Final Review Flashcards
(196 cards)
1
Q
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?
A
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.
2
Q
Compare and contrast peptidoglycan structure and transpeptidation in gram negative and gram positive bacteria.
A
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
3
Q
How do gram negative bacteria acquire resistance to β-lactams? How is this different from gram positive bacteria?
A
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.
4
Q
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
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.
5
Q
How are flagella different in Gram Negative vs Gram Positive bacteria?
A
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.
6
Q
Why do mycobacteria fail to stain in Gram protocols? How do we stain them instead?
A
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.
7
Q
A physician is treating a patient with tuberculosis, a gram positive bacteria. He plans to use Vancomycin. Will his patient be cured?
A
NO.
8
Q
How is peptidoglycan synthesized?
A
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.
9
Q
Why is peptidoglycan synthesized with five amino acids, even though the final form has only four? What purpose does the fifth amino acid serve?
A
The fifth amino acid is cleaved during transpeptidation to provide the activation energy to create the crosslinking peptide bond between two peptidoglycan molecules.
10
Q
How is the newly synthesized peptidoglycan monomer transported to the periplasm or extracellular environment?
A
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.
11
Q
What makes peptidoglycan such a unique molecule?
A
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
12
Q
What does the lactyl group attach the peptide portion to?
A
NAM
13
Q
What is the most basic definition of chemotaxis?
A
Going towards or away from chemicals
14
Q
What happens to the MCP chemotaxis complex when the chemoattractant is present?
A
When the chemoattractant is present, MCP inactivates CheA, allowing the basal body to keep rotating.
15
Q
What happens to the MCP chemotaxis complex when the chemoattractant is absent?
A
When chemoattractant is absent, MCP activates CheA, which starts a phosphorylation cascade and makes the basal body stop rotating, inducing a tumble.
16
Q
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?
A
CheZ removes CheY’s phosphate, which makes it unable to interact with the basal body.
17
Q
What is a random walk and when do bacteria take them?
A
Random tumbling through the environment, happens in the absence of a chemical gradient.
18
Q
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?
A
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.
19
Q
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?
A
The chemorepellent will always trump the chemoattractant. The bacteria will always tumble away.
20
Q
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.
A
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.
21
Q
How do bacteria preserve their ability to tumble even when attractants are predominantly bound to MCP?
A
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.
22
Q
How do bacteria in a gradient of some chemoattractant recognize higher levels of that chemoattractant?
A
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.
23
Q
How do bacteria reset accommodation?
A
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.
24
Q
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?
A
Flagellar rotation switches to counter-clockwise (CCW) rotation.
25
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.
26
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.
27
What is the most common pathway bacteria use to catabolize glucose to pyruvate?
EMP
28
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.
29
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.
30
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.
31
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.
32
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.
33
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
34
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)
35
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).
36
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.
37
If an E. coli bacterium is depleted of ATP, where will it send its G6P?
To EMP and ED to generate more ATP.
38
What does fermentation do?
Regenerates NAD
39
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.
40
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.
41
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.
42
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.
43
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.
44
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.
45
Which uses electrons more efficiently: respiration or fermentation?
Respiration
46
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.
47
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.
48
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.
49
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.
50
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.
51
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.
52
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
53
Which energy production method, fermentation or aerobic respiration, is better? Why?
Respiration because it uses pyruvate and electrons more efficiently.
54
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.
55
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.
56
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.
57
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.
58
Where do human colonocytes get the majority of their energy from?
From butyrate, a fermentation byproduct of the microbes nearby.
59
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.
60
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.
61
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.
62
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.
63
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.
64
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.
65
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
66
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.
67
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.
68
What’s the difference between chemotaxis and response regulators?
Chemotaxis → changes protein function
Response regulators (aka quorum sensing) → changes gene expression
69
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.
70
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.
71
What happens when LexA levels drop?
More and more genes in the SOS response will be transcribed.
72
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.
73
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.
74
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.
75
Why is attenuation unique to prokaryotes?
Because it is dependent on transcription and translation happening in the same place at the same time.
76
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.
77
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.
78
What is the most used global regulatory system in E. coli?
Catabolite repression
79
List some different types of GRS in E. coli.
SOS response, catabolite repression, oxidative stress, aerobic respiration, anaerobic respiration, etc.
