Chapter 2 - Microbial Cell Structure and Function Flashcards
What kind of light does a compound light microscope use?
Visible light
How does a bright-field microscope work?
Specimens are visualized in contrast between specimen and surroundings
What are the lenses a bright-field microscope uses?
Objective and ocular lens
Magnification
The ability to make an object larger
Resolution
The ability to distinguish two adjacent objects as separate and distinct
Limit of resolution for a light microscope
0.2 μm
As wavelength decreases
Resolution improves
Two points are viewed as separate objects when
Light passes between them
What are dyes?
Organic compounds that bind to specific cellular materials
Simple Staining
One dye used to color specimen
Chromophore
Colored portion of dye
Basic dye
Positive charged chromophore
Binds to negatively charged molecule on cell surface
Acidic dye
Negatively charged chromophore
Repelled by cell surface
Used to stain background
Negative stain
Example of basic dye
Crystal violet
Example of acidic dye
Nigrosin
Gram positive
Cells that retain a primary stain - purple
Gram negative
Cells that lose the primary stain and take color of counterstain - red or pink
Acid fast stain
Detects mycolic acid in the cell wall of the genus Mycobacterium - pink, anything else will be blue
Endospore stain
Endospores retain primary - green, cells counterstained - pink
Phase-contrast microscopy
Phase ring amplifies differences in the refractive index of cell and surroundings
Advantages of phase-contrast microscopy
Improves the contrast of sample without the use of stain
Live samples can be seen
Phase-contrast appearance
Dark cells on a light background
Dark field microscopy
Specimen is illuminated with a hollow cone, only refracted light enters the objective
Dark field appearance
Specimen is bright and background is dark
Advantages of dark field microscopy
Observe bacteria that don’t stain well
Fluorescence microscopy
Used to visualize specimens that fluoresce
Fluorescence microscopy appearance
Emit light of one color when illuminated with another color of light. Some cells fluoresce naturally
Chlorophyll fluoresce
Absorbs light at 430 nm (blue-violet)
Emits at 670 nm (red)
DAPI
Fluorescent dye that binds to DNA
Differential interference contrast microscopy
Uses a polarizer to create two distinct beams of polarized light
DIC microscopy appearance
Structures appear three-dimensional
DIC structures that can be seen
Endospores, vacuoles, and granules
Confocal scanning laser microscopy
Uses a computerized microscope coupled with a laser source to generate a three-dimensional image
Advantaged of CSLM
Can focus on a single layer
Layers can be compiled for a three-dimensional image
Resolution is 0.1 μm
Wavelength of electrons
Much shorter than light (better resolution)
Transmission electron microscope
Electron beam focused on specimen by condenser. Electrons pass through the specimen are focused by two sets of lenses. Electrons strike a fluorescent viewing screen.
What is used for a lens on a TEM?
Magnet
Advantages of TEM
High magnification and resolution (0.2 nm)
Specimen requirements for TEM
Must be very thin (20-60 nm)
Must be stained with metal - lead or uranium
Why must a cell be stained with a metal?
To make them more electron dense
Enables visualization of structures at molecular level
Scanning electron microscopy
Specimen is coated with a thin film of heavy metal (e.g., gold). An electron beam scans the object. Scattered electrons are collected by a detector and an image is produced.
SEM image
3D image of a specimen’s surface
Bacteria
Diverse metabolism
Live in a broad range of ecosystems
Pathogens and non-pathogens
Archaea
Diverse metabolism
Live in extreme environments
Non-pathogens
Coccus
Roughly spherical
Bacillus
Rod shaped
Spirillum
Spiral shaped
Spirochete
Spiraled and more flexible
Budding and appendaged bacteria
Have a stalk or hyphae
Filamentous bacteria
Appear like hyphae
Morphology does not predict
Physiology, ecology, phylogency
What shape of cells promote gliding motility?
Filamentous
What shape of cell allows swimming motility?
