Lecture 3 .pdf

General Microbiology 2nd year students Pharm D Lecture 3 1 III) Structures internal to 2 the cell wall A) The Plasma (Cytoplasmic) Membrane B) Cytoplasm C) Nuclear Area (DNA and pla...

General Microbiology 2nd year students Pharm D Lecture 3 1 III) Structures internal to 2 the cell wall A) The Plasma (Cytoplasmic) Membrane B) Cytoplasm C) Nuclear Area (DNA and plasmid) D) Ribosomes E) Inclusions F) Endospores  A) The Cytoplasmic Membrane 3 A thin structure enclosing the cytoplasm. Thickness: approx. 8 nm. Consists of phospholipids bilayer, and proteins.  Each Phospholipid molecule contains: 1. A polar hydrophilic head (phosphate group and glycerol). 2. Non-polar hydrophobic tails (fatty acids). -  Eukaryotic membranes contain sterols, such as cholesterol and ergosterol which makes it more rigid than that of prokaryotes.  A) The Cytoplasmic Membrane There are two types of protein molecules in 4 the cell membrane:  Peripheral proteins sla Lie at the inner or outer surface of the membrane. Easily removed from the membrane by mild · treatments. Support membrane during movement.  Integral proteins Penetrate the membrane completely. bill, Contain channels through which substances enter and exit the cell. - 1906(9 s is & is Can be removed only after disrupting the bilayer stoplasmic membrane - (by detergents, for example).  Functions of the cell membrane * ↳ Chtoplasmic membran 5 A. Selective permeability and uptake nutrient (determine what go in and what go out). ⑤S B. Production of energy, site of oxidative phosphorylation enzymes (respiratory enzymes). ↳ C. Secretion of extracellular hydrolytic enzymes. D. Site of polymerizing enzymes for synthesis of cell wall, cell membrane, and DNA.  Selective permeability 6 A selective barrier through which materials enter and exit the cell. Large molecules (as proteins) cannot pass through the plasma membrane because they are larger than the channels in integral proteins. Smaller molecules (as H2O, 02, CO2, and simple sugars) pass easily. Ions penetrate the membrane very slowly. Lipid-soluble substances (as O2, CO2, and non-polar organic molecules) enter and exit more easily than other substances because the membrane consists of phospholipids. The movement of materials across plasma membranes also may depend on carrier molecules.  Production of energy 7 Plasma membrane contains enzymes catalyze chemical reactions which break down nutrients and produce energy (ATP). This is called oxidative phosphorylation through a chain of electron transport system. Plasma membranes contain one or more large irregular folds called mesosomes. -> energy 2 s !, I. Mesosomes: membranous structures that function in cell wall injury and chromosome replication, and oxidative phosphorylation.  The Movement of Materials Across cell Membrane 8 Large molecules needed by the bacteria are first broken down into simpler molecules. Proteins amino acids, Polysaccharides simple sugars. Such enzymes which are released by the bacteria into the surrounding medium, are called extracellular enzymes. large nahouhys Once the enzymes degrade the large molecules, the subunits are transported by permeases into the cell.  1- Passive Processes energy 2. - * ~ The movment af 9 Substances cross the membrane prest in ions that from an area of high conc. to an afte PIagcric area of low conc. without expending memb ranes energy (ATP) by the cell. a) Simple Diffusion  It is the movement of molecules from an area of high conc. to an area of low conc.  The movement continues until the molecules or ions are evenly distributed.  The point of even distribution is called equilibrium.  Simple diffusion transports certain molecules, as O2 and CO2.  b) Facilitated Diffusion no energy fi The substance (as glucose) to be transported combines with a carrier protein (permeases) in the plasma membrane. - ↳88 91 The mechanism: carrier protein makes a change in its shape that enables it to transport substances from one side to the other.  Facilitated diffusion is like simple diffusion in that the bacterial cell does not need to expend energy because the substance moves from a high to a low conc.  The process differs from simple diffusion in its use of carriers. 