B io 120 lecture 3 2012 2013
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MICROBIAL CELL
BIOLOGY
Biology 120 Lecture 3
Reference: Chapter 3 and 4 TORTORA
Tuesday, July 3, 2012
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GETTING TO KNOW YOUR BACTERIA
• Unit of measure (micrometers or nanometers)
• Microscopy = most essential tool in microbial cell biology
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ESSENTIALS IN MICROSCOPY
• Simple versus Compound
• number of lenses
• Brightfield versus Darkfield
• background
• Light versus Electron Microscopy
• light/ beam of electrons
• SEM versus TEM
• 3D surface/ 2D internal structuresTuesday, July 3, 2012
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BRIGHT & DARK
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SEM & TEM
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WHY OIO?
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PREPARATION OF SPECIMENS FOR MICROSCOPY
WET MOUNT
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PREPARATION OF SPECIMENS FOR MICROSCOPY
WET MOUNT
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PREPARATION OF SPECIMENS FOR MICROSCOPY
FIXED SMEAR = STAININGTuesday, July 3, 2012
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PREPARATION OF SPECIMENS FOR MICROSCOPY
• FIXATION
• Process by which the internal and external structures of cells and microorganisms are preserved and fixed in position
• Inactivates enzymes that might disrupt cell morphology
• Toughens cell structures to prevent changes during staining and observation
• Usually microbes are killed when fixedTuesday, July 3, 2012
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TYPES OF FIXATIONS• preserves overall
morphology but not structures within cells
• Penetrates cells and react with cellular components
• smear ready for staining!!!
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STAINING
•STAINS
•salt of + or - ions, one of which is colored (chromophore)
•+ basic dye (CV, MB, MG, Sf)
•- acidic dye (Ng)
•Types of Staining: SIMPLE, DIFFERENTIAL, SPECIAL
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POSITIVE AND NEGATIVE STAINING
CAN YOU SEE THE
DIFFERENCE?Tuesday, July 3, 2012
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STAINING• SIMPLE
• e.g. MB, CF, CV, Sf
• highlight shapes and arrangements
• DIFFERENTIAL
• e.g. Gram stain and Acid fast stain
• differentiate and distinguish one kind of bacteria from another
• SPECIAL
• e.g. negative, endospore, flagella staining
• staining special structures
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SIMPLE STAINING
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GRAM STAINING
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GRAM STAINING
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ACID FAST STAINING
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ACID FAST STAINING
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NEGATIVE STAINING
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ENDOSPORE STAINING
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FLAGELLA STAINING
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QUESTIONS?Tuesday, July 3, 2012
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RECALL...• PROKARYOTIC ORGANISMS
• small, mostly unicellular
• bacteria and archaea
• DIFFERENCES & VARIATION
• bacteria vs archeae
• prokaryotes vs eukaryotes (microbes)
• FACTORS: morphology, chemical composition, nutritional requirements, biochemical activities, source of energy
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THE PROKARYOTES
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SIZE, SHAPE & ARRANGEMENT
OF BACTERIAL CELLSTuesday, July 3, 2012
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WHAT ARE THE 3 BASIC SHAPES OF BACTERIA?
•Measure: 0.2-2.0µm (diameter) x 2-8µm (length)
•Basic shapes: coccus, bacillus, spiral
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COCCI• usually round, can be oval, elongated or
flattened on one side
• e.g. diplococci (remain in pairs)
• e.g. streptococci (remain in chains)
• e.g. tetrads (remain attached in cube-like group of 4)
• e.g. sarcinae (remain attached in cube-like group of 8)
• e.g. staphylococci (divide in multiple planes, grape-like)
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Tuesday, July 3, 2012
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BACCILI
• divide only across their short axis; mostly single rods
• e.g. diplobacilli (remain in pairs after division)
• e.g streptobacilli (occurs in chains)
• e.g. coccobacilli (cocci-like)
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Tuesday, July 3, 2012
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SPIRALS & CURVED
• have on or more twists
• never staright
• e.g. vibrios (curved rods(
• e.g. spirilla (helical, cork-screw, rigid)
• e.g spirochetes (helical but flexible)
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Tuesday, July 3, 2012
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flagella - rigid
axial filament - flexibleTuesday, July 3, 2012
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THE OTHERS
Stella sp. Haloarcula sp.
