Molecular microbiology Chapter 1

• Consists of phospholipid bilayer, with hydrophobic fatty acids directed inward and phosphoryl (negative chargerd) outwards
• Hopanoids act as a stiffening agents (like cholesterol in eukaryotes)
• 50% of the volume consists of membrane protein

• Membrane lipid composition varies depending on the environment or region of the cell
  • Positive charged head groups are found often in membranes interacting with DNA
  • Cardiolipid increase under starvation
  • Cis unsaturated fatty acids increase membrane fluidity (for low temperature)

• Membrane is semipermeable, soluble protein, small, uncharged molecules (O2, CO2, H2O) go through, polar molecules, charged ions, large compounds do not
• Weak acid and bases can go through in their uncharged state
• Water permeability increases with specific channels (aquaporins)

• Exoskeleton is made out of peptidoglycan, proteins and Lipids, which hold from 5-20 bar)
• Gramm Test: Bakterias fixed with methanol, stained with crystal violet and Iodine à washed with ethanol à counter stained with eosin (pink) à two main types
• Gramm Positive:
  • Stay violet in the Gram test (the Iodine is “trapped” under all the peptidoglycan layers)
  • Thick cell wall, multiple layers of peptidoglycan (Teichoic acids hold them to the cell membrane
• Gramm Negative:
  • Stay collarless after the Gram test (loosely trapped Iodine is easily washed out, since there is only one pept. layer
  • Single layer of peptidoglycan and inner and outer membrane
  • Lipoproteins connect Pept. With outer membrane
  • Outer membrane is more porous
  • Better protected against harsh environment (eg. Toxic compounds)
• Both may have a s-layer on the outside and carbohydrates that form the capsule

• 20-80 nm thick
• Teichoic acid are linear polymers, alternated substituted polyols (glycerol or ribitol) and are covalently bounded to the peptidoglycan
• Lipoteichoic acids have a lipid substituent that anchor into the plasma membrane
• Different proteins are also anchored in the peptidoglycan (eg deradative enzymes)
  they can also be abundant for defense purpose and protect bacteria from complete deposition

• Have members that cause sever human infections (tuberculosis, leprosy (Lepra), Buruli ulcures)
• Based on their 16s rRNA they are grouped to the Gramm Positive, but they don’t led mild staining into their peptidoglycan layers
• They also have glycolipids, which makes mycobacteria specially impermeable to dyes and other chemical (also antibiotic)
• Only in 2008 with cryo EM it was possible to analyze them in their native state and since then they are trying to find the exact structure, it is known so far, that myocilin is required

• Peptidoglycan layer is thinner than gram positive (5-10nm) but also has an outer membrane (lipid bilayer), which consist  of two different lipid layers
  • Inner layer is made of phospholipids
  • Outer layer out of phospholipids and lipopolysaccharide
• Outer layer contains many copies of a few proteins and is attached to the peptidoglycan via specialized lipoproteins, murein lipoprotein
  • In E.coli Braun’s Lipoprotein consist out of 58 AA with 5-7 AA repeats and it is attached to lipids at the N-Terminal Cys with a peptide bridge with the NH₂ group of a Lys
• Space in between is the periplasm

Global structure:

The lipopolysaccharide (LPS) is a hydrophilic polmer composed out of three parts:

• The anchor: Lipid A
• Polysaccharidic part: core polysaccharide
• Variable polysaccharidic part: repetition of tri, tetra or penta saccharides

Lipid A

• Phosphoglycolipid with beta(1-6)-linked hexosamine backbone with phosphoryl groups and fatty acids with ester an amide linkages with acyl resst
• Variation in nature occurs on the hexosamine type, phosphorylation tipe and chain length of the acyl group and its position
• It is one major microbe-associated molecular patter, which is recognized by the mammalian immunity system
• Toll like receptor 4 (TLR4) recognized them and triggers the process to be protected against infection
• If the response happened in the blood, the response can be to large and lead to a shockà organ failure
• TLR4 activation depends on the two acyloxyacyl chain and numbers of phosphate groups (can be more or less effective)

The polysaccharide

• The core is well conserved but varies from each species
• Variable part made of short repetitive units and usually tri-saccharides or tetra-saccharides, diversity is the basis of to “umgehen” the immune system (O-Antigens)

Assembly of the LPS

• Lipid A and the core are assembled at the cytoplasmic side of the plasma membrane and then flipped accros the bilayer by an ABC transporter
• The O-antigen repeat units by glycosyltransferase on a lipid anchor (C55) and then brought through the membrane by another ABC transporter
• On the periplasmic side the two parts are then added together by the WaaL Ligase
• At the end the hole complex is transportet to the outer membrane by a multiprotein system Lpt (lipopolysaccharide transport)

• Phospholipids are similar to the ones in the inner membrane
• LPS give a hydrophobic character to the outside, it has more fatty acids than phospholipids (so more hydrophobic interaction) and the chains are saturated which increases the molecular packing à less fluidity à les permeability à more resistant to hydrophobic toxic compounds and antibiotics compared to Gram-positive
• Ca²⁺ and MG²⁺ neutralize the negative charge of the phospholipids, if they get into a chelate complex with EDTA the repulsion happens and phospholipids translocate form the inner to the outer sideà permeability increses
• There are also a lot of outer membrane proteins (OMP)
  • Most commonly general trimetric porins which allow of unspecific aqueous diffusion, they consist of anti-parallel beta strand forming a barrel, IM porins consist mostly out of alpha helixes
  • FhuA transports iron Fe 3+
  • LamB is a porin specialized on import of malrose

• About 4% of the proteins are found here
• Essential degradative enzymes like nuclease, alkaline phosphatase, beta lactamase, is like the lysosome of eukaryotic cells
• Has also proteins that are for the import of substrate
  • Example: TonB, anchored in the plasma membrane and acts as a reseptor with protein from the OM like FhuA à allows transport across the outer membrane
  • DsBA oxydo-reductase: catalyzes formation or S-S bonds ( no S-S bonds in the cytoplasm) or help folding of proteins that are secreted in unfolded form

• Most cell can secret protective and adhesive (klebrig) layer out of sugar polymers (capsules)
• They protect the bacteria from bacteriophages, or white blood cels
• They are loosely attached to the cell envelop (not anchored like LPS)

Exopolysacharides:

• Associated with multicellular life style (biofilm)
• allow different cells to stick to each other and then build colonize, this also allows communication (via hormones) and sharing of genetic material (horizontal gene transfer) more efficiently
• increase tolerance against mechanical and chemical stress (like antibiotics)
• are often associated with chronic infections in humans and difficult to get rid of