10  jess - & c) Osmosis  Movement of water across a selectively permeable barrier with the concentration gradient. A bacterial cell may be subjected to one of three kinds of osmotic solutions:  Isotonic solution: concentrations of solutes are the same on both sides of the membrane. Water leaves and enters the cell at the same rate (no change).  Hypotonic solution: conc. of solutes outside the cell is lower than inside the cell. Water moves into the cell. Bacterial cells with weak cell walls as (Gm -ve bacteria) may burst because of excessive water intake (osmotic lysis). Lysozyme and certain antibiotics damage cell walls, causing the cells also to rupture or lyse.  Hypertonic solution: concentration of solutes is higher than the cell has. Bacterial cells placed in a hypertonic solution shrink and collapse because water leaves the cells by osmosis (plasmolysis). Keep in mind that these terms describe the concentration of solutions outside the cell relative to the concentration inside the cell. 11  c) Osmosis 12 - 156 a b c $1d : 2506. it js it ⑳> - 11 2 -↑ /st -1. - 1 &1  2- Active eneva $2 : Processes 13 10 ~ The cell must use energy to move substances from areas of low conc. to areas of high conc. - 11 Active processes include active transport and group translocation. > a) Active Transport The cell uses energy in the form of ATP to move substances across the plasma membrane. The movement of a substance is from outside to inside. = 559585 / 55 Depends on carrier proteins in the plasma membrane. There appears to be a different carrier for each transported substance.  b) Group Translocation :D8 $ 14 The substance (as glucose) is chemically altered during transport across the membrane. Once the substance is altered and transported 69-8 to the inside of the cell, the plasma membrane is impermeable to it, so it remains inside the cell. Group translocation requires energy supplied by high-energy phosphate compounds as phosphoenol pyruvic acid (PEP). While a specific carrier protein is transporting glucose molecule across the membrane, a phosphate group is added to the sugar. Destruction of the plasma membrane by antimicrobial agents 15 Plasma membrane is vital to the bacterial cell. In addition to the chemicals that damage the cell wall and indirectly expose the membrane to injury, many compounds specifically damage plasma membranes.  These compounds include certain alcohols and quaternary ammonium compounds (QACs) disrupt the membrane’s phospholipids.  A group of antibiotics as the polymyxins cause leakage of intracellular contents and subsequent cell death.  B) Cytoplasm 16 Cytoplasm is a gel-like matrix. Composed of water (80%). Also contains proteins, enzymes, carbohydrates, lipids, and many low molecular weight compounds. The major structures in the cytoplasm are DNA, plasmid, ribosomes, and inclusions.  C) Nuclear Area 17 Bacterial chromosome: A single long circular moleculeals of double-stranded DNA. - *j IS ⑰ ssbls 1I - ↳ single-stranded DNA This is the cell's genetic information required for the cell's structures and functions. Bacterial chromosomes do not include histones and are not surrounded by a nuclear membrane. In actively growing bacteria, about 20% of the cell volume is occupied by DNA because such cells resynthesize nuclear material for future cells. excrastic I- and Procortic -? 51 Bacteria often contain small circular DNA chromosome molecules called plasmids.  - Plasmids is · Plesmides & - 18  Extrachromosomal genetic elements.  Replicate independently of · chromosomal DNA. -  Carry about 5-100 genes.  Controlling non-essential genetic information.  Can be transferred from one bacterium to another.  Confer properties such as antibiotic resistance.  Manipulated in biotechnology. >  D) Ribosomes ↳ Protine 19 All eukaryotic and prokaryotic cells contain sinthezes ribosomes (the sites of protein synthesis). · big iss - Ribosomes 191 The cytoplasm contains tens of thousands 3551 ass of these very small structures, which give the cytoplasm a granular appearance. 