Jackson: Bacterial Structure and Metabolism

Question 1

BACTERIAL MORPHOLOGY:

What is the Classification Scheme based on?

G+/-

Shapes

Answer 1

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)

Question 2

Gram stain procedure

Answer 2

• 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

Question 3

G+/- colors

Answer 3

Color Distinction: Gram positive are purple, Gram negative are pink

Question 4

Gram staining mechanism

Answer 4

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

Question 5

Acid-Fast Bacteria: genus?

Cell walls contain:

Gram stain?

Methods used to stain cells:

Answer 5

• 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

Question 6

G+/- Cell Envelope

Answer 6

CELL ENVELOPE:
• General:

• Gram Positive: cytoplasmic membrane + thick peptidoglycan
• Gram Negative: cytoplasmic membrane + thin peptidoglycan + outer membrane (2 cell membranes)

Question 7

Teichoic acid =

LPS =

Answer 7

Teichoic acid = lipoteichoic acid

LPS = endotoxin

Question 8

Peptidoglycan aka:

Function:

Answer 8

• Cell Wall (Peptidoglycan/Murein Layer):

• Gives bacterial cell shape and resistance to osmotic changes
• Target for antibacterial agents

Question 9

Composition of Peptidoglycan:

Answer 9

Disaccharide-pentapeptide subunits

Question 10

Disaccaride types:

Answer 10

• N-Acetylglucosamine (NAG)

* N-Acetylmuramic acic (NAM)

Question 11

Peptide bridges between:

G+:

G-:

Answer 11

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

Question 12

B-Lactam MOA:

What form cross-links?

Penicillin structure resembles:

Answer 12

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)

Question 13

Teichoic (Mycolic) Acids (Gram Positive):

Associated with:

Also found in:

Answer 13

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

Question 14

Outer Membrane only found in:

Composed of:

Answer 14

Outer Membrane (Only Gram Negative):

• Composed of bilayer of LPS and phospholipids

Question 15

LPS

Composition (3 parts):

What causes endotoxic shock?

Answer 15

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)

Question 16

Where is periplasmic space found?
What is periplasmic space?
Constituents include:

Answer 16

• Periplasmic Space (Only Gram Negative):

• Space between inner and outer membranes (contains peptidoglycan layer)
• Constituents include enzymes that degrade antibiotics (ie. beta-lactamases)

Question 17

Cytoplasmic (Inner) Membrane:

Found where?

Composed of:

Answer 17

Gram Positive and Gram Negative

• Composed of a lipid bilayer heavily laced with protein

Question 18

Beta-lactams interfere with the formation of (2)

Answer 18

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

Question 19

Cytoplasmic (Inner) Membrane:

Functions

Answer 19

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

Question 20

Capsule:

Location:
Composition:
Production:
Protects cell from:

Answer 20

• 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

Question 21

Pili aka:

Definition:
Composition:
Mediates:
Antigenic and certain bacteria will:

Answer 21

• 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

Question 22

Two Types of Pili

Answer 22

o Common Pili: mediate adhesion to host eukaryotic cells

o Sex Pili: join conjugating bacteria

Question 23

Flagella:

Definition:
Composition:
Function:

Answer 23

Flagella: long, helical filaments extending from cytoplasmic membrane

• Composition: proteins called flagellins (highly antigenic; H antigens)
• Function: cell motility

Question 24

Endospore definition:

Medically Important Endospore Formers:

Answer 24

Endospore: resting phase of bacteria when living conditions get tough

Medically Important Endospore Formers: Bacillus and Clostridium

Question 25

PROTEIN SECRETION: Type I Gram +/-? Sec-dependent or independent pathway? Mechanism: Example:

Answer 25

• 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)

Question 26

PROTEIN SECRETION: Type II Gram +/-? Sec-dependent or independent pathway? Mechanism: Recognizes: Secretes proteins into:

Answer 26

• 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 (+)

Question 27

PROTEIN SECRETION: Type II Secretes proteins into: What form pores in outer membrane? What else is required? Example:

Answer 27

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

Question 28

PROTEIN SECRETION: Type III Gram +/-? Sec-dependent or independent pathway? Contact-Dependent or Independent? Mechanism: Requires: Examples:

Answer 28

• 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

Question 29

Nutrient Transport Passive Diffusion: Active Transport: Group Translocation:

Answer 29

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)

Question 30

EMP Pathway (Glycolysis):

Answer 30

Glucose → pyruate + NAD + CoA → Acetyl CoA

Question 31

Fermentation:

Answer 31

Incomplete glucose oxidation (anaerobic; produces acid or alcohol)

Question 32

TCA Cycle:

Answer 32

Acetyl CoA + citrate → NADH + FADH2

Question 33

ETC:

Answer 33

ETC: redox carriers in cytoplasmic membrane yield ATP | o Substrates → Dehydrogenases → Quinones → Cytochromes → Terminal oxidases

Question 34

Aerobic vs anaerobic vs Facultative Examples

Answer 34

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

Question 35

Lab Tests Based on Metabolic Reactions (4): Catabolism of sugars: H2S production detected as: Oxidase test detects: Catalase test checks for:

Answer 35

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

Question 36

Capnophilic vs Microaerophilic:

Answer 36

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