Microbio Lecture 3
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What are the purposes of the cell wall?
- Withstand osmotic pressure
- Keep cell shape and rigidity
What are the steps of a gram stain?
- 1. Flood slide with crystal violet 1 min
- 2. Flood with iodine 1 min
- 3. Rinse or decolorize with alcohol 20sec
- 4. Flood with safranin 1 min
- Purple = Gram positive
- Pink = Gram negative
What are the layers of the cell wall of a gram positive bacteria?
- 2. Thick layer of peptidoglycan
- 1. Cytoplasmic membrane
What are the layers of the cell wall of a gram negative bacteria?
- 3. Outer membrane
- 2. Peptidoglycan
- 1. Cytoplasmic membrane
What are some of the shapes of prokaryotes?
- Budding and appendaged
What is another name for peptidoglycan?
What are the subunits of peptidoglycan?
- 2 sugars:
- 1. NAG - N-acetylglucosamine
- 2. NAM - N-acetylmuramic acid
What is special about the subunits of peptidoglycan?
- 1. Short peptide chains containing unusual amino acids (D-amino acids instead of the L-amino type found in proteins)
- 2. NAM and DAP (Diaminopimelic acid) have never been found in Archaea or Eukaryotes
What is lysozyme and what is its target?
- Lysozyme is an enzyme secreted in tears, saliva and other body fluid to protect against bacterial pathogens
- The link between NAG and NAM is its target
How is the rigidity of of peptidoglycan maintained?
- Polymerization of the sugar backbone in one direction
- Bond between the peptide chain of 2 adjacent peptidoglycan chains in the other direction
What is the difference between the cross-linking of the peptidoglycan of gram negative and gram positive bacteria?
- Gram negative = direct cross linking
- Gram positive = interbridge cross linking
What is cross-linking also called?
What is an interbridge?
Link between sugars of peptidoglycan of gram-positive bacteria where an interbridge of 5 amino acids (usually Gly) connects the 2 molecules
What is the major component of the cell wall of gram positive bacteria?
What are the components that can be attached to the peptidoglycan?
- Teichoic acid and Lipoteichoic acid (TA/LTA)
- Composed of glycerol (3C) or ribitol (5C) and decorated of amino acids, sugars.
- Covalently bonded to peptidoglycan
- Give cell wall a negative charge
Describe the motifs of the surface proteins in gram positive bacteria
- In the cell wall - motif LPXTG
- In carboxy-terminal region - hydrophobic motif called transmembrane domain
What does sortase do?
Cleaves the protein between the T and G residues and attached the T to the DAP or lysine in the peptide chain of the peptidoglycan
What percentage of the cell wall is peptidoglycan in gram negative bacteria?
What is the periplasm?
- Space delimited by the cytoplasmic membrane and the outer membrane
- Where the peptidoglycan is located in gram negative bacteria
- Contains high concentration of proteins involved in a diversity of functions (sensing, nutrient acquisition etc)
- Very dense, sometimes called "protein gel"
Why is the outer membrane an atypical lipid bilayer?
Phospholipids in the inside layer, phospholipids and lipopolysaccharide in the outside layer (LPS layer)
What are the key benefits of LPS to a bacteria?
Helps protect bacteria against substances like antibiotics and against the host defense system
What are OMPs?
- Outer membrane proteins, found in gram negative bacteria
- Examples: porins, lipoproteins
- Play a structural role
What is LPS composed of?
- Family of complex sugar polymers attached to a lipid moiety known as Lipid A
- O-specific polysaccharide attached to core polysaccharide attached to KDO (2-keto-deoxyoctonate) which links the polysaccharide to Lipid A
What is Lipid A?
Toxic to many animals, can cause acute inflammation and vascular problems (septic shock) that can result in death. It is an endotoxin and it plays a major role in the pathogenesis of gram negative bacteria pathogens. It contains 6 lipid tails that are embedded in the membrane
What is O-specific polysaccharide?
- Also known as the O antigen, consists of repeating sequences of 2-4 monosaccharides. There is a huge diversity of O antigen so it can be used to identify the strains of one species of bacteria
- Example: E. coli O157:H7 means O157 serogroup
Is there any peptidoglycan in the cell wall of Archaea?
Is there usually an outermembrane present in the cell wall of Archaea?
What does penicillin inhibit?
What is pseudomurein?
- It's similar to peptidoglycan, but contains N-acetyltalosaminuronic acid instead of NAM and lacks D-amino acids
- Found in cell wall of of Archaea
- Linkage between sugars is insensitive to lysozyme
What is an S-layer?
- Paracrystalline surface layer
- Have a crystalline appearance
- Great majority of cell wall species of archaea consist of proteins and/or glycoprotein
- S-layer may also be found in some species of bacteria (on top of peptidoglycan in gram positive, on top of outer membrane in gram negative)
What are the capsule and slime layers of prokaryotes?
