lOMoARcPSD|8703963 CH 4 - Lecture notes; chapter 4 Microbiology For Health Professionals (Drexel University) StuDocu is not sponsored or endorsed by any college or university Downloaded by Cool Girl (rossatomelson@gmail.com) lOMoARcPSD|8703963 Chapter 4 ● ● ● ● ● ● Prokaryote comes from the Greek words for prenucleus. Eukaryote comes from the Greek words for true nucleus A prokaryote is around the size of a mitochondrion or a chloroplast Prokaryotes ○ One circular chromosome, not in a membrane ○ No histones ○ No organelles ○ Bacteria: peptidoglycan cell walls ○ Archaea: pseudomurein cell walls ○ Binary fission Eukaryotes ○ Paired chromosomes, in the nuclear membrane ○ Histones ○ Organelles ○ Polysaccharide cell walls ○ Mitotic spindle The three basic shapes of bacteria ○ Bacillus (rod-shaped) ○ Coccus (spherical) ○ Spiral ■ Spirillum ■ Vibrio ■ Spirochete Downloaded by Cool Girl (rossatomelson@gmail.com) lOMoARcPSD|8703963 ● ● ● ● In order to see bacteria, we need to use oil immersion microscopes in order to magnify them a 1000 fold ○ Average size: 0.2 -1.0 µm ´ 2 - 8 µm Most bacteria are monomorphic – single shape A few are pleomorphic – irregular morphology ○ Signaled by changes in their environment Arrangement of cocci ○ Often give rise to the genus names Downloaded by Cool Girl (rossatomelson@gmail.com) lOMoARcPSD|8703963 ● ● ● Arrangement of bacilli ○ Coccobacilli are referred to as rods Coccus ○ Spherical ○ Diplococci ■ Two ○ Streptococci ■ Chains ○ Tetrads ■ groups of four ○ Sarcinae ■ Cubelike ○ Staphylococci ■ grape-like clusters Bacillus ○ Rod ○ Diplobacilli and streptobacilli ○ Coccobacilli ■ oval Downloaded by Cool Girl (rossatomelson@gmail.com) lOMoARcPSD|8703963 ● ● Spiral ○ Vibrios ■ curved, comma-like ○ Spirillum (spiral) ■ helical corkscrew with flagella ○ Spirochetes ■ shaped like a spiral, axial filament Structures external to the cell wall ○ Glycocalyx ■ adherence, avoid phagocytosis, prevent dehydration, prevent loss of nutrients ○ Flagella ■ motility ○ Axial Filaments ■ (endoflagella) corkscrew motility ○ Fimbriae ■ adherence ○ Pili ■ genetic transfer Downloaded by Cool Girl (rossatomelson@gmail.com) lOMoARcPSD|8703963 ● ● ● ● ● ● ● ● Red ○ Present in all bacteria Black ○ Present in idealized bacteria Glycocalyx ○ Outside cell wall ○ Usually sticky ■ adherence ○ A capsule is neatly organized ○ A slime layer is unorganized & loose ○ Extracellular polysaccharide (EPS) allows the cell to attach (biofilms) ○ Capsules prevent phagocytosis Flagella ○ Outside cell wall ○ Motility ○ Made of chains of flagellin (protein) ○ Attached to a protein hook ○ Anchored to the cell wall and membrane by the basal body Different bacteria have a different number of flagella Flagella are made of proteins ○ Can be used for identification ■ Called H antigens Serovar ○ A variant that is able to be recognized by serological techniques ■ Antibodies Motile cells ○ Rotate flagella to run or tumble ○ Move toward or away from stimuli (taxis) ○ Flagella proteins are H Ags (E. coli O157:H7) à serovars Downloaded by Cool Girl (rossatomelson@gmail.com) lOMoARcPSD|8703963 ● ● ● ● Antibiotics kill a lot of bystanders ○ Can kill good bacteria One way to disrupt infections without disrupting all bacteria would be to disrupt the bacteria's motility - to disrupt the flagella Axial filaments ○ Endoflagella ○ In spirochetes ○ Anchored at one end of a cell ○ Rotation causes the cell to move in a corkscrew manner Bacterial structures ○ Fimbriae ■ allow attachment ■ Factor in pathogenicity ■ Numerous, hairlike ■ Protein – pilin Downloaded by Cool Girl (rossatomelson@gmail.com) lOMoARcPSD|8703963 ○ ● ● ● Pili are used to transfer DNA from one cell to another ■ Longer than fimbriae ■ 1 or 2 per cell ■ Referred to as sex pili ● Generates genetic diversity Cell wall ○ Semi-rigid structure giving characteristic cell shape ○ Prevents osmotic lysis ○ Made of peptidoglycan (in