일반미생물 강의 3장 세포생물학(출처:연세대학교 생명공학과)

[colony]

Chapter 3. Cell Biology

Microscopes, a major tool of the microbiologist
Structure and function of cellular components
Comparison between prokaryotic and eukaryotic cells

3.1 Light Microscopy

Resolution limit of light microscope: 0.2 mm
(Resolution of electron microscope: 1000-fold higher than that of light microscope)

(1) The Compound Light Microscope

A. Bright-field
-consists of objective and ocular lens
-low contrast with the surrounding medium (immersion oil on lens can increase the resolution power).
-Staining: increases the contrast for bright-field microscopy.
O Dyes can be used to stain cells and increase their contrast, allowing better visualization by bright-field miroscope.
O Positively charged dyes (cationic): methylene blue, safranin, and crystal blue; binds to the negatively charged cellular constituents such as nucleic acid and polysaccharides
O Procedures: spreading cells on slideàair dryàquick passing over flame to fixàstaining with dyesàexamine
O Gram staining: fixing cells on slideàstaining with crystal violet (purple staining)àdecolorization with ethanol (G+: purple; G-: colorless)àcounterstaining with safranin (G-: pink to red)

B. Phase contrast
-improved contrast between cells and the surrounding
-used to observe wet-mounting (living) preparations

C. Dark-field microscope
-only the light scattered from the specimen reaches to the lens with a dark background.
-often used to observe the mobility of microorganisms.

D. Fluorescence miroscope
-used to visualize specimens that fluoresce (emitting light derived from fluorescent substances such as chlorophyll or dyes used to stain cells)

3.2 Three-Dimensional Imaging: Interference Contrast, Atomic Force, and Confocal Scanning Laser Microscopy

(1) Differential Interference Contrast (DIC) Microscopy
-Two distinct beams generated by passing the polarized light through prism traverse the specimens.
-useful to observe the internal cell structure using unstained cells

(2) Atomic Force Microscopy (AFM)
-useful for three-dimensional imaging of biological structure
-images similar to those obtained from the scanning electron microsope.
-No coating or fixing is required to prepare the specimens.
-used for viewing of living and hydrated specimens.

(3) Confocal Scanning Laser Microscopy
-computerized microscope allowing for the three dimensional images of microorganisms
-Cells can be stained with fluorescent dyes.
-useful to examine microbial content with depth.

(4) Electron microscope
TEM (transmission electron microscope: used for studying the internal structure) and

SEM (scanning electron microscope: for whole organisms and their surface structures)



3.4 Overview of Cell Structure and the Significance of Smallness

(1) The Prokaryotic Cell

A. Cell wall -- cell membrane -- cytoplasm (ribosome; inclusion consisting of storage material; nucleoid, an aggregated form of chromosomal DNA; flagella)

B. Morphology (shape) of Prokaryotes
Coccus (cocci): spherical or ovoid shape
Rod
Spirilla

C. Grouped or clustered or rearranged after cell division
Spirochetes: tightly coiled bacteria
Appendaged bacteria: possess extension of cells as long tubes or stalks
Filamentous bacteria: long, thin cells or chains of cells

(2) The Eukaryotic Cell
-has true nuclei, true spherical membrane-enclosed structure, which are duplicated during nuclear division called mitosis.
-have organelles such as mitochondria (respiration) and chloroplast (contains chlorophyll and is involved in photosynthesis in algae and green plant)

(3) The size of Microbial Cells and the Significance of Being Small
-0.1-0.5 mm to 50 mm (surgeonfish symbiont, Epulopiscium fishelsoni)
-Rod-shaped bacteria, E. coli:1 X 3 mm
-Typical eukaryotes: 2 mm to 200 mm in diameter
-Cells with a smaller diameter have a higher ratio of surface area to volume, leading a more efficient exchange of nutrients.

3.5 Cytoplasmic Membrane: Structure
-about 8 nm thick structure surrounding cells and separating cytoplasm from environment.
-a highly selective barrier, enabling cells to concentrate specific metabolites and excrete waste materials.

(1) Chemical Composition of Membranes
-Phospholipid bilayer consisting of hydrophobic (fatty acid) and hydrophilic (glycerol) moieties, where proteins are embedded (hydrophobic surface is spanned the membrane).

(2) Other Features of the Cytoplasmic Membrane
-has membrane-bound proteins: protein facing the environment (for substrate binding and transport or process of large molecules into the cells), protein facing the cytoplasm (for energy yielding).
-Some of these peripheral membrane proteins are lipoproteins and contain lipid tails on the amino terminus of the protein, which anchors the protein into the membrane.
-quite fluid; lipid and protein molecules have a freedom to move about the membrane surface (fluid mosaics).