5) 2 URNA ⑦ Ribosome is composed of two subunits, and protine each of which consists of protein and ribosomal RNA (rRNA). Prokaryotic ribosomes differ from * rRNA eukaryotic ribosomes in the number of => and prosine proteins and rRNA molecules they contain; they are also somewhat smaller and less dense than ribosomes of eukaryotic cells.  D) Ribosomes 20 Prokaryotic ribosomes (70S ribosomes) while Eukaryotic cells are 80S ribosomes. - - -- The letter S refers to Svedberg units, which indicate the rate of sedimentation during ultra-high-speed centrifugation. Sedimentation rate is a function of the size, weight, and shape of a particle. The subunits of a 70S ribosome are a small 30S subunit containing one molecule of rRNA and a larger 50S subunit containing two molecules of rRNA.  Several antibiotics inhibit protein synthesis on prokaryotic ribosomes.  Antibiotics such as gentamicin attach to the 30S subunit preventing protein synthesis.  Other antibiotics, as erythromycin and chloramphenicol, attaching to the 50S subunit interfere with protein synthesis. Because of differences in prokaryotic and eukaryotic ribosomes, the microbial cell can be killed by the antibiotic while the eukaryotic host cell remains unaffected.  Prokaryotic vs. Eukaryotic ribosomes -> 2 URNA and protine ↳ BURNA and preting -> 2URNA - and ~ Protine l 8 ~ - DrRNA and protine > a re a 21  E) Inclusions -> usls to, jist 98 st - 1514 22 es) s Some inclusions are common to a wide variety of bacteria, whereas others are limited to a small number of species and therefore serve as a basis for identification. of bactrial 1- Metachromatic (volutin) granules ↳ 8 = 91805 Large inclusions that take their name from the fact that they sometimes stains red with blue dyes such as methylene blue. Volutin stores inorganic phosphate that can be used in the synthesis of ATP. They are formed by cells that grow in phosphate-rich environments. These granules are found in many organisms such as algae, fungi, and bacteria. Corynebacterium diphtheriae and Lactobacillus are a common examples. ↳ we his inclusions  2- Polysaccharide Granules 23 Granules contain either glycogen or starch as an energy or carbon reserve. After staining with iodine, glycogen granules appear reddish-brown, while starch granules appear blue. 3- Lipid Inclusions Lipid core surrounded by a monolayer of phospholipids, which protects the inclusions from cytoplasm, preventing denaturation of cytoplasmic proteins due to hydrophobic interactions. Appear in various species of Mycobacterium, Bacillus, Azotobacter, and other genera. Lipid inclusions are revealed using fat-soluble dyes, such as Sudan dyes.  4- Gas Vacuoles 24 Hollow cavities found in many aquatic - prokaryotes, including cyanobacteria and halobacteria. Consists of rows of several gas vesicles, which are hollow cylinders covered by protein. Sos ↑ Gas vacuoles help bacteria in buoyancy and enable them to float at the desired depth in the water appropriate for them to receive enough oxygen, light, and nutrients. They do so by inflating and deflating the vesicles. They help the photosynthetic bacteria in getting optimal light and oxygen.  5- Magnetosomes 25 Inclusions of iron oxide (Fe3O4), formed by several Gm-ve bacteria such as Aquaspirillum magnetotacticum, that act like magnets. Bacteria use magnetosomes to move downward until they reach a suitable attachment site. Magnetosomes protect the cell against H2O2 accumulation. Industrial microbiologists are developing culture methods to obtain large quantities of magnetite from bacteria to use in the production of magnetic tapes for sound and data recording.  F) Endospores > = 26 Certain Gm +ve bacteria, such as Clostridium and Bacillus, form specialized cells called endospores when essential nutrients are depleted. Endospores are highly durable dehydrated cells with thick walls and additional layers. ⑧I ⑪) They are formed internal to the bacterial cell membrane. When released into the environment, they can survive extreme heat, lack of water, and exposure to many toxic chemicals and radiation. For example, million-year-old endospores have germinated when placed in nutrient media. One Gm -ve species, Coxiella burnetii, the cause of Q fever, forms endospores. The process of endospore formation 27 Sporulation or sporogenesis within a vegetative (parent) cell takes several hours. In the first stage of sporulation, a newly replicated bacterial chromosome and a small portion of cytoplasm are isolated by an in growth of the plasma membrane called a spore septum. The spore septum becomes a double-layered membrane that surrounds the chromosome and cytoplasm within the original cell and is called a forespore. Thick layers of peptidoglycan are laid down between the two membrane layers. Then a thick spore coat of protein forms around the outside membrane. This coat is responsible for the resistance of endospores to many harsh chemicals. 