Structure and Function of Prokaryotes
Last revised: Friday, March 7, 2003
Reading: Ch. 4 in text

Microscopy

Resolving Power

• measures the ability to distinguish small objects close together

                	        0.61 (lambda)
               r.p. =   ______________
                         (N sinØ)
  

  where lambda = wavelength of illuminating light.
  
  
  
  
• for light scope, can improve R.P. by making lambda smaller or sinØ larger.
  
  
  
  
• R.P. is smallest for violet light, but because human eye is more sensitive to blue, optimal R.P. is achieved with blue light (~450 nm). Use filters to remove other light in best microscopes
• n sinØ is called numerical aperture. It measures how much light cone spreads out between condenser & specimen. More spread = better resolution. Ø = angle of light cone; maximum value is 1.0
• n = refractive index. n = 1.0 in air. Can increase with certain oils (up to 1.4), called immersion oil. N.A. is property of lens. Look on side of lens to identify.
• Theoretical limit of R.P. for light scope is 0.2 micrometers.

Optical Instrument	Resolving Power	RP in Angstroms
Human eye	0.2 millimeters (mm)	2,000,000 A
Light microscope	0.20 micrometers (µm)	2000 A
Scanning electron microscope (SEM)	5-10 nanometers (nm)	50-100 A
Transmission electron microscope (TEM)	0.5 nanometers (nm)	5 A

Light Microscopes

1. Bright-Field Microscope
   
   
   • Advantages: convenient, relatively inexpensive, widely available
   • Disadvantages: resolving power 0.2 micrometers at best, can recognize cells but not fine details
   • Needs contrast; cells are mainly water and don't contrast with their medium. Easiest way to view cells is to fix and stain.
   • Fixation
     • preserves cells; disrupts proteins, prevents decay/degradation.
     • typical treatments: heat, formalin, glutaraldehyde
   • Staining
     1. Simple Stains
        • adds colored compounds contrast
        • basic dyes: e.g. methylene blue, crystal violet. Cations ( + charges) bind to - charge groups on proteins, nucleic acids
        • acidic dyes: e.g. eosin, acid fuchsin. Anions ( - charges); bind to + charges on proteins, phospholipids
     2. Differential Stains
        • allow differentiation between different organsisms. Examples:
        • Gram stain -- will do in lab this week. Distinguishes 2 major groups of bacteria: + and -.
        • Spore stain -- will perform in lab next week. Malachite green binds specifically to compounds in endospore wall.
        • Acid fast stain -- not done in 229 lab. Allows detection of some bacteria with waxy coat (Mycobacteria).
2. Phase-Contrast Microscope
   
   
   • Cells are mostly water, very little contrast from surrounding medium, so not very visible in light
   • Phase scope converts slight diffs. in refractive index and cell density into variations.
   • Scope uses annular stop below condenser: thin transparent ring in opaque disk hollow light cone. As light passes through specimen, some rays are deviated and retarded by 1/4 wavelength.
   • Have phase plate in objective lens: transparent optical disk with phase ring.
   • Undeviated light passes through ring, is advanced by 1/4 wavelength bright background. But deviated light doesn't pass through phase ring, so is not advanced. When light gets focused, deviated rays cancel out with undeviated rays, producing dark image where objects were.
   • Advantage: can see live material w/o staining
3. Fluorescence Microscope
   
   
   • Fluors are chemicals that adsorb light to produce excited electrons, later reradiate light = flourescence.
   • To use, need special type of microscope -- view photo of fluorescent microscope. Must illuminate with ultraviolet or violet light ( excited fluor). Need filters to remove this light from light traveling to ocular lens; only fluoresced light emitted from object will then appear to eye. Need dark field condenser to create dark background.
   • Can couple flour to specific probe molecules (usually antibodies), bind to preparation. If sample is illuminated with wavelength of exciting light, then filter out that wavelength to prevent reaching sample, see nothing. But if fluorescence occurs, diff. wavelength light produced, see object.
   • good technique to detect specific microbe in complex sample. (e.g. detect gonococcus in vaginal smear). Requires correct microscope, fluors, technical skill.
4. For further information, see "Cell structure and Microscopy"