(3) Membrane Strengthening Agents: Sterols and Hopanoids
-sterols (rigid and planner molecule) making up 5 to 25% of the total lipids of eukaryotic cells: enabling eukaryotic cells (no cell wall in animal cells) to endure greater physical stress on the membrane.
-hapanoids present in several bacteria play a similar role.

(4) Archaeal Membranes
-Ether linkage between fatty acids and the glycerol.
-Glycerol diethers and glycerol tetraethers are the major classes of lipid in Archaea.
-Diglycerol tetraethers yield a lipid monolayer (not bilayer) in Archaea by forming covalent bonds between the phytanyl side chains.
-Resistance to peeling apart the lipid monolayer (conferred by lipid monolayer) allows growth of thermophilic Archaea and other prokaryotes living at very high temperature.

3.6 Cytoplsmic Membrane: Function

-Permeability barrier: by the hydrophobic nature of the membrane; only small hydrophobic molecules can pass through by diffusion, but hydrophilic and charged molecules do not cross the membrane.
-Protein anchoring: proteins involved in transport, bioenergitics, and chemotaxis are anchored in the membrane.
-Energy conservation: proton motive force is generated and used.

(1) The Necessity of transport Proteins
-allow accumulating solutes inside the cell against the concentration gradient.

(2) Structure and Function of Membrane Transport Proteins
-Simple transporter (Symporter)
-Group translocation
-ABC system transport

-In prokaryotes, membrane-spanning transporter contains typically 12 a-helices (spanning in the membrane).

(3) 3 different transport events: Uniporter, Antiporter, and Symporter
-Simple transporter (symporter): driven by the proton motive force
LacY permease: one lactose transport along with one proton

-Group translocation:
requires energy and results in “chemical modification” of the substance transported.
phosphotransferase system: uptake of glucose as G-6-P

-ATP-binding cassette (ABC) system:
Uptake of maltose
Trapping of transported substance with a high affinity by a periplasmic binding proteinà transport occurs by the APT-generated energy.

(4) Types of transport
-Uniporters: proteins that simply transport a molecule in a unidirectional fashion
-Antiporters: proteins that transport a substance across the membrane in one direction while at the same time transporting a second substance in the apposite direction.
-Symporters: proteins that transport a substance along with another substance, frequently a proton (H+)

3.7 The Cell Wall of Prokaryotes: Peptidoglycan and Related Molecules

-Functions cell wall: conferring shape and rigidity to the cell
-Bacteria: G+ and G- ; based of gram staining procedure
G+: thicker single layer of cell wall
G-: a multilayered complex structure

(1) Peptidoglycan
-composed of two major sugar derivatives (N-acetylglucosamine and N-acetylmuramic acid) and a small group of amino acids consisting of (L-alanine, D-glutamic acid, either lysine or diaminopimeric acid)à form repeating structure, the glycan tetrapeptide.
-Sheet formation by peptide cross-links and glycosidic bonds
-G+ cell wall: (a) 90% peptidoglycan (several layers) and small amount of teichoic acid and (b) peptide interlinkage-the kinds and numbers of cross-linking amino acids vary.
-G- cell wall: (a) only about 10% peptidoglycan and (b) a single layer.


(2) Diversity in peptidoglycan
-Main backbone: glucosamine and muramic acid
-Muramic acids are cross-linked with amino acids, which are very diverse in numbers and types.
-More than 100 different peptidoglycan types are known.

(3) Teichoic acids and a summary of the Gram-positive wall
-In G+ cell wall, teichoic acid contributes partially for the negative charge of the cell surface, which may affect passages of ions.
-Sometimes, it binds to lipid to form lipoteichonic acid.

(4) Protoplast formation
-Treatment of cells with lysozyme (cut 1,4-glycosidic bonds) in proper concentration of a solute, such as sucroseà protoplast (no cell wall) or spheroplast (contains still pieces of cell wall)
-Putting the protoplast into low solute solution results in bursting of cells: Cell lysis by osmotic shock

(5) Pseudopeptidoglycan and other cell walls of Archaea.
-Types of Archaea cell wall

O N-acetylglucosamine connected with N-acetyltalosaminuronic acid by a b1à3 linkage
O Some of them contains no peptidoglycan, instead it has polysaccharide, glycoprotein, or protein

3.8 The Outer Membrane of Gram-Negative Bacteria

-Lipopolysacchride layer (LPS) or outer membrane: consisted of phospolipid, polysaccharide, and protein.
-Lipid and polysaccharide are linked in the outer layer to form specific lipopoysacchiride structures.