80
Why do E. coli use glucose to the exclusion of every other carbon source?
Because it is more efficient. It fits directly into the glycolytic pathway. All other sugars need to be modified before entering glycolysis, making it less efficient.
81
Why is catabolite repression sometimes called “the glucose effect”?
Because glucose mediates the catabolite’s repression. It ensures that nothing is wasted making things to break down alternative carbon sources when glucose is present, it ensures that the cell uses its resources efficiently.
82
What happens to cAMP levels when glucose is high?
They are low.
83
What happens to cAMP levels when glucose is low?
They are high.
84
How is catabolite repression mediated?
Adenyl cyclase turns ATP into cAMP
85
How does cAMP control the lac operon?
cAMP binds to CAP, allowing it to bind to the promoter, inducing transcription.
86
What is the function of Catabolite Activator Protein (CAP)?
CAP ensures that genes for the catabolism of less favorable energy sources remain untranscribed until more favorable energy sources are depleted.
87
How does the Phosphotransferase System (PTS), aka Group Translocation, dictate sugar use?
With glucose-specific EIIA phosphorylation. When glucose is present, EIIA’s PO4 is transferred to make G6P. When glucose is absent, EIIA’s pool becomes predominantly phosphorylated, and then induces adenyl cyclase to stop inducer exclusion of other sugar options.
88
What happens to EIIA when glucose is present?
Its pool shifts to being predominantly nonphosphorylated.
89
What happens to EIIA when glucose is absent?
Its pool shifts to being predominantly phosphorylated.
90
What happens to adenyl cyclase when glucose is present?
It is inactive.
91
What is the difference between the CheA histidine kinase and signal transduction histidine kinases?
CheA never interacts with the ligand, instead the signal is mediated by MCPs. This allows CheA to respond to more than one ligand/signal molecule at a time.
92
What is the function of signal transduction?
It allows the bug to sense a change in environment and change its gene expression, thus changing its behavior, in response.
93
How does a phosphate group move through a two component signal transduction system?
It is transferred down a relay system cascade.
94
How are signal transduction pathways different between eukaryotes and prokaryotes?
Eukaryotic cells don’t have two component systems. Two component systems are unique to prokaryotes, yeasts, and a handful of plants.
95
What kinds of bonds/what do phosphate groups typically bind to in bacterial cells? How is this different from eukaryotic cells?
Bacterial cells, when phosphorylating amino acids, typically phosphorylate histidine or aspartic acid, using phosphoimidazole acylphosphate groups to do so respectively. Eukaryotic cells typically phosphorylate serine, threonine, or tyrosine, using phosphodiester bonds to do so.
96
What is the significance of the phosphoimidazole bond used to bind a phosphate group to histidine?
Phosphoimidazole bonds have enough energy in them to transfer that phosphate group to aspartic acid without using ATP.
97
What is the significance of the acylphosphate bond used to bind a phosphate group to histidine?
Acylphosphate bonds are unstable, high energy bonds. They are rapidly hydrolyzed in aqueous environments, no phosphatase needed. This gives the phosphate group on aspartic acid a relatively short half-life, which allows all two component systems to respond quickly when the environmental signal goes away.
98
What is the function of the EnvZ +OmpR system in E. coli?
It allows E. coli to respond to and acclimate to changes in its environment’s osmolarity.
99
What does the Omp stand for in OmpR?
Osmoregulated protein, NOT outer membrane protein.
100
How is E. coli’s two component system different from a generic two component system?
E. coli’s kinase domains can’t phosphorylate their own histidines. Instead, its kinase domains must transphosphorylate.
101
What is the signal in E. coli’s two component EnvZ+OmpR system?
A change in the concentration of solutes, aka osmolarity.
102
What happens to a bacterium’s generic two component system when the environmental signal goes away?
The bacterium needs to remove the phosphate groups off its aspartic acids, because otherwise the bug would remain adapted to the old conditions.
103
What happens to EnvZ when the transduction signal goes away?
It becomes a phosphatase specific for aspartic acids. It chews the phosphate groups off the aspartic acids, allowing the system to reset quite quickly.
104
How is EnvZ different from generic histidine kinases used in signal transduction?
