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