Unit 2 Slides (1) PDF

Size in the Microbial World Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Nucleus Small molecules Atoms Proteins Viruses Mitochondria Prion fibril Lipids Ribosomes Smallest bacteria Most bacteria Most eukaryotic cells Adult roundworm Human he...

Size in the Microbial World Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Nucleus Small molecules Atoms Proteins Viruses Mitochondria Prion fibril Lipids Ribosomes Smallest bacteria Most bacteria Most eukaryotic cells Adult roundworm Human height Electron microscope Light microscope Unaided human eye 0.1 nm 1 nm 10 nm 100 nm 1 µm 10 µm The basic unit of length is the meter (m), and all other units are fractions of a meter. nanometer (nm) = 10 –9 meter = .000000001 meter micrometer (µm) = 10 –6 meter = .000001 meter millimeter (mm) = 10 –3 meter = .001 meter 1 meter = 39.4 inches 100 µm 1 mm 1 cm 0.1 m These units of measurement correspond to units in an older but still widely used convention. 1 angstrom (Å) = 10 –10 meter 1 micron (µ) = 10 –6 meter 1m 10 m Limitations of Light Microscopy LIGHT VS. ELECTRON MICROSCOPES • Light Microscope Ø Uses visible light Ø Magnify objects ~1000x Ø Easy to observe cell size, shape, & motility • Electron Microscope (1931) Ø Magnify in excess of 100,000x Ø Reveal fine details of cell structure Light Microscopy MICROSCOPY PRINCIPLES • Refraction Ø Refraction is a physical phenomenon. It’s when light rays change direction due to a change in the medium through which they travel. Ø In the picture to the right the pencil seems broken because the light was bent differently when it traveled into and out of the water and through the glass. Ø Refractive index – measure of relative speed of light at it passes through a medium (air, water, etc) Ø Lenses use refraction to focus light. MICROSCOPY PRINCIPLES • Refraction Ø Refractive index – measure of relative speed of light at it passes through a medium (air, water, etc). Ø Light bends when it hits a change in materials when the refractive index of each differs significantly. The bend is sharper when the light ray hits the interface between the two materials at an angle. Ø Lenses are shaped to take advantage of refraction to focus light. MICROSCOPY PRINCIPLES • Refraction Ø Lenses bend light based on the principles of refraction. MICROSCOPY PRINCIPLES • Magnification Ø The increase in the apparent size of the object compared to the size of the actual object. Four times the diameter when seen in microscope Original object A A In this example the circle with the letter “A” is actually 1 centimeter in diameter. When viewed through the microscope it appears to be 4 times larger in diameter. Therefore it has been magnified “4x”. When you read a number like “100,000x” it means the image has made the object appear one-hundred thousand times larger than it actually is. MICROSCOPY PRINCIPLES • Resolution Ø The ability to see objects (or points) as distinct, instead of as a blur that combines them. Poor resolution – when magnified they look like only one big object Original objects In this example the there are actually three objects. When viewed through the microscope, however, the three objects blur together to appear as only a single object. Resolution is defined as the minimum distance at which two points can be distinguished as individuals. MICROSCOPY PRINCIPLES • Resolution Ø The ability to see objects as a different objects instead of a blur that combines both. Good resolution – when magnified they look like three smaller objects Original objects In this example the three objects can easily be distinguished. We would say this microscope has “higher resolution” than the one in the past example. Resolution is defined as the minimum distance at which two points