Jackson: Bacterial Structure and Metabolism Flashcards
BACTERIAL MORPHOLOGY:
What is the Classification Scheme based on?
G+/-
Shapes
BACTERIAL MORPHOLOGY:
• Classification Scheme: based on cell envelope and shape
- Gram positive or Gram negative
- Rods, Cocci, Spirilla
- Pairs, tetrads, clusters, chains (not a reliable classification scheme)
Gram stain procedure
• Gram Stain: most commonly used diagnostic technique; color distinction based on bacterial cell wall constituents
- Procedure: ~1 minute each step
o Air dry and heat fix bacteria on slide
o Add crystal violet, rinse
o Add iodine (mordant), rinse
o Add acetone/isopropyl alcohol solution (10 drops), rinse
o Add safranin, rinse and dry
o Examine using oil immersion (1000x) objective
G+/- colors
Color Distinction: Gram positive are purple, Gram negative are pink
Gram staining mechanism
Mechanism:
o Crystal violet penetrates peptidoglycan layer to stain cytoplasm blue
o Iodine + crystal violet complexes, and it is too large to be washed out of Gram positive bacteria (thick cell wall), but is washed out of Gram negative bacteria
o Gram negative then counterstained with safranin (pink); done in order to visualize
Acid-Fast Bacteria: genus?
Cell walls contain:
Gram stain?
Methods used to stain cells:
• Acid-Fast Bacteria (Mycobacterium):
- Need to use a special stain (diagnosis of TB)
- Cell walls contain long chain fatty acids (mycolic acid), which repels dye
o Gram stain poorly
o Hard to decolorize with acetone/alcohol
- Hot (Ziehl-Neelsen) or Cold (Kinyoun) methods are used to stain these cells
G+/- Cell Envelope
CELL ENVELOPE:
• General:
- Gram Positive: cytoplasmic membrane + thick peptidoglycan
- Gram Negative: cytoplasmic membrane + thin peptidoglycan + outer membrane (2 cell membranes)
Teichoic acid =
LPS =
Teichoic acid = lipoteichoic acid
LPS = endotoxin
Peptidoglycan aka:
Function:
• Cell Wall (Peptidoglycan/Murein Layer):
- Gives bacterial cell shape and resistance to osmotic changes
- Target for antibacterial agents
Composition of Peptidoglycan:
Disaccharide-pentapeptide subunits
Disaccaride types:
- N-Acetylglucosamine (NAG)
* N-Acetylmuramic acic (NAM)
Peptide bridges between:
G+:
G-:
Peptide bridges between NAM cross-link subunits
- Gram Positive: pentaglycine bridge between lysine and D-alanine
- Gram Negative: direct link between D-alanine and diaminopimelic acid
B-Lactam MOA:
What form cross-links?
Penicillin structure resembles:
B-Lactam MOA: target peptide bridges
Penicillin binding proteins (PBPs) form cross-link
Penicillin structure resembles D-ala/D-ala intermediate (binds PBP instead, preventing crosslinking)
Teichoic (Mycolic) Acids (Gram Positive):
Associated with:
Also found in:
Techoic and lipotechioic acids provide strength to cell wall
- Associated with inflammatory response in some Gram positive infections
- Also found in Acid Fast Bacilli, making them resistant to phagocytic killing, drying
Outer Membrane only found in:
Composed of:
Outer Membrane (Only Gram Negative):
- Composed of bilayer of LPS and phospholipids
LPS
Composition (3 parts):
What causes endotoxic shock?
o LPS: important virulence factor; key mediator of inflammation (shock)
Composition of LPS:
• Repeating sugar residues (O antigens) useful for bacterial typing
• Core sugars (consistent, not part of pathogenesis)
• Fatty acid moieties (lipid A) is the bioactive portion of LPS (causes endotoxic shock)
Where is periplasmic space found?
What is periplasmic space?
Constituents include:
• Periplasmic Space (Only Gram Negative):
- Space between inner and outer membranes (contains peptidoglycan layer)
- Constituents include enzymes that degrade antibiotics (ie. beta-lactamases)
Cytoplasmic (Inner) Membrane:
Found where?
