
elsewhere in nature, even among other pathogenic bacteria.
Caption: Campylobacter jejuni is a spiral shaped, Gram negative
bacterium with a propeller (or flagellum) at each end. Individual
cells are between one and a half and two and a half microns [1
micron = 1/1000th mm].
Picture courtesy: Professor B.Wren.
Symptoms of Campylobacter food
poisoning are:
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diarrhoea accompanied by fever, nausea, headache
and abdominal pain
illness typically starts 2-5 days after ingestion of the
bacteria
effects may last up to 10 days
a rare but serious complication of Campylobacter
infection can be neuro-muscular paralysis.
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Helicobacter pylori
The gut pathogenic bacterium Helicobacter pylori is implicated in
gastritis and the formation of gastric and duodenal ulcers.
Antibiotic treatment against this organism is used in treating ulcers.
H. pylori is broadly related to Campylobacter jejuni (see also page 3).It
differs however, in the part of the body it occupies to cause disease,
which is the stomach lining.It produces large amounts of an
unusual enzyme, urease, which breaks down the nitrogenous waste
product of the body, urea (which diffuses to the tissue from the
blood stream),to carbon dioxide and ammonia.The ammonia is
alkaline and ir ritant and probably helps cause damage to the
stomach wall.The organism may need the ammonia in order to
counter acid secreted by the stomach and aid its own survival in
such acidic conditions.
Caption: a cell of H.pylori, a bacterial pathogen of the human stomach. The curved shape of the cell, and its bundle of flagella which
enable it to wriggle through mucus covering the cells lining the stomach, can be seen clearly. Picture courtesy: Dr C. Penn.
Lactic acid bacteria
Gram positive lactobacilli have many uses.
and bubbles of this gas account for the holes found in cheeses such
as Gouda.
Cheese
Cheesemaking requires three main ingredients:milk;the proteindegrading enzyme chymosin which clots the milk;and a starter
culture of fermentative bacteria known as lactic acid bacteria
which include species of Lactobacillus and Streptococcus. These
bacteria convert the milk sugar lactose into lactic acid.When this
fermentation is finished, the bacteria begin to die and as they do
so they are digested by their own enzymes - a process known as
autolysis.This digestion releases flavour-generating enzymes,
including peptidases,which are important in cheese maturation or
ripening,i.e. in the development of the characteristic taste of
cheese. Some bacteria produce carbon dioxide during maturation
Scientists at the Institute of Food Research are studying the
genetics and metabolism of lactic acid bacteria in order to improve
their usefulness for the food industry. For example, it may be
possible to accelerate autolysis and so speed up flavour
development.
Other foods
Lactic acid bacteria are also used in the manufacture of fermented
meats such as salami,and vegetables,and in wine fermentation.In
wine fermentation they convert malic acid to lactic acid and
carbon dioxide.This contributes to fla vour development.In
sauerkraut - a type of fermented cabbage - Leuconostoc mesenteroides
ferments the cabbage juices to products that include lactic acid,
acetic acid,carbon dioxide and ethanol. Lactobacillus plantarum and
Lactobacillus brevis are also used.
Yoghurt
Cultures containing two types of lactic acid bacteria, Lactobacillus
bulgaricus and Streptococcus thermophilus, are inoculated into milk to
make yoghurt. In so-called ‘live yoghurt’these bacteria are alive,
but growing only slowly at refrigeration storage temperatures.Live
yoghurt can be used as a starter culture to inoculate more milk to
make more yoghurt.When yoghurt is pasteurised,the lactic acid
bacteria are killed.
Picture courtesy: Institute of Food Research.
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Probiotic cultures
The human intestines contain large numbers of bacteria,most of
which exert beneficial effects.Living lactic acid bacteria are used in
some dairy products specifically to increase the proportion of
beneficial species in the gut.The idea is that these bacteria inhibit
the growth of pathogenic species such as Salmonella. Other health
benefits are also claimed.In order for the bacteria to work they
must be able to survive acid conditions in the upper gut.The lactic
acid bacteria used for yoghurt do not survive well in the gut,
instead such strains as Lactobacillus johnsonii and Bifidobacterium brevis
are used in probiotic foods.
Some health aspects proposed for
Bifidobacteria
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inhibit growth of potential pathogens
produce vitamins
anti-tumour properties
lower blood lipid levels.
Silage
Farmers use the fermentative properties of lactic acid bacteria to make
silage - a sort of pickled or preserved form of grass (or other crops
such as maize).Silage is used as a winter feed for cattle and sheep.
