Prokaryotic Cell Structure - MLAB 213 PDF

General Microbiology MLAB 213 Prokaryotic cell structure Charbel Al-Bayssari, PhD University of Balamand Faculty of Health Sciences Medical Laboratory Sciences Department 1 v Microorganism:is a microscopic organi...

General Microbiology MLAB 213 Prokaryotic cell structure Charbel Al-Bayssari, PhD University of Balamand Faculty of Health Sciences Medical Laboratory Sciences Department 1 v Microorganism:is a microscopic organism that comprises either a single cell (unicellular),cell clusters,or no cell at all (acellular). v Viruses depend on the energy and metabolic machinery of a host cell in order to reproduce 2  Cell Morphology of bacteria q Cocci Cocci may be seen as § Single coccus § Pairs (diplococci) § Chains (streptococci) § Clusters (staphylococci) § Packets of four (tetrads) § Packets of eight (octads) 3 q Bacilli Short,long,thick or thin, pointed or with curved or blund ends They occur in pairs, in chains, in long filaments,or branched. Some rods are short: cocobacilli 4 q Bacilli spiral, curved § Curved (vibrio) § Spirilla (helical shape, like a corkscrew) § Spiral-shaped bacilli (spirochete). Spiral-shaped bacteria usually occur singly, but some species may form pairs The shape of a bacterium could be: Ø Monomorphic: maintain a single shape Ø Pleomorphic: have many shapes (pleomorphic) 5  PROCARYOTIC CELL S TR U C TU R E ⚫ All bacteria are prokaryotes ⚫ They are about 10 times smaller than eucaryotic cells ⚫ Reproduction of procaryotic cells is by binary fission ⚫ Within the cytoplasm of procaryotic cells are a chromosome, ribosomes, and other cytoplasmic particles ⚫ The cytoplasm is surrounded by a cell wall and sometimes a capsule or slime layer 6 7  Glycocalyx ü Glycocalyx is a viscous, slimy, gelatinous material composed of polysaccharide, polypeptide, or both. ü It is produced by the cell membrane and secreted outside of the cell wall. There are two types: ⚫ A slime layer (the substance is unorganized and only loosely attached to the cell wall). Slime layers enable certain bacteria to glide or slide along solid surfaces. It consists mostly of exopolysaccharides, glycoproteins, and glycolipids. A loose, water-soluble glycocalyx is called a slime layer ⚫ Capsule (the substance is organized and is firmly attached to the cell wall. Usually consist of polysaccharides, which may be combined with lipids and proteins). Capsules serve an antiphagocytic function, protecting the encapsulated bacteria from being phagocytized by phagocytic white blood cells. 8 9 The glycocalyx is a very important component of biofilms 10 11  Flagella of prokaryotic cells ⚫ Flagella are threadlike, protein appendages that enable bacteria to move. ⚫ Flagellated bacteria are said to be motile, whereas nonflagellated bacteria are usually nonmotile. ⚫ They are about 10 to 20 nm thick; too thin to be seen with the compound light microscope. ⚫ They consist of three, four, or more threads of protein (called flagellin) twisted like a rope. ⚫ Bacterial flagella do not contain microtubules, and their flagella are not membrane-bound q Axial filament: (two flagella-like fibrils) rap around the organism therefore the movement is spiral or helical (spirochetes) 12 Flagellum: arises from a basal body in the cell membrane and project outward through the cell wall and capsule. Each prokaryotic flagellum is a semirigid, helical structure that moves the cell by rotating from the basal body The rotation of a flagellum is either clockwise or counterclockwise Flagellar rotation depends on the cell's continuous generation of energy. 13 14  Other movement for flagella chemotaxis vs phototaxis q Bacterial cells can alter the speed and direction of rotation of flagella and thus are capable of various patterns of motility q The movement of a bacterium toward or away from a particular stimulus is called taxis q Such stimuli include chemicals (chemotaxis) and light (phototaxis) q Motile bacteria contain receptors in various locations such as in or just under the cell wall.These receptors pick up chemical stimuli, such as oxygen, ribose, and galactose. q In response to the stimuli, information is passed to the flagella Ø If the chemotactic signal is positive, called an attractant, the bacteria move toward the stimulus with many runs and few tumbles. Ø If the chemotactic signal is negative, called a repellent, the frequency of tumbles increases as the bacteria move away from the stimulus 15  Axial filaments Spiochetes:Treponema Pallidium (syphilis) and Borrelia burgdorferi (Lyme disease) q Axial filaments have a structure similar to that of flagella. q These axial filaments extend toward each other, wrap around the organism between the layers of the cell wall q As a result of its axial