Final Review Flashcards
An absent-minded student forgets to de-stain their bacteria with alcohol during the Gram staining period. Assuming all other parts of the protocol were followed perfectly, what kind of bacteria would this forgetful student think they have? Why?
Gram positive, because the purple stain wouldn’t be washed away. If the bacteria was gram negative, the purple dye would still be in their thin peptidoglycan layer, resulting in a purple stain, where if the student had done the procedure correctly there should be a pink stain.
Compare and contrast peptidoglycan structure and transpeptidation in gram negative and gram positive bacteria.
Gram negative:
- The extra amine group on DAP forms a peptide bond with a second peptidoglycan molecule
- The fifth amino acid, D-alanine, is cleaved from the first peptidoglycan molecule to provide the energy used to form the cross-linking peptide bond
- The peptide bond forms a direct interbridge
Gram positive:
- The extra amino group on L-Lysine is used to connect the two peptidoglycan molecules
Five glycine molecules form a pentaglycine interbridge
- These glycine molecules are used to attach various stuff to so it doesn’t diffuse away from the cell. They’re kind of like bonus hands holding keys or misc whatevers
- The fifth amino acid, D-alanine, of the first cleaved to provide the activation energy to form the interbridge
How do gram negative bacteria acquire resistance to β-lactams? How is this different from gram positive bacteria?
Most gram negative bacteria acquire resistance to ꞵ-lactams by acquiring genes for ꞵ-lactamases, enzymes that cleave the ꞵ-lactam antibiotic in two. Most gram positive bacteria, however, acquire resistance to ꞵ-lactams by acquiring genes that alter their transpeptidases, such that they still bind to D-alanyl D-alanine but no longer to the ꞵ-lactam antibiotics.
Why would you use augmentin to treat a β-lactam antibiotic resistant gram negative infection? Why would you not use augmentin to treat MRSA?
A gram negative bacteria that is resistant to beta lactams is likely producing lactamase into the environment, effectively neutralizing the antibiotics. However, when augmentin is added to the treatment plan, it distracts the beta lactamase enzymes. It takes up their time so that the original antibiotics can sneak past and get their job done. However, you would not use this tactic with MRSA, because its resistance isn’t based on beta lactamase. Its resistance is based on modifications to its transpeptidase, transforming into an enzyme called MecA. MecA can still form the crosslinks between peptidoglycan molecules, but it cannot bind to any beta lactam molecule, so augmentin is completely ineffective.
How are flagella different in Gram Negative vs Gram Positive bacteria?
Because gram negative bacteria have two lipid membranes making up their cell wall, the basal bodies of their flagella have two rings, one to anchor into each lipid membrane. The basal bodies of the flagella in gram positive bacteria, because they need to anchor into only one membrane, have only one ring.
Why do mycobacteria fail to stain in Gram protocols? How do we stain them instead?
Mycobacteria are coated in mycolic acid, producing a highly hydrophobic waxy coating that is impervious to many dyes, including those used in typical Gram staining protocols. Instead, we need to use an Acid-Fast Stain to visualize them. The protocol cooks the mycobacteria in the presence of carbolfuchsin, which drives the pink dye past the mycolic acid. Then the bacteria are washed with acidified hydrochloric acid. All other bacteria will give up the carbolfuchsin in the presence of acidified hydrochloric acid, but again because of its waxy mycolic acid coating, the pink dye holds fast.
A physician is treating a patient with tuberculosis, a gram positive bacteria. He plans to use Vancomycin. Will his patient be cured?
NO.
How is peptidoglycan synthesized?
Peptidoglycan is synthesized INTRAcellularly and assembled EXTRAcellularly.
- UDP adds amino acids to NAM
- D-Alanine is synthesized from L-Alanine
- Two D-Alanines are attached to NAM, forming a pentapeptide called Lipid 1
- Lipid 1 uses phosphates to covalently bind to bactoprenol, aka a membrane lipid
- NAG is added to NAM, forming Lipid 2
- Flippase, aka MurJ, takes Lipid 2 + Bactoprenol across the membrane
- Bactoprenol is removed
- One phosphoanhydride bond undergoes hydrolysis to provide the activation energy for bactoprenol to move back across the membrane
- The new peptidoglycan fills in a nick made by an autolysin in the cell wall.
Why is peptidoglycan synthesized with five amino acids, even though the final form has only four? What purpose does the fifth amino acid serve?
The fifth amino acid is cleaved during transpeptidation to provide the activation energy to create the crosslinking peptide bond between two peptidoglycan molecules.
How is the newly synthesized peptidoglycan monomer transported to the periplasm or extracellular environment?
From the answer key: the peptidoglycan monomer is attached to the lipid bactoprenol and by the action of the enzyme flippase/MurJ is transported across the periplasm or cell membrane.
What makes peptidoglycan such a unique molecule?
It is only found in bacteria. No archaea or eukaryotes have peptidoglycan
Uses both L and D isoforms of amino acids.
