Module 3 Flashcards
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.
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.
How does a phosphate group move through a two component signal transduction system?
It is transferred down a relay system cascade.
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.
What kinds of bonds/what do phosphate groups typically bind to in bacterial cells? How is this different from eukaryotic cells?
Bacterial cells:
Histidine - phosphoimidazole
Aspartic acid - acylphosphate
Eukaryotic cells:
Serine
Threonine
Tyrosine
(all phosphodiester)
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.
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.
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.
What does the Omp stand for in OmpR?
Osmoregulated protein, NOT outer membrane protein.
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.
What is the signal in E. coli’s two component EnvZ+OmpR system?
A change in the concentration of solutes, aka osmolarity.
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.
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.
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.
How does OmpR bind to DNA?
It must first dimerize, then it binds to inverted repeats.
What kind of control does a histidine kinase’s sensor arm exert on the genome?
Both positive and negative control.
What part of E. coli’s two component system is the response regulator?
OmpR
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.
True or false: the colon is a very osmotically concentrated environment.
True
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.
What happens to OmpC expression when environmental osmolarity increases?
OmpC expression increases
What happens to OmpF expression when environmental osmolarity increases?
OmpF expression decreases
What happens to OmpC expression when environmental osmolarity decreases?
OmpC expression decreases
What happens to OmpF expression when environmental osmolarity decreases?
OmpF expression increases
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.
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.
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.
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.
Which porin protein is the default in E. coli?
OmpF
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
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
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.
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.
True or false: bacterial ribosomes can bind to dsDNA.
False!
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.
What is the definition of a plasmid?
Plasmids are genetic structures that replicate independently of the bacterial host chromosomes.
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.
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.
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.
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
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.
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.
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.
Which origin sequence is used for theta replication?
OriV
Which origin sequence is used for rolling circle replication?
OriT
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.
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.
How is pR100 spread?
Through sex pilus conjugation.
True or false: F-plasmids drive their own transfer.
True. They do so via sex pili and conjugation.
What mediates pilin polymerization and pilus formation?
The Type IV secretion system.
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.
What initiates pilus depolymerization/retraction?
Contact of the pilus’s tip with the proper outer membrane receptor protein.
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.
What does the TraI gene code for?
Relaxase
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.
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.
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.
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.
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.
What drives homologous recombination?
RecA
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.
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.
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.
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.
How can horizontal gene transfer occur?
Three methods: conjugation, transformation, and transduction.
What is the exact definition of transformation?
Uptake of free or naked DNA from the external environment.