filaments, spirochetes can move in a spiral, helical, or inch- worm manner. Ø This type of movement is similar to the way a corkscrew moves through a cork 16  Flagella of prokaryotic cells cont’d There are four types of flagella moving bacteria: 1. Peritrichous bacteria: Bacteria possessing flagella over their entire surface 2. Monotrichous bacteria: Bacteria possessing a single polar flagellum 3. Lophotrichous bacteria: Bacteria with several flagella at one end 4. Amphitrichous bacteria: Bacteria having one or more flagella at each end Bacteria that lack flagella are referred to as atrichous 17  Pili and Fimbriae ⚫ Often observed on Gram-negative bacteria ü They are composed of polymerized protein molecules called pilin, arranged helically around a central core. ü They arise from the cytoplasm and extend through the plasma membrane, cell wall, and capsule. ⚫ Fimbriae (Adhesin) can occur at the poles of the bacterial cell or can be evenly distributed over the entire surface of the cell ü Fimbriae are important for attachment ⚫ Pili are usually longer than fimbriae and number only one or two per cell. Pili are involved in motility and DNA transfer ⚫ Pili have a gliding motility. ⚫ There are two types of pili: Ø one type enables bacteria to adhere or attach to surfaces Ø The other type (called a sex pilus) enables transfer of genetic material from one bacterial cell to another (conjugation) 18  Bacterial Cell Wall ⚫ The main constituent of most bacterial cell walls is a complex macromolecular polymer known as peptidoglycan (murein), consisting of many polysaccharide chains linked together by small peptide (protein) chains. ⚫ The cell walls of certain bacteria, called “Gram-positive bacteria” have a thick layer of peptidoglycan combined with teichoic acid and lipoteichoic acid molecules. ⚫ The cell walls of “Gram-negative bacteria” have a much thinner layer of peptidoglycan, it is covered with a complex layer of lipid macromolecules, usually referred to as the outer membrane 19 Peptidoglycan is made of disaccharide: The disaccharide portion is made up of monosaccharides called N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) (from murus, meaning wall), which are related to glucose. 20 21 22 The assembly of peptidoglycan on the outside of the plasma membrane is mediated by a group of periplasmic enzymes,which are transglycosylases,transpeptidases and carboxypeptidases 23 q Gram positive cell The cell wall consists of many layers of peptidoglycan, forming a thick, rigid structure. Cell walls of gram-positive bacteria contain teichoic acids ü linearpolymers of polyglycerol or polyribitol substituted with phosphates and a few amino acids and sugars. Teichoic acids and lipoteichoic acid are negatively charged ⚫ Cation movement (positively charged substances) ⚫ Cell growth ⚫ Cell wall antigens 24 q Gram negative cell ⚫ The cell walls of gram-negative bacteria consist of one or a very few layers of peptidoglycan and an outer membrane ⚫ The peptidoglycan is bonded to lipoproteins in the outer membrane ⚫ In the periplasm, there is a gel-like fluid between the outer membrane and the plasma membrane. Ø The periplasm contains a high concentration of degradative enzymes and transport proteins ⚫ The outer membrane consists of lipopolysaccharide, lipoproteins and phospholipids Ø Evade phagocytosis and immune response. Ø Provide a barrier to certain antibiotics, digestive enzymes Ø Porins: passage of nucleotides, disaccharides, peptides, amino acids,vitamin B12 and iron. Ø LPS: Lipid A (endotoxin), core polysaccharide (stability), O polysaccharide (antigen) 25 q Lipid A is the lipid portion of the LPS and is embedded in the top layer of the outer membrane Ø When gram-negative bacteria die, they release lipid A, which functions as an endotoxin Ø Lipid A is responsible for the symptoms associated with infections by gram-negative bacteria such as fever, dilation of blood vessels, shock, and blood clotting q The core polysaccharide is attached to lipid A and contains unusual sugars. Its role is structural-to provide stability. q The O polysaccharide extends outward from the core polysaccharide and is composed of sugar molecules. Ø The O polysaccharide functions as an antigen and is useful for distinguishing species of gram negative bacteria 26  Cell wall function ⚫ Prevent bacterial cells from rupturing when the water pressure inside the cell is greater that that outside the cell. ⚫ Helps maintain the shape of a bacterium and serves as a point of anchorage for flagella ⚫ Cell wall contributes to the ability of some species to cause disease and is the site of action of some antibiotics. 