Microbiology

Front 1

Positive control? Ex.

Back 1

Positive controls confirm that the procedure is competent in observing the effect (therefore minimizing false negatives). ex: known sequence run on gel electrophoresis.

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Negative control? Ex.

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Negative controls confirm that the procedure is not observing an unrelated effect (therefore minimizing false positives). ex: Running the PCR product with no DNA in it on gel electrophoresis.

Front 3

Outer Membrane functions?

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Creates perplasmic space
– a compartment of cell that contains enzymes for nutrient breakdown, scavenging, receptors for chemotaxis and sensing environment
• Protection/Evasion of host immune response
– Can quickly change structure of LPS to “hide” from
immune response
• Barrier
– More permeable than CellMemb but still barrier to some molecules
• Colonization – Attachment to host tissues and surfaces

Front 4

What are fluorophores? How do you use them?

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Fluorophores– molecules capable of fluorescence. Emit certain wavelengths.
Gene Fusions– Fuse GFP (or others) to specific gene of interest

Front 5

Describe the phases of a typical growth curve in broth.

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1) Lag phase- Bact. prepare for growth.
2) Log phase- Exponential growth.
3) Stationary Phase- Cells stop growing, begin stress responses.
4) Death Phase- Cells die. Neg. exponential curve-ish.

Front 6

Caulobacter crescentus?

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Specialized cell division.
Cell division results in the parent cell, which is attached to sediment (with a stalk), and a flagellated daughter cell.

Front 7

Endospore Formation--how different from binary fission?

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Mother cell and forespore formed (Septum is close to 1 pole of cell). Forespore is smaller, and enclosed in a membrane within the cell memb. Mother cell then engulfs the forespore, still inside the cell membrane, so now spore has two membranes. Chemicals are added to spore memb., spore is released.

Front 8

Anabaena heterocysts?

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Specialized cell division.
-These bact. form in chains. -Photosynthesize, except for every 10th cell, which is a heterocyst and performs N fixation.
-N fixation requires ABSENCE of Oxygen, but photosynthesis results in O2.
-Heterocysts are able to exclude O2. (They're specialized).

Front 9

Myxococcus?

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Specialized cell shit!
-Fruiting bodies, formed in response to starvation.
-Chemotactic signals attract cells together.
-Some cells differentiate to form spores.
-Release spores from tip of fruiting body.

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Streptomyces?

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Specialization.
-Many cells. Looks like fungi. Releases spores.

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Biofilms.

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Community of cells that occurs when resources are ABUNDANT.
Cells communicate via chemical signals--Quorum sensing--when a certain number of cells are present, biofilm is formed. Cells differentiate and form special structures--towers, channels.

Front 12

Bacteria in the phylogenetic tree?

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Bacteria is a Domain. (Other 2 are Archaea and Eukarya, which are more closely related to each other). Bacteria and Archaea are all prokaryotes.

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Prokaryotic cell shapes!

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Cocci: round
rod: rod shaped.
Spirochette: corkscrew w/ axial filament.
Spirilla: curved rod (often flagella on each pole)
Filimentous: Stringy.

Front 14

Why are prokaryotes small?

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Larger cells have smaller surface area:volume ratio. Greater SA:V ratio equals higher metabolic rate.

Front 15

Cell membrane structure, please.

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Fluid (depending on environmental temperature!). Phospholipid bilayer with proteins floating in it. Hopanoids decrease fluidity.

Front 16

Phospholipid structure?

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Hydophobic fatty acid (hydrocarbon) tails, hydrophilic phosphate head.

Front 17

Changing cell membrane fluidity.

Back 17

More hopanoids. Saturation of fatty acid tails. (Makes them go all straight)

Front 18

Functions of the membrane?

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Barrier- Physically and chemically. Semi permeable. Regulates transport.
• Protein anchor – Many essential proteins. Transport proteins cross through memb. ATP synthase. Bioenergetics, cell movement, etc.
• Energy conservation (stored potential energy and synthesis of ATP)
– Generation and use of proton motive force
– regulated barrier to H+ ions, movement of ions down concentration gradient used for energy

Front 19

Helicobacter pylori and urease adaptation

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• Stomach ulcers: caused by
bacterial infection, not stress
• StomachpH~1.4
• H.pylori increases local pH by secreting ammonia (a strong base)
• Ammonia is made with an enzyme called urease, which breaks down urea into ammonia.
• Urease is located in the cytoplasm
• Ammonia is only produced when cells are in low pH environment.

