Module 3 Flashcards
(166 cards)
1
Q
What is the difference between the CheA histidine kinase and signal transduction histidine kinases?
A
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.
2
Q
What is the function of signal transduction?
A
It allows the bug to sense a change in environment and change its gene expression, thus changing its behavior, in response.
3
Q
How does a phosphate group move through a two component signal transduction system?
A
It is transferred down a relay system cascade.
4
Q
How are signal transduction pathways different between eukaryotes and prokaryotes?
A
Eukaryotic cells don’t have two component systems. Two component systems are unique to prokaryotes, yeasts, and a handful of plants.
5
Q
What kinds of bonds/what do phosphate groups typically bind to in bacterial cells? How is this different from eukaryotic cells?
A
Bacterial cells:
Histidine - phosphoimidazole
Aspartic acid - acylphosphate
Eukaryotic cells:
Serine
Threonine
Tyrosine
(all phosphodiester)
6
Q
What is the significance of the phosphoimidazole bond used to bind a phosphate group to histidine?
A
Phosphoimidazole bonds have enough energy in them to transfer that phosphate group to aspartic acid without using ATP.
7
Q
What is the significance of the acylphosphate bond used to bind a phosphate group to histidine?
A
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.
8
Q
What is the function of the EnvZ +OmpR system in E. coli?
A
It allows E. coli to respond to and acclimate to changes in its environment’s osmolarity.
9
Q
What does the Omp stand for in OmpR?
A
Osmoregulated protein, NOT outer membrane protein.
10
Q
How is E. coli’s two component system different from a generic two component system?
A
E. coli’s kinase domains can’t phosphorylate their own histidines. Instead, its kinase domains must transphosphorylate.
11
Q
What is the signal in E. coli’s two component EnvZ+OmpR system?
A
A change in the concentration of solutes, aka osmolarity.
12
Q
What happens to a bacterium’s generic two component system when the environmental signal goes away?
A
The bacterium needs to remove the phosphate groups off its aspartic acids, because otherwise the bug would remain adapted to the old conditions.
13
Q
What happens to EnvZ when the transduction signal goes away?
A
It becomes a phosphatase specific for aspartic acids. It chews the phosphate groups off the aspartic acids, allowing the system to reset quite quickly.
14
Q
How is EnvZ different from generic histidine kinases used in signal transduction?
A
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.
15
Q
How does OmpR bind to DNA?
A
It must first dimerize, then it binds to inverted repeats.
16
Q
What kind of control does a histidine kinase’s sensor arm exert on the genome?
A
Both positive and negative control.
17
Q
What part of E. coli’s two component system is the response regulator?
A
OmpR
18
Q
What happens to EnvZ’s sensor arm in high solute concentrations?
A
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.
19
Q
True or false: the colon is a very osmotically concentrated environment.
A
True
20
Q
How do E. coli’s expressions of OmpC and OmpF relate to each other (mathematically)?
A
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.
21
Q
What happens to OmpC expression when environmental osmolarity increases?
A
OmpC expression increases
22
Q
What happens to OmpF expression when environmental osmolarity increases?
A
OmpF expression decreases
23
Q
What happens to OmpC expression when environmental osmolarity decreases?
A
OmpC expression decreases
24
Q
What happens to OmpF expression when environmental osmolarity decreases?
A
OmpF expression increases
25
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.
26
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.
27
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.
28
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.
29
Which porin protein is the default in E. coli?
OmpF
30
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
31
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
32
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.
33
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.
34
True or false: bacterial ribosomes can bind to dsDNA.
False!
35
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.
36
What is the definition of a plasmid?
Plasmids are genetic structures that replicate independently of the bacterial host chromosomes.
37
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.
38
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.
39
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.
40
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
41
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.
42
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.
43
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.
44
Which origin sequence is used for theta replication?
OriV
45
Which origin sequence is used for rolling circle replication?
OriT
46
How does the proteic addiction system seen on F-plasmids work?
Two adjacent proteins are encoded on the plasmid: CcdB and CcdA. CcdB codes for the toxin: a stable protein with a long half-life that inhibits DNA gyrase. CcdA codes for the antitoxin: an unstable protein with a short half-life. The antitoxin is quickly degraded by Lon protease, so the cell needs the plasmid to keep transcribing the antidote.
