Lec#25 .pptx

Lecture #25- Bacillus: friend or foe? Endospore-forming Gram+ bacteria Bacillus, Clostridium Microorganisms and Concepts Bacillus thuringiensis, B. popillae, B. anthracis, B. cereus. Clostridium acetobutylicum, C. botulinum • delta-endotoxin • transgenic crops • endospore • neurotoxin • butanol an...

Lecture #25- Bacillus: friend or foe? Endospore-forming Gram+ bacteria Bacillus, Clostridium Microorganisms and Concepts Bacillus thuringiensis, B. popillae, B. anthracis, B. cereus. Clostridium acetobutylicum, C. botulinum • delta-endotoxin • transgenic crops • endospore • neurotoxin • butanol and acetone -Bacillus represents a genus of Gram-positive bacteria which are ubiquitous in nature (soil, water, and airborne dust). -Some species are natural flora in the human intestines. -When grown on blood agar, Bacillus produces large, spreading, gray-white colonies with irregular margins. -A unique characteristic of this bacterium is its ability to produce endospores when environmental conditions are stressful. -Although most species of Bacillus are harmless saprophytes, two species are considered medically significant: B. anthracis and B. cereus. -Bacillus thuringiensis and B. popillae are used in insect biocontrol. -The only other known endospore-producing bacterium is Clostridium (anaerobic). -C. acetobutylicum used industrially. -C. botulinum culprit of botulism endospore crystal Bacillus thuringiensis crystal endospore B. thuringiensis Bt parasporal crystal -B. thuringiensis (known as 'Bt') is an insecticidal bacterium - caterpillars of the Lepidoptera (butterflies and moths) but also for control of mosquito larvae, and simuliid blackflies that vector river blindness in Africa. - Bt products represent about 1% of the total ‘agrochemical’ market (fungicides, herbicides and insecticides) across the world. -The toxin genes are plasmid encoded and have also been genetically engineered into several crop plants i.e. Bt corn, Bt cotton. Mode of action - summary -The crystals are aggregates of a large protein (about 130-140 kDa) that is actually a protoxin - it must be activated before it has any effect. -safe to humans, higher animals and most insects. - solubilised in reducing conditions of high pH (above about pH 9.5) the conditions commonly found in the mid-gut of lepidopteran larvae. Specific insecticidal agent. -once it has been solubilised in the insect gut, the protoxin is cleaved by a gut protease to produce an active toxin. This toxin is termed delta-endotoxin. -binds to the midgut epithelial cells, creating pores in the cell. As a result, the gut is rapidly immobilised, the epithelial cells lyse. Toxin structure • Domain 1 - Bundle of 7  helices which insert into the gut cell membrane • Domain 2 - 3 antiparallel  sheets this domain binds to receptors in the gut • Domain 3 - tightly packed  sandwich, protects c-terminus from further cleavage -Recent studies on the delta-endotoxin structure show that it has three domains: -Domain I is a bundle of 7 alpha-helices, some or all of which can insert into the gut cell membrane, creating a pore through which ions can pass freely. -Domain II consists of three antiparallel beta-sheets that binds to receptors in the gut. - Domain III is a tightly packed beta-sandwich which is thought to protect the exposed end (C-terminus) of the active toxin, preventing further cleavage by gut proteases. History, products and insect host range -Bacillus thuringiensis was first discovered in 1911 as a pathogen of flour moths from the province of Thuringia, Germany. - Most of these strains have the same basic toxin structure, but differ in insect host range, perhaps because of different degrees of binding affinity to the toxin receptors in the insect gut. - B.t. var kurstaki highly specific to lepidoptera, with no effect on other insects. - B.t. var israelensis are highly active against blackfly and mosquitoe - B.t. var san diego or B.t. var tenebrionis are marketed for control of some coleoptera - especially for control of the important Colorado potato beetle. Milky Spore - Bacillus popillae - Developed by the USDA, Milky Spore attacks the larvae of Japanese Beetles. Anthrax & Biological Weapons Bacillus anthracis -Anthrax is primarily a disease of domesticated and wild animals, particularly herbivorous animals. Humans become infected incidentally when brought into contact with diseased animals - In severe cases, where the blood stream is eventually invaded, the disease is frequently fatal. -Churchill’s biological weapon? http://www.youtube.com/watch?v=TIpB2gV1iyk&feature=related Bacillus anthracis Anthrax and Biological Warfare -U.S. military forces have been vaccinated recently against anthrax, reflecting the concern about the prospect of anthrax spores being used in defense against them. Iraq, Russia and as many as ten nations have the capability to load spores of B. anthracis into weapons. -The spores of B. anthracis can be produced and stored in a dry form and remain viable for decades in storage or after release. When released, the spores are easily dispersed in air for inhalation by unprotected troops (or civilians downwind) and may remain in soil for many years. -Anthrax spores are apparently one of the top choices of weapons for biological warfare. The virulence of Bacillus anthracis is attributable to three bacterial components: 1. Capsular material composed of poly-D-glutamate 2. EF component of exotoxin --binding of toxin to adenylate cyclase 3. LF component of exotoxin--aids in toxin effect http://www.youtube.com/watch?v=7LRGT098lxc Clostridium botulinum -Clostridium botulinum is an anaerobic, Gram-positive, sporeforming rod that produces a potent neurotoxin. - spore is the dormant state of the cell; resistant to heat - vegetative cells produce the toxin. - bacteria and the spore also are inhibited from growing in acid environments (pH 4.6 or below). -spores are heat-resistant and can survive in foods that are incorrectly or minimally processed. -seven types (A, B, C, D, E, F and G) of botulism are recognized, based on the antigenic specificity of the toxin produced by each strain. - death may result from suffocation because the nerves can no longer stimulate breathing--- acetylcholine release is blocked- muscles unable to contract. Antitoxins have been used to reduce the fatality rate. However, one toxin is also used for cosmetic procedures - home canned low-acid foods (vegetables, meat, poultry, fish) are usually the cause of a botulism foodborne illness. - improper temperatures to destroy the spores are not reached, the spores now have the proper environment to grow into vegetative cells and eventually produce the deadly toxin. Clostridium acetobutylicum-creator of Israel -butanol and acetone were discovered as the main end products of fermentation by Clostridium acetobutylicum during the World War I. -This discovery solved a critical problem of explosives manufacture (acetone is required in the manufacture gunpowder) and is said to have affected the outcome of the war. -Acetone was distilled from the fermentation liquor of Clostridium acetobutylicum.