S-LAYER ;- Protoplasts, Spheroplasts, and L Forms The Mycoplasmas 1

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1 S-LAYER ;- A paracrystalline protein or glycolprotein layer has been demonstrated in some bacteria (both G+ and G- bacteria as well as archae bacteria). This layer can be shown by electron microscopy. The functions of the S-layer are : 1-to protect the cell from wall-degrading enzymes, from invasion by other bacteria and from bacteriophages. 1- plays a role in the maintenance of cell shape in some species of archae bacteria, 2- may be involved in cell adhesion to host epidermal surfaces. Protoplasts, Spheroplasts, and L Forms Removal of the bacterial wall by hydrolysis with lysozyme or by blocking peptidoglycan synthesis with an antibiotic such as penicillin,such treatments liberate protoplasts from gram-positive cells and spheroplasts (which retain outer membrane and entrapped peptidoglycan) from gram-negative cells. If such cells are able to grow and divide, they are called L forms, which are produced spontaneously or antibiotic-induced formation(more readily with penicillin than with lysozyme). Some L forms can revert to the normal bacillary form upon removal of the inducing stimulus. Thus, they are able to resume normal cell wall synthesis. Others are stable and never revert. The factor that determines their capacity to revert is the presence of residual peptidoglycan, which normally acts as a primer in its own biosynthesis. L form in the host may produce chronic infections and are relatively resistant to antibiotic treatment, so they present special problems in chemotherapy. The Mycoplasmas The Mycoplasmas are cell wall-lacking bacteria containing no peptidoglycan. Genomic analysis places the mycoplasmas close to the gram-positive bacteria from which they may have been derived. Mycoplasmas lack a target for cell wall-inhibiting antimicrobial agents (e.g., penicillins and cephalosporins) and are therefore resistant to these drugs. Some, like Mycoplasma pneumoniae, an agent of pneumonia, contain sterols in their membranes. The difference between L forms and mycoplasmas is that L forms revert to their original bacteria shape and not resist penicillins, but Mycoplasmas never do and resist penicillins. 1

2 The Bacterial "Life Cycle" Bacteria have been as;- growing (vegetative) phase. stationary (endospore) or dead phase. All bacteria adapt to their environment at both phenotypic and genotypic levels. There are many physiological states(as capacity of the immune system and antimicrobial agents)in which bacteria can exist and alter their morphology as: 1- The cells of bacteria are larger in growing rapidly than in slowly growing. 2- Bacilli appear cocco-bacillary or coccoid in shape. 3- Bacilli exposed to some antibiotic may produce filamentous shape. 4- Filamentous bacteria released coccal forms. Bacterial Endospore Members of bacterial are capable of forming endospores. The two most common are Bacillus and Clostridium,the other bacteria are Thermoactinomyces, Sporolactobacillu.The process, sporulation, is began by near depletion of any of several nutrients (carbon, nitrogen, or phosphorous). Each mother cell forms a single internal spore(endospore) that is liberated when the mother cell undergoes autolysis. The spore is a resting cell, when returned to favorable nutritional conditions and activated (see below), the spore germinates to produce a single vegetative cell. (Figure 10). Sporulating cells of bacillus species. A: Unidentified bacillus from soil. B: B cereus. C: B megaterium. (1)-Sporulation;- Can be thought of as repackaging a copy of bacterial DNA into a new form (core form) that contains very little water, has greatly reduced metabolic activity, does not divided, and has a restructured highly impermeable, multilayered envelope. 2

3 Sporulation begins with the formation of an axial filament (Figure 11). The process continues with an infolding of the membrane so as to produce a double membrane structure whose facing surfaces correspond to the cell wall-synthesizing surface of the cell envelope. The growing points move progressively toward the pole of the cell so as to engulf the developing spore.the spore mother cell will be highly of metabolic activity or protein synthesis to produce cortex, coat and maturation (some spp. have the an additional covering called exosporium,figure -12-),in the same time the protein synthesis of the mother cell will be reduced.when the endospore is completed mature, the mother cell lysis and relase endospore. The stages of endospore formation. By the light microscope : the spore resist coloration by simple stain or gram stain appearing as a clear space within the stained cell protoplasm. (Figure 10) or stained by spore stain. The appearance of the mature spores varies in the spp. ; A- according to the shape of the spore (spherical, ovoid, elongated). 3

4 B- according to the position of the spore (terminal, subterminal or central). C- according to the size of the spore (being narrower than the cell,not bulging or bulging). Figure(12) Bacterial spore Spores are much more resistant than vegetative forms to disinfectants, drying and heating, ultraviolet light. In fact sterilization procedures are assessed by their ability to kill spores,so the moist heat (autoclaving)at c o, min. kill spores( while heating at 60c o kill vegetative cells).this resistance of spore due to several factors in which they differ from vegetative cells;- (1)-the impermeability of their cortex and outer coat. (2)-their high content of calcium and dipicolinic acid. (3)-their low content of water. (4)-their vary low metabolic and enzymic activity. (2)-Germination;- To return to the vegetative state, spores must first be subjected to a various treatment(such as heat(80c o )or chang ph or in nutritious environment), to make the outer coat weakens.then, It beings to germinate by destruction of the cortex by lytic enzymes + take up of water+ release of calcium and dipicolinate from the cell + loses heat resistance + becomes permeable to dyes + is refractivity changes. Enriched culture media are used when testing the sterility of materials that have been exposed to disinfecting procedures. Why? The germination process occurs in three stages: activation, initiation, and outgrowth. Activation Most endospores activated in a nutritionally rich medium, by one or another agent that damages the spore coat, as transient exposure to heat at 80c o, abrasion, acidity, and compounds containing free sulfhydryl groups. 4

5 Initiation Once activated, a spore will initiate germination if the environmental conditions are favorable by receptors that recognize different effectors as signaling a rich medium. Binding of the effector activates an autolysin that rapidly degrades the cortex peptidoglycan. Water is taken up, calcium and dipicolinate is released, and a variety of spore constituents are degraded by hydrolytic enzymes. Outgrowth Degradation of the cortex and outer layers results in the emergence of a new vegetative cell consisting of the spore protoplast with its surrounding wall. The formation of the first vegetative cell and before the first cell division is called outgrowth, which requires a supply of all nutrients essential for cell growth. Exospores or Conidia ;- Some of filamentous bacteria (Actinomycetales) and many filamentous fungi form exospores or conidia, resting spores different from endospoes in ;- (1) the conidia are borne externally by abstraction from the end of the parent cells (conidiaphores). (2) they are disseminated by the air or fresh habitats. (3) they are not especially resistant to heat and disinfectants. Pleomorphism During growth, bacteria may shows considerable variation in size and shape, or form a proportion cells that are swollen, spherical, elongated or pear shaped, this pelomorphism occurs in certain spp.(e.g. Corynebacterium, Haemophilus)in a culture on artificial medium in the presence of substance (penicillin, glycine, lithium chloride, Nacl and organic acid at PH). 5