47
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.
48
How is pR100 spread?
Through sex pilus conjugation.
49
True or false: F-plasmids drive their own transfer.
True. They do so via sex pili and conjugation.
50
What mediates pilin polymerization and pilus formation?
The Type IV secretion system.
51
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.
52
What initiates pilus depolymerization/retraction?
Contact of the pilus’s tip with the proper outer membrane receptor protein.
53
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.
54
What does the TraI gene code for?
Relaxase
55
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.
56
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.
57
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.
58
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.
59
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.
60
What drives homologous recombination?
RecA
61
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.
62
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.
63
What is an example of merodiploidy?
Merodiploidy is when two alleles of the same gene exist in an otherwise haploid genome/background. An example of this is a bacterium having two alleles of the same gene, with one on their chromosome and the other on a plasmid somewhere in its cytoplasm.
64
What is the exact definition of horizontal gene transfer?
The ability to transfer DNA from one bacterium to another such that the recipient/transconjugant instantly changes its phenotype.
65
How can horizontal gene transfer occur?
Three methods: conjugation, transformation, and transduction.
66
What is the exact definition of transformation?
Uptake of free or naked DNA from the external environment.
67
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.
68
How does H. influenzae ensure that it only uptakes DNA from its own species?
H. influenzae only uptakes foreign DNA with a specific 11bp uptake sequence.
69
Under what conditions will S. pneumoniae uptake foreign DNA?
S. pneumoniae will only uptake foreign DNA if it senses a quorum of other S. pneumoniae bacteria.
70
How do the transduction regulation strategies compare between H. influenzae and S. pneumoniae?
H. influenzae regulates its transduction by only uptaking foreign DNA that contains a specific 11bp sequence, whereas S. pneumoniae only uptakes foreign DNA when it senses a quorum of other S. pneumoniae bacteria. Both strategies ensure that they only take in DNA from their own species.
71
Do naturally component bacteria uptake dsDNA or ssDNA?
They will uptake dsDNA, but they will depolymerize one strand upon entry so that only ssDNA is free floating in the cytoplasm.
72
What is the definition of transduction?
The incorporation of foreign bacterial DNA into the genome of another bacterium, mediated by a virus.
73
What life cycles to bacteriophages usually have? How are they different from each other?
Bacteriophages usually undergo one of two life cycles: lytic, or lysogenic.
Lytic: virus undergoes reproduction and lyses the host cell immediately
Lysogenic: if conditions are poor, the virus will insert their DNA into the host chromosome to wait until conditions are better, then it will resurrect its genome and undergo reproduction
74
How do bacteriophages package DNA?
Bacteriophages usually package DNA in one of two ways: headfull, or pack site.
Headfull: DNA is packed into capsids based only on the size of the genetic material
Pack site: DNA is packed into capsids only if a specific signal sequence is recognized in the genetic material before packaging.
75
What kind of replication do viral genomes undergo during infectious reproduction?
Viral genomes, after circularizing upon entering the host cytoplasm, undergo rolling circle replication to reproduce.
76
How does a viral infection facilitate the spread/horizontal gene transfer between bacterial populations?
Either generalized transduction or specialized transduction.
Generalized Transduction
- In the course of a bacterial infection the host’s genome is broken down into randomly sized pieces.
- If one of those pieces of the genome is the same size as the viral genome, and if the bacteriophage packages its DNA into capsids using the headfull strategy, that piece of the bacterial genome could be packaged into a capsid and sent out to infect another bacterium.
- If the new host cell’s genome has some homology with the bacterial genes in the defunct capsid, RecA can mediate a crossover event and incorporate those new genes into its genome
Specialized Transduction
- Very rarely a lysogenic virus will make a mistake during resurrection and pull out bacterial genes adjacent to the plasmid from the bacterial chromosome
- If that happens, a part of the viral genome is left behind.
- I the nascent cell where this occurs, the virus still has all its genes within the cell, they’re just in two different locations
In newly infected cells, the virus will be defective, as it has left a part of its genome behind in the previous host
- If conditions are also poor in the new host, the virus will again incorporate its genetic material into the host’s genome, taking the old host’s genes with it.
77
How is generalized transduction different from specialized transduction?