bacteria) Penicillin was designed to prevent the formation of the peptidoglycan cell wall without harming the human cell ○ Works best on gram-positive bacteria ○ There are organisms resistant to it ○ Has limited toxicity ■ Does not harm the eukaryotic host cell Peptidoglycan ○ Polymer of disaccharide ■ N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) ○ Linked by polypeptides ○ Important for gram-positive organisms Downloaded by Cool Girl (rossatomelson@gmail.com) lOMoARcPSD|8703963 ● Repetitive unit of NAG and NAM will make the carbohydrate backbone of the cell wall ● In a gram-negative cell, the peptidoglycan layer is sandwiched between two membranes ○ Outer: lipopolysaccharide layer Downloaded by Cool Girl (rossatomelson@gmail.com) lOMoARcPSD|8703963 ● ● ● ● ● Gram-negative cell wall ○ Thin peptidoglycan ○ Outer membrane - LPS ○ Periplasmic space Gram-positive cell wall ○ Thick peptidoglycan ○ Teichoic acids ■ Lipoteichoic acid links to the plasma membrane ■ Wall teichoic acid links to peptidoglycan ■ May regulate the movement of cations ■ Polysaccharides provide antigenic variation ○ In acid-fast cells, contains mycolic acid Lipopolysaccharides, lipoproteins, phospholipids Forms the periplasm between the outer membrane and the plasma membrane Protection from phagocytes, complement, antibiotics. Downloaded by Cool Girl (rossatomelson@gmail.com) lOMoARcPSD|8703963 ● ● ● ● ● ● ● ● ● O polysaccharide antigen, e.g., E. coli O157: H7 Lipid A is an endotoxin Porins (proteins) form channels through the membrane Complement is an immune component that is going to allow the lysis of bacterial cells Cells, in general, have a negative charge due to the DNA and other negatively charged proteins while basic dyes have a positive charge Gram staining A mordant is a fixative (places the dye in place) Gram stain mechanism ○ Crystal violet-iodine (CV-I) crystals form in the cell ■ Gram(+) retention of 1o dye ● Alcohol dehydrates peptidoglycan ● Collapses peptidoglycan ● CV-I crystals do not leave ■ Gram(-) loss of 1o dye ● Alcohol dissolves the outer LPS membrane and leaves holes in peptidoglycan ● Cell wall fragments ● CV-I washes out Different antibiotics are used for gram-positive and gram-negative bacteria ○ Gram-positive ■ 2-ring basal body ■ Disrupted by lysozyme ■ Penicillin sensitive ○ Gram-negative ■ 4-ring basal body Downloaded by Cool Girl (rossatomelson@gmail.com) lOMoARcPSD|8703963 ■ ■ Endotoxin Tetracycline sensitive Downloaded by Cool Girl (rossatomelson@gmail.com) lOMoARcPSD|8703963 ● ● ● Sodium azide is an inhibitor of the electron transport chain Atypical cell walls ○ Mycoplasmas ■ Smallest known bacteria that can grow and reproduce outside a host cell ■ Lack cell walls ■ Sterols in the plasma membrane ○ Archaea = Wall-less or walls of pseudomurein (lack NAM and D amino acids) ■ Appear “Gram (-)” ● stain red ○ Acid-Fast Cells ■ The high concentration of mycolic acid (waxy) ■ Mycobacterium and Nocardia ● Carbolfuchsin + heat destain w/ acid-alcohol Damage to cell walls ○ Can be natural or introduced by humans ○ Lysozyme ■ digests disaccharide in peptidoglycan. ○ Penicillin ■ inhibits peptide bridges in peptidoglycan. ■ Most Gram (-) bacteria are not susceptible to penicillin ○ Protoplast ■ is a wall-less cell ○ Spheroplast ■ is a wall-less Gram-negative cell. ■ Protoplasts and spheroplasts are susceptible to osmotic lysis à burst in pure water or very dilute salt or sugar solutions Downloaded by Cool Girl (rossatomelson@gmail.com) lOMoARcPSD|8703963 ○ ● ● ● ● L forms ■ are wall-less cells that swell into irregular shapes A bacteria without a cell wall = protoplast ○ If you dont have a cell wall, you dont have a characteristic feature of what the morphology looks like ■ The cell does not replicate normally ■ Susceptible to osmotic lysis ■ Over time, will turn into L-cells Plasma membrane ○ Phospholipid bilayer ■ Peripheral proteins, Integral