(1) Chemistry of LPS
-O-specific polysaccharide: six carbon sugars such as glucose, galactose, rhamnose, and mannose, as well as unusual dideoxy sugars in 4-to 5 units with a branch occasionally.
-Core polysaccharide: consists of ketodeoxyoctonate (KDO; connected to lipid A), heptose (seven-carbon sugar), glucose, galoctose, and N-acetylglucosamine.
-Lipid A: fatty acids connected by ester amine linkage to a disaccharide composed of N-acetylglucosamine phosphate, which is connected to core-saccharides through KDO.

(2) Endotoxin
-Pathogenic G- bacteria such as Salmonella, Shigella, and Escherichia: lipid A of LPS is responsible for endotoxin activity of these pathogens.

(3) Porins and the periplasm
-Proteins in periplasm: porins, hydrolytic enzymes (function in the initial degradation of food molecules), binding protins (transporting substrates), chemorecptors (involved in chemotaxis response)
-Porins: transmembrane protein in the outer membrane responsible for the uptake of small molecules through the lipid bilayer
(a) specific and nonspecific porins involved in transport of small molecules (b) transmembrane protein consisted of three identical subunits: form small membrane holes about 1 nm in diameter.

(4) Relationship of cell wall structure to the Gram stain
-In G+ bacteria: several layers of peptidoglycan to form thick cell wall: insoluble crystal violet-iodine complex is trapped inside of cell by closing the pores during alcohol destaining.
-In G- bacteria: the complex is washed out by alcohol penetrating the lipid-rich outer layer and the thin peptidoglycan layer.

3.9 Cell Wall Synthesis and Cell Division

-Autolysine-mediated formation of wall-bands across the openings: essential for cell wall splicing

(1) Biosynthesis of Peptidoglycan
-Connection of bactoprenol (C55 isoprenoid) to N-acetylmuramic acid to which a peptapeptide is attached à transport of the building blocks across the membrane (bactoprenol renders sugar intermediates hydrophobic to pass through the hydrophobic cytoplasmic membrane)

(2) Transpeptidation: The penicillin target
-occurs outside of cytoplasmic membrane without requirement of energy.
-cross-linking between the D-Ala and DAP with release of a D-Ala at the end of the peptapeptide residue.
-Penicillin brocks the linkage formation and lyses the cells only when the penicillin is added to the growing cells since the action of autolysin is necessary.

3.10 Arrangement of DNA in Prokaryotes

-In bacteria: genetic information resides in chromosomal DNA and plasmid.

(1) Supercoiling and chromosomal structure
-Circular formàsupercoiling stabilized by structural protein.
-E. coli: 4600 kilobase pairs
-Nucleoid: aggregated form of bacterial chromosomal DNA

(2) Chromosomal copy number
-Prokaryotes: only one copy chromosome (haploid)
3.11 Flagella and Mobility
-Mobility conferred by flagella allows the cell to reach different regions of its environment.

(1) Bacterial flagella
-Different flagella arrangement: polar, lophotrichous (group of flagella at one end of cell), and peritrichous (around the cell surface)

(2) Structure
-made of over 40 gene products, called fla, fli, and flg
-Filament (consisted of flagellin) --- hook --- basal body (across the cell membrane and cell wall; consisted of several rod-shape rings in cell wall and cell membrane, inner rings in cytoplasmic membrane are surrounded by Mot protein responsible for torque generation).
-CW and CCW rotations determine the direction of movement, which is controlled by the Fli protein in between two rings in cytoplasmic membrane.

3.12 Bacterial Behavior: Chemotaxis, Phototaxis, and Other Taxes

(1) Chemotaxis
-Random movement by runs and tumbles in the absence of a chemical attractant or repellant, but swimming toward or backward in the presence of chemical signals.
-Measuring chemotaxis
O Chemoreceptors in the membrane sense the chemical gradient with time and interact with the cytoplasmic proteins to affect flagellar motor direction.
O Measuring of chemotaxis by using a capillary tube containing an attractant or repellant à count the cell number in the tube.

(2) Phototaxis
-Scotophobotaxis: move toward the region of the light wavelengths at which bacterial pigments absorb.
-Phototaxis: a directed movement up a light gradient toward an increasing intensity of light.
-Photoreceptor: able to sense a gradient of light and interact with the proteins that affect flagella rotation to maintain the cell in a run if it is swimming toward an increasing intensity of light.
(3) Other Taxes
-Aerotaxis (depending on oxygen) and osmotaxis (depending on ionic strength)

3.13 Bacterial Cell Surface Structures and Cell Inclusions

(1) Fimbriae
-not involved in motility,
-shorter than flagella but more numerous
-involved in attachment of some of pathogenic bacteria on the surface of animal tissue or in the formation of biofilms on surfaces.