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 process of endospore formation 28 The process of endospore formation 29  Endospores 30 The highly dehydrated endospore core contains only DNA, small amounts of RNA, ribosomes, enzymes, and a few important small molecules. The small molecules include a large amount of dipicolinic acid which is accompanied by a large number of calcium ions. These cellular components are essential for resuming metabolism later.  Their size, shape and position are varied.  Spherical or oval.  Central, subterminal or terminal.  bulging (swollen- expanded) or non-bulging.  Endospores 31 Endospore returns to its vegetative state by a process called germination, which triggered by physical or chemical damage to the endospore's coat. The endospore's enzymes break down the extra layers surrounding the endospore, water enters, and metabolism resumes.  Sporulation in bacteria is not a process of reproduction because one vegetative cell forms a single endospore which after germination remains one cell.  Endospores are important from a clinical point of view and in the food industry because they are resistant to processes that kill vegetative cells. Such processes include heating, freezing, desiccation, use of chemicals, and radiation. Most vegetative cells are killed by temperatures above 70°C, endospores can survive in boiling water for several hours.  Endospores 32 - - *  Resistance of endospores is -I - - due to :- 1. Very low permeability of spore wall. 2. High content of Calcium and dipicolinic acid. 3. Low water content. 4. Very low metabolic activity (dormant). I Structure of endospore -  Structure Predominant chemical composition ⑰ Function(s) -Protection against phagocytosis. Glycocalyx (Capsules or Usually polysaccharide; -Attachment to surfaces slime layers) occasionally polypeptide or both -Reserve of nutrients - Protection against desiccation Flagella - Protein (flagellin) -Swimming movement Pili - -Mediates DNA transfer during Sex pili Protein (pilin) conjugation Pili or fimbriae -Attachment to surfaces Protein (pilin) -protection against phagocytosis Cell wall V Gram-positive bacteria Peptidoglycan (murein) -Prevents osmotic lysis of cell complexed with teichoic acids -confers rigidity and shape of cells Peptidoglycan (murein) -Peptidoglycan prevents osmotic lysis and surrounded confers rigidity and shape Gram-negative bacteria by phospholipid protein -outer membrane is permeability barrier; lipopolysaccharide "outer -associated LPS and proteins have various membrane" functions 33  Predominant Structure Function(s) chemical composition & Permeability barrier; Plasma transport of solutes; membrane Phospholipid and protein energy generation; location of numerous enzyme systems Sites of translation (protein Ribosomes rRNA and protein synthesis) Highly variable; Often reserves of nutrients; Inclusions carbohydrate, lipid, protein additional specialized functions Genetic material of cell Chromosome DNA controlling the main genetic information of the cell. Extrachromosomal genetic Plasmid DNA material controlling non-essential genetic information. 34  Nutrition and Metabolism of microorganisms 35 Chemical composition of microorganism -of  Bacteria contain: bacteria  70 – 80 % water.  