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β-Lactam Antibiotics: Cell Envelopes and Transport Mechanisms

Explore how β-lactam antibiotics cross bacterial cell barriers with detailed diagrams and explanations. Learn about bacterial transport mechanisms, including symport, antiport, and PTS systems. Discover the role of ABC transporters and OM receptors.

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β-Lactam Antibiotics: Cell Envelopes and Transport Mechanisms

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  1. Quiz_20.09.04 1235-1245 h

  2. Quiz_20.09.04 • β lactam antibiotics cross barriers of the bacterial cell to reach their targets. Draw two diagrams cell envelopes: one for G-negative cells, the other for G-positive cells. Label each diagram fully by identifying all macromolecules found in every layer. [5 points] • Show the pathway for traverse of a β lactam across the two cell walls. List the cell’s barriers against antibiotic activity. [3 points] • Inside a Living Cell: coloured diagrams to be submitted [2 points]

  3. Bacterial transport MFS Group translocation ABC transporters OM receptors

  4. MFS • Symport • 2 solutes simultaneously • Same direction across CM • Anion + H+ • Sugar + H+  • LacY of E. coli

  5. Antiport • 2 solutes simultaneously • Opposite directions • NhaA of E. coli • Active uniport • Flow of one ion • Driven directly by ion gradient: K+ • KcsA of E. coli

  6. LacY • lacY of E. coli 46.5 kDa • Integral CM protein • 12 transmembrane helices • Reconsitution • LacY purified in detergent • + P-lipids  • sonicate  • membrane vesicles with LacY

  7. LacY • LacY in vesicles • Add 14C-lactose + H+ • Measurements • 14C inside vesicles • medium: ↓ [H+] ; more basic • Conclusion: • single protein acts as symporter • H.R. Kaback: Science August 1, 2003

  8. Group translocation • Accumulate sugars as P~esters • ~P from PEP • PTS = PEP:carbohydrate phosphotransferase system • Multiple genes • Shared functions • Unique functions

  9. PTS • Sugar1: requires A + B + C  sugar1~P • Sugar2: requires A + B + D  sugar2~P • Sugar3: requires A + B + E  sugar3~P • A and B • Soluble proteins • Cytoplasm • C, D, E • CM proteins

  10. PTS_soluble proteins • enzyme I + PEP  enzyme I~P + pyruvate • E. coli: ptsI locus • 2 identical polypeptides; 58 kDa; dimer • 10,000 copies / cell • His~P • enzyme I~P + HPr  HPr~P + enzyme I • E. coli: ptsH locus • monomer • 100,000 copies / cell

  11. PTS_membrane proteins • HPr~P + enzyme II  HPr + enzyme II~P • enzyme II • Sugar-specific • E II mannitol; also glucitol • E II glucose; also glucosamine, 2-deoxyglucose • E II mannose; also fructose • Fine structure mapping • Binding of sugar • Interaction of HPr~P • Site for transphosphorylation

  12. PTS_membrane proteins • Mechanism of enzyme II • “pore” normally closed • Activated by HPr~P • “pore” opens • P. Maloney: • electron crystallography of enzyme II

  13. ABC transporters • Binding protein-dependent systems • Expt: • cold osmotic shock: 4 °C, sucrose • plasmolysis of E. coli: CM retracts • periplasmic contents: shocked outside cell • 100s soluble proteins: concentrate = shockate • 1. test cell for transport: his, mal • 2. test shockate……..

  14. ABC transporters • shockate • Lyophilize 100s of proteins • Suspend in 1 ml; add to dialysis bag • Buffer, 500 ml • + 14C-histidine • or + 14C-maltose • Proteins in dialysis bag bind radiolabel • Concln: periplasmic contents contain BPs

  15. ABC transporters • Molecular genetics • Maltose transport • malA locus: 74 min; catabolism • malB locus: 91 min; transport • Promoter: malP • malE-malF-malG • MalE = maltose BP (MBP), periplasm • MalF + MalG = CM complex

  16. Promoter: malP • malK-lamB • MalK = ATP hydrolysis, cytoplasmic face of CM • LamB = maltoporin, OM

  17. Maltose transport: mechanism • OM: maltose + LamB • LamB’s greasy slide • Periplasm: maltose finds MBP • MBP: open, bilobar • MBP + maltose  MBP closes; maltose in cleft • Venus fly trap • CM: [MBP+maltose] finds MalF-MalG • Maltose transferred to MalF-MalG cmplx • MalK2: hydrolysis of ATP

  18. OM receptors • Scavenge scarce nutrients • Iron-containing cmpds = siderophores • Regulated by iron supply • Vitamin B12 • IROMPs • FhuA, FepA, Cir, FecA, FhuE • BtuB

  19. OM receptors • Mechanism • Binding of siderophore to IROMP • Signalling to other proteins in periplasm • Movement of siderophore across receptor • Transfer to periplasmic BP • Energy requiring: TonB-ExbB-ExbD

  20. Ferrichrome transport in E. coli ferrichrome OM FhuA TonB CM

  21. OM receptors • Structures of 3 IROMPs • Purify protein • Grow protein crystals • X-ray crystallography  3D of polypeptide chain

  22. OM receptor FhuA: 2 domains • N terminus: cork • C terminus: • βbarrel, 22 strands; • Surface-exposed loops • Short connecting turns: periplasm • Signalling: • Minus siderophore = α helix • Plus siderophore = helix  random coil • Tells energy transducing system to activate

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