proteins, Transmembrane proteins ■ The membrane is as vicious as olive oil. ■ Proteins move to function ● Fluid Mosaic Model ■ Phospholipids rotate and move ● laterally – but, ● don’t flip-flop Integral proteins are embedded in the membrane ○ Going to run completely through the membrane Peripheral proteins associate with the membrane ○ In close proximity to things embedded in the membrane ○ Selective permeability ■ Allows passage of some molecules Downloaded by Cool Girl (rossatomelson@gmail.com) lOMoARcPSD|8703963 ● ● ● Most important function ■ Large molecules (proteins) cannot pass ■ Small uncharged molecules (aa or simple sugars) can pass ■ Lipid-soluble substances pass more easily ■ Enzymes for ATP production ● (ETC) ■ Damage to the membrane by alcohols, quaternary ammonium (detergents), and polymyxin antibiotics causes leakage of cell contents The plasma membrane ○ Photosynthetic pigments on foldings called chromatophores or thylakoids ○ Chromatophores are derived from cell membranes ○ Found in photosynthetic cells ○ They contain pigment used to capture light energy for producing sugar ○ Some bacterial plasma membranes have mesosomes ■ Electron microscope artifact Movement across membrane ○ Simple diffusion: Movement of a solute from an area of high concentration to an area of low concentration ○ Facilitative diffusion: Solute combines with a transporter protein (permease) in the membrane Downloaded by Cool Girl (rossatomelson@gmail.com) lOMoARcPSD|8703963 ● Passive processes ● Diffusion ○ Always going to eventually reach equilibrium ○ A dye will cross a membrane until both solutions have equal concentrations of the dye ■ Equilibrium Downloaded by Cool Girl (rossatomelson@gmail.com) lOMoARcPSD|8703963 ○ Substances diffuse down its own concentration gradient. ■ From higher concentration to lower concentration ● Movement across membrane ○ Osmosis ■ Movement of water across a selectively permeable membrane from an area of high water concentration to an area of lower water. ○ Osmotic pressure ■ The pressure needed to stop the movement of water across the membrane. ● Aquaporins ○ Water channels ○ Osmosis: ■ Passive movement of water across a membrane ○ Occurs two ways: ■ Diffusion and bulk flow (aquaporins) ○ Membranes contain integral proteins (aquaporins) that serve as water-filled “pipes” across the membrane. Downloaded by Cool Girl (rossatomelson@gmail.com) lOMoARcPSD|8703963 ● The principle of osmosis ○ Isotonic ○ Hypotonic ■ The water inside the cell is higher ■ Leads to osmotic lysis ○ Hypertonic ■ Solute inside the cell is higher ■ Leads to shrinkage Downloaded by Cool Girl (rossatomelson@gmail.com) lOMoARcPSD|8703963 ● ● Movement across membrane ○ Active Transport ■ requires a transporter protein + ATP ■ Occurs when bacterial cells are in an environment that is low in nutrients ○ Group translocation ■ occurs only in prokaryotes ○ Requires a transporter protein and PEP (phosphoenolpyruvic acid). ■ Transported substance becomes chemically altered (ex. phosphorylated) ■ Example: glucose transport Movement of materials across membranes ○ Facilitated Diffusion: requires a transporter protein ■ Follows concentration gradient ● High to low ○ Active transport: requires a transporter protein and ATP ■ Goes against the concentration gradient ○ Group translocation: requires a transporter protein and PEP Downloaded by Cool Girl (rossatomelson@gmail.com) lOMoARcPSD|8703963 ● ● ● Structures internal to the plasma membrane ○ Nuclear area (nucleoid) ■ bacterial genome ■ Essential genes for structures and metabolism ○ Plasmids ■ Extrachromosomal ● Because it’s outside of the bacterial chromosome ○ The cytoplasm is the substance inside the plasma membrane Plasmids ○ Small, circular DNA ○ Extrachromosomal ○ Non-essential genes ■ “bonus” genes ■ Helpful in certain environments ○ Antibiotic resistance genes, virulence factors ○ Biotechnology à useful