Most generic histidine kinases used in signal transduction don’t have EnvZ’s phosphatase ability. Instead, the system waits for the phosphate group to fall off/disassociate on its own. While this does still easily and quickly happen in aqueous environments (the bond between an aspartic acid and a phosphate group is an acylphosphate bond, which easily hydrolyzes in aqueous environments), it’s still not quite as fast as EnvZ’s phosphatase activity.
105
How does OmpR bind to DNA?
It must first dimerize, then it binds to inverted repeats.
106
What kind of control does a histidine kinase’s sensor arm exert on the genome?
Both positive and negative control.
107
What part of E. coli’s two component system is the response regulator?
OmpR
108
What happens to EnvZ’s sensor arm in high solute concentrations?
The periplasmic sensor arm changes conformation. That change transduces the information across the periplasmic membrane to EnvZ’s kinase domain, which increases its rate of phosphorylation.
109
What is the EnvZ/OmpR Two component system? How would the loss of function of the high affinity OmpR-PO4 binding sites associated with the ompF gene affect the bacterium’s ability to adapt to a more dilute, watery type environment? What about a higher osmolarity, colonic-type environment?
If the high affinity OmpR-PO4 binding sites lost their function, then the genes for OmpF wouldn’t be transcribed, and the cell wouldn’t be able to express an OmpF protein. In high osmotic conditions, like the colon, the cell would be fine, as the alternate OmpC porin protein is better suited to that environment, however if the cell were in low osmotic conditions the smaller pore of OmpC would make it harder to take in nutrients.
110
True or false: the colon is a very osmotically concentrated environment.
True
110
How do E. coli’s expressions of OmpC and OmpF relate to each other (mathematically)?
Their expressions are inversely proportional. In a perfect world, they’d be mutually exclusive, but life isn’t perfect so we’re stuck with inverse proportions.
110
What happens to OmpF expression when environmental osmolarity increases?
OmpF expression decreases
110
What happens to OmpC expression when environmental osmolarity increases?
OmpC expression increases
111
What happens to OmpC expression when environmental osmolarity decreases?
OmpF expression decreases
111
What happens to OmpF expression when environmental osmolarity decreases?
OmpF expression increases
112
What happens to OmpC and OmpF expressions in the colonic environment?
Because the colon is an osmotically concentrated environment, OmpC expression is high and OmpF expression is low.
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What happens to OmpC and OmpF expressions in the post-colon environment?
Because the environment is no longer osmotically concentrated, OmpC expression is low and OmpF expression is high.
114
Why does E. coli switch to using OmpC in more concentrated environments?
It uses OmpC in more concentrated environments because it has a smaller, low iron flux, pore. The smaller pore helps keep the bad stuff out while still getting enough nutrients.
114
Why does E. coli switch to using OmpF in less concentrated environments?
It uses OmpF in less concentrated environments because it has a larger, high iron flux, pore. The size difference in the pore changes the flow rate of ions and other water soluble molecules across the porin. This larger pore allows for a larger and faster flux rate, thereby increasing the bacterium’s chance of taking in nutrients.
115
Which porin protein is the default in E. coli?
OmpF
116
Which of the following DNA binding sites have the highest affinity for OmpR-PO4: F1, F2, F3, F4, C1, C2, or C3?
F1, F2, F3, and C1
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Which of the following DNA binding sites have the lowest affinity for OmpR-PO4: F1, F2, F3, F4, C1, C2, or C3?
F4, C2, and C3
118
How does OmpR-PO4 halt the transcription of OmpF?
At high enough concentrations, OmpR-PO4 will bind to the F4 low affinity site upstream of the OmpF gene. This induces bending and causes the DNA to fold over on itself, blocking the transcription machinery from transcribing OmpF.
119
What is the function of MicF antisense RNA transcripts?
MicF is a non-protein coding gene. It is transcribed when OmpR-PO4 binds to the OmpC activator site. Once transcribed, its transcripts bind to premade OmpF mRNA transcripts and stop their translation.
120
True or false: bacterial ribosomes can bind to dsDNA.
False!
121
How does MicF stop translation of premade OmpF mRNA transcripts?
