Biology 11 - Lecture 6
Eubacteria Cell Wall Structure
Important Figs: 3.29, 3.32c, 3.33b, 3.36, 3.37, 3.38, 3.42, 3.45, 
	3.50, 3.53, 3.55, 3.63
Optional: S-layers
 
A few sample exam questions:
1. The _________ theory suggests that present-day mitochondria 
and  chloroplasts arose from a primitive bacterial cell.
Select the one BEST answer:
2. Which of the following is true regarding eubacteria 
	and archaeabacteria?
	A. Eubacteria have 70s ribosomes, 
		whereas archaeabacteria have 80s ribosomes.
	B. Eubacteria are eukaryotic and archaeabacteria are prokaryotic.
	C. Both eubacteria and archaeabacteria have 
		peptidoglycan in their cell walls.
	D. Some archaeabacteria are methanogens
3. Which are true  regarding the light microscope 
	we use in laboratory?
	A. Numerical aperature number is the magnification factor of the lens.
	B. Bacterial flagella are too small to be seen without special staining.
	C. The image is light against a dark background.
	D. Maximum magnification is 10,000 times.
	
I. Quick overview of major external structures from last period
Know O:H:K antigenic designation: Ex.  0157:H7:K2
Optional equipment on Gram positive or Gram negatives
A. Flagellum - H Ag - Self aggregating protein = motility.
	Terms: Monotrichous, lophotrichous, peritrichous, 
		axial filament (spirochetes). Fig 3.45
	Detection: Hang drop; flagellar stains, agglutination
	Motility media (0.5 % agar with tetrazolium Cl - 
		reduced to red by NADH)
	Chemotaxis (Fig 3.50) 
	Mutants - mot-, che- 
	Other means of motility - Axial filament, Gliding
	Speed? Faster (per body size) than any animal
	Energy - from PMF
B. Pilus (fimbriae) - self aggregating protein (Fig 3.53, 3.54)
	Function: sex; attachment to mucosal epithelia 
C. Capsule - K Ag. Most are polysaccharide. (Fig 3.55)
	Function - antiphagocytic 
	(hydrophobic with respect to phagocyte surface)
	Ex. N. meningitidis, S. pneumoniae, 
		H. influenzae type b (2,4,6 months vaccine)
	Vaccines --> opsonic antibodies.
	Detection - india ink.
II. Cell wall of Eubacteria -> rigidity, barrier, and 
	protection from osmotic shock
	Shape and arrangement - often reflected in 
		species name. 
	Rod (Bacillus vs bacillus) - vibrio, coccobacilli, coryne, bifido
	Cocci - Strepto (single planeof division), Staphylo (alternating plane)
	Spirillum (spiral) - difficult to grow - syphilis, lyme
III. Gram Positive and Gram Negative cell wall structure
A. Gram stain - Hans Christian Gram (1880s) - 
	dichotomy based upon staining. 

	Crystal Violet	Iodine	Acetone/Alcohol	Safranin
	(basic dye)	(mordant)	(decolorize)	(counterstain)
Gram positive	purple	purple	purple	purple
Gram negative	purple	purple	clear	pink

Biochemical analysis of cell wall -> fundamental difference
Mechanism suggested -->  thicker Peptidoglycan of Gm+
Other: 	Decolorize (25 % acetone in class)
	Acetone dissolves lipids releasing CV-I2 from Gm-
	Problems: 	Thick smear (no penetration)
				Older Gm+ autolysis
				Excess heating
B. Gram positive cell wall structure (Fig 3.32c, 3.33b)
	1. Teichoic acid (sugar phosphate polymer)- attachment
	2. Peptidoglycan (PG) - alternating sugar NAM-NAG 
		linked by amino acids (extensive cross-link)
		40 - 90 % of cell wall in Gm+ 
		Determines shape - remove -> protoplast (Fig 3.34)
		Cell growth and crosslinking of PG
		Site of action for: 
		Penicillins, cephalosporins - Bind transpeptidase
		Vancomycin - Bind d-Ala-d-Ala
		Lysozyme - Split NAM-NAG backbone (tears, saliva, lysosome)
		Other: Muramyl dipeptide (from Mycobacterium) - immunostimulant
	3. Cytoplasmid membrane - Osmotic barrier; 
		defines inside vs outside; site of electron transport.
	4. C55 carrier lipid (Bactoprenol) - 
		transport PG and teichoic building blocks
C. Gram negative cell wall structure (Fig 3.37)
	1. Outer membrane (second membrane)- 
		phospholipid inner layer, LPS outer layer.
	2. Lipopolysaccharide (LPS) or lipooligosaccharide (LOS) 
		= endotoxin) - Fig 3.36
     	a. O-Ag - repeating sugars - Antigenic diversity
	b. core - 10 sugars conserved
	c. Lipid A - Fatty acids (embedded in OM) toxic portion -
		--> Biological meltdown
		100,000 die in US from septic shock (circulatory failure) - 
		i. pyrogen - fever via IL-1 and hypothalamus
		ii. leukocytosis - increase WBC
		iii. generalized clotting - DIC, bleeding, circulatory collapse
		iv. peripheral vasodilation, blood pooling, 
			decrease blood pressure -> 
			multiple organ failure from anoxia.
		Sources: Contaminated saline, vaccine side effects
			 (whoop cough), plague...
		Hot areas of research: Centocore - monoclonal to Lipid A.
		Antibody to Tumor Necrosis Factor (TNF) alpha, 
			Interleukin-2 and other mediators
		TNF receptor bound to Fc.
		Assay - Limulus amoebocyte lysate assay (LAL) 
			- horseshoe crab lysate gels with LPS.
	3. Outer Membrane Proteins - porin; 
		vaccine target (N. meningitidis gp B)
	4. Lipoprotein - anchor Outer Membrane to PG
	5. Peptidoglycan - similar to Gm+ but less thick
		 and less cross-linking.
	6. Periplasmic space - concentration of enzymes
	7. Cytoplasmic membrane, C55 carrier, etc same as Gram +
IV. Acid Fast - Differential stain (specific for Mycobacterium) - p 517.
	Mycolic acids (C90) of Mycobacterium
	Nocardic acids (C50) of Nocardia
	Other bacteria (C18)
	Mycolic acid: Connected to PG -> Waxy appearance of Mycobacteria.
	Procedure: ZCF (baked in); Decolorize (Acid alcohol);
		 Counterstain (LMB)

	Ziel's Carbol Fuchsin	Acid alcohol	LMB
Mycobacterium	red	red	red
Non-acid fast	red	clear	blue

Sputum stain to determine presence of Mycobacterium tuberculosis