(2) Pili
-longer than fimbriae
-involved in conjugation in bacteria.

(3) Paracrystalline surface layers (S-layers)
-associated with a variety of cell wall structures, including the LPS of gram-positive bacteria.
-Functions in (1) external permeability barrier and (2) protection of pathogenic bacteria against host defense.
-found in some bacteria and most of Archaea
-composed of protein or glycoprotein to form a two-dimensional array of protein.

(4) Capsules and slime layers: the glycocalyx
-secreted slimy or gummy material on the cell surface.
-mainly consists of polysaccharides and glycoprotein.

-Capsule: a tight rigid matrix excluding particles such as India ink

-Slime layer: less rigid than capsule
-involved in attachment of pathogenic bacteria to host.
-enabling pathogenic encapsulated-pathogenic bacteria to resist against phogocytic functions of immune system.

(5) Carbon storage polymers
-Poly-b-hydroxybutylic acid (PHB): a lipid-like compound formed from b-hydroxybutylic acid units
-Glycogen: (a) polymer of glucose, (b) can be stained by dilute iodine (red-brown color), and (c) storage depot for carbon and energy

(6) Other storage materials and inclusions
-Polyphosphate
-Sulfur granules in bacteria capable of oxidizing reduced sulfur compounds
-Magnetosomes (crystal particles of the iron mineral magnetite, Fe3O4) in magnetic bacteria

3.14 Gas Vesicles
-confer buoyancy on the cells.
-found in cyanobacteria, purple and green phototrophic bacteria.
-composed of protein to form hollow, rigid, spindle-shaped structures.

3.15 Endospores
-Structures within the cell, which is resistant to heat and chemicals, and non permeable for general staining.
-Calcium-dipicolinic acid represents about 10% of the dry mass of the endospores.
-From out layer: exosporium - spore coat – cortex – core

(1) Properties of the endospore core
-low (10-30%) contents of water
-lower pH of the core cytoplasm compared with that of vegetative cells
-binding of small acid-soluble spore protein (SASPs) to DNA: protect DNA from potential damage from UV, desiccation, and dry heat.

(2) Endospore formation
-when vegetative growth ceases (by limited nutrients such as the carbon and nitrogen source)
à 200 genes are involved.

(3) Germination
-Activation (heating of freshly formed spores for several minutes at a sublethal temperature)àgerminationàoutgrowth (uptake of water and synthesis of new RNA, protein, and DNA)
3.16 The Nucleus and Organelles of Eukaryotic Microorganisms

(1) Nuclear structure
-a pair of unit membranes
-contains many pores composed of several proteins
-Functions of pore: import and export of substances into and out of the nucleus.
-Nucleolus: an area rich in RNA that is the site of ribosomal RNA synthesis.
-Ribosomal proteins synthesized in the cytoplasm are transported into the nucleolus and combined with ribosomal RNA to form the small and large subunits of the eukaryotic ribosome.

(2) Mitochondria
-Site of respiration and oxidative phosphorylation
-covered with phospholipid bilayer, permeable for small organic molecules such as ATP, which is produced in cristae, a folded inner membrane, and transported to cytoplasm to be used in energy-requiring reactions.
-The matrix inside contains a number of enzymes involved in the oxidation of organic compounds, in particular, enzymes if the citric acid cycle.

(3) Chloroplasts
-found in all photosynthetic eukaryotic cells.
-surrounded by very permeable outer membrane and less permeable inner membrane
-Chlorophyll and other components necessary for the photosynthesis is on flattened membrane discs called thylakoids.