These anaerobic bacteria ferment sugars in the plant tissues to
mainly lactic and acetic acid. As a result,the pH of the herbage
falls and this helps to prevent its spoilage by other naturally
occurring bacteria such as enterobacteria and clostridia.
The production of good silage depends on rapidly achieving and
maintaining anaerobic conditions (ie. excluding air from the crop)
and obtaining a low pH. Typically, pH values of less than 4.2 are
needed to keep the silage in good condition.To make silage, grass
is stored in clamps, sealed with black plastic and weighted down
with old car tyres,or as bales which are wrapped tightly in ‘stretch’
film.Both methods prevent air from entering and ensure rapid
creation of anaerobic conditions.If air does get in during the
fermentation process it delays the acidification and more sugars
must be fermented to achieve the required pH level. Air can also
enter silage when it is fed,causing aerobic yeasts,bacteria and
moulds to grow and oxidise
lactic acid and sugars to form
carbon dioxide and water.
Both of these processes can
reduce the nutritional value
of the silage.
Farmers can use additives to
speed up the fermentation
process.These include sugar
(and enzymes that release
sugar from cellulose in grass)
– added so that there is more
for the bacteria to work on.
Organic and inorganic acids
can be used to speed
acidification and reduce the
fermentation needed to lower
the pH, but this is less
popular now as agriculture
becomes more environment
and user-friendly.
Caption: applying inoculant in the field during harvesting of grass
for silage making. Picture courtesy: Institute of Grassland and
Environmental Research.
The most popular biological approach is to add high numbers (up to
1 million/gram) of extra lactic bacteria that have been selected for
their efficiency at producing lactic acid, in order to enhance the
natural fermentation. Examples of species commonly used are
Lactobacillus plantarum, Lactobacillus casei and Pediococcus acidilactici.
These products are called inoculants. They are often freeze dried,
but in recent years scientists have developed a system that uses
freshly-cultured bacteria. In this case the farmer grows up a strain of
Lactobacillus plantarum or other lactic acid bacteria in a special growth
medium at a temperature of 30˚C. This is then diluted with water
and applied to the crop in the field as it is cut and harvested.
The big advantage of this technique is that the bacteria are applied
when they are actively growing and dividing.Thus,they are ready
to start fermenting the crop almost immediately, unlike freezedried inoculants,which tend to take longer to get started. This
year a new strain of Lactobacillus plantarum (isolated and researched
by scientists at the Institute of Grassland and Environmental
Research) has been added to the system.It grows very rapidly and
is extremely efficient at converting fructan,the main sugar in grass,
to lactic acid. This will help the inoculant to work more
effectively, even when sugar in the grass falls to a low level,which
often happens in the autumn.
Left: an electron micrograph of lactic acid bacteria.
Picture courtesy: Institute of Grassland and Environmental Research.
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Bacteria that make insecticides
Some bacteria naturally make toxins that kill insects.One of the
best known examples is the spore-forming organism Bacillus
thuringiensis (Bt) which exists naturally in soil and on plant
surfaces.
The toxins are protein molecules.They are made inside the
cytoplasm of the bacteria as inactive forms known as protoxins.
They are released from the bacterial cell as water-insoluble crystals.
When ingested by insect lar vae, the crystals dissolve in the gut of
the larvae, where enzymes partially degrade the toxins and convert
them to active forms.The active toxins bind to the surface of cells
that line the gut of the larvae.They insert themselves into the cell
membranes forming pores that make the cells ‘leaky’.This
eventually kills the insect lar vae.
The main advantages of Bt insecticides are:
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their specificity: different sub-species of B. thuringiensis
produce different toxins active against different orders of
insect. For example, there are anti-beetle toxins, anti-moth and
butterfly toxins, anti-mosquito toxins, and anti-wasp toxins.
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their biodegradability:because they are proteins, Bt toxins are
readily biodegraded.
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their non-toxicity to humans: none of the Bt toxins tested so far
has proved to be toxic to species other than their insect targets.
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the use of natural Bt toxins can reduce the levels of synthetic
chemical pesticides needed to protect crops.
Bt toxins are made commercially by fermentation and have been
used in sprays for over 40 years.Bt products have accounted for
over 90% of worldwide sales of non-chemical insecticides.They
can be used by organic farmers.
Bt and genetic modification
Some crops, for example, cotton and maize
have been genetically modified to carry
copies of the Bt genes for toxins against
specific pests. So-called Bt maize contains
a gene for a Bt toxin against the cornborer pest.
Other bacterial insecticides
Because pests can become resistant to Bt
toxins (this is a problem with all synthetic
and natural pesticides) scientists are
looking for other bacterial insecticides.
