The procaryotic cell wall

CytoplasmCytoplasm

Oxidative phosphorylation occurs at cell membrane (since there are no mitochondria).

Cell membrane

The cell wall is outside of cell membrane – rigid, protecting cell from osmotic lysis.

Cell Wall

The Cell EnvelopeThe Cell Envelope

Gram PositiveGram Positive Gram NegativeGram Negative

Cell EnvelopeCell Envelope

• Cell membrane + cell wall (+ plus outer ell membrane + cell wall (+ plus outer membrane)membrane)

• Cell wallCell wall

– peptidoglycan peptidoglycan

– attached structuresattached structures.

Gram positiveGram positive

Gram negativeGram negative

CytoplasmCytoplasm

Lipoteichoic acid Peptidoglycan-teichoic acid

Cytoplasmic membrane

GRAM POSITIVE CELL GRAM POSITIVE CELL ENVELOPEENVELOPE

Degradative enzyme

PeptidoglycanPeptidoglycan

• single macromolecule single macromolecule

• highly cross-linkedhighly cross-linked

• surrounds cellsurrounds cell

• provides rigidityprovides rigidity

Peptidoglycan structurePeptidoglycan structure

• glycan backbone– muramic acid(NAM)

– Glucosamine (NAG)

-1,4-bond

• peptide side chain of four alternating D- and L-amino acids

• peptide cross-bridge– D- and L- amino acids

– diaminopimelic acid

• (attention:difference between G+ and G-)

PeptidoglycanPeptidoglycan

Muramic acid

Glucosamine

L-alanineD-glutamic acidL-lysine/Diaminopimelic acidD-alanineD-alanine

Muramic acid, D-amino acids diaminopimelic acid

– not synthesized by mammals.

Gram Positive Cell EnvelopeGram Positive Cell Envelope

• Teichoic acid – polymer– phosphorus– ribitol or glycerol backbone–

• Teichuronic acid– polymer – no phosphorus– glucuronic acid backbone

Teichoic and teichuronic acidsTeichoic and teichuronic acids

• Metal ion uptake

• Direct autolytic enzymes– cell wall digestioncell wall digestion

– cell wall synthesis cell wall synthesis

GRAM NEGATIVE GRAM NEGATIVE CELL ENVELOPECELL ENVELOPE

CytoplasmCytoplasm

Inner (cytoplasmic) membrane

Outer Membrane(Major permeability barrier) LipopolysaccharidePorin

Braun lipoprotein

Periplasmic space Degradative enzyme

Periplasmic binding proteinPermease

Outer MembraneOuter Membrane

• lipopolysaccharidelipopolysaccharide

• phospholipidsphospholipids

• ProteinsProteins

– PorinsPorins

• Braun lipoprotein– binds cell wall to outer membrane

Outer MembraneOuter Membrane Gram negative bacteria Gram negative bacteria functionfunction1. Prevent or slow the entry of toxic substance1. Prevent or slow the entry of toxic substance2. permeable and permits the passage of small molecules p2. permeable and permits the passage of small molecules p

orin protein.orin protein.3. Prevent the loss of constituents like periplasmic enzymes.3. Prevent the loss of constituents like periplasmic enzymes.

periplasmic space:periplasmic space: space between inner and outer membranespace between inner and outer membrane

store degradative enzymesstore degradative enzymes• Gram positive bacteriaGram positive bacteria no periplasmic spaceno periplasmic space

Periplasmic spacePeriplasmic 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

LipopolysaccharideLipopolysaccharide

Lipid A• Glucosamine disaccharide

• Beta hydroxy fatty acids

Core • Heptoses• Ketodeoxyoctonic acid

O-antigenHighly variablen

GRAM POSITIVEGRAM POSITIVE

GRAM NEGATIVEGRAM NEGATIVE

CytoplasmCytoplasm

CytoplasmCytoplasm

Lipoteichoic acid Peptidoglycan-teichoic acid

Cytoplasmic membrane

Inner (cytoplasmic) membrane

Outer Membrane

LipopolysaccharidePorin

Braun lipoprotein

Peri

plas

mic

spa

ce

The mechanism of Gram stainingThe mechanism of Gram staining During the procedure the bacteria are first stained with crystal violetcrystal violet and next treated with iodine iodine to promote dye retention. When gram-positive bacteria then are decolorized with etethanolhanol, 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 Gram-stain.

1. Place one needle of solid bacterial growth or two loopsof 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 watercompletely and spread the mixture out to cover about half of thetotal 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 layersCapsules and slime layers

• outside cell envelopeoutside cell envelope• well defined: capsule well defined: capsule • not defined: slime layer or glycocalyx not defined: slime layer or glycocalyx • usually polysaccharideusually polysaccharide• Be negative stain or special capsule stains Be negative stain or special capsule stains • often lost on often lost on in vitroin vitro culture culture• protective protective in vivoin vivo• Not required for bacteria growth and reproduction Not required for bacteria growth and reproduction

Capsules and Slime Layers Capsules and Slime Layers (Glycocalyx or (Glycocalyx or 多糖包被多糖包被 ))

Glycocalyx:Polysaccharide-co

ntaining material lying outside the cell.

Capsules:Rigid layers of gly

cocalyx.

Slime Layers:Flexible layers of

glycocalyx.

Capsule

The functions of capsuleThe 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 obje

cts in aquatic enviroments or to tissue surfaces in plant and animals hosts.

• 5. Aids in gliding bacteria motility.

Paracrystalline layers (S-layers)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?What we have learned so far?

• Gram Staining procedure? • Differences in cell walls between Gram-positive and Gram-neg

ative 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.