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Lecture_PP frames: Coulton
undergraduate/courses/mimm323/
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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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Proteins of bacterial membranes
Lengeler et al. pp , 37-42
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Cellular fractionation
Lysozyme digestion Separation of cell envelopes from cytoplasm Isopycnic centrifugation
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Cytoplasmic membranes
Composition Proteins Functions
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Outer membranes Composition Proteins: major, minor Functions:
structural integrity diffusion channels nutrient transport: iron, vit B12
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Gram-negative cell envelope
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Lysozyme α – bacterial activity N-acetyl glucosaminidase
Target: peptidoglycan
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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
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Cell lysis G + cell wall: ~ no barrier to lysozyme
G - cell envelope: OM = barrier to lysozyme’s access to peptidoglycan
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Separation of cell envelopes
Differential centrifugation Bacterial cell lysate 15,000 x g, 30 min: Pelleted membranes Soluble cytoplasm
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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
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Composition: CM Phospholipids: G + and G – Fatty acids:
P ethanolamine, 75% P glycerol, 18% cardiolipin, 5% Fatty acids: palmitic acid, 43% palmitoleic acid, 33% cis vaccinic acid, 25%
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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
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Functions: CM proteins
Transporters = permeases Synthesis of Membrane lipids Peptidoglycan: PBPs Electron transport chain DNA attachment: OriC Chemotaxis Secretory apparatus
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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
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Lipid A
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Inner core
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Outer core
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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
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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
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OM proteins 3. receptors: Iron transporters in E. coli
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
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Protein + LPS
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Protein + LPS
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