can be distinguished as individuals. MICROSCOPY PRINCIPLES • Contrast Ø The ability to see objects against the background. low contrast Original objects In this example the three objects cannot be easily seen against the bright white background. We would say this image has low contrast. Notice that we still have good magnification (the objects are bigger) and resolution (we can see three objects). Techniques to Improve Contrast MICROSCOPY PRINCIPLES • Contrast Ø The ability to see objects against the background. high contrast Original objects In this example the three objects are easy to see and even have a three dimensional quality to them. For some types of microscopy contrast is improved by staining the cells with a dye (for light microscopy) or electron-dense material (for electron microscopy). BRIGHT-FIELD MICROSCOPE • Most common microscope in a laboratory • Light passes through specimen, then a series of magnifying lenses Ø Evenly illuminates entire field of view Bright-field Copyright © The McGraw-Hill Companies, Inc. BRIGHT-FIELD MICROSCOPE BRIGHT-FIELD MICROSCOPE Objective lens • Magnification Ø Compound microscope has 2 types of magnifying lenses Ø Ocular (10x) Ø Objective (4x, 10x, 40x & 100x) Ø Condenser focuses light from the lamp but does not magnify object Ocular lens Condenser Courtesy of Leica, Inc., Deerfield, FL Copyright © The McGraw-Hill Companies, Inc. BRIGHT-FIELD MICROSCOPE Condenser Lens Lamp BRIGHT-FIELD MICROSCOPE Objective Lenses (4 X to 100 X) BRIGHT-FIELD MICROSCOPE Occular Lens (10 X) BRIGHT-FIELD MICROSCOPE The total magnification is the product of the magnifying power of the ocular lens and the objective lens. EXAMPLE: 40 X objective lens 10 X occular lens 400 X total magnification. BRIGHT-FIELD MICROSCOPE • Resolution • 0.2µm resolution maximum for light microscope • This is a physical limitation of visible light and cannot be improved upon by simply making better or more powerful lenses. • Can see the shape of bacteria but cannot see any details. Objective lens Ocular lens Condenser • Cannot see viruses. Courtesy of Leica, Inc., Deerfield, FL Copyright © The McGraw-Hill Companies, Inc. BRIGHT-FIELD MICROSCOPE The 100x lens on a bright field microscope requires that a drop of immersion oil is placed between the slide and the lens. Ø Displaces air between lens & specimen Ø Oil has same refractive index as glass Ø Prevents light from missing objective lens Ø Objective lens Air Slide Oil Light source © W. A. Jensen Copyright © The McGraw-Hill Companies, Inc. BRIGHTFIELD PROBLEMS WITH CONTRAST MICROSCOPES THAT INCREASE CONTRAST • Invaluable when examining characteristics of living organisms (e.g. motility) • Specimen prepared as a wet mount Ø Slide + Drop of liquid + Cover www.microscopy-uk.org.uk/mag/imgsep09/3a-porta-dedo-gota-2-450.jpg DARK-FIELD MICROSCOPE • Directs light towards specimen at an angle • Only light scattered by specimen enters objective • Cells stand out as bright against a dark background Filamentous alga (Spirogyra sp.) Colonial alga (Volvox sp.) 25 µm Copyright © The McGraw-Hill Companies, Inc. © T. E. Adams/Visuals Unlimited ELECTRON MICROSCOPES • Comparable to light microscopy Ø Ø Ø Ø Electromagnetic lenses, electrons, & fluorescent screen replace glass lenses, visible light, and eye Image can be captured on film to create an electron micrograph Light Microscope Electron gun Lamp Electromagnet Condenser lens Glass Electron beams Light rays Specimen Glass Objective lens Wavelength of electrons ~1,000 shorter than light Resolving power ~1,000-fold greater than BF microscope: ~0.3 nm Transmission Electron Microscope Electromagnet Image Ocular lens Glass Eye Electromagnet Viewing screen Copyright © The McGraw-Hill Companies, Inc. ELECTRON MICROSCOPES