(4) Relationships of organelles to prokaryotes
-Endosymbiosis theory: mitochondria and chloroplasts are descendants of ancient prokaryotic cells.
-Evidences supporting the hypothesis:
A. Mitochondria and chloroplasts contain a covalently closed circular form DNA seen in prokaryotic cells
B. Mitochondria and chloroplasts have 70S (but not 80S ribosome found in eukaryotic cells) ribosomes.
C. Antibiotics interfering with 70S ribosome function also inhibit protein synthesis in mitochondria and chloroplasts.
D. Molecular phylogeny: similar rRNA sequences
3. 17 Comparison of the Prokaryotic and Eukaryotic Cell: Table 3.3









General Microbiology DU115-01 Lecture Note 3

Chapter 3. Cell Biology

Microscopes, a major tool of the microbiologist
Structure and function of cellular components
Comparison between prokaryotic and eukaryotic cells

3.1 Light Microscopy

Resolution limit of light microscope: 0.2 mm
(Resolution of electron microscope: 1000-fold higher than that of light microscope)

(1) The Compound Light Microscope

A. Bright-field
-consists of objective and ocular lens
-low contrast with the surrounding medium (immersion oil on lens can increase the resolution power).
-Staining: increases the contrast for bright-field microscopy.
O Dyes can be used to stain cells and increase their contrast, allowing better visualization by bright-field miroscope.
O Positively charged dyes (cationic): methylene blue, safranin, and crystal blue; binds to the negatively charged cellular constituents such as nucleic acid and polysaccharides
O Procedures: spreading cells on slideàair dryàquick passing over flame to fixàstaining with dyesàexamine
O Gram staining: fixing cells on slideàstaining with crystal violet (purple staining)àdecolorization with ethanol (G+: purple; G-: colorless)àcounterstaining with safranin (G-: pink to red)

B. Phase contrast
-improved contrast between cells and the surrounding
-used to observe wet-mounting (living) preparations

C. Dark-field microscope
-only the light scattered from the specimen reaches to the lens with a dark background.
-often used to observe the mobility of microorganisms.

D. Fluorescence miroscope
-used to visualize specimens that fluoresce (emitting light derived from fluorescent substances such as chlorophyll or dyes used to stain cells)

3.2 Three-Dimensional Imaging: Interference Contrast, Atomic Force, and Confocal Scanning Laser Microscopy

(1) Differential Interference Contrast (DIC) Microscopy
-Two distinct beams generated by passing the polarized light through prism traverse the specimens.
-useful to observe the internal cell structure using unstained cells

(2) Atomic Force Microscopy (AFM)
-useful for three-dimensional imaging of biological structure
-images similar to those obtained from the scanning electron microsope.
-No coating or fixing is required to prepare the specimens.
-used for viewing of living and hydrated specimens.

(3) Confocal Scanning Laser Microscopy
-computerized microscope allowing for the three dimensional images of microorganisms
-Cells can be stained with fluorescent dyes.
-useful to examine microbial content with depth.

(4) Electron microscope
TEM (transmission electron microscope: used for studying the internal structure) and

SEM (scanning electron microscope: for whole organisms and their surface structures)



3.4 Overview of Cell Structure and the Significance of Smallness

(1) The Prokaryotic Cell

A. Cell wall -- cell membrane -- cytoplasm (ribosome; inclusion consisting of storage material; nucleoid, an aggregated form of chromosomal DNA; flagella)

B. Morphology (shape) of Prokaryotes
Coccus (cocci): spherical or ovoid shape
Rod
Spirilla

C. Grouped or clustered or rearranged after cell division
Spirochetes: tightly coiled bacteria
Appendaged bacteria: possess extension of cells as long tubes or stalks
Filamentous bacteria: long, thin cells or chains of cells

(2) The Eukaryotic Cell
-has true nuclei, true spherical membrane-enclosed structure, which are duplicated during nuclear division called mitosis.
-have organelles such as mitochondria (respiration) and chloroplast (contains chlorophyll and is involved in photosynthesis in algae and green plant)

(3) The size of Microbial Cells and the Significance of Being Small
-0.1-0.5 mm to 50 mm (surgeonfish symbiont, Epulopiscium fishelsoni)
-Rod-shaped bacteria, E. coli:1 X 3 mm
-Typical eukaryotes: 2 mm to 200 mm in diameter
-Cells with a smaller diameter have a higher ratio of surface area to volume, leading a more efficient exchange of nutrients.

3.5 Cytoplasmic Membrane: Structure
-about 8 nm thick structure surrounding cells and separating cytoplasm from environment.
-a highly selective barrier, enabling cells to concentrate specific metabolites and excrete waste materials.

(1) Chemical Composition of Membranes
-Phospholipid bilayer consisting of hydrophobic (fatty acid) and hydrophilic (glycerol) moieties, where proteins are embedded (hydrophobic surface is spanned the membrane).

(2) Other Features of the Cytoplasmic Membrane
-has membrane-bound proteins: protein facing the environment (for substrate binding and transport or process of large molecules into the cells), protein facing the cytoplasm (for energy yielding).
-Some of these peripheral membrane proteins are lipoproteins and contain lipid tails on the amino terminus of the protein, which anchors the protein into the membrane.
-quite fluid; lipid and protein molecules have a freedom to move about the membrane surface (fluid mosaics).