Helical or spiral-shaped
Advantages of small cells or those with high surface-to-volume ratio
Optimization for nutrient intake
Size range for prokaryote cells
0.2 μm to >700 μm
Size range for eukaryote cells
10 μm to >200 μm
Advantages of small cells
Higher surface area relative to cell volume
Support greater nutrient exchange per unit cell volume
Tend to grow faster
Lower limits of cell size
Small cells are found in
Open oceans
Cytoplasmic membrane
Thin structure that surround the cell, it separates the cytoplasm from the environment
Highly selective permeable barrier
Enables concentration of specific metabolites and excretion of waste products
General structure of membranes
Phospholipid bilayer
Phospholipid bilayer
Hydrophobic (fatty acids) and hydrophobic (glycerol-phosphate) components
Location of fatty acids and hydrophilic portions
Fatty acids point inward to form hydrophobic environment; hydrophilic portion remains exposed to external environment
Ester phospholipids
Glycerol, 2 fatty acids, phosphate, and optional side chain
Amphipathic
Has both polar and non-polar characteristics
Polar
Molecule carries a charge
Hydrophilic
Non-polar
Molecule is uncharged
Hydrophobic
Gram negative membrane proteins
Interacts with a variety of proteins (periplasmic proteins) that bind substrates or process large molecules for transport
Inner surface of cytoplasmic membrane
Interacts with proteins involved in energy-yielding reactions and other cellular functions
Integral membrane proteins
Firmly embedded in the membrane
Peripheral membrane proteins
One portion anchored in the membrane
Archaeal membrane linkages
Ether linkages in phospholipids
Bacterial and Eukarya membrane linkages
Ester linkages
Archaeal lipids lack and have what instead
Fatty acids; have isoprenes
Archaeal major lipids
Glycerol diethers and triethers
Structure of archaeal lipid
Monolayers, bilayers, or mixture
Advantage of monolayer lipid
Extremely heat resistant
Where are monolayer lipids usually found?
Hyperthermophilic archaea
Permeability barrier
Polar and charged molecules must be transported
Transport proteins accumulate solutes against the concentration gradient
Protein anchor
Holds transport proteins in place
Energy conservation
Site of generation of proton motive force
Carrier-mediated transport systems
Show saturation effect
Highly specific
Three major classes of transport systems in prokaryotes
Simple transport
Group translocation
ABC system
Simple transport
Driven by the energy in the proton motive force
Group translocation
Chemical modification of the transported substance driven by PEP (phosphoenolpyruvate)
What does all transport systems require?
Energy in some form, usually proton motive force or ATP
ABC system
Chaperone protein is used to lead the protein to the port (periplasmic binding)
Three transport events
Uniport, symport, antiport
Uniport
One direction across the membrane
Symport
Co-transporters (two molecules moves across membrane in same direction)
Antiporters
One molecule into the membrane, one molecule out
Example of simple transport
Lac permease of E. coli
Lac permease
Helps transport lactose and H+ into E. coli
Group translocation
Sugar is phosphorylated during transport across the membrane
Moves glucose, fructose, mannose
Phosphoenolpyruvate (PEP) donates a P to a phosphorelay system
P is transferred through a series of carrier proteins and deposited onto the sugar as it is brought into the cell
ABC transport systems
Involved in uptake of organic compounds (sugars, amino acids), inorganic nutrients (sulfate, phosphate), and trace metals
ABC transport systems display
High substrate specificity
ABC transport systems (gram-negative)
Employ periplasmic-binding proteins and ATP-driven transport proteins
ABC transport systems (gram positive)
Employ substrate-binding lipoproteins (anchored to external surface of cell membrane) and ATP driven transport proteins
ABC transports
Solute binding proteins, integral membrane proteins, ATP-hydrolyzing proteins
Solute binding protein
Periplasm
Binds specific substrate
ATP-hydrolyzing proteins
Supply energy for the transport event
Cell walls of bacteria and archaea
Rigid - help maintain cell shape
Porous to most small molecules
Protects cell against osmotic changes
Role of cell wall
Prevent cell expansion - protects against osmotic lysis
Protects against toxic substances - large hydrophobic molecules (detergents, antibiotics)
Pathogenicity
Partly responsible for cell shape
Pathogenicity
Helps evade host immune system
Helps bacterium stick to surfaces
Gram-negative cell wall
Two layer: LPS (lipopolysaccharide) and peptidoglycan
Gram-positive cell wall
One layer: peptidoglycan
Peptidoglycan
Rigid layer that provides strength to cell wall
Polysaccharide composed of
N-acetylglucosamine and N-acetylmuramic acid (NAG and NAM sugars)
Amino acids
Lysine or DAP
Polysaccharide form
Glycan tetrapeptide
Number of peptidoglycan structures identified
More than 100
How do peptidoglycan differ?
In peptide cross-links and/or interbridge
Where are interbridges found?
In gram-positive bacteria, none in gram-negative
How many interbridges does S. aureus have?
5 glycine residues
How much peptidoglycan do gram-positive cell walls have?
Up to 90%
What do gram positive bacteria have in their cell wall?
Teichoic acid
Lipoteichoic acid
Teichoic acids covalently bound to membrane lipids
Backbone of peptidoglycan
NAM and NAG connected by glycosidic bonds
Glycoside bonds
Crosslinks formed by peptides
Shape of peptidoglycan strand
Helical
Why is the peptidoglycan strand helical?
Allows 3-dimensional crosslinking?
How many layers of peptidoglycan does E. coli have?
1
How many layers of cell walls does Bacillus species have?