Light Microscope Ø Can magnify images 100,000x Ø One drawback is that lenses and specimen must be in vacuum Ø Ø Ø Air molecules would interfere with electrons Results in large, expensive unit and complex specimen preparation Transmission Electron Microscope Electron gun Lamp Electromagnet Condenser lens Glass Electron beams Light rays Specimen Glass Objective lens Electromagnet Image Ocular lens Glass Electromagnet Two major types Eye Viewing screen Copyright © The McGraw-Hill Companies, Inc. TRANSMISSION EM OR TEM • Used to observe fine detail of cell structure • Works by directing electrons that either pass through or scatter • Dark areas on image correspond to dense por tions of specimen • Thin-sectioning: allows you to see internal details, but process can distor t cells SCANNING EM OR SEM • Used to observe surface details • Surface coated with thin film of metal • Beam of electrons is scanned over surface of specimen • Electrons released from specimen are reflected & observed in viewing chamber • Relatively large specimens can be viewed • Yields 3-D effect © David M. Phillips/Visuals Unlimited 2 µm Copyright © The McGraw-Hill Companies, Inc. Advantages/Disadvantages of Electron Microscopy DYES & STAINING TECHNIQUES IN MICROSCOPY USED BRIGHT-FIELD PROBLEMS WITH CONTRAST CELL STAINS TO IDENTIFY ORGANELLES FLUORESCENT STAINING http://www.microscopyu.com/galleries/fluorescence/cells.html STEPS TO STAINING • Fixation • Permeabilization • Mounting • Staining • Addition of mordant • Washing • Counterstaining (optional) Copyright © The McGraw-Hill Companies, Inc. WHY STAIN IN MICROSCOPY? • Problem: Observing cells with BF microscope is difficult Ø Cells move around & are nearly transparent • Solution: Samples can be immobilized & stained to visualize • Method: Heat smear Drop of liquid containing specimen is spread (thinly) over slide. Allow to air dry. Forms a film or smear Pass slide through flame to heat-fix specimen. Flood the smear with stain, rinse, and dry. Copyright © The McGraw-Hill Companies, Inc. Examine with microscope. DYES AND STAINS • Basic dyes (positive charge) Ø Attracted to negatively charged cellular components • Acidic dyes (negative charge) Negative staining: cells repel, so colors background Ø Can be done as wet mount Ø Drop of liquid containing specimen is spread (thinly) over slide. Allow to air dry. Forms a film or smear Pass slide through flame to heat-fix specimen. Flood the smear with stain, rinse, and dry. Copyright © The McGraw-Hill Companies, Inc. Examine with microscope. DYES AND STAINS • Basic dyes (positive charge) Ø Type of dyes most commonly used Ø Attracted to negatively charged cellular components Ø E.g. methylene blue, crystal violet • Acidic dyes (negative charge) Do not stain cells Ø Negative staining: colors background dark while cells appear as clear or white Ø Can be done as wet mount Ø Ø Avoid heat-treating slide – which can distort cell shape DYES AND STAINS • Simple staining - involves one dye • Differential staining - distinguish different types of bacteria Copyright © The McGraw-Hill Companies, Inc. GRAM STAIN • Most commonly used stain for bacteria • Developed by Dr. Hans Christian Gram • Distinguishes Gram +ve from Gram –ve bacteria Ø Fundamental difference in cell wall composition © Leon J. Le Beau/Biological Photo Service 10 µm Copyright © The McGraw-Hill Companies, Inc. GRAM STAIN Steps in Staining State of Bacteria 1 Crystal violet Cells stain purple. 2 Iodine Cells remain purple. 3 Alcohol Gram-positive cells remain purple; Gram-negative cells become colorless. 