(3) Membrane Strengthening Agents: Sterols and Hopanoids
-sterols (rigid and planner molecule) making up 5 to 25% of the total lipids of eukaryotic cells: enabling eukaryotic cells (no cell wall in animal cells) to endure greater physical stress on the membrane.
-hapanoids present in several bacteria play a similar role.

(4) Archaeal Membranes
-Ether linkage between fatty acids and the glycerol.
-Glycerol diethers and glycerol tetraethers are the major classes of lipid in Archaea.
-Diglycerol tetraethers yield a lipid monolayer (not bilayer) in Archaea by forming covalent bonds between the phytanyl side chains.
-Resistance to peeling apart the lipid monolayer (conferred by lipid monolayer) allows growth of thermophilic Archaea and other prokaryotes living at very high temperature.

3.6 Cytoplsmic Membrane: Function

-Permeability barrier: by the hydrophobic nature of the membrane; only small hydrophobic molecules can pass through by diffusion, but hydrophilic and charged molecules do not cross the membrane.
-Protein anchoring: proteins involved in transport, bioenergitics, and chemotaxis are anchored in the membrane.
-Energy conservation: proton motive force is generated and used.

(1) The Necessity of transport Proteins
-allow accumulating solutes inside the cell against the concentration gradient.

(2) Structure and Function of Membrane Transport Proteins
-Simple transporter (Symporter)
-Group translocation
-ABC system transport

-In prokaryotes, membrane-spanning transporter contains typically 12 a-helices (spanning in the membrane).

(3) 3 different transport events: Uniporter, Antiporter, and Symporter
-Simple transporter (symporter): driven by the proton motive force
LacY permease: one lactose transport along with one proton

-Group translocation:
requires energy and results in “chemical modification” of the substance transported.
phosphotransferase system: uptake of glucose as G-6-P

-ATP-binding cassette (ABC) system:
Uptake of maltose
Trapping of transported substance with a high affinity by a periplasmic binding proteinà transport occurs by the APT-generated energy.

(4) Types of transport
-Uniporters: proteins that simply transport a molecule in a unidirectional fashion
-Antiporters: proteins that transport a substance across the membrane in one direction while at the same time transporting a second substance in the apposite direction.
-Symporters: proteins that transport a substance along with another substance, frequently a proton (H+)

3.7 The Cell Wall of Prokaryotes: Peptidoglycan and Related Molecules

-Functions cell wall: conferring shape and rigidity to the cell
-Bacteria: G+ and G- ; based of gram staining procedure
G+: thicker single layer of cell wall
G-: a multilayered complex structure

(1) Peptidoglycan
-composed of two major sugar derivatives (N-acetylglucosamine and N-acetylmuramic acid) and a small group of amino acids consisting of (L-alanine, D-glutamic acid, either lysine or diaminopimeric acid)à form repeating structure, the glycan tetrapeptide.
-Sheet formation by peptide cross-links and glycosidic bonds
-G+ cell wall: (a) 90% peptidoglycan (several layers) and small amount of teichoic acid and (b) peptide interlinkage-the kinds and numbers of cross-linking amino acids vary.
-G- cell wall: (a) only about 10% peptidoglycan and (b) a single layer.


(2) Diversity in peptidoglycan
-Main backbone: glucosamine and muramic acid
-Muramic acids are cross-linked with amino acids, which are very diverse in numbers and types.
-More than 100 different peptidoglycan types are known.

(3) Teichoic acids and a summary of the Gram-positive wall
-In G+ cell wall, teichoic acid contributes partially for the negative charge of the cell surface, which may affect passages of ions.
-Sometimes, it binds to lipid to form lipoteichonic acid.

(4) Protoplast formation
-Treatment of cells with lysozyme (cut 1,4-glycosidic bonds) in proper concentration of a solute, such as sucroseà protoplast (no cell wall) or spheroplast (contains still pieces of cell wall)
-Putting the protoplast into low solute solution results in bursting of cells: Cell lysis by osmotic shock

(5) Pseudopeptidoglycan and other cell walls of Archaea.
-Types of Archaea cell wall

O N-acetylglucosamine connected with N-acetyltalosaminuronic acid by a b1à3 linkage
O Some of them contains no peptidoglycan, instead it has polysaccharide, glycoprotein, or protein

3.8 The Outer Membrane of Gram-Negative Bacteria

-Lipopolysacchride layer (LPS) or outer membrane: consisted of phospolipid, polysaccharide, and protein.
-Lipid and polysaccharide are linked in the outer layer to form specific lipopoysacchiride structures.