for cloning ○ There is a high copy number not just a couple of plasmids Prokaryotic ribosomes ○ Site of protein synthesis ○ Determined by the measure of buoyancy in a heavy gradient ■ Mimics the size but not the usual measure of weight ○ The individual subunits do not add up to the buoyancy of the complete ribosome Downloaded by Cool Girl (rossatomelson@gmail.com) lOMoARcPSD|8703963 ● Inclusions ○ Aggregates of various compounds that are normally involved in energy reserves or as the building blocks for the cell ○ Accumulate when cells are grown in the presence of excess nutrients ■ Stored for a later use ● Inclusions ○ Reserves deposits ○ Metachromatic granules (volutin) ■ Phosphate reserves ○ Polysaccharide granules ■ Energy reserves ○ Lipid inclusions ■ Energy reserves ○ Sulfur granules ■ Energy reserves ○ Carboxysomes ■ Ribulose 1,5-diphosphate carboxylase for CO2 fixation ○ Gas vacuoles ■ Protein covered cylinders ○ Magnetosomes ■ Iron oxide (destroys H2O2) Downloaded by Cool Girl (rossatomelson@gmail.com) lOMoARcPSD|8703963 ● ● ● ● ● ● Endospores ○ Not found in every single cell ○ Resting cells that allow for the survival of certain cells under adverse conditions ○ Resistant to desiccation, heat, chemicals ○ Occurs in ■ Bacillus is an aerobic gram-positive rod ■ Clostridium is an anaerobic gram-positive rod Sporulation ○ Endospore formation ○ Not reproduction Germination ○ Return to a vegetative state A gram stain does not stain the spore Note ○ Coxiella burnetti ○ forms “endospore-like” structures ○ Gram (-) rod ■ causes Q fever Endospore stain ○ Pink ■ Vegetative cell ○ Green ■ Endospore Downloaded by Cool Girl (rossatomelson@gmail.com) lOMoARcPSD|8703963 ● Formation of endospores by sporulation ● Organelles ○ Nucleus ■ Contains chromosomes ○ ER ■ Transport network ○ Golgi complex ■ Membrane formation and secretion ○ Lysosome ■ Digestive enzymes ○ Vacuole ■ Brings food into cells and provides support ○ Mitochondrion ■ Cellular respiration ○ Chloroplast ■ Photosynthesis ○ Peroxisome ■ Oxidation of fatty acids; destroys H2O2 ○ Centrosome ■ Consists of protein fibers and centrioles Downloaded by Cool Girl (rossatomelson@gmail.com) lOMoARcPSD|8703963 ● The evolution of eukaryotes ○ Endosymbiotic Theory pioneered by Lynn Margulis ■ Larger bacterial (prokaryotic) cells are presumed to have engulfed smaller bacterial cells ■ The endosymbiotic relationship eventually evolved into the eukaryotic cell ■ Mitochondria and chloroplasts are considered evidence for the theory ■ They resemble prokaryotic cells and can reproduce independently of their eukaryotic host cell Downloaded by Cool Girl (rossatomelson@gmail.com) lOMoARcPSD|8703963 ● A model of the origin of eukaryotes Downloaded by Cool Girl (rossatomelson@gmail.com) lOMoARcPSD|8703963 Week #1A HW Chapter 4 – Prokaryotic and Eukaryotic Cells Chapter 4 Concepts: ● List the distinguishing characteristics of prokaryotic vs eukaryotic cells. o Prokaryotes have one circular chromosome, not in a membrane. They have no histones and no organelles. Bacteria have peptidoglycan cell walls and Archaea have pseudomurein cell walls. They reproduce by binary fission. On the other hand, eukaryotes have paired chromosomes, in the nuclear membrane. They contain a nucleus and organelles. They have polysaccharide cell walls. They divide by mitosis or meiosis. ● Define the basic shape and arrangement of bacterial cells. o Basic shapes § Bacillus (rod-shaped) § Coccus (spherical) § Spiral o Arrangement § Cocci · diplococcus, streptococcus, tetrad, sarcinae, and staphylococcus § Bacilli · ● Single bacillus, diplobacilli, streptobacilli, coccobacillus § Spiral · Spirillum · Vibrio · Spirochete For the following bacterial structures external to the cell wall, define their composition and function. Which ones are virulence factors? ○ Glycocalyx ■ It is a coating that covers the outside of bacteria. ■ It provides