4 Safranin Gram-positive cells remain purple; Gram-negative cells appear pink. (primary stain) (mordant) (decolorizer) (counterstain) Appearance Copyright © The McGraw-Hill Companies, Inc. ACID-FAST STAIN • Detect small group of organisms that do not readily take up stain Ø E.g. Mycobacterium (genus) • Cell wall contains high concentrations of mycolic acid Waxy fatty acid that prevents uptake of dyes Ø Harsh methods needed Ø • Used to presumptively identify clinical specimens STAINS TO OBSERVE CELL STRUCTURES C APSULE STAIN • Some microbes surrounded by gel-like layer (i.e., capsule) Ø Protective role & increases pathogenicity • Capsules stain poorly Negative stain often used Ø India ink added to wet mount is common method Ø 10 µm © Dr Gladden Willis/Visuals Unlimited/Getty Copyright © The McGraw-Hill Companies, Inc. ENDOSPORE STAIN • Members of genera including Bacillus, Clostridium form resistant, dormant endospore Ø Resists Gram stain, often appears as clear object • Endospore stain Ø Ø Uses heat to facilitate uptake of primary dye (usually malachite green) by endospore Counterstain (usually safranin) used to visualize other cells © Jack M. Bostrack/Visuals Unlimited 10 µm Copyright © The McGraw-Hill Companies, Inc. FLAGELLA STAIN • Flagella used for prokaryotic motility Ø Too thin to be seen with light microscope Ø Flagella stain coats flagella to thicken and make visible Ø Presence and distribution of flagella can aid with identification © E. Chan/Visuals Unlimited 1 µm Copyright © The McGraw-Hill Companies, Inc. FLUORESCENT DYES AND TAGS Auramine dye • Some fluorescent dyes bind to compounds found in all cells Ø Acridine orange, Ethidium Bromide – DNA • Some bind to structures found on certain microbes Ø Auramine – mycolic acid in Mycobacterium sp. © Richard L. Moore/Biological Photo Service Living vs. Dead cells • Some are changed by cellular processes Ø Distinguish between living and dead cells Courtesy of Molecular Probes, Eugene Copyright © The McGraw-Hill Companies, Inc. IMMUNOFLUORESCENCE Immunofluorescence Fluorescent Ab – S. pyogenes • Technique used to tag specific proteins with a fluorescent compound • Uses an antibody to deliver the fluorescent tag • Tagging a protein unique to a microbe can detect that organism © Evans Roberts Copyright © The McGraw-Hill Companies, Inc. FLUORESCENCE MICROSCOPES Actin = red CEACAM = green DNA = blue Bacteria = cyan Image from Anna Sintsova, Lab of Dr.. Scott Gray-Owen Published in PLoS Pathogens (2014) DOI: 10.1371/journal.ppat.1004341 INTRODUCTION TO BACTERIA SHAPES Coccus Rod (bacillus) • Two types most common Coccus: spherical Ø Rod (bacillus): cylindrical Ø 1 µm 11.4 µm © SciMAT/Photo Researchers, Inc. • Variety of other shapes Ø © Dennis Kunkel Microscopy inc. Copyright © The McGraw-Hill Companies, Inc. Vibrio, spirillum, spirochete Vibrio Spirochete Spirillum 15 µm 15 µm 7.5 µm Copyright © The McGraw-Hill Companies, Inc.; © Dennis Kunkel Microscopy inc. STAPHYLOCOCCUS AUREUS SALMONELLA TYPHIMURIUM Images from the Public Health Image Library (phil.cdc.gov) Bacteria Modified from the “interactive tree of life” (http://itol.embl.de) Staphylococcus aureus Listeria E. coli Haemophilus = coccus = rod Bacteria Modified from the “interactive tree of life” (http://itol.embl.de) GROUPINGS Chains • Most prokaryotes divide by binary fission Ø Ø Cells often stick together following division Diplococcus © George Musil/Vis uals Unlimited Cell divides in one plane. Chain of cocci Form characteristic groupings © David M. Phillips /Visuals Unlimited Ø Neisseria gonorrhoeae Packets (diplococcus) Ø Streptococcus Ø Sarcina (long chains) (cubical packets) Ø Staphylococcus clusters) Cell divides in two or more planes perpendicular to one another. Packet © R. Kess el & C. Shih/Visuals Unlimited Clusters (grapelike Cell divides in several planes at random. Cluster © Oliver Mecks /Photo Researchers, Inc. Copyright © The McGraw-Hill Companies, Inc STAPHYLOCOCCUS