27  Cell wall damage Lysozyme Ø Active against gram positive. Ø catalyzes hydrolysis of the bonds between the sugars in the repeating disaccharide "backbone" of peptidoglycan v Protoplasts (gram positive): Have their cell wall entirely removed v Spheroplasts (gram negative): Have their cell wall only partially removed 28 Cell Membrane (Plasma membrane) 29  Plasma membrane (PM) ⚫ Chemically, the plasma membrane consists of proteins (peripheral and integral proteins and phospholipids).The phospholipids and proteins are arranged as a mosaic form: Fluid mosaic phospholipid model. ⚫ It is flexible and so thin Ø PM is a selective barrier through which materials enter and exit the cell (Passive and active movement): selective permeability Chromatophores Ø Many enzymes are attached to the cell membrane, and a variety of metabolic reactions take place there.( infoldings of the plasma membrane) Ø PM is important to the breakdown of nutrients and the production of energy Ø The semiliquid cytoplasm of prokaryotic In some bacteria, pigments and cells consists of water, enzymes, enzymes involved in photosynthesis dissolved oxygen (in some cases), waste are found in infoldings of the products, essential plasma membrane that extend into the cytoplasm. nutrients, proteins, carbohydrates, and lipids—a complex mixture of all the materials required by the cell for its metabolic functions. 30  Movement of material across the PM Passive Processes include Ø Simple diffusion: movement of molecules (O2 and CO2) or ions from an area of high concentration to an area of low concentration Ø Facilitated diffusion: integral membrane proteins transporters or permeases function as channels or carriers that facilitate the movement of ions or large molecules across the plasma membrane 31  Movement of material across the P M (cont’d) Although ions (for example Na+, K+, H+, CaH , and CI-), amino acids, and simple sugars can move by passive processes, their movement by active processes can go against the concentration gradient, allowing a cell to accumulate needed materials Active Processes § The cell uses energy in the form of ATP to move substances (ions, amino acids and sugars) across the plasma membrane § The movement of a substance in active transport is usually from outside to inside § Active transport depends on transporter proteins in the plasma membrane § Active transport enables microbes to move substances across the plasma membrane at a constant rate, even if they are in short supply 32  Osmosis ØOsmosis: the net movement of solvent molecules across a selectively permeable membrane from an area with a high concentration of solvent molecules (low concentration of solute molecules) to an area of low concentration of solvent molecules (high concentration of solute molecules) 33  Osmotic pressure § Osmotic pressure is the pressure needed to stop the flow of water across the selectively permeable membrane § High osmotic pressure (hypertonic) removes water causing plasmolysis – inhibits growth i.e. salt as preservative § Low osmotic pressures (hypotonic) cause water to enter and can cause lysis § When the concentration of solutes outside a cell equals the concentration of solutes inside the cell, the solution is said to be isotonic § Bacteria are more tolerant to osmotic variations because of the mechanical strength of the cell wall 34 Osmotic pressure is the pressure that is exerted on a cell membrane by solutions both inside and outside the cell Hypertonic solution: A solution having a larger concentration of a substance than is found within the cells themselves An isotonic solution is a medium in which the overall concentration of solutes equals that found inside a cell A hypotonic solution outside the cell is a medium whose concentration of solutes is lower than that inside the cell (hypo means under or less Osmosis: It is the movement of a solvent (e.g., water), through a permeable membrane, from a solution having a lower concentration of solute to a solution having a higher concentration of solute Plasmolysis: Plasmolysis is the process where bacterial the cell membrane and cytoplasm shrink away from the cell wall due to the loss of water through osmosis Plasmoptysis: It occurs when pressure becomes so great that the cell ruptures hence causing the cytoplasm to escape from the cell. 35  Prokaryotic cytoplasm Cytoplasm is thick. aqueous, semitransparent, and elastic. It contains: ⚫ 80% water ⚫ Proteins (enzymes) ⚫ Carbohydrates ⚫ Lipids ⚫ inorganic ions. The major structures in the cytoplasm: ⚫ Nucleoid (containing DNA) ⚫ Ribosomes (smaller then eucaryotic ribosomes (A 70S prokaryotic)) ⚫ inclusions (reserve deposits). ⚫ Protein filaments in the cytoplasm are most likely responsible for the rod and helical cell shapes of bacteria. 