Module 2 Flashcards
How do sugar molecules like glucose or mannose enter a bacterial cell?
They must use some form of active transport, whether its simple active transport, group translocation/phosphotransferase, or ABC transporters
What are the three different types of simple active transport and what makes them different from each other?
Symport - protons in, sugars in
Antiport - protons in, sodium out
Uniport - potassium in
Compare the three different types of active transport.
Simple - driven by PMF
Phosphotransferase - driven by PEP, substrate is phosphorylated
ABC Transporter - driven by ATP, cargo is bound to a periplasmic or extracellular protein and transported inside, similar to Type I Secretion systems
Why can’t most microbes ever be cultured in isolation?
Syntrophy
Bacteria produce waste products, and eventually they will accumulate in their environment and inhibit the bacteria that live there. They need other organisms in that environment to metabolize and get rid of those waste products.
What kind of bug undergoes photosynthesis, reduces hydrogen sulfide for electrons, and gets its carbon from carbon dioxide?
A photolithic autotroph
What kind of bug undergoes photosynthesis, gets its electrons from methane, and its carbon from glucose?
A photoorganotrophic heterotroph
What kind of bug oxidizes hydrogen sulfide, gets its electrons from hydrogen sulfide, and its carbon from carbon dioxide?
A chemolithic autotroph
What kind of bug oxidizes, gets its electrons, and gets its carbons from maltose?
A chemoorganotrophic heterotroph
Describe the mutualistic relationship between Riftia pachyptila and their endosymbiotic thermophiles.
Riftia pachyptila’s endosymbiotic microbes are chemolithotrophic autotrophs. They live in deep sea hydrothermal vents, under extreme pressure and heat. R. pachyptila exchanges H2S and O2 through its gill plume, allowing the chemolithoautotrophs in its trophosomes to take in those nutrients and produce CO2 as a waste product.
Why do bacteria need to use active transport to get their nutrients?
They need to use active transport because they almost never live in an environment where there are enough nutrients that they can enter the cell via simple diffusion.
Why do bacteria need iron to survive?
They need it as a cofactor for their redox reactions. It’s necessary to sustain their life.
How has our human biology evolved to take advantage of bacteria’s iron requirements? How have bacteria evolved in response?
We scavenge virtually all the iron out of our environment and hold it tightly in proteins in our cells. In addition to helping us for our purposes, this deprives potentially pathogenic bacteria of a necessary cofactor for their redox reactions. In response, though, the bacteria have developed their own high-iron-binding-affinity molecules, siderophores.
How do gram negative bacteria import iron into their cells?
They use a cargo-specific transport protein to move the iron-bound enterobactin into the periplasm, and then they use a type of ABC transport specific to iron-bound enterobactin to move it from the periplasm to the cytoplasm.
What happens to the iron-bound enterobactin once it enters the cytoplasm?
The cytoplasm is a reducing environment, so the Fe3+ is reduced with the addition of an electron to Fe2+. But enterobactin can bind only to Fe3+, so the iron is released from it, and it is now released back outside the cell.
What are the two means of ATP production?
Substrate level phosphorylation and oxidative phosphorylation.
What does catabolism generate?
Energy, reducing power, and precursor molecules for anabolic pathways
Why does the cell need reducing power? What is it used for?
Reducing power gives the cell the ability to use electrons taken from the reduction of NADP → NADPH to construct new organic molecules. They need it for their anabolic pathways.
What is the most common pathway bacteria use to catabolize glucose to pyruvate?
EMP
How is glycolysis regulated in bacteria?
Pyruvate kinase, in addition to its two binding sites for ADP and PEP, has an allosteric site that binds to fructose 1,6-bisphosphate. This gives the pyruvate kinase an incentive to make pyruvate from PEP only when there’s a high presence of fructose (meaning high levels of glycolytic activity) within the cell.
Describe the dual role PEP plays in group translocation and glycolysis.
PEP powers group translocation (phosphotransferase) systems in addition to being dephosophorylated to create pyruvate and ATP in glycolysis. So if you’re a bacterium that’s starving for glucose, you’re going to use that PEP in group translocation, importing more glucose instead of breaking it down in glycolysis.
Why is glucose favored over other sugars?
Because it fits directly into the glycolytic pathway. Everything else needs to be modified before it can enter glycolysis, making it less efficient.
Why must bacteria use their reducing power?
Because there’s only a small pool of NAD+ in the cell. If all of that shifts to NADH and can’t be reduced back to NAD+, the oxidation in EMP glycolysis will suffer.
How do bacteria synthesize precursor molecules for use in anabolic pathways?
Intermediate molecules are pulled off from the different pathways. Not every carbon that enters these pathways will go all the way through, there are siphons at different points.
Yields of EMP
2 NADH, 2 ATP
Yields of ED
1 NADH, 1 NADPH, 1 ATP
Yields of PPP
2 NADPH and hella carbon skeletons, precursors for anabolic pathways
How does EMP generate ATP?
Substrate-level phosphorylation
Where are EMP, ED, and PPP found in nature?