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Electron Microscope

• Physicists discovered electrons have wave properties. Can use magnetic coils like lenses to focus beams of electrons. Basic design of EM similar to light scope
• But: electrons don't scatter from H, C, O, N: must add heavy atoms (e.g. Pb, Ur, Os, Gold) as stains.
• Also, electrons are scattered by air molecules. So must remove air from microscope with vaccum pump. But water in specimen will evaporate, so must be removed by dehydration after fixation. Cannot view living specimens.

Transmission Electron Microscope (TEM)

• see slide. R.P. approx. 1000x better than light; 0.2 nm, instead of 0.2 micrometers.
• excellent for seeing internal detail. But cannot use with large/thick specimens.
• View TEM of cyanobacterium
• Specimen Preparation: specimen must be thin. Use grids with thin film supports. Prepare thick materials by sectioning with glass knives sections about 20-100 nm thick. Prepare small preparations (viruses, or subcellular particles) by negative staining.

Scanning Electron Microscope (SEM)

• Same principle as TV screen, except reflected (secondary) electrons used to produce magnified image.
• complementary to TEM. Only see surface view --no internal detail visible. Infinite depth of focus, in contrast to light scopes.
• R.P. around 2 nm at best, usually a bit poorer. (100x better than light scope, not as powerful as TEM)
• View SEM of E. coli
• Specimen Preparation: fix & dry specimen. Shadow with thin metal film (e.g. gold). Mount on block and scan. (Note: sometimes possible to use ordinary air-dried material; but charge builds up on surface, distorts image).

Prokaryote Anatomy

Composition of a bacterial cell

• Important points:
  1. Water makes up ~ 70%
  2. Macromolecules make up ~ 26% ( ~90% of dry wt)
  3. The most abundant molecules other than water are proteins (~50% of dry wt)
  4. Small molecules & ions make up 4% (~10% of dry wt)

How big are bacteria?

• View graphic of bacterial sizes
• Smallest bacteria are Mycoplasmas, as small as 0.2 micrometers (almost as small as largest poxviruses!)
• Accepted wisdom is that bacteria are smaller than eukaryotes. But certain cyanobacteria are quite large; Oscillatoria cells are 7 micrometers diam, size of red blood cells. And certain eukarotes (e.g. Nanochlorum eukaryotum) are very small, only 1 to 2 micrometers, but true eukaryotes (nucleus, chloroplast, mitochondrion are present). So size difference is, like many generalizations, only a useful yardstick, not an absolute truth.
• Epulopiscium fishelsoni, discovered in 1985 in intestinal tract of sturgeonfish, is an enormous, cigar-shaped cell, as large as 80 x 600 micrometers (that's .6 mm, large enough to be seen by the naked eye). Amazingly, this cell is prokaryotic! Initial evidence by EM was hard to believe, but confirmed ty rRNA comparisons with other organisms, a cousin of Gram-positive Clostridium genus.

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Basic Structures of Prokaryotic Cells.

1. Overview: view model of the bacterial cell

Cell membrane

• function: permeability barrier, prevents cell contents from leaking away. Very impermeable to polar & charged molecules (most biomolecules, also small ions such as K⁺, H⁺). Only water, lipid-soluble materials pass freely.
• But cells have many specific protein carriers in membrane, carry out selective transport.

