1. The procaryotic cell wall

2. Cytoplasm 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

3. The Cell Envelope Gram Positive Gram Negative

7. Cell Envelope ell membrane + cell wall (+ plus outer membrane) Cell membrane + cell wall (+ plus outer membrane) Cell wall Cell wall – peptidoglycan – attached structures – attached structures.

8. Gram positive Gram negative

9. Cytoplasm Lipoteichoic acid Peptidoglycan-teichoic acid Cytoplasmic membrane GRAM POSITIVE CELL ENVELOPE Degradative enzyme

10. Peptidoglycan single macromolecule single macromolecule highly cross-linked highly cross-linked surrounds cell surrounds cell provides rigidity provides rigidity

11. Peptidoglycan 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 - )

12. Peptidoglycan Muramic acid Glucosamine L-alanine D-glutamic acid L-lysine/Diaminopimelic acid D-alanine

13. Muramic acid, D-amino acids diaminopimelic acid – not synthesized by mammals.

14. Gram Positive Cell Envelope Teichoic acid –polymer –phosphorus –ribitol or glycerol backbone – Teichuronic acid –polymer –no phosphorus –glucuronic acid backbone

15. Teichoic and teichuronic acids Metal ion uptake Direct autolytic enzymes –cell wall digestion –cell wall synthesis

16. GRAM NEGATIVE CELL ENVELOPE GRAM NEGATIVE CELL ENVELOPE Cytoplasm Inner (cytoplasmic) membrane Outer Membrane (Major permeability barrier) Lipopolysaccharide Porin Braun lipoprotein Periplasmic space Degradative enzyme Periplasmic binding protein Permease

17. Outer Membrane lipopolysaccharidelipopolysaccharide phospholipidsphospholipids ProteinsProteins –Porins Braun lipoprotein –binds cell wall to outer membrane

18. Outer Membrane Gram negative bacteria Gram negative bacteria function 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 bacteriaGram positive bacteria no periplasmic space no periplasmic space

19. 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

21. Lipopolysaccharide Lipid A Glucosamine disaccharide Beta hydroxy fatty acids Core Heptoses Ketodeoxyoctonic acid O-antigen Highly variable n

22. GRAM POSITIVE GRAM NEGATIVE Cytoplasm Cytoplasm Lipoteichoic acid Peptidoglycan-teichoic acid Cytoplasmic membrane Inner (cytoplasmic) membrane Outer Membrane Lipopolysaccharide Porin Braun lipoprotein Periplasmic space

23. The mechanism of Gram staining crystal violetiodine ethanol 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.

24. 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 loops of liquid bacterial growth in the center of a clean slide.

25. 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.

26. 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)

27. 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.

28. 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.

29. Capsules and slime layers outside cell envelopeoutside cell envelope well defined: capsulewell defined: capsule not defined: slime layer or glycocalyxnot defined: slime layer or glycocalyx usually polysaccharideusually polysaccharide Be negative stain or special capsule stainsBe negative stain or special capsule stains often lost on in vitro cultureoften lost on in vitro culture protective in vivoprotective in vivo Not required for bacteria growth and reproductionNot required for bacteria growth and reproduction

30. Capsules and Slime Layers (Glycocalyx or 多糖包被 ) Glycocalyx: Polysaccharide- containing material lying outside the cell. Capsules: Rigid layers of glycocalyx. Slime Layers: Flexible layers of glycocalyx.

31. Capsule

32. 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.

33. 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.

34. 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.

35. What we have learned so far? Gram Staining procedure? Differences in cell walls between Gram-positive and Gram- negative 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.