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Lecture_PP frames: Coulton

Lecture_PP frames: Coulton. http://www.mcgill.ca/microimm/ undergraduate/courses/mimm323/. 4. Disruption of bacterial cells. Boiling in 2% SDS Cell lysate: all contents dissolved Proteins: SDS-PAGE Sonic oscillation Pop bacteria French pressure cell 16,000 psi.

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Lecture_PP frames: Coulton

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  1. Lecture_PP frames: Coulton http://www.mcgill.ca/microimm/ undergraduate/courses/mimm323/

  2. 4. Disruption of bacterial cells • Boiling in 2% SDS • Cell lysate: all contents dissolved • Proteins: SDS-PAGE • Sonic oscillation • Pop bacteria • French pressure cell • 16,000 psi

  3. Proteins of bacterial membranes Lengeler et al. pp. 30-33, 37-42

  4. Cellular fractionation • Lysozyme digestion • Separation of cell envelopes from cytoplasm • Isopycnic centrifugation

  5. Cytoplasmic membranes • Composition • Proteins • Functions

  6. Outer membranes • Composition • Proteins: major, minor • Functions: • structural integrity • diffusion channels • nutrient transport: iron, vit B12

  7. Gram-negative cell envelope

  8. Lysozyme • α – bacterial activity • N-acetyl glucosaminidase • Target: peptidoglycan

  9. Lysozyme digestion • G + / lysozyme; dilute medium lysis • G + / lysozyme; iso-osmotic  protoplasts • Protoplasts in dilute medium  lysis • G - / lysozyme; dilute medium  no lysis • G - / + EDTA + lysozyme; iso-osmotic  spheroplasts • Spheroplasts in dilute medium  lysis

  10. Cell lysis • G + cell wall: ~ no barrier to lysozyme • G - cell envelope: OM = barrier to lysozyme’s access to peptidoglycan

  11. Separation of cell envelopes • Differential centrifugation • Bacterial cell lysate  15,000 x g, 30 min: • Pelleted membranes • Soluble cytoplasm

  12. Separation of cell envelopes • Isopycnic centrifugation • Gradient of densities of sucrose: • 70%, 55%, 50%, 45%, 40%, 35%, 30%, 25% • Sample: pelleted membranes • 100,000 x g • 2 bands: 55% - 50% interface = OM vesicles 40% - 35% interface = CM vesicles

  13. Composition: CM • Phospholipids: G + and G – P ethanolamine, 75% P glycerol, 18% cardiolipin, 5% • Fatty acids: palmitic acid, 43% palmitoleic acid, 33% cis vaccinic acid, 25%

  14. Composition: CM • Proteins: • 1 D SDS-PAGE • 2 D SDS-PAGE: pI and molecular mass (Mr): • > 1500 proteins in CM of E. coli • MALDI – mass spectrometry: • Identification of spots on 2D gels

  15. Functions: CM proteins • Transporters = permeases • Synthesis of • Membrane lipids • Peptidoglycan: PBPs • Electron transport chain • DNA attachment: OriC • Chemotaxis • Secretory apparatus

  16. Composition: OM • P lipids: similar to CM • Lipopolysaccharide (LPS): • Lipid A: diglucosamine disaccharide; P or PP • Lipid: β OH myristic acid • Core sugars: • 2-keto-3-deoxyoctonate (KDO) • L-glycero-D-mannoheptose • N-Ac glucosamine, glucose, galactose

  17. Lipid A

  18. Inner core

  19. Outer core

  20. OM proteins • Identified by SDS-PAGE: pI, Mr • Major proteins: 105 copies / cell • Minor proteins: 103 copies / cell • ~ 50 different species • 1. Braun’s lipoprotein; lpp gene • Most abundant protein in E. coli • Mr 7500 da • 2/3 bound to p/g; 1/3 free • Stabilizes OM: structural integrity

  21. OM proteins • 2. porins: water-filled channels • 100,000 copies / cell • Diffusion of nutrients • Trimers • Non-specific channels: Mr 35 kDa E. coli: OmpF, OmpC H. influenzae: OmpP2 • Specific porin channels: E. coli: LamB for maltose

  22. OM proteins • 3. receptors: • 1000 copies / cell • Transport of scarce nutrients • Iron transporters in E. coli FepA: ferric enterochelin; catechol FhuA: ferric hydroxamate uptake FhuE: coprogen FecA: citrate iron • Other nutrients: BtuB: vitamin B12

  23. Protein + LPS

  24. Protein + LPS

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