Molecular Microbiology Chapter 3

• Made of a bilayer of phospholipids and embedded proteins ( 1:1 amount)
• Three major phospholipid species depending on the species, strain (Belastung) and growth condition in different proportions: Phosphoatidylethonalamine (PE) (70-80%), phosphatidylgycerol (PG) (15-20%) and diphosphatidylglycerol (cardiolipin) (5-10%) (example E.Colu) but no cholesterol or sphingomyelin (typical in Eukaryots)
• To solve membranes and membrane proteins, detergents with long hydrophobic tails can be used. IM and OM (also Proteins) habve different solubility in detergents. Example Triton x-100 solves IM (protein) and not OM à possible to determine if it is from the OM or IM

• IM has many different proteins (20%≈ of total proteins ) compared to OM (≈2%), but in smaller and more regulated amounts
• Proteins are essential for Respiration (energy production, transport and motility)
• IMPs have 1-18 a-Helices, linked by hydrophilic loops and one helix is made out of 20-27 residues (length needed to span the bilayer) (can be perpendicular or tilted)
• Loops can easily be identified, but orientation (cytosol or Periplasm) more difficult, this can be done experimentally with the phosphatase fusion strategy
  • PhoA only gets active if it can make disulfide bonds between internal cysteine residues and this oxidation only happens in the periplasm
  • Gene sequence of PhoA can be inserted to artificial transponsons, which allows them to jump and fuse randomly in the gene if the IMP à PhoA will either be in the periplasm or cytoplasm (active or not active)
  • Active PhoA can be monitored
  • If many colonies are screened and insertion site later determined by DNA sequencing

It was found that generally positive charged residues are on the cytosol side (positive inside rule)

• Respiration: Transfer of electrons to an oxidizing substrate like O₂ or also NO₃^- (can be anaerobic)
• Alternative way to produce Energy is fermentation: Means transfer of electrons from one part of the substrate to another part of the same substrate. The electron are then eliminated by reduced metabolites (H₂, acids or alcohols)
• Many bacteria can both production ways, depending on the environment

• Rotary Nano machine (consist out of F_O, F₁) that can produce ATP with letting Protons trough the membrane. If needed it can be reversed.
• Here only small differences between mitochondria, bacteria’s and chloroplast
• F_O Part consist out of three subunits (a, b, c; 1:2:9) translocates the proton across the membrane and induces a rotation of the c unit (very hydrophobic part), when Protons enter into it over the a part form the outside. B part holds the F₁
  F₁ consist of 5 subunits (a,-Delta). a and b part catalyce 1 ATP for each rotation

• OM is not in contact with ATP (in contrast to the IM) and thus the OMP have no acess to ATP (no ATP in periplasm). Transport of specific solutes though specialized prions need ATP (example Iron is transported by siderophores)
• Siderophore goes through OM porin FhuA and this is energized by coupling protein TonB (energy transducer from IM to OM). The exact process in not fully understood

Import into the IM is done by permeases, three main groups:

• 1 Symporter/antiporter
  • use concentration gradients to move substrate across the membrane
  • example: Lactose permase LacY works with proton force to move lactose
  • have many TMs, that form a pore for the substrate and proton
• 2 Phosphotransferase system (PTS)
  • imports sugar like glucose, fructose, mannose, galactose etc…
  • uses the energy from PEP that is bound on the substrate (made by enzymes)
• 3 ABC transporter
  • Multiprotein complex: 1 protein binding substrate ind periplasm, 1 ind cytoplasm binding the ATP and 1 or 2 tmIMP
  • Can control the flux (abfluss) of toxic substances
  • Example Siderophore-iron complex is taken up trhough the OM  and then by Tol-coupeled system shuttled to the inner membrane by  a ABC transported, then in the inside the iron is reduced and released

• Mobility apparatuses are complex machineries attached to the membrane
• Are powered either by Protons or ATP
• 3 Main mechanism: Flagellar-basses swimming, pili-based twitching (on surface) and gliding (also on surface)
• Some bacteria evolved more than one mechanism
• Advantages: move towards nutrients and away from toxins, repellants or general stress conditions

• Different type: Monotrichous (one motor at the pole), Lophotrichous (several at the pole), amphitrichous (one at each pole) and peritrichous (several allover)

Example (best studied Motor) Salmonella Typhimurium consist of:

• Bals body that is in the membrane
  • 4 Rings (C and MS are the rotating once)
  • Motor consists of two proteins MotA/B that form 11 stators units
• 55nm long hook
  • 130 copies of FlgE protein
• several um long filament
  • act as a propeller
  • 20’000 copies of flagellin
• Rotation is powered by Protons that move through MotAB channel (similar to ATPase) (in marine bacteria often Na⁺ gradient
• 258 Protonsà one rotation (350Hz) a and can reach a speed of 25um/sec (10 body legth/sec) wich is extremely fast and efficient
• Basal body also contaisn an export function the Type-III secretion system
• Rod and hook are hollow that transport new subunits to the end, where they grow
• Some bacterias can rotate the propeller CW or CCW. If rotating CCW the flagella form a bundle that act as a propeller. If CW they all rotate independenly and start  “tumbling” à bacterium spins randomly. Used to follow increasing consetration of attractant detected by specialized chemoreceptors (or opposite) (called chemotax)

• Slower motion (1um/min), still not fully understood but two different mechanisms:
• 1. Motors and focal adhesion complex assemble at a helical cytoskeletal structure and move backward, since the adhesion complex is attached with the substratum (unterlage) the bacteria rotates and moves. At the end the Proteins disassemble
• 2. Only few protein embedded in the cell and powered by ATP

• Number of antimicrobial peptides cann attack the IM or OM, and are produced by other bacteria or multicellular organism (including humans)
• Defensins are + charged peptides with amphiphilic structure
  • + charge interacts with – charged phospholipids and hydrophobic par penetrates the membranes
• Several human cells produce those proteins which work also against eukaryotes but efficiency decreases with increasing cholesterol
• Can also be made as precursors and then later be activated
• Lantibiotics: peptides that transform lactose into lactic acid in dairy (Milch) products
  • heat stable and used for food preservation
• Polymyxins: are bacterial cyclic non ribosomal peptides active on gram – negative
  • Could get important against multi-resistant gramm – bacteria
  • Interacts electrostatically wit OM and displaces cations (2 *- charged) from phosphate groups of membrane lipids
• Daptomycin: is a lipopetdide that disrupts the plasma membrane of gramm + bacteria