Faculty: Dr. Alvin Fox

1. CELL ENVELOPE, SPORES AND MACROMOLECULAR BIOSYNTHESIS Faculty: Dr. Alvin Fox Suggested reading: Murray, Third edition Chapter 3

2. KEY WORDS Cell envelope Lipopolysaccharide (endotoxin) Cell wall Teichoic acid Cell membrane Teichuronic acid Outer membrane Lipoteichoic acid Peptidoglycan Mycolic acid Braun lipoprotein Undecaprenol (bactoprenol) Porins Endospore

3. Topics for discussion: • The structure of the Gram negative, Gram positive and acid fast cell envelopes. • The composition and function of unique cell envelope macromolecules and their biosynthesis. • Endospores, which are unusual in many ways.

4. Cell Envelope The cell envelope may be defined as the cell membrane and cell wall plus an outer membrane if one is present. The cell wall consists of the peptidoglycan layer and attached structures.

5. Bacterial cell envelopes fall into two major categories: Gram positive and Gram negative Gram staining is based on characteristics that reflect major structural differences between the two groups.

7. Peptidoglycan A single bag-shaped, highly cross-linked macromolecule that surrounds the bacterial cell membrane and provides rigidity.

8. Peptidoglycan consists of: • 1. Glycan (polysaccharide) backbone consisting of • N-acetyl muramicacid (Mur) • N-acetyl glucosamine (Gln) • 2. Peptide side chains containing • A. D- and L- amino acids • B. Diaminopimelic acid (in some cases) • C. The side chains are cross-linked by peptide bridges which vary in structure among bacterial species.

11. Muramic acid, D-amino acids and diaminopimelic acid are notsynthesized by mammals. PG is found in all eubacteria exceptChlamydiaandMycoplasma.

12. r r r r r r r r r r Gram Positive Cell Envelope Lipoteichoic acid Peptidoglycan-teichoic acid Cytoplasmic membrane Cytoplasm

13. Gram Positive Cell Envelope • Covalently bound to the thick peptidoglycan are: • Teichoic acid their backbones are usually phosphorus-containing polymers of ribitol or glycerol or • Teichuronic acid which are glucuronic acid- containing polysaccharides. These negatively charged molecules are believed to be involved in concentrating metal ions from the surroundings. Teichoic acids can also direct autolytic enzymes to sites of peptidoglycan digestion (autolysis), one of the steps in cell wall biosynthesis.

14. r Gram Negative Cell Envelope Lipopolysaccharide Porin Outer Membrane Braun lipoprotein Peptidoglycan Cytoplasm Inner (cytoplasmic) membrane

15. Gram Negative Cell Envelope • Covalently linked to the thin peptidoglycan is • Braun lipoprotein which binds the outer membrane to the cell wall. • Proteins and phospholipids • Lipopolysaccharide whichconsists of three regions: • an outer O antigen, • a middle core which contains several sugars (heptoses and ketodeoxyoctonic acid), not found elsewhere in nature • an inner lipid A region which contains ß hydroxy fatty acids (uncommon in nature). The molecule displays endotoxin activity. • Porins in the outer membrane help form channels to allow passage of • small hydrophilic nutrients (such as sugars) through the outer membrane.

16. Structure of Lipopolysaccharide

17. Acid fast and related bacteria (mycobacteria, nocardia and corynebacteria) The cell envelopes of these organisms are considerably more complex than other bacteria. Mycolic acid (long, branch chained fatty acids) is covalently bound via a polysaccharide to peptidoglycan. Additional mycolic acid-containing compounds and other complex lipids form a thick waxy membranous layer outside the peptidoglycan layer.

18. Synthesis of cell envelope macromolecules Peptidoglycan: 1. The precursor subunit (muramyl pentapeptide attached to uridine diphosphate, UDP) is synthesized in the cytoplasm and passed to the cell membrane. 2. The subunit is moved enzymatically from the nucleotide to a lipid carrier(undecaprenol/bactoprenol) and built into a completed subunit (disaccharide pentapeptide with attached bridge peptide). 3. The completed subunits are then exported to the cell wall.

19. 4. The undecaprenol is recirculated in the cell membrane and used again. 5. The glycan backbones of the existing cell wall is enzymatically broken (by autolysins) to allow insertion of the newly synthesized subunit. 6. Cross-linking of the peptide side-chain of the inserted subunit to the existing chain then occurs enzymatically (penicillin binding proteins). 7. Completed subunits of teichoic and teichuronic acids are also synthesized in the cell membrane (on lipid carriers) before transport and insertion into the existing cell wall.

20. Lipopolysaccharide 1. Lipid A is assembled in the cell membrane and the core sugars attached sequentially. 2. O-antigen subunits are independently synthesized (on a lipid carrier as in peptidoglycan synthesis). 3. The fully synthesized O-antigen is then attached to the lipid A-core (generating lipopolysaccharide) in the cell membrane before passage/insertion into the outer membrane.

21. Endospores 1. These modified Gram positive bacterial cells have an unusual cell envelope that contains a cell membrane and an outer membrane. 2. The peptidoglycan layer is less cross-linked than in most bacterial cells and contains a dehydrated form of muramic acid. 3. The spore peptidoglycan is referred to as a cortex and is found between the two membranes.

22. 4. A coat consisting of highly cross-linked keratin is found around the outside of the cell. 5. The bacterial spore is highly resistant to chemical agents because of this coat. 6. When sporulation occurs, cell division is unequal and the larger so-called "mother cell" envelops the daughter cell. 7. The cell membrane of the daughter cell constitutes the inner membrane of the spore and the cell membrane of the mother forms the outer membrane.