Lecture 3 - Bacterial Cell Wall.docx

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Department
Cell and Systems Biology
Course
CSB328H1
Professor
William Navarre
Semester
Winter

Description
MGY377H © Lisa | Page 13 L E C T U R E 3 : B A C T E R I A L C E L L WA L L GRAM STAIN 1. is a differential staining procedure 2. bacteria can be divided into 2 major groups based on their rx to Gram staining: Gram- positive & Gram-negative 1. diff rxs are due to differences in the cell wall structure of Gm+ & Gm- cells, particularly the composition of peptidoglycan (PG) 1. in Gm+ bacteria, as much as 90% of the cell wall consists of peptidoglycan layers (up to ~30 layers) 2. in Gm- bacteria, only ~10% of cell wall is peptidoglycan (1 layer) PEPTIDOGLYCAN (MUREIN) 3. chemically unique, rigid structural component of cell wall found in bacteria 1. composed of peptides & sugars 4. largest molecule in bacterial cell & constitute 50-90% of Gm+ cell wall & <10% of Gm- cell wall dry weight 5. strengthening element, provides shape of cells & preserves integrity of the cytoplasmic membrane from rupture in medium/low osmolarity (see picture) 1. low solute condition → w/o PG, imbalance allows H O to2enter cell MGY377H © Lisa Z| Page 2 1. lysozyme is added in buffer for lab experiments requiring extraction of bacteria proteins 2. isotonic solution → lysozyme digests PG, but OM remains intact 6. contains compounds unique to the microbial world: ᴅ-amino acids, diaminopilemic acid (DAP), N-acetyl muramic acid MGY377H © Lisa | Page 33 7. each cell wall peptidoglycan subunit has 2 similar sugars: 1. N-acetyl-glucosamine (NAG, GlcNAc) is found in a lot of species including animals 2. N-acetyl-muramic acid (NAM, MurNAc) is a derivative of NAG that has an extra carboxyl group that can link it to the N-terminus (amino group) of the wall peptide (it is unique to bacteria) 8. major peptide subunits: 1. L-alanine 2. lysine 3. mDAP 1. unusual ᴅ-amino acids (config is determined by amino acid location [L/R], ᴅ imp in resisting degradation by enzymes) 4. ᴅ-isoglutamate (Gm-) 5. ᴅ-isoglutamine (Gm+) 6. ᴅ- alanine MGY377H © Lisa Zh| Page 4 9. the subunits are attached to one another via 2 diff rxs 1. transglycosylation (via transglycosylase, joins sugar subunits) 2. transpeptidation (via transpeptidase, joins peptide subunits from diff sugar chains) 10. the polymerization of subunits via their sugars leads to the formation of long glycan strands that alternate bw NAG & NAM 11. the transpepdation rx links these together to make the “net”-like structure of peptidoglycan rd 12. the 3 amino acid (lysine or mDAP) residue has a free amino group (N) that can form a peptide bond w another AA 13. wall peptides end w 2 ᴅ-alanine residues (4 , 5 ) at their C-terminal end 14. transpeptidation rx (peptide bond bw PG chains) 1. transpeptidase cleaves the C-terminal (5 ) ᴅ-alanine of one subunit 2. transpeptidase then links the free amino group of the neighbouring wall peptide (L- th lysine or mDAP) to the newly liberated C-terminus (4 ) ᴅ-alanine of the first wall peptide 3. 2 glycan chains have been linked by transpeptidation bw each of their 3 subunits 1. when carboxylase cleaves the last ᴅ-alanine, it terminates the chain ENZYMES ACTIVE ON PEPTIDOGLYCAN 1. carboxypeptidase (carboxylase): cleaves the terminal (5 ) ᴅ-Ala from the peptide rd subunit but doesn’t form a bond bw the remaining ᴅ-Ala & the 3 amino acid of the peptide subunit of the N-acetylmuramic acid of another glycan strand 1. functions in regulating the degree of cross-linking of glycan strands 2. amount of cross-linking depends on enzymatic action of transpeptidase vs. carboxypeptidase 15. types of wall peptide cross-linking: 1. Gm- bacteria: link peptides bw the AA on the side chain of the mDAP (m- A2pm) to the carboxy terminus of the 4 ᴅ-Ala of the adjacent wall peptide *the epsilon (ԑ) symbol means that the linkage is occurring off the epsilon (5 ) carbon of the mDAP side chain *the gamma (γ) symbol means that the linkage is occurring off the gamma (3rd) carbon of the ᴅ- glutamic acid 2. Gm+ bacteria: link peptides bw the amino group on the side chain of a the 3 rd lysine to a chain of AAs (interbridge; ex. in S. aureus, its 5 glycines) 1. the N-terminus of this cross-bridge connects to the carboxy terminus of the ᴅ- Ala in position 4 of the adjacent wall peptide 2. Gm+ cell wall precursors often have additional peptide cross-bridges (interbridges) that are attached to a lysine at position 3 in penicillin targets the wall peptide 2. unusual variants in transpeptidase: crossbridges have mimics the rx of been observed: 1. Micrococcus luteus transpeptidase, binds ᴅ-Ala (Gm+), the cross-bridge is derived from a wall and blocks transpeptidase rx peptide that was cleaved off of a pre-existing cell wall subunit 1. the cross-bridge tetrapeptide is cleaved from the N-acetyl muramic acid by an enzyme (N-acetyl- in Micrococcus muramyl-L-alanine amidase) luteus 2. an odd case: Corynebacterium poinsettiae – there is no free amino group (h
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