It is the antisense, complementary strand to OmpF mRNA transcripts, so when it binds it creates a dsDNA RNA molecule. Bacterial ribosomes can’t translate dsDNA, halting translation. Additionally, because dsDNA is broken down quickly in E. coli cells, it also signals for the transcript’s degradation.
122
What makes OmpC and OmpF more suited for specific environments?
OmpF has 2 DNA binding sites for OmpR-P. There is a high affinity site, which turns the gene ON, and a low affinity site, which turns the gene OFF. OmpC has 1 DNA binding site, which is low affinity and turns the gene ON; this site also functions to activate transcription of MicF, a non-protein coding antisense RNA gene that binds to OmpF mRNA transcripts and stops them from being translated and signals for their degradation.
123
What do you think would be the effect of having a high-affinity transcriptional activation site binding OmpR-P present for both the ompF and ompC genes, if a gram-negative bacterium were to be in a low-solute environment? High-solute environment? Assume all the other usual components of the EnvZ/OmpR system are present and working fine, and that the expression of micF is separate from ompC, but still controlled by a low-affinity activation site.
If the genes for both porin proteins had high affinity ON sites, both proteins would be expressed at high solute concentrations and neither would be expressed at low concentrations. The cell would most likely die in either condition.
123
What is the definition of a plasmid?
Plasmids are genetic structures that replicate independently of the bacterial host chromosomes.
124
In one sentence, describe what a plasmid is, and name the two main ways they can replicate.
A plasmid is a parasitic genetic structure that can replicate independently of its bacterial host genome, using either theta replication or rolling circle replication.
125
Explain what is meant when we describe plasmids as being high copy number. For most plasmids in nature, are they usually at high copy numbers?
Being high copy number means that there’s a relatively high number of copies of the plasmid within a single cell. Most plasmids aren’t high copy number, because it takes a lot of energetics and resources from the host cell to maintain all those copies, which would put the host, and subsequently the plasmid, at a slight evolutionary disadvantage.
126
What is the advantage of being a low copy number plasmid?
Being a low copy number plasmid ensures that the host uses only a minor amount of its resources maintaining the plasmid’s replicates, which ensures the host isn’t completely energetically disadvantaged compared to bacteria that don’t have the plasmid.
127
What is the disadvantage of being a low copy number plasmid?
The downside of being a low copy number plasmid is that the risk of stochastic loss during host cell division greatly increases.
128
Plasmids are selfish, parasitic DNA that will do anything to survive and regenerate. Considering this, how does a plasmid ensure that both of a bacterium’s daughter cells end up with at least one copy of the plasmid during division?
Active partitioning.
Plasmids containing both the ParC and ParR genes will transcribe ParR to create many ParR proteins, which attach to the ParC gene and then to each other, acting as monomer building blocks creating a long filament. If the filament contacts a filament from another copy of the plasmid, they will push the two plasmids to opposite ends of the cell, then depolymerize so that the plasmids stay there. This whole process is linked to the host cells’ creation of the Ftz ring, so this only happens during cell division.
129
How do plasmids ensure that no daughter cells escape without at least one copy of the plasmid?
Active partitioning.
If the plasmid has both the ParC and ParR genes, it can transcribe ParR to create ParR proteins. Those proteins, with the addition of ATP, will bind to the ParC sequence to form long filaments. When the filaments of two plasmids contact each other, they push each other away, pushing one copy towards the north end of the host and the other copy towards the south end. The plasmids then dissociate from the filaments, ensuring that the host will divide with at least one plasmid in each of the two daughter cells.
129
Which of the following strategies for maintaining low copy number plasmids is an active strategy: active partitioning, or post-segregational killing/addiction modules?
Active partitioning.
130
Which of the following strategies for maintaining low copy number plasmids is a passive strategy: active partitioning, or post-segregational killing/addiction modules?
Post-segregational killing/addiction modules.
131
What mediates post segregational killing of bacterial cells? How does it do this?
Post-segregational killing is mediated by addiction modules.
The plasmid encodes both a toxin and the antidote to the toxin. The toxin has a much longer half-life than the antidote, and is therefore present in the cell for longer periods of time. If the cell were to lose the plasmid, it would lose the ability to make the antidote, and as the antidote would be degraded faster than the toxin, the cell would die because it lost the plasmid.