Front 20

Transport across memb. using proteins.

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Membrane proteins:
-Highly specific, saturable, regulate transport.
Concentration gradients=potential energy.
-Proton Motive Force: when more H+ outside cell, they want to come in. Can't because of charge. This can be used to make ATP.
-Passive diffusion is w/o protein help, if molecule can just cross memb.
-Facilitated diff=passive transport w/ a protein channel.
-Active transport is against conc grad. Need pumps.

Front 21

Urease Experiments

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Question: How does urea get in to the cell so that urease can make ammonia?
• Hypothesis? – Urea enters the cell using a specific membrane localized transporter
• Researchers identified a candidate transporter, Ure1
– Testable Hypothesis: Ure1 is a membrane bound urea transporter.
• Ure1 is regulated by pH – it opens only when pH is low.

Hypothesis: UreI is found in the membrane
• Fractionate cells
– TM = total membrane
– (busted cells apart and separated the membrane from the rest of the cell)
• Run on a gel to detect proteins
• Western blot
• UreI will show up as a band if it is present
• What other fractions would be valuable to see?

Hypothesis: UreI allows urea to enter the cell.
• UreI-cells lack Ure1.
• WT cells are normal.
• Is urease active in UreI- cells?
• What would the curve for WT homogenate look like?
• What can we conclude about UreI from this data?

Radioactively label urea (14C)
• Add UreI to cells
• Measure amount of 14C inside cells

Front 22

Gram + v gram -

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gram+ have 1 memb, then thick cell wall. Purple with gram stain. Gram- have memb, thin cell wall, 2nd memb with LPS layer. Pink.

Front 23

Cell Wall functions

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Prevents lysis by turgor pressure (high conc of solutes in cell) and prevents shrinkage w/ dehydration.

Present in almost all prokaryotes except:
Mycoplasmas
Thermoplasma (archaea)
Have tougher membrane - sterols, lipoglycans.
Live in osmotically regulated environments.

Front 24

Sacculus

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Cell wall emptied of cytoplasm. Retains shape.

Front 25

H. pylori--cause of shape?

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-helical rod.
Hypothesis: – Cell shape is determined by the cell
wall.
-If cell wall determines cell shape, then a carefully isolated
intact sacculus should have the same shape as the cell.

Front 26

Cell wall structure? Relation to cell shape.

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• Multiple layers (sheets) of peptidoglycan
• Repeating carbohydrate units:
– N-acetylglucosamine (NAG)
– N-acetylmuramic acid (NAM)
• Cross linked with tetra or pentapeptides.

Peptidoglycan structure is maintained by peptide crosslinking
• Csd1 is a peptidase and cells lacking csd1 have rod shape
• Does csd1 cleave peptide crosslinks in peptidoglycan
• Hypothesis:
– Helicity: caused by less crosslinking of peptidoglycan (therefore rod shaped cells should have more crosslinked peptidoglycan than helical cells)
• Results: csd1 deficient rod shaped cells have almost 50% more crosslinking than helical cells.

Front 27

Advantage of helical shape to H.pylori?

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• Hypothesis:
– Helical shape is necessary for efficient colonization of the stomach.
• InfectmicewithamixofbothWTandcsd1deficientcells
• Cellswillcompeteinthestomachandonlythemostfitwill be ably to colonize and infect
• Samplestomachcontentsafter1week – What cells are there?
• Do we know it is cell shape that is affecting colonization? Could it be something else?

Front 28

Salmonella.

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-No capsule.
-Can make you sick even when it's dead.
-Does part of the cell make you sick?
Hypothesis: The LPS causes Salmonella food poisoning. Specific part of LPS?

-Mutant Salmonella strains that fail to make normal LPS--something wrong with the lipid A.
-Are not pathogenic.