Membrane Components and Structure

• Chemical Composition of membranes: roughly ~50% lipid, 50% protein
• Membrane lipids are typically phospholipids, contain two fatty acids joined to glycerol by ester bonds. See text Fig. 4.34.
• View interactive pdb file of phospholipid. (Note: to view this file on your computer, the Chime plug-in must be installed in your plug-in folder.)
• Note: Archaea have different type of lipids; not fatty acids, instead built of isoprene units. Typical membrane lipids are called diglycerol diethers or glycerol tetraethers, and produced produces lipid monolayers or bilayers. See text Fig. 4.36.
• See figure contrasting lipid linkages in bacteria and archaea.
• Phospholipids (and some archaeal lipids) assemble into a lipid bilayer.
• Biological membranes contain lipid bilayer + many embedded proteins. Proteins "float" in fluid lipid layer = "fluid mosaic model" of membrane structure.
• Sterols (e.g. cholesterol) usually absent in bacterial membranes (common in eukaryotic membranes, account for 5-20% of total lipid). But hopanoids present in some bacteria, probably stabilize membranes. Enormous % of fossil fuels = hopanoids (global mass est. at 10^11-12 tons, about same as total mass of organic carbon in all living organisms, ~ 10¹² tons!) See structure of hopanoid.
• Membrane proteins contribute stability and many functions, such as transport.

Inside the Cell

Cytoplasm

• largely water (~70%)
• contains as many as 1000 different enzymes, organized into metabolic pathways
• many ribosomes (occupy up to 25% of cell volume, use up to 90% of cell energy), used to make new proteins (mostly enzymes)
• Ribosome size measured in Svedberg (S) units, orginally from ultracentrifugation studies.
• Bacterial ribosomes = 70 S; but eukaryotic ribosomes larger, = 80S
• Ribosomes dissociate into subunits. 70S = 30S + 50S. (prokaryote)
  80S = 40S + 60S (eukartyote)
• View diagram comparing ribosome structure in prokaryotes and eukaryotes

Nucleoid

• Bacteria lack nucleus; DNA contained in cytoplasm in form of tighly coiled circular single DNA molecule, called the bacterial chromosome.
• View TEM of bacterial cell showing DNA localization
• View bacterial DNA "nucleoid"
• Length is about 1000x longer than cell. Total size for smallest bacteria less then 1,000,000 base pairs. For E. coli (fairly sophisticated bacterium), 4,700 kilo base pairs (kbp).
• Bacterial DNA organized in dense staining region = nucleoid. DNA is supercoiled
• Replication of DNA in procaryotes is fundamentally different from eukaryotes: no mitosis, no cytoskeleton. Instead, DNA division occurs, two copies separated to opposite ends of cell, cell pinches off into two copies.
• Plasmids (small circular DNA elements) are found in virtually all bacterial cells.

Inclusion bodies (found in some, not all, cells)

1. stored energy
   • poly-beta-hydroxybutyric acid (fatty material, very high energy). View TEM of bacteria with PHB granules.
   • glycogen (polysaccharide, made of glucose sugare; good energy source)
   • polyphosphate (used to store phosphate, often a limiting nutrient). View structural diagram of polyphosphate.
   • sulfur granules (common in some photosynthetic bacteria)
   • magnetosomes (common in magnetotactic bacteria); allow cells to orient to magnetic fields. View Magnetotactic Bacteria Photo Gallery
2. gas vesicles
   • found in many photosynthetic bacteria and cyanobacteria
   • involved in flotation: form rigid air-filled sac
   • surrounded by rigid protein (not lipid) membrane. Impermeable to liquids, but permeable to gas.

Cell wall

• Rigid layer, preserves shape when rest of cell is digested.
• Made of peptidoglycan = polymer of peptides (typically 4 amino acids long, cross-linked to other chains) and glycans (made of alternating amino sugars)
• Sugars found in peptidoglycan
  1. N-acetylglucosamine (NAG).
  2. N-acetylmuramic acid (NAM).
• NAG-NAM sugars are linked by ß-1,4 linkage). See Fig. 4.42 in text. Note: glycan chain is similar to chitin (= lobster shell), a polymer of NAG.

chemical structure of peptidoglycan (symbols: G = NAG, M = NAM, DAP = diaminopimelic acid)

Unusual properties of PG:

• contains D-amino acids (proteins are made only of L-amino acids), such as D-alanine, D-glutamic acid; also unusual amino acid diaminopimelic acid (or lysine). These are diamino acids, contain an additional -NH₂ group that is needed to cross-link to another peptide chain.
• synthesized not on ribosomes, but by series of enzymes

Mechanism of synthesis of Peptidoglycan

• (NOTE: analogous to cutting open tire, adding more rubber, sealing up, without losing pressure!)
• enzyme needed to cut PG = autolysin
• two Carrier molecules: uridine diphosphate (related precursor of RNA synthesis also) and bactoprenol (55-C atom alcohol)
• synthesis begins inside cell with construction of NAG-NAM-peptide unit.
• carry across membrane, insert into growing wall.
• PENICILLIN: blocks cross peptide linkage. Wall loses all rigidity (only affects growing cells).
• View effects of penicillin on bacteria

Structural Basis for the Gram Stain

• Christian Gram recognized two different types of bacteria based on reaction to certain staining procedure.
  
  
  
  
  
• View cartoon diagram of gram+ and gram- cell wall
• With EM, could see that Gram⁺ cells have thicker walls.
• Gram⁺ cells have thick peptidoglycan layer, also have teichoic acids = polymers of polyalcohols (ribitol or glycerol) + phosphate; also attached D-ala or sugars. Function not clear: highly - charged, probably project out through PG layer.
• Gram^- cells have much thinner peptidoglycan, but also have outer membrane (OM) made of lipid, protein, and LPS (lipopolysaccharide). OM is porous because of porin proteins that create pores, allow small molecules to pass freely.
• Periplasmic space = space between Inner membrane and OM. May occupy as much as 30% of cell volume. Not just an entryway: contains many specialized proteins, often involved in nutrient acquisition. Ex: phosphatases, enzymes that cleave organic molecules containing phosphate, make it available for cell nutrition.
• Can "shock" periplasmic proteins out by sudden immersion in ice cold water.

Osmotic effects

• Water moves freely across membranes (= osmosis) to try to equalize concentration. Interior of cell = ~ 0.9% salt.
• If surrounding fluid = 0.9% salt, Isotonic. Equal water flow in & out
• If surrounding fluid < 0.9% salt, Hypotonic. Water flows in, presses against wall. No problem if wall is intact (can withstand 20 atmospheres of pressure, vs 2 for car tire)
• If surrounding fluid > 0.9% salt, Hypertonic. Water flows out; cell shrinks --> plasmolysis. Stops metabolism.

Effects of lysozyme and pencillin

• Growing cell must break peptidoglyan at certain spots, insert new material, seal back together to grow new wall
• Cell wall contains bacterial enzymes called autolysins to make these breaks, but carefully controlled. Mechanism not well understood.
• View animation of role of autolysin and transpeptidase enzymes
• Lysozyme = enzyme that attacks PG, breaks glycan chain. Found in animal secretions, e.g. tears, saliva, mucous membranes. More active against Gram⁺ than Gram^- cells.
• Penicillin = fungal antibiotic, blocks formation of new peptide links in growing cells walls (= transpeptidation). Note particular advantage of penicillin: no harmful effects on human cells, since PG is unique to bacteria. Penicillin G (the natural isolate) is less effective against many gram-negative cells, since it has difficulty penetrating the outer membrane.
• View animation of effects of penicillin in blocking transpeptidation

Spheroplasts, Protoplasts L-forms, and Mycoplasmas

• Lysozyme and penicillin can lead to cells without walls. Cells lack rigid shape, assume spherical shape = spheroplast (if gram-negative cell) or protoplast (if gram-positive parent). Still able to live if isotonic, but will lyse if put in hypotonic environment.
• L-forms = stable spheroplasts, grow indefinitely w/o wall, but derived from parents with normal walls.
• Mycoplasmas = group of bacteria that evolved to lack wall. Typically found in environments with high osmotic strength (e.g. sewage, vagina. Names like M. orales, M. genitalium, M. pneumoniae.