Generalized transduction can take any piece of bacterial DNA and incorporate it into the viral capsid and subsequently the new bacterial host’s genome. Specialized transduction can transfer only bacterial DNA that was adjacent to the viral genome during its lysogenic cycle. Specialized transduction is only possible in lysogenic infections.
78
Compare and contrast homologous recombination with site specific recombination.
Homologous recombination
- Relies on RecA
- Relies on sequence homology/sequence similarity/identical sequences
- Used by bacteria
Site specific recombination
- Independent of sequence homology
- Used by viruses, lambda, and transposons
79
Compare and contrast intercellular mobile genetic elements with intracellular mobile genetic elements.
Intercellular:
- Direct their own movement between bacterial genomes
- Ex: bacteriophages, plasmids
Intracellular
- Direct their own movement within bacterial genomes
- Ex: insertion sequences, transposons
80
How are insertion sequences different from all other types of transposons?
Insertion sequences contain only one gene between their inverted repeats: transposase.
81
What is transposase?
The enzyme that drives the movement of a transposon from one locus to another.
82
How are composite transposons created?
Composite transposons are the result of Outside End Transposition, what happens when transposase (by random change) misses the transposon’s inner inverted repeat and binds only to the outer inverted repeat, moving the sequences making up the transposon around.
83
Why are there often mutations in the inner inverted repeats of composite transposons?
Because composite transposons are the result of Outside End Transposition. For OET to happen, the transposase needs to miss/not bind to the inner inverted repeat; this is more likely to happen if the inner inverted repeat is mutated to the point where transposase can no longer recognize it.
84
Compare the two different methods of transposition.
Conservative (Cut & Paste):
- Copy # stays the same
- Transposon is cut out of the DNA and is inserted into a new location
- Used by insertion sequences
- Less successful, therefore less common
Replicative (Copy & Paste):
- Copy # increases
- Transposon is replicated
- One copy remains in the original location, the other is inserted into a new location
- More successful, therefore more common
85
What’s worse than accumulating all seven genes for high level vancomycin resistance in a transposon?
Placing that transposon on a conjugative plasmid.
86
Why is transposition of insertion sequences relatively rare?
If insertion sequences had a high rate of transposition, they would almost certainly eventually land in a necessary gene, disrupting it, and leading to the cell’s death.
87
Why is replicative transposition more common than conservative transposition?
Because replicative transposition is more successful.
88
What is a cointegrate? How are they created?
A plasmid within the chromosome. Cointegrates are created when a plasmid with a transposon on it is integrated into the host’s genome during reproduction, and as a result have two copies of the transposon on them.
89
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.
90
What is the function of resolvase in replicative transposition?
Resolvase recognizes the Internal Resolution Sites (IRS) on either side of the transposon. It makes a cut in one strand of each of these sites, and resurrects the original, unchanged plasmid, leaving one copy of the transposon behind.
91
What happens to the transposon’s copy number during replicative transposition?
It increases by one.
92
Why is transposition/integration/site specific recombination important?
Because there’s continuity amongst genetic parasites. There’s interdigitation, similarity, between a lot of these things. For example, HIV1 drugs were tested on Tn5 transposase because of how similar it is, both in structure and in function, to HIV1-integrase. Several successful and widely used HIV1 drugs were developed this way.
93
What are integrons?
Mobile DNA elements that can acquire novel genes.
94
Integrons don’t normally mobilize themselves. Why then are they classified as mobile DNA elements?
Because they can mobilize other DNA and insert it into themselves.
95
True or false: integrase gene expression is random.
False. Integrase’s promoter (Pint) is part of the SOS response. LexA remains bound to Pint, repressing integrase’s transcription, until the SOS response is activated. Only under stressful conditions will integrase be expressed, therefore making its expression non-random.
96
Which gene on an integron is more likely to be expressed: one that is in the primary platform, or one that is in the low cost memory cassette reservoir?
One that is in the primary position. Expression levels in integrons are based on how close a particular gene is to the promoter. Genes in the primary/stable platform are closest to the promoter, so they are expressed at higher levels than genes in the low cost memory cassette reservoir, which is farthest away from the promoter.
97
Why are archaea prokaryotes?
Because they’re not eukaryotes.
98
Where are methanogens found?
