1. Lecture_PP frames: Coulton
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 , 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 – Fatty acids:
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: 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

23. Protein + LPS

24. Protein + LPS