VS. STREPTOCOCCUS Images from the Public Health Image Library (phil.cdc.gov) STRUCTURAL FEATURES Pilus Ribosomes Cytoplasm Chromosome (DNA) Nucleoid Cell wall Flagellum Copyright © The McGraw-Hill Companies, Inc. Capsule Cell wall Cytoplasmic membrane 0.5 µm Courtesy of L. Santo et al., J. Bacteriology 99:824, 1969. American Society for Microbiology THE CYTOPLASMIC MEMBRANE • Thin, delicate membrane that surrounds the cytoplasm & defines the boundary of the cell • Critical permeability barrier between cell & external environment • Structure Ø Phospholipid bilayer embedded with proteins Phospholipid bilayer Hydrophobic tails face in Ø Hydrophilic tails face out Ø Serves as semipermeable membrane Ø Hydrophilic head Proteins Hydrophobic tail PERMEABILITY OF THE LIPID BILAYER • Cytoplasmic membrane is selectively permeable O2, CO2, N2, small hydrophobic molecules, and water pass freely Ø Some cells facilitate water passage with aquaporins Ø Other molecules must be moved across membrane via transport systems Ø Pass through easily: Passes through: Gases (O2, CO2, N2) Water Small hydrophobic molecules Do not pass through: Sugars Ions Amino acids ATP Macromolecules Water Aquaporin Copyright © The McGraw-Hill Companies, Inc. STRUCTURAL FEATURES Pilus Ribosomes Cytoplasm Chromosome (DNA) Nucleoid Cell wall Flagellum Copyright © The McGraw-Hill Companies, Inc. Capsule Cell wall Cytoplasmic membrane 0.5 µm Courtesy of L. Santo et al., J. Bacteriology 99:824, 1969. American Society for Microbiology DIRECTED MOVEMENT OF MOLECULES ACROSS CYTOPLASMIC MEMBRANES • Most molecules must pass through proteins functioning as selective gates Termed transport systems (permeases or carrier) Ø Move nutrients, small molecules, waste & other compounds Ø Membrane-spanning Ø Highly specific: carriers transport certain molecule type Ø 1 Transport protein recognizes a specific molecule. 2 Binding of that molecule changes the shape of the transport protein. Copyright © The McGraw-Hill Companies, Inc. 3 The molecule is released on the other side of the membrane. TYPES OF TRANSPORT SYSTEMS Facilitated Diffusion Active Transport Transported substance Binding protein H+ H+ H+ Group Translocation H+ P P P ATP P P ADP H+ Copyright © The McGraw-Hill Companies, Inc. R P + Pi P R P P TYPES OF TRANSPORT SYSTEMS Transported substance FACILITATED DIFFUSION • Form of passive transport • Movement down gradient; no energy required • Not useful in low-nutrient environments • Rarely used by prokaryotes Facilitated diffusion Transporter allows a substance to move across the membrane, but only down the concentration gradient. Copyright © The McGraw-Hill Companies, Inc. TYPES OF TRANSPORT SYSTEMS ACTIVE TRANSPORT • Movement against a concentration gradient • Requires energy Use proton motive force Ø Use ATP (ABC transporter) Binding protein H+ H+ H+ H+ H+ Ø • Commonly used by bacteria Active transport - using proton motive force as an energy source. P P P ATP P P ADP + Pi Active transport - using ATP as an energy source. A binding protein gathers the transported molecules. Transporter uses energy (ATP or proton motive force) to move a substance across the membrane and against a concentration gradient. Copyright © The McGraw-Hill Companies, Inc. PROTEIN SECRETION • Active movement of proteins out of cell Examples: extracellular enzymes, external structures Ø Proteins tagged for secretion via signal sequence of amino acids Ø Prokaryotes use variety of secretion systems Ø Macromolecule Extracellular enzyme Subunit of macromolecule Signal sequence Preprotein P P P ATP P P ADP + Pi The signal sequence on the preprotein targets it for secretion and is removed during the secretion process. Once outside the cell, the protein folds into its functional