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SHAPE = heredity* Monomorphic, maintain a single shape
** Pleomorphic more than one shape
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ORGANIZATION IN A TYPICAL PROKARYOTE STRUCTURE:
1. Structures external to the cell wall
2. the cell wall
3. structures internal to the cell wall
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STRUCTURES EXTERNAL TO THE
CELL WALLTuesday, July 3, 2012
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GLYCOCALYX or SUGAR COAT• secreted on prokaryotic surface
• viscous, sticky, gelatinous polymer
• composed of polysaccharide, polypeptide or both
• made inside the cell and secreted outside
• CAPSULE: organized and firmly attached to the cell wall
• SLIMY LAYER: unorganized and loosely attached to the cell wall
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FUNCTIONS:
• contributory to virulence (degree of pathogenicity)
• protect pathogen from phagocytosis
• attachment to various surfaces for survival
• prevent cell from dehydration
• viscosity = inhibits movement of nutrients out of the cell
• EPS (extracellular polysaccharide) = capsules made up of sugars
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EXAMPLES
•Bacillus anthracis (anthrax)
•Streptococcus pneumoniae (bacterial pneumonia)
•Streptococcus mutans (dental caries)
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OBSERVATION OF GLYCOCALYX
•Bacillus anthracis (anthrax)
•Streptococcus pneumoniae (bacterial pneumonia)
•Streptococcus mutans (dental caries)
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FLAGELLA• long filamentous appendages that
propel bacteria
•3 basic parts:
•filament = long outermost region (flagellin)
•hook = where filament is attached (various protein)
•basal body = anchors flagellum to cell wall and plasma membrane
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FLAGELLA
•atrichous = lacks flagellum
•monotrichous = single polar
•amphitrichous = tufts at both ends
• lophotrichous =two or more on one or both ends
•peritrichous = distributed over the entire cell
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FLAGELLA
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DO NUMBERS MATTER?
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DO NUMBERS MATTER?
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DO NUMBERS MATTER?
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DIFFERENCES IN BASAL BODY (Gram + vs Gram -)
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MOTILITY
•ability of an organism to MOVE by itself
•RUN-TUMBLE-RUN routine
•TAXIS: move away or towards a stimuls (chemotaxis, phototaxis)
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FLAGELLA & MOTILITY
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FLAGELLAR PROTEINS
•H antigen: useful for distinguishing among serovars of bacteria
•e.g. E. coli O157:H7
•NOTE: there are at least 50 fifferent H antigens for E. coli
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AXIAL FILAMENTS•locomotory structure for
spirochetes
•also called “endoflagella”
•bundles of fibrils that arise at the ends of the cell beneath an outer sheath
•spirals around the cell
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AXIAL FILAMENTS
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FIMBRIAE & PILI
•hair-like appendages
•shorter, straighter and thinner than flagella
•used for attachment (F) and transfer of DNA/conjugation (P)
•essential for colonization in Neisseria (F)
•NOT for motility!
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FIMBRIAE & PILI
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THE CELL WALL
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CELL WALL• complex, semi-rigid structure
• gives the shape of the cell
• surrounds the plasma membrane and protects interior from adverse changes in the outside environment
• prevents rupture of bacterial cells
• contributes to ability of some species to cause disease
• site of action of some antibiotics
• ALMOST ALL prokaryotes have cell walls
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CELL WALL: Composition & Characteristics
• peptidoglycan or murein
• N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM)
• Linked by polypeptides
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CELL WALL: Composition & Characteristics
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GRAM + CELL WALL
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GRAM + CELL WALLNote: lipoteichoic acid
provide antigenic variation
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GRAM - CELL WALL
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GRAM - CELL WALLNote: outer membrane polysaccharides provide antigenic variation; Lipid
A is an endotoxin
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GRAM + VS GRAM -
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GRAM + VS GRAM -
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ATYPICAL CELL WALL
• e.g. Mycoplasma
• no walls or have little wall materials
• plasma membrane have sterols (help protect from lysis vice the CW)
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ATYPICAL CELL WALL• e.g. Archaea
(Halobacterium sp)
• may lack walls or have unusual walls composed of polysaccharides and proteins not peptidoglycan (pseudomurein)
• PSEUDOMUREIN = lacks the D-amino acids found in bacterial CWs
• NOTE: Gram stain not applicable
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ATYPICAL CELL WALL
• e.g. Mycobacterium and Nocardia
• high concentrations of mycolic acids in CWs (60%)
• prevents the uptakes of dyes
• Note: Gram stain will work only if mycolic acids removed
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DAMAGING CELL WALL
• antimicrobial drugs (e.g. penicillin) = halts CW synthesis
• lysozyme = targets PG backbone
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STRUCTURE INTERNAL TO THE
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PLASMA MEMBRANE• primarily phospholipids
• lacks sterols thus LESS rigid
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PLASMA MEMBRANE
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IMPORTANT STRUCTURES•GLYCOPROTEINS & GLYCOLIPIDS
•help protect and lubricate the cell
• involved in cell-to-cell interactions (e.g. pathogen binding in inlfuenza)
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CM FUNCTIONS
•selective barrier
•breakdown of nutrients and production of energy
•What happens when CM destroyed?