Again, only bacteria can do this, archaea and eukaryotes can only use L isoforms
NAM isn’t found in eukaryotic cells
Gram negative bacteria use DAP, DAP isn’t found in eukaryotes either
What does the lactyl group attach the peptide portion to?
NAM
What is the most basic definition of chemotaxis?
Going towards or away from chemicals
What happens to the MCP chemotaxis complex when the chemoattractant is present?
When the chemoattractant is present, MCP inactivates CheA, allowing the basal body to keep rotating.
What happens to the MCP chemotaxis complex when the chemoattractant is absent?
When chemoattractant is absent, MCP activates CheA, which starts a phosphorylation cascade and makes the basal body stop rotating, inducing a tumble.
It is in the bacteria’s best interest to tumble for only brief periods of time. How does the bacteria regulate chemotaxis for this interest?
CheZ removes CheY’s phosphate, which makes it unable to interact with the basal body.
What is a random walk and when do bacteria take them?
Random tumbling through the environment, happens in the absence of a chemical gradient.
A sneaky bacteria is out on a random walk and sees some maltose. What happens next? As this bacterium acclimates to the new increased maltose concentration, what happens?
The MCP is engaged by maltose. CheA kinase activity is suppressed, the pool of CheY shifts toward the non-phosphorylated form, and the flagella rotate longer in the counterclockwise rotation.
An E. coli bacteria travels towards an increasing concentration of maltose, but on the way there also encounters an increasing concentration of acetate (a waste product of fermentation). What happens when these two environmental signals encounter the bacterium’s chemotactic apparatus? What will the final result be?
The chemorepellent will always trump the chemoattractant. The bacteria will always tumble away.
Explain the process of accommodation, or how bacteria acclimate to a high concentration of a chemoattractant such that they can still reorient themselves and find areas of even higher concentrations of that chemoattractant.
At an intermediate presence of a chemoattractant, CheR gradually methylates MCP’s glutamic acids over time, which stimulates the CheA histidine kinase to phosphorylate itself, starting the tumbling cascade.
How do bacteria preserve their ability to tumble even when attractants are predominantly bound to MCP?
They use accommodation to preserve their ability to tumble. Essentially, if the bacteria is in an intermediate presence of an attractant, CheR will gradually methylate the glutamic acids of MCP over time, changing its confirmation and making it harder for attractants to bind to it, so that CheA is activated more and induces the phosphorylation cascade that results in a tumble.
How do bacteria in a gradient of some chemoattractant recognize higher levels of that chemoattractant?
CheR’s gradual methylation of MCP’s glutamic acids changes the receptor’s confirmation so that the attractant doesn’t bind as well to MCP. It needs higher and higher levels of the chemoattractant to bind at the same frequency+length as before.
How do bacteria reset accommodation?
To reset accommodation, CheA transfers its phosphate group to CheB. CheB is now free to chew the methyl groups off the glutamic acids in the MCPs, allowing the bacteria to respond to low chemoattractant levels again.
In a 10 second run through an aqueous environment, a bacterium encounters an increasing amount of a noxious chemo-repellent compound. What is the bacterium’s most likely response?
Flagellar rotation switches to counter-clockwise (CCW) rotation.
What is the function of CheW?
CheW allows a signal from one MCP receptor to permeate through the loud array of all the different MCP proteins in the cell membrane. It silences the other CheAs so that its signal can be expressed.
How do bacterial cells undergo glycolysis? What are the advantages and disadvantages to each pathway?
Bacteria can use one of two glycolytic pathways to produce ATP: the Embden-Meyerhof-Parnas (EMP) pathway, and the Entner-Doudoroff (ED) pathway.
EMP is the main glycolytic pathway, and yields 2 ATP and 2 NADH. ED is a secondary, more ancient pathway, and while it is less efficient than EMP (yields only 1 ATP, 1 NADH, and 1 NADPH), it is essential for bacterial growth (although we don’t know why yet). It also may be advantageous because it produces both NADH and NADPH, whereas EMP produces only NADPH for its reducing power.
Bacteria will alternate their use of EMP and ED depending on whether they need more ATP or more reducing power.
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 to fit into glycolysis, so it’s 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.
Compare and contrast the EMP, the ED, and the PPP.
EMP:
All ATP comes from substrate-level phosphorylation
Yields 2 NADH
Yields 2 ATP
Found in all organisms
ED:
Yields 1 NADH
Yields 1 NADPH
Yields 1 ATP
Found only in bacteria
PPP:
Does NOT generate energy!
No substrate-level phosphorylation
Not ATP or GTP generated
Yields 2 NADPH
Yields critical precursors for anabolism, all possible carbon skeletons
Found in all organisms
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 (through shoving electrons onto things, hopeful the ETC)
Does the PPP use substrate level phosphorylation?
No.
What are the yields of the PPP? How does this compare to the yields of the EMP and the ED?