(1) Chemistry of LPS
-O-specific polysaccharide: six carbon sugars such as glucose, galactose, rhamnose, and mannose, as well as unusual dideoxy sugars in 4-to 5 units with a branch occasionally.
-Core polysaccharide: consists of ketodeoxyoctonate (KDO; connected to lipid A), heptose (seven-carbon sugar), glucose, galoctose, and N-acetylglucosamine.
-Lipid A: fatty acids connected by ester amine linkage to a disaccharide composed of N-acetylglucosamine phosphate, which is connected to core-saccharides through KDO.

(2) Endotoxin
-Pathogenic G- bacteria such as Salmonella, Shigella, and Escherichia: lipid A of LPS is responsible for endotoxin activity of these pathogens.

(3) Porins and the periplasm
-Proteins in periplasm: porins, hydrolytic enzymes (function in the initial degradation of food molecules), binding protins (transporting substrates), chemorecptors (involved in chemotaxis response)
-Porins: transmembrane protein in the outer membrane responsible for the uptake of small molecules through the lipid bilayer
(a) specific and nonspecific porins involved in transport of small molecules (b) transmembrane protein consisted of three identical subunits: form small membrane holes about 1 nm in diameter.

(4) Relationship of cell wall structure to the Gram stain
-In G+ bacteria: several layers of peptidoglycan to form thick cell wall: insoluble crystal violet-iodine complex is trapped inside of cell by closing the pores during alcohol destaining.
-In G- bacteria: the complex is washed out by alcohol penetrating the lipid-rich outer layer and the thin peptidoglycan layer.

3.9 Cell Wall Synthesis and Cell Division

-Autolysine-mediated formation of wall-bands across the openings: essential for cell wall splicing

(1) Biosynthesis of Peptidoglycan
-Connection of bactoprenol (C55 isoprenoid) to N-acetylmuramic acid to which a peptapeptide is attached à transport of the building blocks across the membrane (bactoprenol renders sugar intermediates hydrophobic to pass through the hydrophobic cytoplasmic membrane)

(2) Transpeptidation: The penicillin target
-occurs outside of cytoplasmic membrane without requirement of energy.
-cross-linking between the D-Ala and DAP with release of a D-Ala at the end of the peptapeptide residue.
-Penicillin brocks the linkage formation and lyses the cells only when the penicillin is added to the growing cells since the action of autolysin is necessary.

3.10 Arrangement of DNA in Prokaryotes

-In bacteria: genetic information resides in chromosomal DNA and plasmid.

(1) Supercoiling and chromosomal structure
-Circular formàsupercoiling stabilized by structural protein.
-E. coli: 4600 kilobase pairs
-Nucleoid: aggregated form of bacterial chromosomal DNA

(2) Chromosomal copy number
-Prokaryotes: only one copy chromosome (haploid)
3.11 Flagella and Mobility
-Mobility conferred by flagella allows the cell to reach different regions of its environment.

(1) Bacterial flagella
-Different flagella arrangement: polar, lophotrichous (group of flagella at one end of cell), and peritrichous (around the cell surface)

(2) Structure
-made of over 40 gene products, called fla, fli, and flg
-Filament (consisted of flagellin) --- hook --- basal body (across the cell membrane and cell wall; consisted of several rod-shape rings in cell wall and cell membrane, inner rings in cytoplasmic membrane are surrounded by Mot protein responsible for torque generation).
-CW and CCW rotations determine the direction of movement, which is controlled by the Fli protein in between two rings in cytoplasmic membrane.

3.12 Bacterial Behavior: Chemotaxis, Phototaxis, and Other Taxes

(1) Chemotaxis
-Random movement by runs and tumbles in the absence of a chemical attractant or repellant, but swimming toward or backward in the presence of chemical signals.
-Measuring chemotaxis
O Chemoreceptors in the membrane sense the chemical gradient with time and interact with the cytoplasmic proteins to affect flagellar motor direction.
O Measuring of chemotaxis by using a capillary tube containing an attractant or repellant à count the cell number in the tube.

(2) Phototaxis
-Scotophobotaxis: move toward the region of the light wavelengths at which bacterial pigments absorb.
-Phototaxis: a directed movement up a light gradient toward an increasing intensity of light.
-Photoreceptor: able to sense a gradient of light and interact with the proteins that affect flagella rotation to maintain the cell in a run if it is swimming toward an increasing intensity of light.
(3) Other Taxes
-Aerotaxis (depending on oxygen) and osmotaxis (depending on ionic strength)

3.13 Bacterial Cell Surface Structures and Cell Inclusions

(1) Fimbriae
-not involved in motility,
-shorter than flagella but more numerous
-involved in attachment of some of pathogenic bacteria on the surface of animal tissue or in the formation of biofilms on surfaces.