132
Explain how the CcdB CcdA toxin-antitoxin system works in post-segregational killing, and the specific reason why bacteria lacking a plasmid are killed. Why must there be so much more CcdA than CcdB being produced in order for the bacterial cell with a plasmid to continue to survive?
CcdB is the toxin, CcdA is the antidote. Both are encoded for on the plasmid, and when the plasmid is present both are made and present in the cell. However, CcdA is quickly degraded by Lon protease, and as such has a very short half-life. CcdB, however, has a very long half-life. If the cell were to lose the plasmid, it’s CcdA levels would decrease and the antidote would disappear before its CcdB levels would. Without the antidote, the toxin would kill the cell.
133
What are the two points/sites at which the F plasmid can begin to replicate itself?
OriV and OriT
134
What kind of replication begins at the OriV site? Under what conditions does this occur?
OriV is the start site for theta (vegetative) replication. This site is used under normal conditions, it is the default mode of replication.
135
What kind of replication begins at the OriT site? Under what conditions does this occur?
OriT is the start site for rolling circle replication. This site is used only when the plasmid needs to undergo conjugation.
136
Suppose a plasmid can do both theta replication and rolling circle replication. Under normal conditions (i.e. either no other bacteria are present or the other bacteria present all contain copies of the plasmid), which replication strategy will the plasmid use?
Theta replication.
137
Under what conditions will a plasmid that can replicate via both theta replication and rolling circle replication choose to undergo rolling circle replication?
When the plasmid senses that bacteria without the plasmid are present, and subsequently needs to undergo conjugation.
138
Which origin sequence is used for theta replication?
OriV
139
Which origin sequence is used for rolling circle replication?
OriT
140
After an F plasmid becomes part of the bacterial chromosome via recombination, what is the cell referred to as?
The Hfr cell
141
What type of replication is used by the F plasmid during conjugation with an F- cell? What type of replication does it use during vegetative bacterial growth in the presence of only F+ cells?
RCR in the presence of F- cells, theta in the presence of F+ cells
142
In a mating between an F- (no F plasmid) E. coli with any of the following E. coli cells, which donor would potentially transfer the greatest amount of bacterial DNA to the transconjugant?
F' E. coli cell
143
What’s the difference between a F’ cell and an Hfr cell?
The difference between an F’ cell and an Hfr cell is the location of the plasmid DNA. An F’ cell arises from the Hfr cell, where the plasmid has arisen from the Hfr cell but has made an error and taken a piece of adjacent bacterial DNA with it.
144
True or false: DNA is transferred through the sex pilus.
False. The pilus pulls the victim closer and opens a pore, allowing the DNA to be transferred inside. The pilus is not a tube that the DNA moves through.
145
How is pR100 spread?
Through sex pilus conjugation
146
True or false: F-plasmids drive their own transfer.
True. They do so via sex pili and conjugation.
147
What mediates pilin polymerization and pilus formation?
The Type IV secretion system.
148
How does the F-plasmid ensure that it never engages with a cell that already contains a copy of its plasmid?
One of the genes on the F plasmid codes for a protein that represses the expression of the host cell’s outer membrane receptor protein that would normally interact with the incoming sex pilus. The pilus can’t bind to the cells that already contain the plasmid.
149
What initiates pilus depolymerization/retraction?
Contact of the pilus’s tip with the proper outer membrane receptor protein.
150
What’s the difference between an F positive cell and an Hfr cell?
An F positive cell has a copy of the plasmid inside it, free floating in the cytoplasm. An Hfr (high frequency recombination) cell has integrated the plasmid into the bacterial host chromosome.
151
What is the main replication mechanism used in conjugal transfer?
Rolling circle replication.
152
What does the TraI gene code for?
Relaxase
153
The role of the protein TraI (aka "relaxase") in F plasmid transfer between bacterial cells is:
It nicks F plasmid at oriT
154
True or false: relaxase creates a double stranded break in the DNA.
False. Relaxase creates a knick only one strand of the DNA, it breaks a phosphodiester bond.
155
How is the F-plasmid conjugated into a new victim cell?
One phosphodiester bond is broken at the OriT site on the plasmid, causing a knick and allowing DNAP to initiate rolling circle replication. Relaxase, now covalently attached to one strand of the plasmid, is pumped across the Type IV secretion system, taking that plasmid strand with it.
