Gram-Positive vs. Gram-negative
G+ have PG layer and cytoplasmic membrane (Periplasmic)
G- have Outer membrane made of lipopolysaccharide and protein, Periplasm, PG layer, and cytoplasmic membrane
Differences between G+ and G- wall
Structure of G- cell wall
Outer membrane has LPS, phospholipid, lipoprotiens and PG
Periplasm
Ctyoplasmic membrane
Bacterial Inner (Cytoplasmic) Membrane
Inner membrane is mostly phospholipids that vary in composition and relative ratios
75% Phosphatidylethanolamine in E. Coli
20% Phosphatidylglycerol in E. Coli
5% Cardiolipin (Disphosphatidylglycerol)
Structure of typical bilayer membrane
Fatty acids of phospholipids associate with each other and help form bilayer
Polar group faces outside
Phospholipids have glycerol, and phosphate group
Membrane serve Many functions
1. Permeability Barrier - Prevents leakages and gateway for nutrient transport in/out of cell
2. Protein Anchor - Transport, bioenergetics and chemotaxis protein associated with it
3. Energy Conservation - Site of generation and using protein motive force
Permeability of Membrane
Water permeable to membrane
Hydrophobic molecules can cross it
Large hydrophobic, and polar molecules can not
Periplasm (Periplasmic Space)
Only in G-
Between the outer and inner membrane and includes spaces between strands of PG
Dynamic changeable matrix of materials
Iso-osmotic with cytoplasm, both have same number of molecules and ions
Solutes have difficulty diffusing out
Easier in G+ because no periplasm
Content of Periplasm
Mostly PG and is hydrated and expanded into gel-like
Proteins associated with and attaced to PG
Contains Detoxifing enzymes: B-lactamase and others that inactivate antibiotics
Scavenging hydrolytic enzymes: degrade compounds too large or charged to pass CM like alkaline, phosphatase and Asp
Nucleases: ribonucleases, deoxynucleases
Periplasmic binding proteins: soluble that binds to specific solutes, large group of transporter
Lipoprotein (braun's) and structural proteins (OmpA)
Outer Membrane
Only in G-
Asymmetric lipid bilayer: Phospholipid (inner leaflet), lipopolysaccharides (LPS, outer leaflet) and protein
*8 nm thick
Impermeable to hydrophobic compounds and large hydrophilic agents
Responsible for resistance to some antibiotics and cehemotherapeutic agents
Outer membrane content
Phospholipids
LPS: Unique consitutient of bacterial OM
LPS has three parts: Proximal hydrophobic lipid A region, Distal hydrophilic O-antigen polysaccharide region that sticks into the medium and core oligosaccharide region that connects the 2
Lipid A
contains hyrophobic membrane achoring region of LPS
Consists Phospho-NAG dimer with 4-7 saturated FA
FA chains associated with inner leaflet phospholipids and form outer leaflet of OM
Attached to core through 2-keto-3-deoxyoctonic acid (KDO) which is - charged
Proximal part of core and lipid A contain charged anionic groups to bind to Mg and Ca
Core Oligosaccharide of LPS
Made of short chains of sugars
Unsual heptose and KDO in core polysaccharide
KDO unqiue to LPS, used as indicator of LSP poisoning
Little virations
LPS structure similair to all for all members in a genus but distict in other genera
O-polysaccharide of LPS
Made of repeating oligosaccharide subunits of 3-5 sugars
Long as 40 repeat units; much longer than core polysaccharide
maintains the hydrophilic domain of LPS
Great variation between species and even strains of G-
20 different sugar kown to occur mostly are dideoxyhexoses found only in G-
Variation allows diverse antigen types
Summery of LPS
Braun's Lipoprotein
Most abundant protein in G- cells (7 x 10^5)
N-terminal Cysteine modified to carry glycerol molecule with two fatty acids
N-terminal also attached to FA
Helps glue OM to PG later and stabilize it
Fatty acyl in OM and Σ N group of C-terminal lysine from the protein peptide bond with Σ carboxyl of DAP in PG
Murein (Braun's) Lipoprotein
N terminal Cystein is modifed with lipids that insert into inner leaflet of OM
Side chain of C-terminal Lysine resiude attached to PG via free mDAP carboxyl group
Synthesis of LPS is reminiscent of PG syntehsis
LPS biosynthesis starts in cytoplasm with nucleotide linked to sugat and transferred to lipid carrier
Lipid A/core is made separately from O-antigen
Both are transfered to periplasm (RFbx or MsbA, 1 and 2) then ligated together
O-antigen subunits polymerized onto LPS core of complete molecule then stransfered to OM and flipped outside
Lpt protein flips LPS occur two ways
Soluble Intermediate Model: Lpt A transfer LPS over
Trans-envelope complex model: Chain of LptA transport LPS outside
Serological classifcation System of E. Coli
Difference in immunological determinants used for Kauffmann-White O- serogrouping for E. Coli
2 Surface compnents used: O atnigen of LPS (O) and flagella (H)
Component has to bo present in all strains, has to be antigenic and different between strains
LPS and virulence of G- Bacteria
LPS is endo toxin
Both lipid A (toxic part) and polysac side chains used as determinants of virulence in G-
Injection of LPS causes nonspecific pahtophysiological reactions. EG: Fever, changes in WBC cound, disseminated intracascular coagulation, tumor necrosis, hypotension, shock and death
O polysach act as water soluble carriers for lipid A
OM as permeability barrier
LPS exclusive in outer leaflet make it less fluid
Phospholipid in inner leaflet highly dynamic
FA chain (6-7) in LPS all saturated and tightly packed
Proximal part of core that has KDO and Lipid A have large negative chare to attract Mg and Ca
OM bilayer is less permeable to hydrophobic solutes than cytoplasmic membrane
OM as a defense against Antibiotics
G- naturally resistant to hydrophobic antibiotics, detergents and hydrogphobic dyes
G- in general more resistant to antibiotics and hemotherapeutics than G+
Perturbation of OM structure
Deep rough mutations of LPS
Lack of LPS parts because of defective enzymes in their synthesis
Deep rough mutants Rd and RE are less resistant to hydrophobic dyes, detergents and antibiotics
TransmembranePorin content in OM of mutant were decreases, lead to incorporaton of phospholipids(PL) in outer leaflet of OM
PL bilayer patches allows tapid transmembrane diffusion og hydrophobic solutes
Effects of Divalent cations
Addition of Mg stablizes OM
Divalent Cations helps tightly packing lipids by neutralizing - charge
Treatment of Ca at 0 degrees causes E. Coli cells into recpipients of DNA in transformation
CA binds less strongly to H20 than MG and precpitates with anions causing LPS to solubilize
Effect of Chelators
EDTA treatment of E. Coli release half of LPS from cell
Removes Mg and Ca
EDTA-treated cells behave like deep rough mutants
Increases permability likely due to filling of space by PL patches