Methanogens, bugs that synthesize methane, are found in soil sediments, digestive tracts, and sewage treatment plants.
99
Briefly describe the process of methanogenesis.
- Methanogenesis is the process by which methanogens use simple substrates to produce methane under oxygen-free conditions.
- This is a type of anaerobic respiration.
The substrates used in it are byproducts of bacterial fermentation taken from the environment.
- It’s a highly complex biochemical process and involves over six unusual enzymes and around 200 gene products to do so.
100
In the process of methanogenesis, where do the carbons from carbon dioxide end up?
Into the methane, NOT biomass!
101
Which process is more complex, methanogenesis or bacterial electron transport chains?
1000% Methanogenesis.
102
In anaerobic food chains, who catalyzes the terminal reactions?
Methanogens!
103
Are methanogens aerobes or anaerobes?
Anaerobes, specifically obligate anaerobes.
104
Why do methanogens need to live in syntrophy with other microorganisms?
Because they are obligate anaerobes. They need their syntrophic partners not only to substrate fodder but also to get rid of any oxygen in their environment. Oxygen isn’t just useless to methanogens, it’s toxic to them.
105
What benefit do methanogens’s syntrophic partners gain from this syntrophic relationship?
The methanogens use and subsequently get rid of the H2 in the environment, which at 1 atm inhibits fermentation reactions. They also get rid of other bacterial waste products to use as substrates for methanogenesis, which could otherwise build up to toxicly high concentrations.
106
Describe the syntrophic relationships that exist in a peat bog.
Organic matter in the peat bog is broken down, and any methane produced is dissolved into the water. In a well-balanced bog, methanotrophic bacteria live in symbiosis with sphagnum moss. Most of the CO2 the methanotrophs release is trapped by the moss as photosynthate.
107
Describe the syntrophic relationships that exist in cold seeps.
Cold seeps are a chemosynthetic ecosystem. They are dependent on methanotrophic bacteria to contribute to the nutrition of filter feeders (mussels, clams, tube worms, etc). These methanotrophs live in the gills of the filter feeders, and harvest the methane from the water as it passes through. The methane is then used to produce biomass both for the methanotroph and for their syntrophic friends.
108
What’s the difference between a methanogen and a methanotroph?
Methanogens create methane, methanotrophs eat methane.
109
Where are halophilic archaea found?
In environments with high salt concentrations. Ex: Great Salt Lake, Dead Sea, Soda lakes, man made salt evaporation ponds.
110
Why do solar salt pools change colors?
They change color when a bloom of halophilic archaea grows in it, which only happens once the salt concentrations rise to a certain level.
111
An organism requires a minimum 4% NaCl to function. Is the organism a halophile? Why or why not?
This organism is not a halophile because the defined minimum NaCl concentration required to be classified as a halophile is 9%, which is much higher than this organism’s 4%.
112
Why are some halophiles mixotrophs? What about being a halophile lends to being a mixotroph?
Because halophiles live at high salt concentrations, they often encounter oxygen deprived environments. Naturally, they need to adapt quickly to these conditions to survive.
113
What nutritional type are halophilic archaea when oxygen levels are high?
Chemoorganotrophic heterotrophs (oxidizes organic molecules, feeds its reducing power into the ETC for PMF generation)
114
What nutritional type are halophilic archaea when oxygen levels are low?
Photoorganotrophic heterotrophs (undergoes photosynthesis instead of oxidizing organic molecules)
115
How do these halophilic mixotrophs switch between nutritional states?
When oxygen levels dip low enough, bacteriorhodopsin is stimulated by this stress. The pigment in the bacteriorhodopsin now becomes a photo-driven proton pump and generates PMF for ATP synthesis and other work. Light in the 570 nm range converts the non-protein, protonated retinal pigment of the bacteriorhodopsin from the trans form to the cis form, translocating a proton to the outer surface of the membrane in which the bacteriorhodopsin lives in the process. Thus a PMF is established.
116
Why are halophilic microbes easier to culture in a lab environment than non-halophilic microbes?
Because they require so much salt, they have to be cultured in very salty mediums. Any contaminant bacteria have a hard time surviving in such a salty environment, they usually just dehydrate and dry out. It’s very easy to maintain a pure culture of halophiles because of this.