EMP and PPP are found in all organisms, prokaryotes and eukaryotes.
ED is found only in bacteria
What makes PPP different from the other glycolytic pathways?
It doesn’t generate energy and it doesn’t involve substrate level phosphorylation
What’s the difference between NADPH and NADH? (not looking for biochemical differences here!)
NADPH is better at biosynthetic reactions, while NADH is better at producing energy (it does so by shoving electrons onto things, hopefully those things are the ETC)
If an E. coli bacterium has a high amount of ATP generated from ED, where will it send its G6P and why?
To the PPP to synthesize the precursor molecules for new amino and nucleic acids, in preparation to commence replication
If an E. coli bacterium is depleted of ATP, where will it send its G6P?
To the EMP to generate more ATP
What does fermentation do?
Regenerates NAD+
What happens to a bacteria if NAD is depleted? How is this problem solved?
If NAD is depleted, glyceraldehyde-3-phosphate (G3P) oxidation stops. The solution is to regenerate NAD by oxidizing NADH and reducing pyruvate.
Why does Strep. need to live in very nutritious environments? Another question with this same answer is “Why are strep often involved in the production of fermented foods?
Because they’re not able to use the TCA cycle, they need a constant energy source, i.e. a nutritious environment.
How/why does our enamel decay?
The bugs in our mouth produce lactic acid as their reduced pyruvate byproduct, which is secreted out of their cells where it collects on our teeth and eats through our enamel.
Describe the importance of fermentation to human health.
All our dietary fiber is food for our microbiome. They eat this fiber, which we are unable to digest, and produce and secrete their fermentation end products, the most important being butyric acid. Butyric acid is the #1 energy source for all our cells in immediate contact with the microbiome, stretching from the lumen to the colon. Those cells aren’t fed by our bloodstream. They need that butyric acid to diffuse directly into their cytoplasm, where it diffuses further to their mitochondria where it’s used in our TCA cycle. Butyric acid also gets into our lamina propria and binds to G-protein coupled receptors on macrophages, dendritic cells, T-cells, etc, and promotes less inflammation in our GI tract.
Almost all of our cells in immediate contact with the interior of our GI tract, stretching from our lumen to our colon, aren’t fed by our bloodstream. How are they fed instead?
Butyric acid, a fermentation endproduct from microbes in our microbiomes, diffuses into those colonocytes.
How do high fiber diets prevent colon cancer?
The Warburg Effect.
Precancerous cells stop relying so heavily on the mitochondria and instead ramp up the glycolytic pathway, generating their ATP through substrate level phosphorylation. In shutting down the mitochondria, the butyrate diffusing into the cells is not being hydrolyzed, and as the butyrate concentration increases it inhibits histone deacetylase (HDAC) which changes the gene expression and induces apoptosis.
Which uses electrons more efficiently: respiration or fermentation?
Respiration
Compare how pyruvate is used in respiration versus fermentation.
Respiration –> pyruvate is decarboxylated to acetyl-CoA and shunted along the TCA cycle through various oxidation and reduction stops
Fermentation –> pyruvate is an electron dump
Describe the genesis of proton motive force.
The NADH generated by membrane proteins in TCA, PPP, and ED moves protons from the cytoplasm across the cell membrane.
List the three most important things PMF can do.
- Generate rotation for the flagella
- Drive active transport
- Gated across ATP synthase, drives oxidative phosphorylation to create ATP from phosphates free in the environment
What metabolic type of organism are E. coli? What does that mean for their biochemistry?
Facultative anaerobes. This means that they use oxygen to the exclusion of any other electron acceptor, until all their oxygen is gone.
What happens if E. coli is placed in an oxygen-deprived environment?
They will continue respiration by reducing nitrate or other forms of nitrogen to nitrate. However, less PMF is generated from this, which is why they spend so much of their resources making a branched ETC so they can use every bit of oxygen.
How and why does our atmosphere maintain its 78% N2(g) state? Why isn’t our planet’s nitrogen tied up in organic material?
Our atmosphere maintains its N2(g) state because N2 is the final endproduct of the ETC in soil-microbe Paracoccus denitrificans. These bug respirate anaerobically and can use all different forms of nitrogen, including NO3, NO2, NO, N2O. Once it finishes with one product, unless it has reached its final-final endproduct, N2, it can shunt that nitrogen molecule back into its ETC until it is finally reduced down to N2.
Who are the true masters of the glycolytic pathways?
Salmonella
How does Salmonella enterica mediate infectious population growth?
They use their Type III secretion systems to directly inject proteins into our cells. These proteins (re: toxins) change our epithelial cell’s cytoskeleton and cause the salmonella to be engulfed by the cell. Salmonella induces its own uptake. From there, our host cells sense danger and release cytokines to attract help from the white blood cells. The WBCs arrive and release ROS to try and kill the salmonella, but salmonella are incredibly resistant to ROS so all it really does is kill other competing bacterial species. They intentionally induce inflammation to kill competing microbes.
The end goal of this is to change sulfates into tetrathionate, which the salmonella can use as a terminal electron acceptor, allowing them to do anaerobic respiration, which is more efficient than fermentation and allows the salmonella to multiply quite rapidly.