Front 29

LPS structure.

Back 29

Lipid A -fatty acids linked to N-acetylglucosamine
• Core Polysaccharide
• O-specific polysaccharide

Front 30

Capsules-structure and function

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Structure:
– Diverse
– Generally rigid layers of polysaccharide.
• Function:
– Attachment to surfaces
– Protect from desiccation
– Protect from immune system (phagocytosis).
– Barrier

Front 31

Capsules-role in pathogenesis experiment.

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Function of capsule: role in pathogenesis?
• (S.pneumonia) isolates from patients found to consistently be of the smooth morphology.
• Question: Is smooth morphology related to ability to cause disease?
• Hypothesis: Smooth, but not rough, S.pneumonia causes disease.
• Experiment?

Front 32

Cell Walls of Archaea

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• Pseudopeptidoglycan-- Resistant to lysozyme digestion.
• Polysaccharide layers
• S-layer-- Probably acts as barrier.

Front 33

Fimbriae, Pili

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• Protein fibrils extending from cell surface.
• Function: Adhesion and motility
• Attachment pili often called fimbriae to distinguish from sex pilus.
• Extremely rare in gr+

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Twitching motility

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-Grapple and retraction motion.
-Using type IV pili.
---3 step cycle:
–Extension
– Attachment to surface
– Retraction
• Retraction is by
disassembly from the base.
• Extension is by assembly, probably using recycled monomers.

Front 35

Flagella

Back 35

• Long thin, helical appendage
• Function: motility
-Can't decide on direction==>Run and Tumble movement.
-Directed movement is result of tumbling more when going the wrong direction and tumbling less when going the right direction. (chemotaxis).
• Arrangement varies.
– Polar
– Lophotrichous (clump in one spot)
– Peritrichous (everywhere)
• Flagellar structure
– Flagellin subunit highly conserved
among bacteria.
– Different structure in archaea, eukarya.

-Uses proton motive force: electrochemical gradient of H+ ions.

Front 36

Bacterial Cell Division

Back 36

-Binary fission
-Daughter and mother cell are equal in size.
-Rods: lengthen (double) w/o change in diameter.
– Increase total cytoplasm and
other contents
– Replicate DNA
– Synthesize new cell wall
-Septum forms in center to divide cell

Front 37

Cell wall during division?

Back 37

• Growing cell needs new cell wall
• Cut wall with autolysins
• Add new peptidoglycan
(Transport of peptidoglycan subunits across membrane, Transpeptidation, Transglycosylation)
• Leaves wall band (scar) (Gr +)

Front 38

Transpeptidation?

Back 38

Making new cell wall.
Crosslinking of peptides by transpeptidases.
Inhibited by penicillin and many other antibiotics.

Front 39

Septation

Back 39

-Formation of cell wall at midpoint of cell.
How does the cell know where septation should occur?
-Mutant strain of E. coli identified that fails to septate. Mutation is in FtsZ.
What does FtsZ do? What is its role in cell division?
• Fluorescent FtsZ
• Forms ring at equator
• But how is FtsZ ring formation regulated?
• Min proteins identified because mutations cause
“minicells”
– Small cells usually without DNA as a result of septation off center
• MinC and MinD acting together can inhibit FtsZ ring
formation
• Hypothesis: MinC, MinD, and MinE help establish the FtsZ ring in the centre of the cell.
-More fluorescence, this time on the Mins. Oscillations and coils observed. FtsZ goes where Mins aren't.

Front 40

DNA Replication

Back 40

• Most bacterial genomes are circular.
• Replication begins at Ori, anchored to membrane at equator.
• Forms replication bubble.
• Replisome: large complex of proteins involved in replicating DNA, located at replication fork.
• DNA threaded through replisome
• Next round of replication can start before septation occurs.

Front 41

Goldilocks Zone?

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Every bact. has one--the perfect temp, pH, oxygen level, solute concentrations, and pressure for growth.

Front 42

Bacteria types--temperature

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Psychrophile (down to -5 deg)
Mesophile
Thermophile
Hypermophile (up to 115 deg)

Front 43

How does temp affect bacteria?

