بسمه تعالی. spores clostridium perfringens spores vegetative cells

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  • SporesClostridium perfringensSporesVegetative cells

  • The Bacterial Endospore

  • Bacterial spores A few species of bacteria have the ability to produce highly resistant structures known as endospores (or simply spores). These resist a range of hazardous environments, and protect against heat, radiation, and desiccation. Endospores form within (hence endo-) special vegetative cells known as sporangia (singular sporangium).

  • Historical Developement & Importance

    Spore which can germinate have been found from structures 7200 year old temples have been found and recently from GI tract of a bee preserved in amber (1 million years old)100's of species mainly of the genera Bacillus (aerobic rods, facultative anaerobes), and Clostridium (anaerobic rods); Few others include Sporosarcina (aerobic cocci), Desulfotomaculum (anaerobic rods, sulfate-reducers)

  • Historical Developement & ImportanceFood industries (canning, milk etc) heat treat products to reduce microbial spoilage & kill pathogens;) Mainly found in soils --> vegetables --> meat where spores germinate to produce toxinsMainly found in soils --> infect wounds (problem with farm associated workers)Some strains were being developed for biological warfare eg B. anthracis (anthrax)Some strains produce important biopesticides (biotechnology) eg B. thuringiensis var. israelensis produces toxic proteins against mosquito & blackfly larvae.

  • Some sporeformers are pathogens of animals, usually due to the production of powerful toxins. Bacillus anthracis causes anthrax, a disease of domestic animals (cattle, sheep, etc.) which may be transmitted to humans. Clostridium botulinum causes botulism, a form of food poisoning. Clostridium tetani is the agent of tetanus.

  • sporesEndospore within the cellsExospore external to the cells

  • Location and shape of endospres within the vegetative cellTerminal (Clostridium tetani)Subterminal (Clostridium botulinum )Central (Bacillus cereus)

    Spore shapes may be round oval

  • There are various types of endospore formation in bacteria Round Ellipsoidal Oval Cylindrical Kidney shaped Banana shaped

  • Clostridium tetaniClostridium botulinum

  • Bacterial endospores. Phase microscopy of sporulating bacteria demonstrates the refractility of endospores, as well as characteristic spore shapes and locations within the mother cell.

  • formation of endospores Under conditions of starvation, especially the lack of carbon and nitrogen sources, a single endospores form within some of the bacteria. The process is called sporulation

  • The developmental cycle of the Endospore.

  • Early stages of endospore formationFirst the DNA replicates and a cytoplasmic membrane septum forms at one end of the cell. A second layer of cytoplasmic membrane then forms around one of the DNA molecules (the one that will become part of the endospore) to form a forespore.

  • Middle stages of endospore formationBoth of these membrane layers then synthesize peptidoglycan in the space between them to form the first protective coat, the cortex.

  • Completion of endospore formationCalcium dipocolinate is also incorporated into the forming endospore. A spore coat composed of a keratin-like protein then forms around the cortex. Sometimes an outer membrane composed of lipid and protein and called an exosporium is also seen Finally, the remainder of the bacterium is degraded and the endospore is released.

  • Morphologic and biochemical event in Bacillus subtilis

    StageMorphological eventBiochemical event1Vegatative cell2Chromatin filamentExoenzyme Antibiotic3Spore septumAlanine dehydrogenase4Spore protoplastAlkaline phosphataseGlucose dehydrogenaseAconitaseHeat-resistant catalase5Cortex formation(refractility)RibosidaseAdenosine deaminaseDipicolinic acid6Coat formationCysteine incorporationChemical resistance7MaturationAlanine racemaseHeat resistance

  • Structure of spore

  • Bacillus cereus

  • The Spread of MicroorganismsSporeBacillus thuringeinsis

  • The endospore4- cortex1-exosporium2- coat5- Inner membrane3- outer membrane

  • Structure of spore Protoplast Core : Calcium Dipicolinate dehydrated . innen membrane : .Spore Cortex Dipicolonic acid .Bacillus megaterium

  • Structure of spore Spore wall Cell wall ( ) peptidoglycan ( ) . Spore Coat : Polypeptide ( ) . exosporium : Lipoprotein Bacillus cereus Bacillus megaterium