117
What are some examples of biotechnological applications of haloarchaea and bacteriorhodopsin?
- Because bacteriorhodopsin is so resilient and can withstand and maintain its function in all kinds of extreme environments (high temps, high and low pH, nonpolar organic solvents, etc) it’s being looked at for use in holographic media, artificial retinas, and photovoltaic cells. It’s a material of interest for anything related to optical-electrics.
- Haloarchaea have been engineered to express the typhoid vaccine antigen. The antigen retains its potency even when the haloarchaea are stabilized into salt crystals. Normally typhus vaccines need to be stored cold, making them difficult to ship and deliver. This shelf-stable form of the vaccine makes it much easier to ship and deliver the vaccine to wherever needed.
118
In what other ways is bacteriorhodopsin used in these archaeal cells?
Halorhodopsin
- Uses light and the retinal isomerization with a different protein to actively transport chlorine ions in from the environment to the cytoplasm
Phototaxis
- Halophiles ability to move towards and away from light
- Very similar to chemotaxis, but CheA isn’t conjoined with MCPs, they’re conjoined with Sensory Rhodopsin proteins
- Sensory Rhodopsin I senses red light, allows motor rotation to move the bug forwards
- Sensory Rhodopsin II senses blue light, increases phosphorylation, reverses the motor, induces a tumble and a change in direction
119
Why would a haloarchaea want to move towards red light?
Because they are more likely to find photosynthetic organisms, and thus higher concentrations of oxygen, in the presence of red light.
120
Why would a haloarchaea want to move away from blue light?
Because the shorter wavelengths of blue light are more likely to damage the bug, it's more advantageous to avoid those environments.
121
One of the challenges to living in a hypersaline environment is water’s natural tendency to move out of the cell to the highly concentrated extracellular environment. This puts a tremendous osmotic pressure on the cell. How do halophilic archaea that live in such environments solve this problem?
They match their interior solute concentration to the exterior solute concentration. They do this by using halorhodopsin to actively transport chlorine ions in from the environment to their cytoplasms. They also pump in potassium ions inside, but those aren’t pumped in by halorhodopsin.
122
One of the challenges to living in a hypersaline environment is water’s natural tendency to move out of the cell to the highly concentrated extracellular environment. This puts a tremendous osmotic pressure on the cell. How do halophilic bacteria that live in such environments solve this problem?
They match their interior solute concentration to the exterior solute concentration. They do this by synthesizing or accumulating from the environment “compatible solutes”, organic compounds that help achieve a water balance without interfering with normal cellular enzyme function
123
Compare the compatible solutes used by halophilic archaea and halophilic bacteria.
Halophilic archaea use inorganic solutes, halophilic bacteria use organic solutes.
124
Why are compatible solutes called compatible?
They’re called compatible because they don’t interfere with the cell’s biochemical reactions and functions.
125
What’s the difference between a chemically defined media and a complex media?
A chemically defined media is one in which we know the exact molarity of each chemical within the media. Total pain in the ass to make, so we use complex media more often instead. Complex media contain undefined components. We know what’s in it, but we don’t know the exact molarity of each chemical in the media.
126
What causes Crown Gall tumors?
Caused by Agrobacterium tumefaciens inserting the TDNA portion of a parasitic Ti plasmid into wounds in the stem and roots of dicotyledonous plants.
127
How does the plasmid transfer its TDNA into the plant cell?
It undergoes rolling circle replication to generate a single stranded copy of the TDNA, then is bound by proteins and pumped out by a Type IV secretion system (all of which is also encoded on the Ti plasmid). The plasmid co-opts its conjugation abilities to transfer the TDNA into a eukaryotic cell.
128
What genes are contained on the TDNA region of the Ti plasmid? What genes aren’t contained on the TDNA region of the Ti plasmid? What is the significance of this?
TDNA region contains genes for auxin, cytokinin, and opine. It does not contain genes for the virulence region, the ori site, or opine catabolism.
Auxin and cytokinin are formerly plant genes that encode proteins that drive the plant cell’s replication, and the opine genes code for a series of fake amino acids (they’re really similar to amino acids, but they’re not and can’t be used by the plant) that are transported out to feed the original bacterium.