shape. Extracellular enzymes degrade macromolecules so that the subunits can then be transported into the cell using the mechanisms shown in figure 3.29. THE CELL WALL • Cell wall is strong, rigid structure that prevents cell lysis • Architecture distinguishes two main types of bacteria Ø Gram-positive Ø Gram-negative • Made from peptidoglycan Ø Found only in bacteria Copyright © The McGraw-Hill Companies, Inc. BACK TO THE GRAM STAIN… © Leon J. Le Beau/Biological Photo Service Copyright © The McGraw-Hill Companies, Inc. THE GRAM STAIN DISTINGUISHES THE MAJOR TYPES OF BACTERIA Copyright © The McGraw-Hill Companies, Inc. Peptidoglycan (cell wall) Gel-like material Cytoplasmic membrane Copyright © The McGraw-Hill Companies, Inc. GRAM-NEGATIVE CELL ENVELOPE • Has thin peptidoglycan layer between two membranes • Outer membrane is unique lipid bilayer embedded with protein Porin protein Peptidoglycan Outer membrane Lipopolysaccharide (LPS) Cytoplasmic membrane Periplasm Outer membrane (lipid bilayer) Lipoprotein Periplasm Outer Cytoplasmic Peptidoglycan membrane Periplasm membrane Peptidoglycan Cytoplasmic membrane (inner membrane; lipid bilayer) 0.15 µm © Terry Beveridge, University of Guelph Copyright © The McGraw-Hill Companies, Inc. Porin protein Lipopolysaccharide (LPS) Outer membrane (lipid bilayer) Lipoprotein Periplasm Peptidoglycan Cytoplasmic membrane (inner membrane; lipid bilayer) Copyright © The McGraw-Hill Companies, Inc. CELL WALL TYPE & THE GRAM STAIN • Crystal violet stains inside of cell, not cell wall • Gram-positive cell wall retain dye Ø Prevents crystal violet–iodine complex from being washed out Ø Decolorizing agent thought to dehydrate thick layer of peptidoglycan Ø Desiccated state acts as barrier, preventing dye from leaving • Gram-negative cell wall lose dye Ø Solvent action of decolorizing agent damages outer membrane of Gram-negatives Ø Thin layer of peptidoglycan cannot retain dye complex © Leon J. Le Beau/Biological Photo Service 10 µm Copyright © The McGraw-Hill Companies, Inc. Modified from the “interactive tree of life” (http://itol.embl.de) Mycobacterium* is a “Grampositive” but it produces a waxy coat that repels the Gram stain. The acid-fast stain must be used for these bacteria. Staphylococcus (Staph) aureus Clostridium (C diff) Listeria Bacillus (anthrax) Streptococcus (strep throat, “flesh eating” bacteria, childbed fever) E. coli Salmonella Yersinia (plague) Haemophilus (pneumonia) Vibrio (cholera) Mycobacterium* (leprosy, tuberculosis) Bordetella (whooping cough) Neisseria (gonnorrhea) Legionella (Legionaire’s disease) Corynebacterium (diphtheria) Bacteria Borellia (Lyme disease) Treponema (syphilus) THE CELL ENVELOPE IS IMPORTANT BECAUSE… THE BACTERIAL CELL ENVELOPE Architecture distinguishes two main types of bacteria Ø Gram-positive Ø Gram-negative • Rigid cell wall is made from peptidoglycan Ø Found only in bacteria Copyright © The McGraw-Hill Companies, Inc. PEPTIDOGLYCAN N-acetylmuramic acid (NAM) CH 2 OH CH 2 OH O H O O HC C Alternating series of subunits form glycan chains Ø N-acetylmuramic acid (NAM) Ø N-acetylglucosamine (NAG) Ø Tetrapeptide chain (string of four amino acids) links glycan chains O H O H • Cell wall is made from peptidoglycan N-acetylglucosamine (NAG) H CH 3 O H NH C OH H H NH O O H C CH 3 Chemical structure of N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM); the ring structure of each molecule is glucose. O CH 3 OH NAG NAM Ø NAG NAM Glycan chain Peptidoglycan Peptide interbridge (Gram-positive cells) Tetrapeptide chain (amino acids) Glycan chains are composed of alternating subunits of NAG and NAM. They are cross-linked via their tetrapeptide chains to create peptidoglycan. NAM NAG NAM NAM NAG NAG Glycan chain Interconnected glycan chains