adherence, helps cell avoid phagocytosis, prevents dehydration, and prevent loss of nutrients. ■ A virulence factor ○ Flagella ■ § They are made of chains of flagellin (protein) and are anchored to the cell wall and membrane by the basal body. Used for motility Downloaded by Cool Girl (rossatomelson@gmail.com) lOMoARcPSD|8703963 ○ Axial filaments ■ Flagella that run lengthwise between the bacterial inner membrane and outer membrane. ○ ● ● ● ● ● Fimbriae § A series of threads or other projections resembling a fringe. ■ A virulence factor ○ Pili § They are used to transfer DNA from one cell to another. What type of flagellar arrangements can bacteria have? ○ Peritrichous, Monotrichous and polar, Lophotrichous and polar, Amphitrichous and polar What is the composition of peptidoglycan? ○ It is a polymer of disaccharide [N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM)] Describe differences in cell wall composition for Gram (+) and Gram (-) cells. o Gram-positive bacteria have cell walls composed of thick layers of peptidoglycan. Gram-negative bacteria have cell walls with a thin layer of peptidoglycan sandwiched between two membrane layers. Describe the Gram Staining procedure. ○ Explain why Gram (+) vs Gram (-) cells stain differently. ■ Due to the varying thickness of the peptidoglycan layer. ○ What is the coloration for a Gram (+) cell? Gram (-)? ■ Gram (+) ● Crystal violet ■ Gram (-) ■ Safranin ○ What would the coloration be if you switched the 1o and 2o dyes in the procedure? ■ The Gram (+) cells would be colored correctly in the end. ○ Give two examples of both Gram (+) and Gram (-) organisms. ■ Gram (+) · Staphylococcus · Streptococcus ■ Gram (-) · Escherichia coli (E. coli) · Salmonella Differentiate Gram (+) and Gram (-) cells by relevant characteristics (See table 4.1) ○ Peptidoglycan layer ■ Thick in Gram (+) (multilayered) and thin in Gram (-) (single-layered) Downloaded by Cool Girl (rossatomelson@gmail.com) lOMoARcPSD|8703963 ○ ○ Cell wall components ■ Gram (+) ■ Thick peptidoglycan layer, contains teichoic acids, lacks periplasmic space, and outer membrane, and LPS ■ Gram (-) ■ Thin peptidoglycan layer, lacks teichoic acids, contains periplasmic space, and outer membrane, and high amount of LPS Toxins produced ■ Gram (+) produce primarily exotoxins ■ Gram (-) produce primarily endotoxins ○ ● ● Resistance to physical disruption ■ Gram (+) cells have a high resistance ■ Gram (-) cells have a low resistance ○ Drying and lysozyme ■ Gram (+) cells have a high resistance to drying and disruption by lysozyme ■ Gram (-) cells have a low resistance to drying and disruption by lysozyme ○ Susceptibility to antibiotics ■ Gram (+) cells have a high susceptibility to penicillin and sulfonamide while they a low susceptibility to streptomycin, chloramphenicol, and tetracycline ■ Gram (-) cells have a low susceptibility to penicillin and sulfonamide while they a high susceptibility to streptomycin, chloramphenicol, and tetracycline ○ Basic dyes ■ Gram (+) cells can easily be inhibited by basic dyes ■ Gram (-) cells cannot easily be inhibited by basic dyes ○ Anionic detergents ■ Gram (+) cells have a high susceptibility to anionic detergents ■ Gram (-) cells have a low susceptibility to anionic detergents Which bacteria do not have cell walls? What do they have in their cell membranes to help stabilize the membrane? ○ Mycoplasmas. They have sterols on their cell membranes to help stabilize the membrane What are acid-fast bacteria? Why do these cells not stain the Gram stain procedure? ○ Give an example of a pathogenic acid-fast bacteria. § Acid-fast stain binds strongly only to bacteria that have a waxy material in their cell walls. § The acid-fast microorganisms retain the pink or red color because the carbolfuchsin is more soluble in the cell wall lipids than in the acid-alcohol. § Mycobacterium tuberculosis belonging to the genus Mycobacterium. Downloaded