156
In addition to being part of the F-plasmid’s conjugation system, where else are Type IV secretion systems found?
Type IV secretion systems have been co-opted by pathogenic bacteria, like H. pylori, to pump toxins into host/victim cells.
157
What is an episome?
A genetic sequence that can move in and out of the bacterial chromosome. Episomes can replicate independently, i.e. outside of the chromosome, but they can also replicate as part of the host chromosome.
158
How do insertion sequences, also known as mobile DNA cassettes, insert themselves into their host's chromosome?
They undergo homologous recombination by having shared homologous sequences with the bacterial chromosome.
158
What drives homologous recombination?
RecA
158
If a plasmid in an Hfr cell is resurrected and reenters the cytoplasm, but leaves a part of its plasmid genome behind on the bacterial chromosome, will the plasmid still be able to function in that cell? What about in a newly infected cell?
The plasmid would undergo its normal functions in the original Hfr cell, as it still has all of the plasmid’s genetic material, they’re just in two different locations. However, the plasmid would be nonfunctional in any newly conjugated/infected cells, as the new cells would receive a copy of the plasmid that’s missing a part of its genome.
158
Describe the process of horizontal gene transfer as it relates to Hfr cells.
Hfr cells initiate conjugation the same way that F positive cells do, however their OriT sequences are now in a larger circle (the bacterial chromosome). When relaxase creates the knick at the OriT site and pumps the plasmid out through the Type IV secretion system, a chunk of the bacterial chromosome will be taken with it. Theoretically, it’s possible for the entire bacterial chromosome to be transferred, but ssDNA is pretty fragile and almost always breaks somewhere during the conjugation process.
159
What is the exact definition of transformation?
Uptake of free or naked DNA from the external environment.
159
How are plasmids transferred between bacteria in nature? How is this different from lab environments?
In nature, plasmids are almost always transferred by conjugation. Most bacteria are not naturally competent, meaning that they don’t naturally uptake free DNA from the environment. In the lab, we are able to force DNA across the membranes of the naturally incompetent bacteria by subjecting them to some stressful treatment.
159
What is the definition of transduction?
The incorporation of foreign bacterial DNA into the genome of another bacterium, mediated by a virus.
159
What’s worse than accumulating all seven genes for high level vancomycin resistance in a transposon?
Placing that transposon on a conjugative plasmid.
160
Aside from location in the cell, how is the cointegrate different from its original plasmid form?
The cointegrate contains two copies of the transposon, whereas the original plasmid contains only one.
161
What gives pioneer species such as lichens and legumes their ability to grow in nitrogen poor environments?
The rhizobacteria that they’re in a mutualistic relationship with fix nitrogen into ammonia for the plants, allowing them to grow and thrive in nitrogen poor environments.
162
True or false: all legumes require mutualistic relationships with rhizobial partners to fix nitrogen.
False. Some legumes can fix nitrogen on their own, without the help of mutualistic friends.
163
True or false: all bacteria require a mutualistic relationship with some plant to fix nitrogen.
False. Some cyanobacteria in marine ecosystems can fix nitrogen while free-floating, no symbiotic relationship required.
164
How is nitrogen fixation important to farmers?
The bacterially generated ammonia allows farmers to save money on fertilizers. Crop rotation allows soil nutrients to be replenished throughout many growing seasons.
165
True or false: nitrogen fixation can occur in the presence of high concentrations of oxygen.
False. Nitrogen fixation can only occur in anaerobic environments.
166
How do marine cyanobacteria undergo nitrogen fixation, a process that cannot happen in the presence of oxygen, when they themselves require oxygen to live?
Marine cyanobacteria exist in colonial filaments, and they specialize some of their cells in the filaments into heterocysts, specialized oxygen deprived anaerobic environments for nitrogen fixation.
167
What role do flavonoids play in the dialogue between the plant and its symbiotic rhizobia?
Flavonoids are molecules secreted by the plants' root hair cells. They act as both the chemotactic signal for the rhizobium and also as the allosteric modifier for NodD, allowing it to bind to a promoter sequence and induce increased transcription of both Nod genes and Rhicadhesin protein.
168
What role do Nod factors play in the dialogue between the plant and its symbiotic rhizobia?