•cell leakage
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PHOTOSYNTHETIC STRUCTURES IN THE CM
purple non-sulfur bacteria
purple sulfur bacteria
green sulfur bacteria
PHOTOSYNTHETIC PIGMENTS IN MEMBRANE FOLDINGS:
1. Chromatophores2. Chlorosomes3. Photosynthetic lamellae
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BACTERIA VS ARCHAEA CM
•EUBACTERIA
•Ester linkage
•Weaker linkage
•ARCHAEBACTERIA
•Ether linkage
•Stronger linkage
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READING ASSIGNMENT: MEMBRANE TRANSPORT SYSTEMS
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THE NUCLEAR AREA
• “NUCLEOID”
• contains the bacterial chromosome
• not surrounded by a nuclear envelope (membrane)
• do not include histones
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THE NUCLEAR AREA• PLASMIDS
• extrachromosomal genetic element
• replicate independently
• Gene: antibiotic resistance, tolerance to toxic metals, toxin production and synthesis of enzymes
• can be transferred from one bacterium to another via conjugation
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BACTERIAL RIBOSOMES
• ALL PROKARYOTES & EUKARYOTES HAVE RIBOSOMES!!!
• site of protein synthesis
• composed of two units:
• protein sub-unit
• ribosomal RNA subunit
• NOTE: differ from EUK ribosomes in the number of proteins and rRNA molecules they contain and they are less dense
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BACTERIAL RIBOSOMES• PROK = 70S ribosomes while EUK = 80S
ribosomes
• The 70S = 30S (1 rRNA molecule) + 50S (2 rRNA molecules)
16S ribosomal DNA = prokaryotes
18S ribosomal DNA = eukaryotes
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BACTERIAL RIBOSOMES• Antimicrobials:
• streptomycin and gentamicin = attach to 30S and interfere with protein synthesis
• erythromycin and chloramphenicol = attach to 50S and interfere with protein synthesis
• THUS only prokaryotes are affected by these antimicrobials
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INCLUSIONS• inclusions = reserve deposits used when
supply are deficient
• METACHROMATIC GRANULES
• POLYSACCHARIDE GRANULES
• LIPID INCLUSIONS
• SULFUR GRANULES
• CARBOXYSOMES
• GAS VACUOLES
• MAGNETOSOMESTuesday, July 3, 2012
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METACHROMATIC GRANULES
• “volutin”
• large inclusions
• stain red with certain blue dyes (e.g. MB)
• inorganic phosphate/polyphosphate reserves
• used for ATP synthesis
• BACTERIA, ALGAE, FUNGI & PROTOZOA
• Corynebacterium diphtheriae (diagnostic)Tuesday, July 3, 2012
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METACHROMATIC GRANULES
“chinese characters”
diagnostic for C. diptheriae
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POLYSACCHARIDE GRANULES
• consist of glycogen and starch
• demonstrated when iodine is applied to cells
• appear reddish brown (Glycogen)
• appear blue (Starch)
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LIPID INCLUSIONS
• Mycobacterium, Bacillus, Azotobacter, Spirillum etc
• e.g. poly-B-hydroxybutyric acid (PHBs)
• revealed using Sudan dyes (fat soluble dye)
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SULFUR GRANULES
• Thiobacillus spp, Beggiatoa
• they derive energy by oxidizing sulfur and sulfur-containing compounds
• deposit sulfur granules as energy reserves
Beggiatoa sp.