  • Lysine biosynthetic pathway

  • Resistant to harsh environments-Heat (owing to high concentration of calcium dipicolinic acid)UV radiation (increased cysteine amino acids)DessicationDisinfectants (impermeable cell coat)Mechanical stressSurvival strategy when nutrients are limiting

  • Mechanism of heat ressistancePhysical (sporecoat): Ressistance to staining demonstrates imperability & therefore ressistant to dehydration & effects of toxins (multilayered thick peptidoglycan) Chemical (core): Low water content (15% instead of 80% found in cells) makes proteins & nucleic acids more ressistant. CDPA complexes with proteins & other labile components & makes them more ressistant. Medium lacking calcium or mutant strains that do not form CDPA produce less "tolerant" spores

  • Heat ressistance Endospore-forming cell Time required to kill a suspension in boiling water (100 C)B. Anthracis 1-2 min (not very heat ressistant)C. botulinum 2-6 hoursC. tetani 1-3 hoursE. coli & S. aureus (non-endospore formers) 30 minutes at 70 C

  • The heat resistance of endospores is due to a variety of factors Calcium-dipicolinate, abundant within the endospore, may stabilize and protect the endospore's DNA. Specialized DNA-binding proteins saturate the endospore's DNA and protect it from heat, drying, chemicals, and radiation. The cortex may osmotically remove water from the interior of the endospore and the dehydration that results is thought to be very important in the endospore's resistance to heat and radiation. Finally, DNA repair enzymes contained within the endospore are able to repair damaged DNA during germination

  • Generation of active vegetative cellActivation

    Germination

    Outgrowth

  • Generation of active vegetative cellActivationReversible processPrepares for germinationOften heat-induced process or by chemical treatL-alanine is the most commonSeveral amino acids, nocleoside and glucose

  • Generation of active vegetative cellGerminationEnd of dormantIrreversible processIn early stageLoss of refractilitySwelling of the cortexAppearance of fine nuclear fibrilsBreaks out of spore coatLost of resistance Increase in the sulfhydryl levelRelease spore componentIncrease metabolic activityNot inhibit by antibiotic that perturb protein and nucleic acid synthesis

  • Generation of active vegetative cellOutgrowthActive growth vegetative stateSopre core membrane develops into the cell wallActive biosynthetic activityInhibit by antibiotic that inhibit cell wall, protein , or nucleic acid synthesis

  • : . . - .

  • Most endospore cannot germinate immediatelyAfter they have rest for several daysAfter heated briefly (5 min, at 60 C).If proper nutrient are presentspore takes in water and swellsspore coat cracksvegetative cell grow

  • Differences Between Endospores And Vegetative Cells1. Endospores Are Dormant (For At Least 100 Years)

    2. Endospores Are Unable To Multiply.

    3. Endospores Do Not Degrade Components To Generate Energy

    4. Endospores Do Not Synthesize Cell Components

  • Endospore stain of Bacillus megaterium Note red endospores inside colorless streptobacillus

  • Spore Stain Purpose A differential stain used to detect the presence or location of spores in bacterial cells. Only a few genera produce spores such as Bacillus and Clostridium Note the endospore within the rod gives the bacterium a "tennis racquet" shape

  • Spore Stain Principle Spores have a touch outer covering made of the protein keratin and are resistant to heat and chemicals. The keratin also resists staining, so extreme measures must be taken to stain the spore. A primary stain of malachite green is forced into the spore by steaming the bacterial emulsion. Malachite green is water soluble and has a low affinity for cell material, so vegetative cells and spore mother cells may be decolorized with water and counterstained with safranin.

  • exosporesCells of the methane-oxidizing genus Methylosinus form exospores i.e. spores external to the vegetative cells, by budding at one end of the cellThese are desiccation and heat resistant, but unlike endospores they do not contain DPA.

  • Endosporesproduced within the cell, the cell wall does not contribute to the spore coateg Bacillus and Clostridium speciesActinomycete exosporesproduced by division of a filamentous cell, the cell wall is modified to form the spore coateg Actinomyces and Streptomyces species

  • Functions of sporesEndosporessurvival and dispersal, very resistant to extreme environmental conditionsonly one spore is produced per cell therefore not significant in reproductionExosporessurvival and dispersal, moderately resistant to extreme environmental conditionsmany spores produced per cell therefore v

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