The Nod factors are secreted by the rhizobia, and once they enter the plants’ root hair cell, they induce a change in the cell’s gene expression, causing a change in the cell’s morphology.
In addition to creating the nodule itself, stimulation by the proper Nod factor causes an “infection thread” to form, a hollow tube through which the rhizobium can migrate to the root tissue.
169
What is the function of Rhicadhesin protein in nodule formation?
Rhicadhesin protein, when secreted by the rhizobium, allows it to physically stick to the root hair cell.
170
What benefit does the plant gain from its symbiotic relationship with its rhizobium?
It gains the ability to create a nodule, something that it cannot do without the Nod factors changing its gene expression. Only in this nodule can nitrogen be fixed into ammonia, which goes on to feed the plant.
171
Why are rhizobia living free in the soil incapable of nitrogen fixation? Why do they need their symbiotic plant partner?
Because the rhizobia can’t generate the appropriate anaerobic environment for nitrogen fixation on their own. Rhizobia are obligate aerobes; they must live in oxygenated environments because they use it as their terminal electron acceptor in their electron transport chains.
172
Individual legume species form symbiotic relationships with only one species of rhizobia each. How is this specificity maintained?
Some flavonoids of some legumes will bind to the NodDs of other rhizobia species, ones that it’s not symbiotically paired with. They do so because it inhibits NodD’s transcriptional activator abilities, effectively inhibiting a competitive species.
173
True or false: the rhizobia, upon entry via the infection thread, enters inside the plant root cells to float free in the cytoplasm.
False. The rhizobia aren’t free in the cytoplasm, they’re bound by a peribacteroid membrane.
174
True or false: the rhizobia, once bound by the peribacteroid membrane, are now free to fix nitrogen.
False. The rhizobia must first divide and undergo a developmental phase to turn into symbiosomes. Symbiosomes are specialized rhizobial cells that spend most of their energy on nitrogen fixation, and as such don’t undergo replication and division.
175
Where in the legume root nodule system is nitrogenase expressed?
Only in the symbiosomes
176
Because the symbiosomes are bound by a peribacteroid membrane, they can’t forage for food free in the environment. How do they get their nutrients instead?
They are fed organic acids by their plant host. These organics are intermediates from the plant’s citric acid cycle.
177
Why do symbiosomes use their TCA pathways far more than they use their glycolytic pathways?
Because their source of nutrients is intermediates from their host plants’ citric acid cycle. These organic acids fit immediately into their own TCA cycle. This is also beneficial because the TCA cycle generates hella NADH (aka hella reducing power), which can then be fed into the ETC to generate PMF and is necessary for nitrogen fixation.
178
Compare and contrast the plasmids used in the crown gall tumor system and the legume root nodule system.
Sym plasmid
Doesn't enter the plant cell
Much larger in size
Ti plasmid
Enters the plant cell
Much smaller in size
Both contain all the genes necessary for their tumor/nodule/infections formation processes
179
What are Nod factors made of and what part of their biology allows them to pass through both the bacterial and plant cell membranes?
Nod factors are polymers of 3-5 NAG subunits, covalently modified by various substituents. One of those substituents is a long hydrocarbon chain. This is what allows them to pass through the necessary membranes.
180
How do rhizobia maintain the anaerobic conditions necessary for nitrogenase to function even though they need oxygen as a necessary terminal electron acceptor in its ETC?
Leghemoglobin
Leghemoglobin delivers O2 directly to the endpoint of the ETC, prevents it from roaming free in the cytoplasm and keeps cellular O2 levels low.
181
Describe the symbiosis between the legume and the rhizobium as it relates to leghemoglobin.
Leghemoglobin, which delivers O2 directly to the endpoint of the rhizhobia’s ETC, is necessary for the rhizobia to survive under the anaerobic conditions required for nitrogen fixation. Leghemoglobin’s protein domain is coded for and synthesized by the host legume, but the heme prosthetic group is coded for and synthesized by its symbiotic rhizobia.
182
What kind of secretion system do rhizobia use to mediate rhizobium-legume symbiosis?
Type III (remember that it’s related to flagella)
183
How do rhizobia mediate rhizobium-legume symbiosis?
They use a Type III secretion system to secrete effector proteins into the host legume, downregulating the host’s defense system.