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CARBOXYSOMES
• contain the enzyme 1,5-diphosphate carboxylase (for carbon dioxide fixation)
• photosynthetic bacteria, Nitrifying bacteria, Cyanobacteria, Thiobacillus
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GAS VACUOLES
• hollow cavities in aquatic prokaryotes
• cyanobacteria, anoxygenic photosynthetic bacteria and halobacteria
• maintain buoyancy
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MAGNETOSOMES• inclusion of iron oxide
• Magnetospirillum magnetotacticum
• used to move downward until they reacha suitable attachment site (act like magnets)
• can decompose hydrogen peroxide (to protect cells from its accumulation)
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ENDOSPORES
• Clostridium, Bacillus (Bacteria)
• Thermoactinomyces vulgaris (Archaea)
• specialized resting cells
• resistant to adverse conditions (extreme heat, lack of water, exposure to toxic chemicals and radiation)
• Dipicolinic acid (DPA) with calcium ions directly involved in spore heat resistance
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SPORULATION/SPOROGENESIS
SPORULATION: SPORE FORMATION
GERMINATION: SPORE TO VEGETATIVE CELL
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ENDOSPORES
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THE EUKARYOTES
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Tuesday, July 3, 2012
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Tuesday, July 3, 2012
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EUKARYOTIC FLAGELLA & CILIA
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For cellular locomotion
• Flagella: projections that are few and long in relation to the size of the cell (e.g. Euglena)
• Cilia: projections that are numerous and short in relation to the size of the cell (e.g. Tetrahymena)
• Difference between prokaryotic flagella:
• PROK = rotates
• EUK = moves in a wave-like manner
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For cellular locomotion
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HOW THEY PROPEL THE CELL
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THE 9 + 2 ARRAY
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EUKARYOTIC CELL WALL &
GLYCOCALYXTuesday, July 3, 2012
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CELL WALLS
• EUK have simpler cell walls
• Algae: cellulose
• most Fungi: chitin
• Yeasts: glucan and mannan
• Protozoa: DO NOT HAVE a typical cell wall = pellicle (flexible outer protein covering)
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GLYCOCALYX
• strengthens the cell surface
• helps attach cells together
• involved in cell to cell recognition
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EUKARYOTIC PLASMA MEMBRANE
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PLASMA MEMBRANE
• similar in function and basic structure with prokaryotes
• differences are the proteins found in the membranes
• also contain carbohydrates which serves as attachment sites for bacteria and as receptor sites for cell-to-cell recognition
• contains sterols (resist lysis due to osmotic pressure)
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PLASMA MEMBRANE• NOTE: group
translocation do not occur in eukaryotic membranes
• instead ENDOCYTOSIS (e.g. pinocytosis and phagocytosis)
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CYTOPLASM
• substance inside the plasma membrane and outside the nucleus
• cytosol = fluid portion of the cytoplasm
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CYTOPLASM• Major difference:
• EUK have complex internal structures (microfilaments, intermediate filaments, microtubules) which forms the cytoskeleton (provides support for cytoplasmic streaming)
• many enzymes fund in cytoplasmic fluid of PROK are sequestered in the organelles of EUK
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EUKARYOTIC RIBOSOMES & ORGANELLES
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RIBOSOMES• same function as in PROK
• larger and denser than PROK (80S = 60S with 3 molecules of rRNA; and 40S with 1 molecule of rRNA)
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RIBOSOMES• free ribosomes: unattached, protein
synthesis used inside the cell
• membrane-bound ribosomes: attached to nuclear membrane and ER, protein synthesis for insertion in the plasma membrane or for export from the cell
• polyribosome: located within mitochondria, synthesis of mitochondrial proteins (10-20 ribosomes joined together in a string-like arrangement)
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ORGANELLES
• organelle: structure with specific shapes and specialized functions; absent in prokaryotes
• Nucleus, ER, golgi complex, lysosomes, vacuoles, mitochondria, chloroplasts, peroxisomes and centrosomes
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ORGANELLES
•Nucleus = houses the chromosome
•ER = transport and storage
•Golgi complex = membrane foration and protein secretion
•Lysosomes = store digestive enzymes
•Vacuoles = storage and rigidity
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ORGANELLES• Vacuoles = storage and rigidity
• Mitochondria = site of ATP production
• Chloroplasts = contain chlorophyll and enzymes for photosynthesis
• Peroxisomes = oxidation of organic compiunds (e.g. catalase) destroying hydrogen peroxide)
• Centrosomes = contains centrioles for mitotic spindle formation
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ORGANELLES
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THE EVOLUTION OF EUKARYOTES
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HOW THEY CAME ABOUT...
• 3.5-4B years ago = simple organisms (similar to prokaryotes)
• 2.5B years ago = eukaryotes from prokaryotes
• Lyn Margulis: The Endosymbiotic Theory
• larger bacterial cells lost their CW and engulfed smaller bacterial cells
• endosymbiosis = lives within another
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ENDOSYMBIOTIC THEORY
• ancestral EUK developed a rudimentary nucleus when the plasma membrane folded around the chromosome (NUCLEOPLASM)
• Nucleoplasm ingested aerobic bacteria and lived inside it
• evolved into a symbiotic relationship (host supply nutrients, while bacteria produce the energy from supplied nutrients
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ENDOSYMBIOTIC THEORY
• CHLOROPLASTS = descendants of photosynthetic prokaryotes ingested by the nucleoplasm
• FLAGELLA & CILIA = motile spiral bacteria/spirochetes
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NEXT MEETING: JOURNAL REPORTING
Tuesday, July 3, 2012