20 – 30% Dry weight, which consists of 80 % Macromols (Protein – fat – Polysaccharide – nucleic acid), 10 % Micromols (Sugar – amino acid – vitamin) and 10% inorganic salts. Requirements for bacterial growth (or any living Organisms) 1. Source of energy (from food or solar radiation) 2. Source of elements (from food) 3. Suitable environmental conditions (pH, temperature, oxygen, and moisture). -> >  * =0 ; -> -> A Cetabolism Anabolism * Fe >. =.. large sell Eusus -> rehensing energy energy smelt-large * 36 releasing enevery Que E - g energy 37 38  Metabolism of microorganisms e No 02 39 - ⑨ Respiration (aerobic) and Fermentation (anaerobic) generally results in: Energy, Micromolecules and Reducing power of H atom which is required in biosynthetic reactions. They are utilized in: A. Biosynthesis of cell components (macromolecules) e.g. DNA, RNA, proteins, enzymes, lipids, carbohydrates and structural parts of the cell as cell wall, cell membrane, flagella, etc. B. Cell movement. C. Cell multiplication and growth. D. Transport of nutrients. E. Repair of cell damage.  Metabolism of microorganisms 40 * All metabolic processes are performed by enzymes. > => Enzymes recognize and catalyze the specific metabolic pathways used by bacteria for the breakdown and synthesis of organic substrates are shared by all living cells. These similarities form the basis for the concept of the unity of biochemistry i.e. the chemistry of all life forms is essentially the same. Bacteria provide excellent models to study metabolism as they are more suitable for handling in the laboratory than plant or animal cells.  Metabolism of microorganisms 41 Metabolic processes begins with hydrolysis of large macromolecules in the extracellular environment by specific extracellular enzymes. Micromolecules (monosaccharides, short peptides and fatty acids) transported into the cytoplasm where they converted to pyruvic acid.- - 81s From pyruvic acid, the carbons derived from the imported nutrients may be channeled toward either energy production or synthesis of new carbohydrates, amino acids, lipids and nucleic acids.  42 Photosynthetic Utilize photons from the sun like plants e.g. cyanobacteria and Sulfur - bacteria Source of Energy Chemosynthetic Utilize energy produced from the -rom oxidation of organic compounds like animals e.g. most bacteria  Link between energy and reducing power> 43 - All cells require a constant supply of energy to survive. This energy is in the form of ATP. This energy is derived from the breakdown of various organic substrates (carbohydrates, fats and proteins) in a process called catabolism. an Most energy producing reactions are oxidation reaction, which involve release of H+ ion, electron (e-) and Energy. The oxidation reaction is accompanied by a reduction one i.e. one compounds is oxidized and the other is reduced. - $3s 81 In biological systems - oxid./red. reactions may be "un-coupled“ through intermediates acting as carrier for electron and energy.  Types of energy releasing mechanisms · 1- e Substrate phosphorylation ⑧ 44- Where phosphorylated substrate donates phosphate group directly to -nee ADP e.g., phosphoenol pyruvate +does - ADP ATP + pyruvate. - 2-· Oxidative phosphorylation Is carried in the cytoplasmic membrane through electron carriers (NAD, NADP) and return of protons across the membrane is coupled to phosphorylation of ADP to ATP through energy released and inorganic ↳ phosphate. ADP + energy + inorganic PO4 ↳ ATP. i i.e. oxidation and phosphorylation reactions are - coupled. i - eee Oxidative phosphorylation is more energetic than phosphorylation. substrate  45 ~give more energy Cytoplasmic 53 membrane -  ⑨ "- S 38 mols ATP. 46 In oxidative phosphorylation 1 mole glucose gives - - In substrate phosphorylation 1 mole glucose gives a 2 mols ATP. In photosynthetic bacteria: the electron donor is m -> H2O (oxidized), and the electron acceptor is& - CO2 (reduced). > In Chemosynthetic bacteria: Electron acceptor Oxygen Inorganic Organic Electron donor NH4, N, NO2NO3 H2S S Inorganic O2  H2O Fe3+  Fe2 Not possible Sugar –H Glucose  CO2 Lactic  CO2 Pyruvic Organic O2 H2O SO4  H2S Pyruvic +H  lactic  47 Hydrogen bacteria: derive energy form oxidation of hydrogen H2 (e- donor) H2O. mee a Methane bacteria: derive energy from oxidation of methane (e- mean donor) CO2. 2 Nitrifying bacteria: derive energy form oxidation of ammonia (e- - donor) NO2 or NO3. nee Sulfur bacteria: derive energy form oxidation of H2S (e- donor) > S or SO4. -