lecture_pp frames: coulton
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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. - PowerPoint PPT PresentationTRANSCRIPT
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
Proteins of bacterial membranes
Lengeler et al.
pp. 30-33, 37-42
Cellular fractionation
• Lysozyme digestion
• Separation of cell envelopes from cytoplasm
• Isopycnic centrifugation
Cytoplasmic membranes
• Composition
• Proteins
• Functions
Outer membranes
• Composition
• Proteins: major, minor
• Functions: – structural integrity– diffusion channels– nutrient transport: iron, vit B12
Gram-negative cell envelope
Lysozyme
• α – bacterial activity
• N-acetyl glucosaminidase
• Target: peptidoglycan
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
Cell lysis
• G + cell wall: ~ no barrier to lysozyme
• G - cell envelope: OM = barrier to lysozyme’s access to peptidoglycan
Separation of cell envelopes
• Differential centrifugation
• Bacterial cell lysate 15,000 x g, 30 min:– Pelleted membranes– Soluble cytoplasm
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
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%
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
Functions: CM proteins
• Transporters = permeases
• Synthesis of – Membrane lipids– Peptidoglycan: PBPs
• Electron transport chain
• DNA attachment: OriC
• Chemotaxis
• Secretory apparatus
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
Lipid A
Inner core
Outer core
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–
OM proteins• 2. porins: water-filled channels
– 100,000 copies / cell– Diffusion of nutrients– Trimers
• Non-specific channels: Mr 35 kDaE. coli: OmpF, OmpC
H. influenzae: OmpP2
• Specific porin channels: E. coli: LamB for maltose
OM proteins• 3. receptors:
– 1000 copies / cell– Transport of scarce nutrients
• Iron transporters in E. coliFepA: ferric enterochelin; catechol
FhuA: ferric hydroxamate uptake
FhuE: coprogen
FecA: citrate iron
• Other nutrients:BtuB: vitamin B12
Protein + LPS
Protein + LPS