These new insecticides would increase the
range of pests which may be controlled by
non-chemical insecticides.
Recently, potentially useful natural toxins
have been identified in the soil bacteria
Photorhabdus luminescens and Xenorhabdus
nematophilus. Different toxins from the
latter are able to kill mosquito larvae and
the larvae of the cabbage root fly.
Caption: the molecular structure of the
beetle toxin protein from Bacillus
thuringiensis tenebrionis. The action of Bt
toxins is being studied by scientists at the
University of Cambridge.
Picture courtesy: Dr. D. Ellar.
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Bacteria as sources of enzymes
Bacterial enzymes are used in a number of applications in the food
and other industries. For example, starch degrading amylases can be
used to make glucose and fructose as sweeteners in foods, fat
degrading enzymes can be used in detergents,and xylanases which
breakdown cellulose may be used as a tool for pulp bleaching.
Some bacteria produce enzymes which enable them to breakdown
unusual compounds and use them as a source of energy.
Researchers at the Institute of Biotechnology at the University of
Cambridge have identified some bacteria which are able to do this
with the natural painkilling substances codeine and morphine. In
the process,these bacteria produce molecules which might be used
by the pharmaceutical industry as a basis to make new painkilling
drugs.One of the bacterial species used in this research is
Pseudomonas putida which produces five valuable breakdown
products from codeine.
Bacterial enzymes can also be useful in biosensors - i.e. in devices
which use a biological reagent to detect or monitor the presence
of a specific substance. Scientists at the Institute of Biotechnology
have developed a biosensor for the drug heroin.They have isolated
an enzyme from Rhodococcus bacteria which can convert heroin
into morphine, and one from Pseudomonas bacteria which can
breakdown the morphine. During the action of the second
enzyme, a chemical change occurs which causes a colourless dye to
turn red.
The biosensor works like this.The two enzymes are fixed onto a
strip of gel which contains the dye. Samples of substances
suspected of containing heroin are placed onto the strip.When a
sample contains heroin,the drug is broken down by the bacterial
enzymes and the sample spot on the gel turns red.
Caption: a sensor indicating the presence of heroin.
Picture courtesy: Dr. N. Bruce.
Bacteria as sources of food additives
Some bacteria are used industrially to
produce food additives.Examples
include:lactic acid (from Lactobacillus,
see page 4) which is used as an acidifier
for jams,jellies, sweets, soft drinks and
other products;xanthan (from
Xanthamonas) used as a food stabiliser;
dextran (from Klebsiella, Acetobacter and
Leuconostoc) used as a stabiliser in
icecream and other products; and
glutamic acid (from Corynebacterium
and other spp) used as a flavour
enhancer.
Picture courtesy: Institute of Food
Research.
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Bacteria as factories
Plants,animals and bacteria share a common genetic code.This
means that DNA codes for the same messenger RNA,which in
turn,codes for the same amino acids, building blocks of proteins,
regardless of whether the DNA is in a bacterial,plant or animal
cell.So genes can be transferred from animal and plant cells into
bacterial cells and made to work there.This is a type of genetic
modification.
Simplified diagramatic representation of
the use of recombinant DNA technology
to produce human insulin
plasmid
chromosome
Insulin
This approach is now used to make insulin to treat people with
diabetes.In the past,patients received animal versions of insulin
which were extracted from cattle and pig pancreatic tissues.New
technology means that they can receive the human form of the
hormone.This reduces the likelihood of allergic reactions.
A copy of the DNA coding for the human gene for insulin is
isolated and inserted into a loop of DNA (a plasmid) which has
been removed from the bacterium Escherichia coli . Typically the
strain of E.coli used is E.coli K12.This is a harmless organism that
resembles the E.coli bacteria which are found normally in the
human gut, but does not colonise the gut.
The gene is introduced into the plasmid using a restriction enzyme
which opens the loop of DNA,and DNA ligase enzyme which
rejoins the DNA strands of the loop after the gene has inserted.
The plasmid is then replaced in an E. coli bacterium.Plasmids exist
in the cytoplasm of bacteria and replicate at the same time as the
chromosome. So genes on the plasmids are passed on to daughter
bacteria in the same way as those on the chromosome.
The bacteria are grown in large fermenters.The insulin gene on
the plasmid is transcribed and translated to make insulin which is
then extracted from the bacteria.
ribosomes (where
protein is synthesized)
E. coli cell
Plasmids are isolated and enzymes are used to cut the DNA ring
An insulin gene is isolated and is inserted in the plasmid
The plasmid is placed in a new E. coli cell by mixing in the
presence of calcium chloride
INSULIN EXTRACTION
AND PURIFICATION
Fermentation of E. coli containing
grafted plasmid
Synthesis of insulin from
instructions on plasmid
Diagram: gene insertion into a bacterium. This illustration first
appeared in the booklet Human Insulin from Recombinant DNA
and is reproduced courtesy of Eli Lilly Company Limited.