36  Nucleoid ⚫ The nucleoid contains the bacterial chromosome ⚫ The procaryotic chromosome usually consists of a single, long, supercoiled, circular DNA molecule. It is embedded in the cytoplasm. ⚫ A procaryotic cell contains neither nucleoplasm nor a nuclear membrane and do not include histone ⚫ A bacterial chromosome contains sufficient genetic information to code for between 850 to 6500 gene products (enzymes, other proteins, rRNA and tRNA molecules) ⚫ Small, circular molecules of double-stranded DNA that are not part of the chromosome (referred to as extrachromosomal DNA or plasmids) may also be present in the cytoplasm of procaryotic cells. Ø A plasmid may contain anywhere from fewer than 10 genes to several hundred genes. They replicate independently ØA bacterial cell may contain one plasmid, multiple copies of the same plasmid. 37  Ribosomes ⚫ Ribosomes are composed of two subunits which consists of protein and ribosomal RNA (70S ribosomes) ⚫ Smaller and less dense than ribosomes of eukaryotic cells 38  Inclusions Metachromatic granules (Volutin):reserve of inorganic phosphate (polyphosphate) that can be used in the synthesis ofATP (methylene blue) Polysaccharide granules.They consist of glycogen and starch (iodine stain) Lipid inclusions (Sudan dye) Sulfur Granules: sulfur granules in the cell,where they serve as an energy reserve Carboxysomes: ribulose I.5-diphosphate carboxylase Gaz vacuoles Magnetosomes are inclusions of iron oxide (may protect the cell again hydrogen peroxide accumulation) Metachromatic granules Magnetosomes 39  Spores (Endospores) ⚫ A few genera of bacteria (e.g., Bacillus and Clostridium) are capable of forming thick-walled spores as a means of survival when their moisture or nutrient supply is low ⚫ Bacterial spores are referred to as endospores, and the process by which they are formed is called sporulation ⚫ Spores are resistant to heat, cold, drying, and most chemicals. some are quite resistant to disinfectants and boiling ⚫ When the dried spore lands on a moist, nutrient-rich surface, it germinates, and a new vegetative bacterial cell (one capable of growing and dividing) emerge 40 Sporulation or sporogenesis 41 ⚫ Most of the water present in the forespore cytoplasm is eliminated by the time sporulation is complete, and endospores do not carry out metabolic reactions. ⚫ The highly dehydrated endospore core contains only DNA, small amounts of RNA, ribosomes, enzymes, and a few important small molecules. ⚫ Endospores can remain dormant for thousands of years. ⚫ An endospore returns to its vegetative state by a process called germination. ⚫ Germination is triggered by physical or chemical damage to the endospore's coat. ⚫ The endospore's enzymes then break down the extra layers surrounding the endospore, water enters, and metabolism resumes. 42  How to differentiate between Gram positive and negative bacteria? Gram staining: ⚫ Crystal violet: the dye enters the cytoplasm of both types of cells ⚫ Iodine: forms large crystals with the crystal violet dye.The latter are too large to escape through the cell wall ⚫ Alcohol dehydrates the peptidoglycan of gram-positive cells to make it more impermeable to the crystal violet-iodine ⚫ Safranin provides a contrasting color to the crystal violet heat-fixation and methanol-fixation. Fixation serves three purposes: § It kills the organisms § It preserves their morphology (shape) § It anchors the smear to the slide 43 44 45 Purple color Pink color 46 47  Acid fast staining Mycobacterium and pathogenic species of Nocardia. These bacteria contain high concentrations (60%) of a hydrophobic waxy lipid (mycolic acid) in their cell wall that prevents the uptake of dyes, including those used in the Gram stain. The carbolfuchsin penetrates the cell wall, binds to cytoplasm,and resists removal by washing with acid-alcohol 48  Special staining for cell wall Negative Staining for Capsules: India ink or nigrosin Many microorganisms contain a gelatinous covering called a capsule ⚫ Place a loopful of India ink on the side of a clean slide. ⚫ A small portion of the solid culture is suspended in saline on the slide near the ink and then emulsified in the drop of ink, or else, mix a loopful of liquid culture of specimens like CSF with the ink. ⚫ Place a clean cover slip over the preparation avoiding air bubbles. ⚫ Press down, or blot gently with a filter paper strip to get a thin, even film. The stain the bacteria with Safranin (optional) ⚫ Examine under dry objectives followed by oil immersion 49  Endospore (Spore) Staining : Schaeffer-Fulton endospore stain ⚫ Malachite green will stain the spores ⚫ The vegetative cells will appear red (counterstain with 0.5% safranin) Flagella Staining (Leifson stain) Tannic acid and the dye form a colloidal precipitate Stain with Leifson stain for ten min and counterstain with methylene blue 50