Composed of:
Gram Positive and Gram Negative
- Composed of a lipid bilayer heavily laced with protein
Beta-lactams interfere with the formation of (2)
Beta-lactams interfere with the formation of D-ala from L-ala
Beta-lactams interfere with the formation of D-ala + Lys or DAP peptide bond
Cytoplasmic (Inner) Membrane:
Functions
Functions:
o Transport of solutes
o Housing enzymes involved in outer membrane and cell wall synthesis
o Assembly and secretion of extracytoplasmic and extracellular substances
o Generation of ATP
o Housing molecular sensor that monitor environmental changes
Capsule:
Location:
Composition:
Production:
Protects cell from:
• Capsule:
Exterior to peptidoglycan layer (Gram positive) or outer membrane (Gram negative)
- Composition: high molecular weight polysaccharides or amino acids
- Production: depends on environment and growth conditions
- Important Virulence Factor: protects cell from complement mediated killing
Pili aka:
Definition:
Composition:
Mediates:
Antigenic and certain bacteria will:
• Pili (Fimbriae): hair like proteinaceous structures extending from cell membrane
- Composition: subunits of protein called pilin, tipped with adhesins
- Important Virulence Factor: mediate adhesion to host tissue
- Antigenic and certain bacteria (gonococcus) will vary the antigenic composition
Two Types of Pili
o Common Pili: mediate adhesion to host eukaryotic cells
o Sex Pili: join conjugating bacteria
Flagella:
Definition:
Composition:
Function:
Flagella: long, helical filaments extending from cytoplasmic membrane
- Composition: proteins called flagellins (highly antigenic; H antigens)
- Function: cell motility
Endospore definition:
Medically Important Endospore Formers:
Endospore: resting phase of bacteria when living conditions get tough
Medically Important Endospore Formers: Bacillus and Clostridium
PROTEIN SECRETION:
Type I
Gram +/-?
Sec-dependent or independent pathway?
Mechanism:
Example:
• Type I: GRAM (-)
- Sec-independent pathway
Mechanism:
o No amino-terminal signal sequence
o ABC transporters (ATP-binding cassette); therefore ATP dependent
o Bypasses periplasm, and is extruded through a hole in cytoplasmic and outer membranes
Example: E.coli hemolysin (alpha toxin)
PROTEIN SECRETION:
Type II
Gram +/-?
Sec-dependent or independent pathway?
Mechanism:
Recognizes:
Secretes proteins into:
• Type II: GRAM (-) and GRAM (+)
- Sec-dependent pathway
Mechanism:
o Recognizes proteins with signal sequence (~20 aa with conserved cleavage site)
Secretes proteins into:
• Periplasm of Gram (-)
• Extracellular environment of Gram (+)
PROTEIN SECRETION:
Type II
Secretes proteins into:
What form pores in outer membrane?
What else is required?
Example:
Secretes proteins into:
• Periplasm of Gram (-)
• Extracellular environment of Gram (+)
From the periplasm, move through outer membrane to exit cell
• Secretins form pores in outer membrane
• Chaperone proteins and ATP are required
Example: several bacterial toxins
PROTEIN SECRETION:
Type III
Gram +/-?
Sec-dependent or independent pathway?
Contact-Dependent or Independent?
Mechanism:
Requires:
Examples:
• Type III: GRAM (-)
- Sec-independent pathway
- Contact-Dependent
- Mechanism:
o Pore formed that injected proteins directly into eukaryotic cells
o Requires ATPase and chaperone proteins
Examples: Yesinia pestis (plague) injects Yops to disrupt eukaryotic cell function
Nutrient Transport
Passive Diffusion:
Active Transport:
Group Translocation:
Nutrient Transport
Passive Diffusion: enter by concentration gradient (water, CO2, O2, simple sugars)
Active Transport: energy dependent pumps in the cell envelope (sugars, aa, organic acids, inorganic ions)
Group Translocation: nutrient chemically modified before being brought into the cell (sugars, nucleic acids, FAs)
EMP Pathway (Glycolysis):
Glucose → pyruate + NAD + CoA → Acetyl CoA
Fermentation:
Incomplete glucose oxidation (anaerobic; produces acid or alcohol)
TCA Cycle:
Acetyl CoA + citrate → NADH + FADH2
ETC:
ETC: redox carriers in cytoplasmic membrane yield ATP
o Substrates → Dehydrogenases → Quinones → Cytochromes → Terminal oxidases
Aerobic vs anaerobic vs Facultative
Examples
o Aerobic: terminal electron acceptor is oxygen (most efficient); Mycobacteria
o Anaerobic: use fermentation pathway; terminal acceptor is nitrate, nitrite, fumarate or sulfate; Clostridia
o Facultative: can use both of the above pathways; E.coli
Lab Tests Based on Metabolic Reactions (4):
Catabolism of sugars:
H2S production detected as:
Oxidase test detects:
Catalase test checks for:
o Catabolism of sugars to produce acid
o H2S production detected as gas or FeS
o Oxidase test detects cytochrome oxidase c
o Catalase test checks for enzyme that breaks down toxic O2 during respiration
Capnophilic vs Microaerophilic:
o Capnophilic: organism requiring CO2 at a level exceeding that of air
o Microaerophilic: organism requiring O2 at a level below that found in air