How do bugs that lack electron transport chains drive their active transport and flagella (if they have them)?
They rotate their ATP synthase proteins backwards to pump protons out. It burns the ATP generated in glycolysis, but the PMF gained is more advantageous.
Which energy production method, fermentation or aerobic respiration, is better? Why?
Aerobic respiration.
Uses electrons more efficiently
Why does E. Coli, a facultative anaerobe, have a branched Electron Transport chain rather than simply shifting to the use of NO3 when O2 becomes limiting?
Less PMF is generated from reducing nitrate than from branching the ETC to continue reducing oxygen.
What is a branched electron transport chain?
The protein that reduces oxygen in normal ETC can work only when oxygen levels are high. A branched electron chain solves this problem. The branching occurs when a new protein is substituted for the old one when oxygen levels are low, allowing for PMF to continue being produced, albeit at lower levels.
What makes a microbe a facultative anaerobe?
The ability to use oxygen to the exclusion of any other electron acceptor, until all the oxygen is gone.
What would happen if a mutant e. Coli lost its ability to use O2 as a terminal electron acceptor? What if it lost its ability to use NO3 as an electron acceptor instead? If one of each of these two mutants were in an environment with adequate supplies of both O2 and NO3, what would happen?
The mutant that can’t use oxygen will use nitrate as its terminal electron acceptor.
The mutant that can’t use nitrate will use oxygen as its terminal electron acceptor, and in the absence of oxygen it will use other terminal electron accepts such as nitrite, TMAO, DMSO, etc.
The mutant that can use oxygen will proliferate and thrive more than the mutant that can’t, as it will be able to generate more PMF and will have and advantage over the other.
Why don’t infectious cells secrete toxins at all times? Why do they wait until their population has reached a certain size?
Many infectious cells don’t want to start secreting toxins until they’ve reached a large enough population size because if they did, they’d risk attracting the host’s immune system while their population is still small enough to be easily wiped out.
Where would you find higher levels of AHL, inside the cell wall or outside the cell wall? Why?
Neither.
The acyl side chain allows the molecule to diffuse freely across membranes, so there should be equal concentrations of AHL inside and outside the cell.
How do AHL levels regulate gene expression?
When AHL levels become high, they bind to LuxR, a transcription factor, activating them. Once activated, LuxR changes conformation and becomes dimers that bind to specific genes coding for quorum-dependent proteins and increases the transcription of AHL synthase, which drastically increases AHL levels.
Can a bacterium recognize the AHSL produced by another species? Why or why not?
Sometimes, mostly no. Each species produces its own unique AHSL molecules, and recognizes the molecule based on those minor structural variations. A bacterium of one species is effectively blind to the AHSL molecules from other species. However, there are some specific cases in which two AHSL molecules are similar enough that the can confuse other populations.
What feature of quorum sensing demonstrates its importance to bacterial survival?
The fact that it evolved twice: once in gram negative bacteria, and again in gram positive bacteria.
It hadn’t evolved yet when the ancestral prokaryotes split into gram positive and gram negative domains, but it became so important to survival that all bacterial species needed to evolve some sort of population density sensing mechanism to survive, hence why it evolved twice.
Describe the life cycle of Vibrio fisherii within the Hawaiian Bobtail Squid.
Vibrio fisherii live in a mutualistic relationship with the nocturnal Hawaiian Bobtail Squid. During the day, the HBS buries itself into the sand and sleeps. During this time V. fisherii populations within its stomach increase until they reach quorum. At night, the HBS wakes up and enters the water column, the bioluminescence now being produced by the V. fisherii now at quorum in the stomach protects the HBS from predators. The predators sense their prey by sensing decreases in moonlight from fish swimming in front of the moon, casting a shadow. That bioluminescence prevents the HBS from casting a shadow, allowing it to survive the night. When morning comes, and the HBS goes back into the sand to sleep, the stomach is full of waste product, so it squeezes its stomach and expels 95% of its stomach contents, leaving behind only a small amount of the V. fisherii to spend the day growing and reaching quorum once again.
Describe the AHSL levels present extracellularly of a bacterial population during its different life/growth phases.
Lag Phase → Low AHSL levels
Log Phase → Increasing AHSL levels
Stationary Phase → High AHSL levels
Death Phase → Decrease AHSL levels
What phenotype do Vibrio fisherii present when LuxR levels are low? Why?
No bioluminescence. When LuxR proteins are low, the population is likely low, so there wouldn’t be enough AHSL to interact with the LuxR to turn on the bioluminescence.
What phenotype do Vibrio fisherii present when LuxR levels are high? Why?
Bioluminescence. When LuxR levels are high, there is enough to interact with the AHSL to turn on the bioluminescence.
What’s the difference between chemotaxis and response regulators?
Chemotaxis → changes protein function
Response regulators (aka quorum sensing) → changes gene expression
Why/how can prokaryotic species survive and thrive at much higher temperatures than us multicellular eukaryotes?
The more complex you are, the more ways there are for heat to kill you.
