The procaryotic cell wall
Oxidative phosphorylation occurs at cell membrane
(since there are no mitochondria).
Cell Wall
Cytoplasm
Cell membrane
The cell wall is outside of cell membrane
– rigid, protecting cell from osmotic lysis.
The Cell Envelope
Gram Positive
Gram Negative
Cell Envelope
• Cell membrane + cell wall (+ plus outer
membrane)
• Cell wall
– peptidoglycan
– attached structures.
Gram positive
Gram negative
GRAM POSITIVE CELL
ENVELOPE
Degradative enzyme
Lipoteichoic acid
Peptidoglycan-teichoic acid
Cytoplasmic membrane
Cytoplasm
Peptidoglycan
• single macromolecule
• highly cross-linked
• surrounds cell
• provides rigidity
Peptidoglycan structure
• glycan backbone
– muramic acid(NAM)
– Glucosamine (NAG)
-1,4-bond
• peptide side chain of four alternating D- and Lamino acids
• peptide cross-bridge
– D- and L- amino acids
– diaminopimelic acid
• (attention:difference between G+ and G-)
L-alanine
D-glutamic acid
L-lysine/Diaminopimelic acid
D-alanine
D-alanine
Peptidoglycan
Muramic acid
Glucosamine
Muramic acid, D-amino acids
diaminopimelic acid
– not synthesized by mammals.
Gram Positive Cell Envelope
•
Teichoic acid
– polymer
– phosphorus
– ribitol or glycerol backbone
–
• Teichuronic acid
– polymer
– no phosphorus
– glucuronic acid backbone
Teichoic and teichuronic acids
• Metal ion uptake
• Direct autolytic enzymes
– cell wall digestion
– cell wall synthesis
GRAM NEGATIVE
CELL ENVELOPE
Outer Membrane
(Major permeability barrier)
Porin
Lipopolysaccharide
Braun lipoprotein
Degradative enzyme
Inner (cytoplasmic) membrane
Periplasmic binding protein
Cytoplasm
Permease
Outer Membrane
• lipopolysaccharide
• phospholipids
• Proteins
– Porins
• Braun lipoprotein
– binds cell wall to outer membrane
Outer Membrane
Gram negative bacteria
function
1. Prevent or slow the entry of toxic substance
2. permeable and permits the passage of small molecules
porin protein.
3. Prevent the loss of constituents like periplasmic enzymes.
periplasmic space: space between inner and outer membrane
store degradative enzymes
• Gram positive bacteria
no periplasmic space
Periplasmic space
• G-: a space between the plasma
membrane and the outer membrane
• G+: smaller gap between the plasma
membrane and wall
• Periplasmic enzymes and other
proteins
Lipopolysaccharide
n
O-antigen
Highly variable
Core
• Heptoses
• Ketodeoxyoctonic acid
Lipid A
• Glucosamine disaccharide
• Beta hydroxy fatty acids
GRAM POSITIVE
Lipoteichoic acid
Peptidoglycan-teichoic acid
Cytoplasmic membrane
Cytoplasm
GRAM NEGATIVE
Porin
Outer Membrane
Braun lipoprotein
Inner (cytoplasmic) membrane
Cytoplasm
Lipopolysaccharide
The mechanism of Gram staining
During the procedure the bacteria are first stained with
crystal violet and next treated with iodine to promote dye
retention. When gram-positive bacteria then are decolorized
with ethanol, the alcohol is thought to shrink the pores of the
thick peptidoglycan. Thus the dye-iodine complex is retained
during the short decolorization step and the bacteria remain
purple. In contrast, gram-negative bacteria peptidoglycan is
very thin, not as highly cross-linked, and has larger pores.
Alcohol treatment also may extract enough lipid from the
gram-neaative wall to increase its porosity further. For these
reasons, alcohol more readily removes the purple crystal
violet-iodine complex from gram-negative bacteria.