(2) Pili
-longer than fimbriae
-involved in conjugation in bacteria.

(3) Paracrystalline surface layers (S-layers)
-associated with a variety of cell wall structures, including the LPS of gram-positive bacteria.
-Functions in (1) external permeability barrier and (2) protection of pathogenic bacteria against host defense.
-found in some bacteria and most of Archaea
-composed of protein or glycoprotein to form a two-dimensional array of protein.

(4) Capsules and slime layers: the glycocalyx
-secreted slimy or gummy material on the cell surface.
-mainly consists of polysaccharides and glycoprotein.

-Capsule: a tight rigid matrix excluding particles such as India ink

-Slime layer: less rigid than capsule
-involved in attachment of pathogenic bacteria to host.
-enabling pathogenic encapsulated-pathogenic bacteria to resist against phogocytic functions of immune system.

(5) Carbon storage polymers
-Poly-b-hydroxybutylic acid (PHB): a lipid-like compound formed from b-hydroxybutylic acid units
-Glycogen: (a) polymer of glucose, (b) can be stained by dilute iodine (red-brown color), and (c) storage depot for carbon and energy

(6) Other storage materials and inclusions
-Polyphosphate
-Sulfur granules in bacteria capable of oxidizing reduced sulfur compounds
-Magnetosomes (crystal particles of the iron mineral magnetite, Fe3O4) in magnetic bacteria

3.14 Gas Vesicles
-confer buoyancy on the cells.
-found in cyanobacteria, purple and green phototrophic bacteria.
-composed of protein to form hollow, rigid, spindle-shaped structures.

3.15 Endospores
-Structures within the cell, which is resistant to heat and chemicals, and non permeable for general staining.
-Calcium-dipicolinic acid represents about 10% of the dry mass of the endospores.
-From out layer: exosporium - spore coat – cortex – core

(1) Properties of the endospore core
-low (10-30%) contents of water
-lower pH of the core cytoplasm compared with that of vegetative cells
-binding of small acid-soluble spore protein (SASPs) to DNA: protect DNA from potential damage from UV, desiccation, and dry heat.

(2) Endospore formation
-when vegetative growth ceases (by limited nutrients such as the carbon and nitrogen source)
à 200 genes are involved.

(3) Germination
-Activation (heating of freshly formed spores for several minutes at a sublethal temperature)àgerminationàoutgrowth (uptake of water and synthesis of new RNA, protein, and DNA)
3.16 The Nucleus and Organelles of Eukaryotic Microorganisms

(1) Nuclear structure
-a pair of unit membranes
-contains many pores composed of several proteins
-Functions of pore: import and export of substances into and out of the nucleus.
-Nucleolus: an area rich in RNA that is the site of ribosomal RNA synthesis.
-Ribosomal proteins synthesized in the cytoplasm are transported into the nucleolus and combined with ribosomal RNA to form the small and large subunits of the eukaryotic ribosome.

(2) Mitochondria
-Site of respiration and oxidative phosphorylation
-covered with phospholipid bilayer, permeable for small organic molecules such as ATP, which is produced in cristae, a folded inner membrane, and transported to cytoplasm to be used in energy-requiring reactions.
-The matrix inside contains a number of enzymes involved in the oxidation of organic compounds, in particular, enzymes if the citric acid cycle.

(3) Chloroplasts
-found in all photosynthetic eukaryotic cells.
-surrounded by very permeable outer membrane and less permeable inner membrane
-Chlorophyll and other components necessary for the photosynthesis is on flattened membrane discs called thylakoids.

(4) Relationships of organelles to prokaryotes
-Endosymbiosis theory: mitochondria and chloroplasts are descendants of ancient prokaryotic cells.
-Evidences supporting the hypothesis:
A. Mitochondria and chloroplasts contain a covalently closed circular form DNA seen in prokaryotic cells
B. Mitochondria and chloroplasts have 70S (but not 80S ribosome found in eukaryotic cells) ribosomes.
C. Antibiotics interfering with 70S ribosome function also inhibit protein synthesis in mitochondria and chloroplasts.
D. Molecular phylogeny: similar rRNA sequences
3. 17 Comparison of the Prokaryotic and Eukaryotic Cell: Table 3.3