The genes for opine catabolism isn’t encoded on the transfer region because it ensures that the plant can’t use the fake amino acids, and the ori site and virulence region aren’t included because that way only the bacterium can transfer the plasmid, not the plant.
129
What advantage do the plasmid and the host bacterium gain from creating a crown gall tumor?
They gain a nutritional source from the fake amino acids produced and secreted by the plant, encoded on the opine genes on the TDNA.
130
What does the virulence region of the Ti plasmid code for?
A two component system (VirA, VirG) and a Type IV secretion system.
VirA → histidine kinase, senses the secondary molecules secreted by the wounded plant
VirG → response regulator, initiates expression of the genes necessary to move the bug closer to the source of the secondary molecules (the wound)
VirB-F → Type IV secretion system
131
What gene encoded on the Ti plasmid is homologous to TraI?
VirD2
132
Why is Agrobacteria tumefaciens and its Ti plasmid important?
Because they’re the basis for all transgenic plants. We can modify the Ti plasmids and put whatever genes we want in the transfer region. It’s the platform for plant transgenesis.
133
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.
134
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.
135
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.
136
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.
137
True or false: nitrogen fixation can occur in the presence of high concentrations of oxygen.
False. Nitrogen fixation can only occur in anaerobic environments.
138
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.
139
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.
140
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.
141
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.
142
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.
143
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.
144
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.
145
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.
146
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.
147
Where in the legume root nodule system is nitrogenase expressed?
Only in the symbiosomes
148
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.
149
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.
150
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
Larger in size
Ti plasmid:
Enters the plant cell
Smaller in size
Both contain all the genes necessary for their tumor/nodule/infectious formation process
151
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.
152
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
It delivers O2 directly to the endpoint of the ETC, prevents it from roaming free in the cytoplasm and keeps cellular O2 levels low.
153
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.
154
On the nitrogenase enzyme, where specifically does N2 reduction occur?
In the Fe-Molybdenum subunit
155
What kind of secretion system do rhizobia use to mediate rhizobium-legume symbiosis?
Type III (related to flagella)
156
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.
157
Which reactions in the nitrogen cycle are anaerobic?
Denitrification and nitrogen fixation
158
Which reactions in the nitrogen cycle are aerobic?
Oxidation and nitrosofication
159
Describe the steps to biofilm formation/development.
1. A pioneer bacterium finds a suitable surface (one with appropriate carbon and energy sources)
2. Those pioneer bacteria undergo a minor developmental program and lose their ability to produce flagella, becoming non-motile
3. Attachment begins. The bacteria begin to produce capsules, and as population density increases those capsules enclose the entire population
4. Water channels form through the capsule architecture
5. Seeding begins. The biofilm releases planktonic bacteria to hopefully colonize new areas.
160
True or false: biofilms that are made up of only a single bacteria species are uniform.
False. The bacteria in a biofilm exist in various physiological states, regardless of whether they’re all the same species or not.
161
How is antibiotic resistance different from antibiotic recalcitrance?
Resistance refers to either aquired genetic changes or natural resistance (usually due to anatomical features).
Recalcitrance refers to physiological changes only, no genetic changes involved.
162
Why are biofilm infections often recalcitrant to antibiotics?
Drugs might not permeate the extracellular polymeric matrix (though this isn’t very likely as most antibiotics will just diffuse in)
The interiors to biofilms often have low O2 concentrations (some antibiotics don’t work very well without O2)
Some of the bacteria in the biofilm become persister cells, and because they’re not growing aren’t very susceptible to most antibiotics. They can wait out the treatment course and recolonize once the antibiotic is gone
163
What is a persister cell?
A bacterium, usually found in biofilms, that has undergone a minor developmental program and has become physiologically inert. Similar to sporulation.
164
Why are bacteria in biofilms better at modulating their gene expression than free floating bacteria?
Because their quorum sensing molecules are much more concentrated. The quorum sensing molecules don’t diffuse away as easily in a biofilm.
165
Why is transformation easier to undergo in a biofilm vs in free floating bacteria?
Because the free/environmental DNA isn’t washed away/doesn’t diffuse away as easily in a biofilm, and because the DNA is held in closer proximity to the bacteria.
166
What is the benefit to the bacteria to form a biofilm?
It allows them to adhere to nutrient sources and control nutrient diffusion during degradation.