Archaea: different cell walls

• Cell walls in certain bacteria made of other materials, not peptidoglycan. Can be protein, glycoprotein, or polysaccharide. Some archaea have pseudopeptidoglycan, similar architecture, but N-acetyltalosaminuronic acid instead of N-acetylmuramic. All archaeal walls are resistant to lysozyme.
• These groups collectively called Archaea (because habitats resemble those common of primitive earth); appear to be as different from other bacteria (Eubacteria) as either are from Eukaryotes!

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What are the Archaea?

• When the distinction between prokaryotes and eukaryotes was made as a result of extensive electron micrographic studies of cell types (~ 1940's and 1950's), it was at first thought that all bacteria (prokaryotes) were fundamentally more similar to each other than to eukaryotes.
• However, certain groups of prokaryotes turned out do differ profoundly from the rest. These organisms fell into four broad groups:
  1. Methanogens; anaerobic methane producers
  2. Extreme Halophiles; salt-dependent organisms
  3. Hyperthermophiles; heat-dependent organisms
  4. Thermoplasma; a group of heat and acid-dependent organisms lacking cell walls
• Because many of these habitats resembled conditions of the early (archaic) earth, such as hot, acidic, or salty, these organisms were collectively called Archaea. The remaining bacteria were collectively called the Bacteria (sometimes referred to as Eubacteria, or "true bacteria").
• We now recognize that the Archea are taxonomically as different from Bacteria as either are from eukaryotes, which collectively are called Eukarya .
• These three groups of organisms are called the 3 Domains of Life:
  1. Archaea
  2. Bacteria
  3. Eukarya

Structural Differences between Archaea and Bacteria

1. Cell Wall Architecture
   • Bacterial walls are made of peptidoglycan, a polymer of N-acetyl glucosamine and N-acetyl muramic acid (glycan chain) with short peptides containing both D- and L-amino acids.
   • Archaeal walls differ widely, and are made from different materials. Walls of methanogens often contain pseudopeptidoglycan, similar to peptidoglycan, but with slightly different sugars and archictecture. Other archaea use polysaccharides, proteins, or glycoproteins as wall materials.
   • Many eukaryotes have cell walls. These are built of a variety of materials, but never peptidoglycan.
2. Fatty Acid Linkages
   • In Bacteria and Eukaryotes, membrane fatty acids are linked to glycerol by ester bonds.
   • In Archaea, membranes are built from different types of lipids, polymers of the highly unsaturated molecule isoprene. These lipids are linked by ether bonds, not ester bonds.
3. Structure of RNA Polymerase
   • RNA polymerase is a crucial enzyme required for the synthesis of new RNA molecules. In bacteria, there is a single type of this enzyme, and it is built of four subunits.
   • In eukaryotes, there are three different enzymes, and they each possess 8-12 subunits.
   • Archaea have intermediate properties; they have only a single enzyme, like bacteria, but it is made of 8-12 subunits, like eukaryotes.
4. Initiation Codon
   • Proteins are synthesized on ribosomes, with the precise sequence of amino acids dictated by the genetic code.
   • Ribosomes recognize a unique codon, called the initiation codon (AUG), as the correct location to begin synthesizing a protein.
   • In eukaryotes and archaea, the AUG codon always specifies the amino acid methionine
   • In Bacteria, the AUG codon specifies N-formylmethionine, a modified form of methionine.