50-100
Prokaryotes that lack cell walls
Mycoplasmas
Thermoplasmas
Mycoplasmas
Group of pathogenic bacteria
Have sterols in cytoplasmic membrane - adds strength and rigidity to membrane
Thermoplasma
Species of archaea
Contain lipoglycans in membrane that have strengthening effect
How much peptidoglycan do gram negative bacteria have?
10%
What does the lipopolysaccharide layer consist of?
Core polysaccharide and O-polysaccharide
What does LPS replace?
Most of phospholipids in outer half of outer membrane
Endotoxin
Toxic component of LPS
Periplasm
Space located between cytoplasmic and out membrane
Size of periplasm
~15 nm wide
Consistency of periplasm
Gel-like
What does the periplasm contain?
Proteins
Porins
Channels for movement of hydrophilic low-molecular weight substances
Gram-positive bacteria cell walls
Thick consisting mainly of peptidoglycan
What happens to gram-positive bacteria cell walls during alcohol step of staining?
Pores in wall close and prevent crystal violet from escaping
What happens to gram-negative bacteria cells wall during alcohol step of staining?
Alcohol penetrates outer membrane, crystal violet is extracted out, and cells appear invisible until counterstained with second dye
Archael cell walls
No peptidoglycan and typically no outer membrane
Pseudomurein
Polysaccharide similar to peptidoglycan
What is pseudomurein composed of
NAG and N-acetylalosaminuronic acid (NO NAM)
Where is pseudomurein found?
Certain methanogenic archaea
S-layers
Most common cell wall type among archaea
S-layers consist of
Protein or glycoprotein
S-layer structure
Paracrystalline structure
True/false: some archaea only have S-layer (no other cell wall components)
True but most have additional cell wall elements
Cell wall structure function in archaea
Prevent osmotic lysis and give shape
Lack of peptidoglycan means archaea are resistant to
Lysozome and penicillin
Cytoplasm
Material bounded by plasma membrane
Protoplast
PM and everything within:
Macromolecules, soluble proteins, DNA and RNA, ribosomes, inclusions
Enzymes
Catalyze chemical reactions
Transport proteins
Move other molecules across membranes
Structural proteins
Help determine shape of cell and are involved in cell division
Proteins are made of
Polypeptides
Polypeptides
A long polymer of amino acids joined by peptide bonds
Nucleoid
Region that contains the genome
Typical bacterial genome
Single circular double stranded DNA chromosome and may have one or more plasmids
Plasmid
Small circular double stranded DNA that is self-replicating and carry non-essential genes
DNA
Carries genetic info of all living cells
Polymer of deoxyribonucleotides
Bacterial ribosomes
Site of protein synthesis
What are the parts of the 70S ribosome?
30S subunit - 16S rRNA
50S subunit - 23S and 5S rRNA
Cytoplasmic ribosomes
Cytoplasmic proteins
PM associated ribosomes
Membrane proteins
Proteins to be exported from the cell
Capsules and slime layers
Polysaccharide/protein layers that assist in attachment to surfaces
Capsule and slime layer appearance
Thin or thick, rigid or flexible
Benefits of capsule and slime layer
Protect against phagocytosis and resist desiccation
Fimbriae
Filamentous protein structure that enable organisms to stick to surfaces or form pellicles
Pili
Filamentous protein structure that assist in surface attachment
Which is longer fimbriae or pilli
Pili
What does the pili facilitate?
Genetic exchange between cells (conjugation)
What type of pili are involved in twitching motility?
Type IV
Cell inclusion bodies
Visible aggregates in cytoplasm
Types of cell inclusion bodies
Carbon storage polymers: poly-beta-hydroxybutyric acid, glycogen
Polyphosphates
Sulfur globules
Magnetosomes
What are carbon storage polymers?
poly-beta-hydroxybutyric acid (lipid) and glycogen (glucose polymer)
Polyphosphates
Accumulations of inorganic phosphate
Sulfur globules
Composed of elemental sulfur
Magnetoaomes
Magnetic storage inclusions
Inorganic inclusions
Polyphosphate granules and sulfur golbules
Polyphosphate granules
Volutin - storage of phosphate and energy
Sulfur globules
Storage of sulfur used in energy generation
Magnetosomes
Intracellular granules of Fe3O4 or Fe3S4
Magnetosomes ability
Gives the cell magnetic properties that allow it to orient itself in a magnetic field
Magnetotaxis
Bacteria migrate along Earth’s magnetic field
Gas vesicles
Confer buoyancy in planktonic cells
Gas vesicle appearance
Spindle-shaped, gas-filled structures made of proteins
Gas vesicle function
Decreasing cell density
Endospores
Highly differentiated cells resistant to heat, harsh chemicals, and radiation
What stage are endospores for a bacterial life cycle?
Dormant
How do endospores travel?
