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What am I responsible for? George Ferguson

What am I responsible for? George Ferguson. Quiz #1 September 20, 2004. Outer membranes of G - bacteria. Lengeler et al. Chapter 23 pp. 555-570 Assembly of cell structures Proteins LPS Export of biopolymers. Escherichia coli. Gram-negative cell envelope. Proteins.

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What am I responsible for? George Ferguson

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  1. What am I responsible for?George Ferguson Quiz #1 September 20, 2004

  2. Outer membranes of G - bacteria • Lengeler et al. • Chapter 23 pp. 555-570 • Assembly of cell structures • Proteins • LPS • Export of biopolymers

  3. Escherichia coli

  4. Gram-negative cell envelope

  5. Proteins • Braun’s lipoprotein (LP) • Mr 7500 da • Synthesized as precursor: pro-LP • Signal sequence: 20 aa • Mature species: 56 aa • NH3-Met1-…Leu-Ala-Gly20-↓-Cys1- ..…….Lys58-COOH

  6. Braun’s LP • Pro-LP ---glyceryl transferase  Pro-LP-Cys1-glycerol………….COOH • ---transacylase(s)  Pro-LP-Cys1-diglyceride…………COOH • ---signal peptidase II  Cys1-diglyceride……..COOH + signal seqantibiotic = globomycin

  7. Braun’s LP (2) • --N-acylase acyl-Cys1-diglyceride……………. = free form of LP • --ligase  acyl-Cys1-diglyceride…………….Lys58-ε-NH2-DAP-peptidoglycan =bound form of LP

  8. Porins • Mr 35 to 40 kDa • Non-specific diffusion channels • Surface-exposed • Trimers: each monomer = channel • Purify, reconstitute • Mr exclusion limits • 2° structure: amphipathic strands, loops, turns • 3° structure: 16-stranded β barrel

  9. Porins • Long, variable loops: surface • Short, connecting turns: periplasm • 8 x 10 A°: constriction • Open • Not gated • No internal binding sites for substrates

  10. Specific porins • LamB, E. coli; maltoporin • 18-stranded β barrel • Greasy slide: 6 aromatic aa • Internal binding site for maltose

  11. Iron regulated OM proteinsIROMPs • Mr 75 to 105 kDa • Induced by low iron • Multi-component systems • High-affinity ligand binding site • Require energy to import iron-siderophore

  12. IROMPs • FhuA: E. coli • 78 kDa • 22 stranded βbarrel • cork = internal globular domain

  13. LPS • 3 regions: • Lipid A, Mr 2000 • Inner core: 3 x KDO; Mr 900 • Outer core: Hep, Gal, Glc, NAcGlc; Mr ~1800 • O side chain: • tetrascccharide repeat • Mr ~4000 for n = 6 • Complete LPS Mr ~8700

  14. LPS genetic lesions • Glycosyl transferases • Extend chain lengths • Deep rough [Re] = lipid A + KDOs • Core mutants [Ra to Rd] = missing core sugars • Complete LPS = smooth

  15. Endotoxin • Small amounts: ng / kg body weight • Targets: • macrophages, monocytes, B lymphocytes, complement • Activity: activation pathways • Effects: • Fever, increased antibody synthesis, inflammation

  16. Endotoxin • Large amounts: μg / kg body weight • Targets: • macrophages, monocytes, platelets • Activity: • increase in IL-1, TNF, clotting factors • Effects: • Septic shock • disseminated intravascular coagulation

  17. LPS and anti-microbials

  18. LPS and anti-microbials • Conclusions: • LPS • barrier to hydrophobic agents • Hydrophilic compounds are not impeded in exerting toxic effects

  19. Inside a living cell • David Goodsell • TIBS 1991 • Cutout 3 figs: 1a, 1c, 1d • Trim figs to edge; no border • Name on back of each figure • Paper clipped • Submit with Quiz #1

  20. Inside a living cell • Colour scheme: • Ribosomes = red • Proteins = blue • DNA = green • LPS / P lipids = brown • Peptidoglycan = red • mRNA, tRNA = yellow

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