- Doesn't confer structural strength on the cell, just chemical stability - very important virulence determinant for capsulated bacterial pathogens, capsule protects against host defense system
- Composed of polysaccharide (vast majority) or protein: Heteropolysaccharide (majority of bacteria) or homopolysaccharide (some gram negative)
- May be covalently bound to the outer membrane or to the peptidoglycan layer
What are the 2 types of surface appendages?
Flagella and fimbriae
What are flagella used for?
How long are flagella typically?
About 15-20 micrometers long
What is the main purpose of fimbriae?
To attach to surfaces
How long are fimbriae typically?
About 4 micrometrs long
Fimbriae are found primarily on ____________ bacteria
What are the 3 types of flagella?
- 1. Monotrichous (1 flagellum)
- 2. Peritrichous (Many flagella all around the cell surface)
- 3. Lophotrichous (Many flagella originating from one end of the cells, polar flagellation)
How many rings does a gram negative bacteria flagellum have?
4: L, P, MS, and C rings
How many rings does a gram positive bacteria flagellum have?
3: P, MS and C rings
What are the 4 different possible ring types in flagella?
- L - LPS
- P- Peptidoglycan
- MS - Membrane superficial
- C - Cytoplasm
What moves the flagella of a bacteria?
The proton motive force
How are flagella synthesized?
- In the cytoplasmic membrane, MS ring and C rings are synthesized.
- Then the Mot (motor) proteins are synthesized
- P ring is synthesized in the peptidoglycan
- L ring is synthesized in the outer membrane
- Early hook is synthesized
- Cap on late hyook is synthesized
- Filament is synthesized from the cap down to the hook filament junction
How does a peritrichous bacteria move?
Bundled flagella in CCW rotation to move forward, to change direction "tumble" (flagella pushed apart) in CW rotation, this flips it then it can move forward in the other direction with flagella bundled in CCW rotation
How does a lophotrichous/monotrichous bacteria move?
- If the flagella are reversible it can spin CCW to move forward, CW to move backwards
- If the flagella are unidirectional, CW to move forward, stop in fluid which will reorient the bacteria, then CW to move forward in the new direction
What is taxis?
Directed movement toward or away from a gradient of chemical or physical agents
What is chemotaxis?
Movement towards or away from chemicals, nutrients, antibiotics, etc
What is phototaxis?
Movement towards or away from light (phototrophic organism)
What is aerotaxis?
Movement towards or away from oxygen
What is osmotaxis?
Movement towards or away from ionic strength (high/low salt concentration)
In a gram positive bacteria, where are fimbriae anchored?
In the cell wall
In a gram negative bacteria, where are fimbriae anchored?
In the outer membrane
What are fimbriae made up of?
In a gram positive bacteria, how are fimbriae linked together?
In a gram negative bacteria, how are fimbriae linked together?
Non covalently bound to one another by strand exchange
In a gram positive bacteria, how are fimbriae assembled?
Assembled enzymatically by sortases
In a gram negative bacteria, how are fimbriae assembled?
Accessory proteins are needed, encoded with the fimbriae pillin genes (chaperone and usher)
Which side do fimbriae of gram negative bacteria grow from?
What is the chaperone (shuttle) in gram negative bacteria fimbriae assembly?
What is the usher (pore) in gram negative bacteria fimbriae assembly?
What is the adhesin (binds to a surface protein on a kidney cell) in gram negative bacteria fimbriae assembly?
What form the fibril in gram negative bacteria fimbriae assembly?
PapE, PapF, and PapK
What is the major subunit and forms the shaft in gram negative bacteria fimbriae assembly?
What is the anchor protein (termination) in gram negative bacteria fimbriae assembly?
How is adhesion to surfaces carried out in gram positive bacteria fimbriae assembly?
Adhesion to surfaces is generally carried out by surface adhesins, consisting of only one protein. Such adhesins mediate very close attachment.
How are the fimbriae of gram positive bacteria assembled?
By sortase and attach to the peptidoglycan
What are endospores?
- Highly differentiated cells that are extrememly resistant to harsh environmental conditions (such as heat, chemicals, radiation, nutrient depletion, desiccation, etc)
- The endospore is a dormant stage in the life cycle and is easily dispersed by wind, water, animal gut, etc.
- Can remain dormant for 100s of years
What are the layers of the endospore?
- Starting at the outermost layer:
- Spore coat
What is the exosporium of the endospore?
Made up of proteins, outermmost layer
What is the spore coat of the endospore?
Layers of spore-specific proteins (keratin-like)
What is the cortex of the endospore?
Made up of peptidoglycan
What is the core of the endospore?
- Contains Ca2+, dipicolinic acid (DPA), SASPs (acid soluble spore proteins), DNA
How does the endospore get ready to be active?
- SASPs bind to DNA and help protect it
- DPA and Ca2+ bind water, dehydrate the core
- Core also contains proteins necessary for germination
What is special about the formation of the endospore?
Endospore germinates into a cell, cell undergoes asymmetric cell division to create a prespore which becomes the endospore. Once the cell matures and lysis occurs, it becomes a free endospore again
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