Wind, water, or animal gut
Bacterial endospores are only produced by
Gram positives
Vegetative cell
Capable of normal growth - metabolically active
Endospore
Dormant cell, formed inside of a mother cell
Endospore: metabolically active or inactive
Inactive
How are endospores triggered?
By lack of nutrients
How long does it take for an endospore to form?
8-10 hours
Layers of endospore
Spore coat and cortex and two membranes
Spore coat and cortex
Protect against chemicals, enzymes, physical damage, and heat
Two membranes of endospores
Permeability barriers against chemicals
Endospore core
Dehydrated - protects against heat
Endospore core is made of
Ca-dipicolinic acid and SASPs that protect against DNA damage
Endospores can resist
Boiling for hours UV, gamme radiation Chemical disinfectants Dessication AGe
First stage of spore forming bacterium
Assymetric cell division - DNA replicates and identical chromosomes are pulled to opposites end of the cell
Second stage of spore forming bacterium
Septation - divides into 2 unequal compartments: the forespore and mother cell
Third stage of spore forming bacterium
Mother cell engulfs the forespore - the forespore is now surrounded by two membranes
Fourth stage of spore forming bacterium
Formation of cortex - thick layers of peptidoglycan form between the two membranes
- highly cross-linked layer - core wall
- loosely cross-linked layer - cortex
Fifth stage of spore forming bacterium
Coat synthesis - protein layers surround the core wall (spore coat and exosporium) to help protect the spore from chemicals and enzymes
Sixth stage of spore forming bacterium
Endospore matures
- core is dehydrated
~ 10-30% of vegetative cell’s water content
Seventh stage of spore forming bacterium
Mother cell is lysed
- mother cell disintegrates
- mature spore is released
Flagella
Hollow protein filaments
Flagella can be viewed
Only when stained
Monotrichous
Single flagellum - polar or subpolar
Amphitrichous
Flagella at opposite ends
Lophotrichous
Multiple flagella in a single tuft
Peritrichous
Flagella distributed around cell
Flagella structure
Filament, hook, and basal body
Flagella filament
Rigid helical protein - 20 micrometers long
Composed of identical protein subunits - flagellin
Flagella hook
Flexible coupling between filament and basal body
Basal body
Consist of central rod that passes through series of rings
Basal body rings
L ring - LPS layer
P ring - peptidoglycan
MS ring - membrane
C ring - cytoplasm
Where does the energy comes from to turn the flagella?
Proton motive force
Proton motive force
Gradient of protons across the cytoplasmic membrane
- high [H+] outside
- low [H+} inside
Mot proteins
Form a channel that allows H+ to move into the cytoplasm
Provides the energy to turn the flagellum
How does the flagellum turn?
Like a propeller to drive the cell forward
Flagellar synthesis
MS ring is made first, other proteins and hook are made next, filament grows from tip
Peritrichously flagellated cell movement
Slowly in a straight line
Polarly flagellated cell movement
Rapidly and typically spin around
Gliding motility
Flagella-independent motility that is slower and smoother than swimming
Gliding motility requires
Surface contact
Mechanisms of gliding motility
Excretion of polysaccharide slim
Type IV pili
Gliding-specific proteins
Taxis
Directed movement in response to chemical or physical gradients
Chemotaxis
Response to chemicals
Phototaxis
Response to light
Aerotaxis
Response to oxygen
Osmotaxis
Response to ionic strength
Hydrotaxis
Response to water
Chemotaxis is best studied in
E. coli
Chemotaxis response
To temporal not spatial differences in chemical concentration
Chemotaxis behaviour
Run and tumble behaviour
Chemoreceptors
Used to sense attractants and repellants - biased random walk
What happens if E. coli senses that glucose is increasing?
Tumble is delayed and the run lasts longer
Chemotaxis is measured by
Inserting a capillary tube containing an attractant or a repellent in a medium motile bacteria
It can be seen under a microscope
Eukaryotic cell size
Lower surface area to volume ratio
- Need more sophisticated transport mechanisms
- Grow slower
Eukaryote nucleus
True nucleus that houses the genetic material
Eukaryote internal structures
Membrane bound organelles
Intracytoplasmic membranes used for transport
Cytoskeleton
Nucleus DNA
Multiple linear dsDNA chromosomes
Chloroplasts
Site of photosynthesis for chlorophyll
How many membranes is the chloroplast surrounded by?
2 membranes
Mitochondria
Site of respiration and oxidative phosphorylation
Endosymbiotic hypothesis
Mitochondria and chloroplasts evolved from bacteria
What is the evidence for the endosymbiotic hypothesis?
Semi-autonomous Circular chromosomes - lack histones 70S ribosomes Two membranes Outer membrane has porins
Mitochondria are most related to
Rickettsia - proteobacteria (obligate intracellular pathogens)
Chloroplasts are most closely related to
Cyanobacteria - blue-green algae