E. coli bacteria have also been used to make the antiviral protein
interferon and human growth factor.
Limitations
Bacterial cells are not always suitable for making proteins
that are normally made in plants or animals. Sometimes
a protein made in plant or animal cells is chemically
modified by enzymes in the cell after it has been
synthesised in order for the protein to function properly.
Bacterial cells may lack these enzymes and so be unable
to make the correct form of the protein.
Some plant proteins contain repeated sequences of
amino acids, and the genes coding for them contain the
corresponding repeat sequences of coding bases in
their DNA. Repeat sequences like these are not usually
found in bacterial DNA. Bacteria contain enzymes that
remove such sequences if they are introduced into the
bacterial cell.
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Caption: inside one of the fermenter houses at the Eli Lilly and
Company factory in Liverpool. Human insulin produced by genetic
modification was first manufactured at this factory in the early 1980s.
Picture courtesy: Eli Lilly Company Limited.
Bio-synthesis
A different way of using bacteria as
factories is to use their natural enzymic
capabilities to replace conventional
chemical methods of synthesis.This
involves finding bacteria that can carry out
the required chemical reactions,and then
devising a way of fixing them inside a
fermenter so that they can be retained and
reused.Scientists at the University of
Huddersfield have demonstrated that
bacteria can be used to convert
acrylonitrile into ammonium acrylate, a
compound used by the polymer industry in
the manufacture of, for example, thickeners
for paints,and absorbents for nappies.
Strains of Rhodococcus and Corynebacteria
bacteria have enzymes with the required
catalytic activities.These bacteria have been
cultured to produce kilogram quantities of
cells.These cells are embedded in
crosslinked polyacrylamide beads which are
then suspended in the fermenter. In pilot scale tests,the
bacteria have produced ammonium acrylate of the same quality
as that made by conventional syntheses.
Bacteria as sensors
Caption: microbes embedded in polyacrylamide bead.
Picture courtesy: Professor D. Ramsdon.
Caption: luminescent bacteria being used as sensors to detect pollution.
Picture courtesy: Professor K. Killham.
Bacteria can be genetically
modified to contain genes which
make them emit light.The genes
are the lux genes from naturally
luminescent marine bacteria.
Organisms modified in this way
are being developed as biological
sensors of environmental pollution
by scientists at the University of
Aberdeen. The higher the level of
pollution,the less light the
bacteria emit. Species such as
Rhizobium and Pseudomonas,
modified to car ry the lux gene,
have been used to measure
toxicity levels in water and in
organic wastes such as sewage.
11
Current research
Scientists are sequencing the entire genetic information (the genome) of important bacteria.
This will help them to develop new ways of combating disease. For example, if the genes
that make a bacterium able to cause disease can be identified,scientists should be able to
design drugs to block their activity.
Scientists at the Sanger Centre near Cambridge are sequencing the genome of a strain
of Streptomyces. This family of bacteria is a major source of antibiotics and other useful
compounds (see also page 1 ). Streptomyces is thought to have about 7000 genes.
Scientists are particularly excited that they have found hundreds of Streptomyces genes
which seem to be involved in regulating metabolism.Many of these can be expected
to control the production of antibiotics.
Scientists at the Sanger Centre have recently sequenced the Campylobacter genome which
contains 1654 genes (see also page 3). Eventually scientists working on Campylobacter hope to
determine how each gene works.
By comparing the genomes of different
types of bacteria,scientists will be able to identify new strains that might be
useful for particular purposes. For example, if scientists know the amino
acid sequence of an enzyme, they can make molecular ‘probes’that will
seek out sequences of DNA which code for similar amino acid sequences,
that are expected to code for similar enzymes.The probes can then be used
to screen different types of bacteria to see if any of them have the genes to
produce these enzymes.
Signalling
Recently, and surprisingly, it has been discovered that bacteria ‘talk to each
other’using chemical signals.This is helping scientists to understand some
important features of bacterial lifestyles. For example, it is revealing how
disease-causing bacteria are able to co-ordinate their efforts to attack their
host.When Erwinia bacteria (see also page 2) invade plant tissue, they grow
and divide without alerting the plant’s defences until the point when their
number is large enough to be effective.Then the cells send signal molecules
to each other so that they all release enzymes to attack their host.