Back 43

--Cold--makes cell memb solid.Therefore, psychrophiles have super-fluid membs. Unsaturated. Also can form ice crystals and damage everything. Also proteins need to be flexible.
--Hot--cell wall can melt. -Thermophiles saturate their phospholipids (hydrogenation). Also more hopanoids.
-Archaeal Hyperthermophiles have Lipid monolayer, where tails extend all the way across.
• Isoprene polymer, not fatty acid
• Ether bond instead of ester bond.
-Other heat adaptations!
Thermosome: a protein complex that re-folds proteins.
Heat stabilizing solutes-stabilize proteins.
Reverse DNA gyrase - enzyme that induces positive supercoils in DNA (twists helix more tightly).
Archaeal histones-bind DNA more tightly.

Front 44

What does pH do to cells and their contents?

Back 44

-Can change charges of proteins, affecting shape and activity.
-pH range of organism is not same as pH range of its enzymes
• Survival is dependent on ability to maintain neutral internal pH.

Front 45

Bact. types--pH

Back 45

**Low H+ = Base, high pH.**
Acidophiles
-membranes reduce proton
permeability
–Proton pumping mechanisms
Alkylophiles
-Modifications to cell wall.
–Proton motive force? They do Sodium Motive force or use a charge gradient.

Front 46

How does solute conc. affect bact?

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-To be usable, water must not be attached to ions or solutes.
• High osmolarity (high conc.) environments have low water availability.
• Can also result in cell shrinkage or high turgor pressure.

Front 47

Types of bact-salinity

Back 47

Nonhalophile
Halotolerant
Halophiles
-Must change the direction of osmosis so water enters the cell, despite conc. gradient.
–Increase the total concentration of solutes inside with small molecules that won't interrupt cell function at high conc.
• K+ • Glycerol • Amino acids or amino acid derivatives.
Extreme halophiles (10-20%)
Most bacteria: 0.2-5%
Seawater: 3.5% NaCl

Front 48

Types of bact.--Oxygen

Back 48

Obligate aerobe
- needs O2
Microaerophile
- needs O2 at lower conc.
Facultative aerobe
- grows better with O2 but not required.
Aerotolerant anaerobe
- tolerate O2 but don’t use it
Obligate anaerobe
- die in presence of O2

Front 49

How does O2 affect cells?

Back 49

-Vital for aerobic respiration. Something else is used for anaerobic respiration. Another option without O2 is fermentation.
-Presence of O2 creates very reactive molecules. Cells exposed to O2 must have mechanisms to reduce the damage. Ex: catalase, which breaks down H peroxide.

Front 50

Cell Fractionation?

Back 50

Lysing cell and separating its parts with ultracentrifuging. Ex: If you want to know if a protein is in the cell memb, look for it in the cell memb parts afterward.

Front 51

Western Blot

Back 51

A gel. Large proteins will move more slowly. Stain specific proteins using antibodies. Used to see if certain proteins are in a sample.

Front 52

Gram staining.

Back 52

Gram + purple, gram - pink. Purple crystal violet is easily washed out of gram -. The counterstain dyes the g- pink.

Front 53

What's the nature of this protein?

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-Create a mycell (empty cellmemb) and put protein in, experiment.
-xray crystalography
-compare its DNA seq to other proteins

Front 54

Endospore

Back 54

Survival structure. Heat resistant. Dehydrated.

Front 55

PCR

Back 55

-Use primers and Taq Polymerase to amplify DNA before a gel.
-Make primers. Forward primer is identical to a bit of the 5'-3' sequence, while reverse primer is the reverse complement to a bit of the 5'-3' sequence.

Front 56

Streak plating

Back 56

Done to get isolated colonies, so you can separate mixed plates, observe colony morphology, or to have a supply of bacteria for future experiments.

Front 57

Spread plating

Back 57

Spread already diluted mixture over entire plates.

Front 58

Serial Dilutions

Back 58

Used to get plates of different dilutions so at least one is obtained that allows us to count the CFUs.

Front 59

Bacterial growth in agar tubes...what for?

Back 59

An oxygen gradient forms. See where in the tube your bacteria grow.