by Cool Girl (rossatomelson@gmail.com) lOMoARcPSD|8703963 ● ● ● ● ● ● ● ● What is the function of the plasma membrane? ○ What is the Fluid Mosaic Model? ■ Selective permeability ■ The Fluid Mosaic Model is the dynamic arrangement of phospholipids and proteins in the plasma membrane. What macromolecules can move across a membrane? o Small, uncharged substances such as oxygen and carbon dioxide, and hydrophobic molecules such as lipids. Define osmosis and osmotic pressure. o Osmosis is the net movement of water molecules across a selectively permeable membrane from an area with a high concentration of water molecules (low concentration of solute molecules) to an area of low concentration of water molecules (high concentration of solute molecules). o Osmotic pressure is the pressure required to prevent the movement of pure water (water with no solutes) into a solution containing some solutes. What happens to a cell when placed in an isotonic, hypertonic, or hypotonic solution? ○ Isotonic: the cell’s contents are in equilibrium with the solution outside the cytoplasmic membrane – no change/ effect. ○ Hypertonic: the bacterial cell shrinks or collapses (plasmolyzes) ○ Hypotonic: the bacterial cell may burst or undergo osmotic lysis What is the difference between simple and facilitated diffusion? ○ Simple diffusion: Movement of a solute from an area of high concentration to an area of low concentration. ○ Facilitated diffusion: Solute combines with a transporter protein (permease) in the membrane. What is active transport? When would a bacterial be likely to use energy for active transport? ○ Active transport is the movement of ions or molecules across a cell membrane into a region of a higher concentration, assisted by transporter protein and requiring energy. o The cell uses energy in the form of ATP to move substances across the plasma membrane What is group translocation? ○ A special form of active transport where the substance is chemically altered during transport across the membrane. What are plasmids? What is their function? o Plasmids are small usually circular, double-stranded DNA molecules. o Plasmids may carry genes for such activities as antibiotic resistance, tolerance to toxic metals, the production of toxins, and the synthesis of enzymes. Downloaded by Cool Girl (rossatomelson@gmail.com) lOMoARcPSD|8703963 ● ● ● ● What are endospores? What is the function of endospores? ○ Name two genera of bacteria that form endospore. ■ Endospores are resting cells that allow for the survival of certain cells under adverse conditions. they can survive extreme heat, lack of water, and exposure to many toxic chemicals and radiation. § Clostridium and Bacillus form endospores. What are inclusions? How do they benefit bacterial cells? ○ Inclusions are reserve deposits. o They benefit bacterial cells in the sense that cells may accumulate certain nutrients when they are plentiful and use them when the environment is deficient. Why do antibiotics that inhibit protein synthesis only affect bacterial cells? o They block bacterial protein synthesis by interfering with the processes at the 30S subunit or 50S subunit of the 70S bacterial ribosome. Explain the endosymbiotic theory. ○ What is the evidence in support of this theory? § According to this theory, larger bacterial cells lost their cell walls and engulfed smaller bacterial cells. § Evidence supporting the theory includes the fact that both mitochondria and chloroplasts resemble bacteria in size and shape. Moreover, mitochondrial and chloroplast ribosomes resemble those of prokaryotes, and their mechanism of protein synthesis is more similar to that found in bacteria than eukaryotes. Finally, the same antibiotics that inhibit protein synthesis on ribosomes in bacteria also inhibit protein synthesis on ribosomes in mitochondria and chloroplasts. Downloaded by Cool Girl (rossatomelson@gmail.com)