Sources of further information
Society for General Microbiology (SGM)
Marlborough House
Basingstoke Road
Spencers Wood
Reading RG7 1AE
www.socgenmicrobiol.org.uk
For the SGM web-based resource ‘Microbes and Food’
covering food safety and food biotechnology with
accompanying worksheets and questions see:
www.socgenmicrobiol.org.uk/PA/ed_car/mf/mf_index.htm
National Centre for Biotechnology Education
Animal and Microbial Sciences
University of Reading
Whiteknights,PO Box 228
Reading RG6 6AJ
www.rdg.ac.uk
NCBE publish a list of microorganisms for investigation in
schools and colleges, which present minimum risk given good
practice.The list is supported by SGM and the Microbiology in
Schools Advisory Committee (MISAC).
Institute of Food Research (IFR)
Norwich Research Park
Colney Lane, Norwich NR4 7UA
www.ifrn.bbsr c.ac.uk
12
Glossary
Aerobic
an oxygenated environment
Anaerobic
an environment without oxygen
Antibiotic
a substance produced by one microorganism that is able to destroy or inhibit the growth of another
microorganism
Autolysis
the breakdown of living matter caused by the action of enzymes produced in the cells concerned
i.e. self digestion
Bacteria
simple microorganisms enclosed by a cell wall or membrane, lacking fully differentiated nuclei
Biofilm
a community of microorganisms forming a layer on a surface
Biosensor
a device that uses a biological element to detect the presence of small amounts of specific compounds
Enzyme
a natural catalyst (usually a protein)
Eukar yote
a compartmentalised cell with a nucleus containing its DNA
DNA
Deoxyribonucleic Acid - the chemical material of which genes are made
Gene
a unit of inherited information
Genome
an organism’s total complement of DNA
Gram positi ve bacteria
bacteria that hold the colour of the (blue) Gram’s stain when treated and then washed.They have a
thick peptidoglycan cell wall that traps the blue crystal dye
Gram negati ve bacteria
bacteria that do not retain the colour of the (blue) Gram’s stain when treated.They have a thin
peptidoglycan cell wall that does not retain the stain when treated and then washed
Medium
a substance used to provide nutrients for cell growth. It may be liquid (e.g. broth) or solid (e. g .a g a r )
Mutant
an altered cell or organism resulting from a change in the DNA sequence of the original wild
(normal) type
Pasteurisation
of milk
maintaining milk at between 62.8˚C and 65.5˚C for 30 minutes in order to reduce the number
of microorganisms
Patho gen
a disease-causing organism
pH value
a measure of acidity. A pH value below 7 indicates acidity, one above 7 alkalinity
Probiotic
cultures that are used to provide ‘beneficial’bacteria to the human gut
RNA
Ribonucleic Acid - a long chain of nucleotides.Its main functions are in protein synthesis
Strain
a group of organisms of the same species that possess distinctive characteristics that set them apart
from others within the same species but which are not great enough for them to be considered a
different breed or variety of that species
Toxin
a poisonous substance
Acknowledgments
BBSRC is grateful to the following for the use of
illustrations and for checking text:
Mr Derek Anthony - Eli Lilly and Company Limited
Dr Neil Bruce - Institute of Biotechnology, University of Cambridge
Dr Jaquie Burt - Lasswade High School
Mrs Jean Bushell - Blackpool and the Fylde College
Professor Keith Chater FRS - John Innes Centre
Dr David Ellar - University of Cambridge
Dr Barry Freeman - Institute for Animal Health
Professor Steven Homans - University of Leeds
Professor Sir David Hopwood FRS - John Innes Centre
Mrs Janet Hurst - Society for General Microbiology
Professor Ken Killham - University of Aberdeen
Dr Mike Leggett - Institute of Grassland and Environmental Research
Dr Roger Merry - Institute of Grassland and Environmental Research
Dr Charles Penn - University of Birmingham
Professor David Ramsdon - University of Huddersfield
Mrs Catherine Reynolds - Institute of Food Research
Dr Sue Riley - BBSRC
Professor George Salmond - University of Cambridge
Dr Liz Sockett - University of Nottingham
Dr Paul Williams - University of Huddersfield
Dr Martin Waller - University of Nottingham
Professor Brendan Wren - London School of Hygiene and Tropical
Medicine.
13
Lactic acid bacteria. Picture courtesy: Institute of Food Research.
Campylobacter jejuni. Picture courtesy: Professor B.Wren.
BBSRC, Polaris House, North Star Avenue, Swindon SN2 1UH www.bbsrc.ac.uk