Table summarizing differences between Bacteria and Archaea

Property	Bacteria	Archaea	Eukarya
Cell wall	Made of peptidoglyan	Made of various materials, not peptidoglyan	(If present) cellulose, others
Lipids	Fatty acids present, linked by ester bonds	Isoprenes present, linked by ether bonds	Fatty acids present, linked by ester bonds
RNA polymerase enzyme	Single small enzyme; 4 subunits	Single large enzyme; many subunits	Three large enzymes; many subunits
Protein synthesis	1st amino acid = formylmethionine	1st amino acid = methionine	1st amino acid = methionine

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Outside the envelope

1. glycocalyx (also called slime layer, capsule). Not found in all bacteria.
   • varies in thickness, rigidity.
   • important in adhesion, ability to avoid phagocytosis
   • some suggestion that many bacteria lose layer when cultured in laboratory.
   • May be much more prominent in nature than thought.
   • Bacterial adhesion promotes formation of biofilms, masses of bacteria encased in large aggregates of extracellular matrix. Biofilms are not well-understood, but incredibly important. Most bacteria may live largely in biofilms rather than as free organisms (the dispersal stage). Biofilms are harder to get rid of, more resistant to antibiotics.
2. fimbriae & pili
   • short, rigid protein rods, similar in size to flagella, but not involved in motility.
   • function in adhesion, formation of pellicles at liquid surfaces. Function not entirely clear. Pili sometimes involved in pathogenic adhesion (e.g. gonnorhaea)
   • View electron micrograph of Neisseria gonnorhaea with frimbriae
3. Flagella
   • curved filament made of flagellin protein: travels through hollow tube, assembles at external end.
   • can be arranged in two ways:
     
     
     1. polar flagellation: flagella attached at one (monopolar) or both (bipolar) ends. Ex: Pseudomonas aeruginosa
     2. peritrichous flagellation: flagella attached at many sites around cell periphery. Ex: E. coli
   • attached to cell via basal region
   • flagellar rotor can rotate in either direction: CW or CCW. Signals from cell control direction of rotation. See Motility below for application
   • flagellar rotor is the only circular rotor found in nature, aside from human artifact

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Motility

1. Flagellar Motility & Chemotaxis
   • View Bacterial motility from "Cells Alive"
   • Flagella can rotate clockwise (CW) -- in peritrichous cells, flagella then become limp, cell TUMBLES or TWIDDLES -- or countercloskwise (CCW) -- flagellar bundle then becomes rigid, cell RUNS. Rotor is always spinning one direction or other.
   • Energy for rotation comes from Proton gradient.
   • flagellated bacteria move through gradients, TOWARD ATTRACTANTS, AWAY from REPELLANTS.
   • How? detect temporal gradient. If moving towards attractant, suppress tumbles. If moving away, increase frequ. of tumbles.
   • complex mechanism in cell membrane: (1) protein receptors bind chemical; (2) membrane proteins (Methyl-accepting chemotaxis proteins) transmit signal.
2. Other forms of motility
   • some bacteria are motile w/o flagella. GLIDING MOTILITY. move slowly across surfaces, involves sulfur-containing lipids.

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Structural adaptations to inhospitable environments

• Many bacteria can survive for extended periods in absence of food, water, conditions for growth.
• One adaptation: cells entering poor environments shut down normal metabolic pathways, make many new proteins to protect cell. Can survive extended periods of dehydration.
• Some bacteria produce dormant structures: spores, cysts. Allow prolonged survival, dispersal. These are not especially heat tolerant.
• A few genera of bacteria produce highly specialized survival structures = Endospores. View TEM of endospore in bacterial cell. View light micrograph of endospores stained with malachite green.
• Endospores can survive boiling for up to several hours, can be revived after decades of dormancy. One recent report suggests reviving dormant endospores from insect gut trapped in million year old amber, the fossilized remains of tree resins -- view piece of amber with preserved insects (and invisible bacteria).
• Endospore pose major source of difficulty for sterilization. Some resist boiling up to several hours.
• Spore is inert storage form. Found in certain species, most abundantly Bacillus and Clostridia. Formed when cells age, run out of nutrients. View life cycle of an endospore.
• Very refractile, difficult to stain. Coated with thick layers, reduced water content. All cell materials present inside coats, but metabolically inactive.
• Spore coat conatins DIPICOLINIC ACID (DPA) and high Ca. Responsible for part of heat-resistance
• Germination can happen in minutes: spore loses refractility, swells visibly, vegetative cell pushes out of coat.

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