PREPARATION
The preparation of a smear is required for many laboratory procedures,
including the Gram-stain. The purpose of making a smear is to fix the
bacteria onto the slide and to prevent the sample from being lost during a
staining procedure. A smear can be prepared from a solid or broth
medium. Below are some guidelines for preparing a smear for a Gramstain.
1. Place one needle of solid
bacterial growth or two loops
of liquid bacterial growth in the
center of a clean slide.
2. If working from a solid medium, add
one drop (and only one drop)
of water to your specimen with a water
bottle. If using a broth medium, do not
add the water.
3. Now, with your inoculating loop,
mix the specimen with the water
completely and spread the mixture out
to cover about half of the
total slide area.
4. Place the slide on a slide warmer and
wait for it to dry. The smear is now
ready for the staining procedure.
Gram-staining Procedure
Gram-staining is a four part procedure which uses certain
dyes to make a bacterial cell stand out against against its
background. The specimen should be mounted and fixed on a
slide before you procede to stain it. The reagents you will need
to successfully perform this operation are:
•Crystal Violet (the Primary Stain)
•Iodine Solution (the Mordant)
•Decolorizer (ethanol is a good choice)
•Safranin (the Counterstain)
•Water (preferably in a squirt bottle)
STEP 1: Place your slide on a
slide holder or a rack. Flood
(cover completely) the entire
slide with crystal violet. Let
the crystal violet stand for
about 60 seconds. When the
time has elapsed, wash your
slide for 5 seconds with water.
The specimen should appear
blue-violet when observed
with the naked eye.
STEP 2: Now, flood your slide with the
iodine solution. Let it stand about a
minute as well. When time has expired,
rinse the slide with water for 5 seconds
and immediately procede to step three.
At this point, the specimen should still
be blue-violet.
Capsules and slime layers
•
•
•
•
•
•
•
•
outside cell envelope
well defined: capsule
not defined: slime layer or glycocalyx
usually polysaccharide
Be negative stain or special capsule stains
often lost on in vitro culture
protective in vivo
Not required for bacteria growth and reproduction
Capsules and Slime Layers
(Glycocalyx or 多糖包被)
Glycocalyx:
Polysaccharidecontaining
material lying
outside the cell.
Capsules:
Rigid layers of
glycocalyx.
Slime Layers:
Flexible layers of
glycocalyx.
Capsule
The functions of capsule
• 1. Resist phagocytosis by host phagocytic cells.
Streptococcus pneumooniae
• 2. Protect bacteria against desiccation.
• 3. Exclude bacterial viruses and most hydrophobic
toxic materials such as detergents.
• 4. aid bacterial attachment to surfaces of solid
objects in aquatic enviroments or to tissue surfaces
in plant and animals hosts.
• 5. Aids in gliding bacteria motility.
Paracrystalline layers (S-layers)
• Many prokaryotes contain a
cell surface layer composed
of a two-dimensional array
of protein called S-layer.
• S-layer has been detected in
representatives of virtually
every phylogenetic grouping
of bacteria and nearly
universal among Archaea.
• In some species of Archaea,
the S-layer is also the cell
wall.
S-layer
Exist in G+, G-, archaea.
G+: adhere directly to the
outer membrane.
G-: associated with the
peptidoglycan surface.
Functions:
1. protect the cell against ion
and pH fluctuations,
osmotic stress, enzymes.
2. Help maintain the shape
and envelope rigidity.
3. Promote cell adhesion to
surfaces.
What we have learned so far?
• Gram Staining procedure?
• Differences in cell walls between Gram-positive and Gramnegative bacteria?
• The structure of peptidoglycan?
• What are the chemical reasons for the rigidity that is conferred
on the cell wall by the peptidoglycan structure?
• List several functions for the outer wall layer in Gram-negative
bacteria?
• The structure of lipopolysaccharide?
• What types of Bacteria have a periplasm and of what significant
is the periplasmic space?
• The mechanism of Gram staining?
• What is capsule? Describe its function.
• What is S-layer? Describe its function.