* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Friends Foes Bacterial Friends and Foes
Survey
Document related concepts
Metagenomics wikipedia , lookup
History of virology wikipedia , lookup
Traveler's diarrhea wikipedia , lookup
Hospital-acquired infection wikipedia , lookup
Trimeric autotransporter adhesin wikipedia , lookup
Quorum sensing wikipedia , lookup
Microorganism wikipedia , lookup
Phospholipid-derived fatty acids wikipedia , lookup
Antibiotics wikipedia , lookup
Horizontal gene transfer wikipedia , lookup
Disinfectant wikipedia , lookup
Human microbiota wikipedia , lookup
Triclocarban wikipedia , lookup
Marine microorganism wikipedia , lookup
Bacterial cell structure wikipedia , lookup
Transcript
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 ● ● ● ● ● ● ● ● 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: ● ● ● 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 ● ● ● 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. ● ● 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. 1 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.) 2 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: ● ● ● ● 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. 3 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. 4 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 ● ● ● ● 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. 5 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: ● 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. ● their biodegradability:because they are proteins, Bt toxins are readily biodegraded. ● 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. ● 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. 8 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. 9 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. 10 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