PPP yields 2 NADPH and critical precursors for anabolism. This is very different from the EMP and ED, which generate energy (ATP).
If an E. coli bacterium has a high amount of ATP generated from ED and EMP, where will it send its G6P and why?
PPP, to synthesize new amino and nucleic acids to start replicating itself.
If an E. coli bacterium is depleted of ATP, where will it send its G6P?
To EMP and ED 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 (Krebs) cycle, they need a constant energy source, ie. 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.
In respiration, pyruvate is decarboxylated to acetyl-CoA and shunted along the Krebs/TCA cycle through various oxidation-reduction stops.
In 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.
Generates rotational energy for flagella, drives active transport, and is gated across ATP synthase, driving oxidative phosphorylation and creating 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.
Compare and contrast fermentation-based energy production and respiration based-energy production.
Fermentation:
Pyruvate is an electron dump.
Regenerates NAD
Aerobic Respiration:
Pyruvate is decarboxylated and shunted along the oxidation-reduction stops of the TCA cycle
Yields 4 NADH, 1 FADH, 1 GTP, and some anabolic precursor molecules
Which energy production method, fermentation or aerobic respiration, is better? Why?
Respiration because it uses pyruvate and 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? And what even is a branched Electron Transport chain anyways?
Less PMF is generated from reducing nitrate than from branching the ETC to continue reducing oxygen.
ETC → Normally, when oxygen levels are high, the ETC puts protons onto oxygen. This generates hella PMF, but when oxygen levels are low, the protein responsible for normal ETC can’t work because it only works when oxygen levels are high. So a new protein is substituted in, and oxygen is still being reduced, although it produces less PMF.
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 bacteria a facultative anaerobe?
The ability to use oxygen to the exclusion of any other electron acceptor until all their 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.
The specific chemical process of fermentation may differ across different bacteria species, but the general idea is always the same. What is that general idea?
The oxidation of NADH to regenerate NAD+ pools.
Where do human colonocytes get the majority of their energy from?
From butyrate, a fermentation byproduct of the microbes nearby.
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. Each species produces its own unique AHSL molecules, and recognizes the molecule based on those minor structural variations. So 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
What is a regulon?
A series of unconnected genes controlled by one regulatory protein.
What is an operator?
DNA sequences that bind to repressor proteins to hold back/decrease transcription.
Why is ssDNA an indicator of DNA damage?
Lesions, such as pyrimidine dimers, cause replication to pause, leaving ssDNA free floating for longer than usual. When there’s too much ssDNA free floating or the ssDNA has been out for a long time, that’s a sign that the damage is severe.
What happens when RecA initiates LexA’s self-destruction?
There’s less LexA to bind to the operator sequences of the uvrABC repair system, allowing those genes to be transcribed to then find and replace the damaged DNA.
What happens when LexA levels drop?
More and more genes in the SOS response will be transcribed.
What is the last gene to be expressed in SOS response? Why this gene?
umuDC. It codes for DNA Polymerase IV, the “sloppier copier.” This DNAP fixes lesions with very poor fidelity, however unlike more fidelous polymerases it can actually fix those lesions. Fixing the larger problems is worth the bp mistakes.
Why do the operator sequences of all the genes controlled by the SOS response have different binding affinities for the protein LexA?
There’s a whole series of repair mechanisms at your disposal and they’re all being repressed by lexA. You want the express the most gentle repair mechanism first and hope that it takes care of the problem. NER is usually first, the sloppier copier is usually last. If the damage isn’t fixed, LexA levels will continue to drop and the next repair pathway will be expressed, then the next, then the next, and then umuDC basically comes in and performs some altruistic fixing, with very low fidelity, so when the bacteria dies (which is likely to happen soon) some new, mutated dna will be released into the environment to hopefully be taken up by some other microbe.
What signal detected inside a bacterium initiates the activation of the SOS response? What molecule detects this signal? What does this molecule then do?
Single stranded DNA. that’s the abnormal situation inside the cell, it’s usually a rare commodity. When replication forks can’t be resolved, that’s when there’s problems, so the ssDNA really is the SOS signal.
Why is attenuation unique to prokaryotes?
Because it is dependent on transcription and translation happening in the same place at the same time.
What about the tryptophan attenuation gene makes it a barometer for cellular tryptophan levels?
It contains a leader gene with two trp amino acids in a row. If the ribosome has to stall and wait for a trp-charged tRNA, that’s an indicator that cellular tryptophan levels are low and the cell needs to make more.
How does a ribosome affect transcription of the tryptophan operon?
Attenuation.
The attenuation gene contains two trp codons in a row → if the ribosome stalls when translating this transcript, Rho will catch up to the ribosome and terminate transcription. That transcript’s protein won’t be made, and the absence of this protein signals that cellular trp levels are low and that the trp operon needs to be transcribed.
What is the most used GRS in E. coli?
Catabolite repression