form a large sheet. Multiple connected layers create a three-dimensional molecule. Copyright © The McGraw-Hill Companies, Inc. Tetrapeptide chain (amino acids) Peptide interbridge The bacterial cell wall peptidoglycan is built from subunits made of sugar (glycan) and peptides wall glycan N-acetyl-glucosamine (NAG) N-acetyl-muramic acid (NAM) This is one “cell wall subunit”. Each subunit has two similar sugars, NAG and NAM. wall peptide N NAG is found in a lot of species including mammals. NAM is a derivative of NAG that can link to the Nterminus of the wall peptide. It is unique to bacteria. C-terminus The wall peptides are short and have unique “D” amino acids L-alanine D-iso-glutamine L-Lysine OR mDAP (meso-diaminopimilic acid) D-alanine D-alanine C-terminus N wall peptide The polymerization of subunits via their sugars leads to the formation of long glycan strands with alternating NAM and NAG sugars – the strands can comprise dozens of subunits N NAG NAM NAG NAM NAG NAM N N N N N Transpeptidation: the side-chain amino group of an amino acid in one wall peptide (lysine or mDAP) is linked to the C-terminus of a neighboring wall peptide (after a D-alanine is removed) A new bond is formed making a link between neighboring wall subunits. C-terminus The final result is a large molecular net that covers the entire cell These two drawings show the same thing in different ways. On the right (from the textbook) the NAM and NAG sugars are shown as light and dark spheres and the crosslinked wall peptides are the sticks between them. = GRAM-POSITIVE CELL WALL • Has thick peptidoglycan layer As many as 30 layers of glycan chains Ø Permeable (e.g. sugars, amino acids) Ø Peptidoglycan and teichoic acids Gel-like material NAG NAM Teichoic acid Peptidoglycan (cell wall) Cytoplasmic membrane Gram-positive Peptidoglycan 0.15 µm Gel-like material Cytoplasmic membrane © Terry Beveridge, University of Guelph Cytoplasmic membrane Copyright © The McGraw-Hill Companies, Inc. GRAM-POSITIVE CELL WALL • Teichoic acids -ve charged chains of ribitol-phosphate or glycerolphosphate subunits Ø Attached to sugars & D-alanine NAG NAM Ø Stick out above peptidoglycan Ø Function not understood Ø Reservoir for cations Ø Cell wall construction Ø Cell Division Peptidoglycan (cell wall) Ø Ø Ø Gel-like material Link to pepdidoglycan Ø Cytoplasmic membrane Wall teichoic acid Linked to cytplasmic membrane Ø Teichoic acid Lipoteichoic acid Copyright © The McGraw-Hill Companies, Inc. GRAM-NEGATIVE CELL WALL • Has thin peptidoglycan layer • Outer membrane is unique lipid bilayer embedded with proteins Ø Joined to peptidoglycan by lipoproteins Porin protein Peptidoglycan Outer membrane Lipopolysaccharide (LPS) Cytoplasmic membrane Periplasm Outer membrane (lipid bilayer) Lipoprotein Periplasm Outer Cytoplasmic Peptidoglycan membrane Periplasm membrane Peptidoglycan Cytoplasmic membrane (inner membrane; lipid bilayer) 0.15 µm © Terry Beveridge, University of Guelph Copyright © The McGraw-Hill Companies, Inc. ANTIBACTERIAL SUBSTANCES THAT TARGET PEPTIDOGLYCAN • Peptidoglycan makes good target since unique to bacteria Ø Can weaken to point where unable to prevent cell lysis • Penicillin interferes with peptidoglycan synthesis Prevents cross-linking of adjacent glycan chains (transpeptidation) Ø Usually more effective against Gram-positive bacteria than Gramnegative bacteria Ø Outer membrane of Gram-negatives blocks access of drug Ø Derivatives have been developed that can cross O.M. Ø • Lysozyme breaks bonds linking glycan chain Enzyme found in tears, saliva, other bodily fluids Ø Destroys structural integrity of peptidoglycan molecule Ø OUTER MEMBRANE Porin protein Lipopolysaccharide (LPS) Outer membrane (lipid bilayer) Lipoprotein • Bilayer made