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Flashcards in Lecture 4 Deck (26):
1

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

2

Differences between G+ and G- wall

A image thumb
3

Structure of G- cell wall

Outer membrane has LPS, phospholipid, lipoprotiens and PG

Periplasm

Ctyoplasmic membrane

4

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)

5

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

6

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

7

Permeability of Membrane

Water permeable to membrane

Hydrophobic molecules can cross it

Large hydrophobic, and polar molecules can not

8

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

9

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)

10

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

11

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

12

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

13

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

14

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

15

Summery of LPS

A image thumb
16

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

17

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

18

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

19

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

20

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

21

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

22

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

23

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+

24

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

25

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

26

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