from lipopolysaccharide (LPS), not phospholipid • Medically important: Can cause symptoms characteristic of infections by live bacteria LPS portion of gram-negative bacteria recognized by immune system Ø Small levels elicit appropriate immune response to eliminate threat Ø Large amounts accumulating in bloodstream can yield deadly response Ø LPS is called endotoxin Ø OUTER MEMBRANE Porin protein O antigen (varies in length and composition) Lipopolysaccharide (LPS) Core polysaccharide Outer membrane (lipid bilayer) Lipid A Lipoprotein • Notable components of LPS Ø Lipid A (part of LPS recognized by immune system) Ø Ø Anchors LPS to lipid bilayer O antigen (can be used to identify species or strains) Ø Composed of sugar molecules (number & type vary) • Outer membrane blocks passage of damaging molecules Includes certain antibiotics – less sensitive to such medication Ø Small molecules and ions can cross via porins Ø LPS structure from Salmonella (Hep) L-glycerol-D-manno-heptose (Gal) galactose (Glc) glucose (KDO) 2-keto-3-deoxyoctonic acid (NGa) N-acetyl-galactosamine (NGc) N-acetyl-glucosamine Ohno and Morrison 1989 KEY POINTS ABOUT O-ANTIGEN PERIPLASM OF GRAM-NEGATIVE BACTERIA Periplasm Peptidoglycan • Between cytoplasmic membrane and outer membrane is the periplasmic space Ø Filled with gel-like periplasm Ø Periplasm filled with proteins because exported proteins accumulate unless specifically moved across outer membrane – porin protein channels are too small for proteins to pass through BACTERIA THAT LACK PEPTIDOGLYCAN • Some bacteria lack a cell wall Ø Mycoplasma species have extremely variable shape Ø Penicillin, lysozyme do not affect Ø Cytoplasmic membrane contains sterols that increase strength 2 µm Courtesy of Dr. Edwin S. Boatman Copyright © The McGraw-Hill Companies, Inc. C APSULES & SLIME LAYERS • Gel-like layer outside cell wall that protects or allows attachment to surface 2 µm Capsule: distinct, gelatinous Ø Slime layer: diffuse, irregular Ø Most composed of glycocalyx (sugar shell) although some are polypeptides Ø • Allow bacteria to adhere to surfaces Once attached, cells can grow as biofilm Ø Polysaccharide encased community Ø Example: dental plaque Ø Some capsules allow bacteria to evade host immune system Ø Cell in intestine Capsule Courtesy of K.J. Cheng and J. W. Costerton 1 µm Courtesy of A. Progulske and S.C. Holt, J. Bacteriology, 143:1003-1018, 1980 Copyright © The McGraw-Hill Companies, Inc. BIOFILMS FILAMENTOUS PROTEIN APPENDAGES • Appendages not essential, but give advantage FLAGELLA • Involved in motility Spin like propellers to move cell Ø Some important in disease Ø Numbers and arrangements help with characterization Ø © Fred Hossler/Visuals Unlimited • Peritrichous: distributed over entire surface • Polar flagellum: single flagellum at one end of cell Ø Some bacteria have tuft (multiple) at one or both ends 1 µm © Science VU/Visuals Unlimited Copyright © The McGraw-Hill Companies, Inc. 1 µm FILAMENTOUS PROTEIN APPENDAGES FLAGELLA STRUCTURE Flagellin FILAMENT • Filament HOOK Composed of flagellin Ø Hollow core Ø Flagellum • Hook Ø Connects filament to surface • Basal body Ø E. coli BASAL BODY Anchors to cell wall & cytoplasmic membrane Harvests the energy of the proton motive force to rotate the flagellum. FILAMENTOUS PROTEIN APPENDAGES PILI • Shorter & thinner than flagella Sex pilus Flagellum • Functionally different than flagella • Types that allow surface attachment termed fimbriae Other pili Courtesy of Dr. Charles Brinton, Jr. 1 µm Epithelial cell • Twitching motility, gliding motility involve pili Bacterium • Sex pilus used to join bacteria for DNA transfer Bacterium with pili U.S. Dept of Agriculture/Harley W. Moon 5 µm Copyright © The McGraw-Hill Companies, Inc.