Integrative Microbiology - Final Flashcards
(79 cards)
1
Q
What is the most conserved gene, and why? Say in bacteria and funghi.
A
[Eukaryote] 16S rRNA. Since ribosomal RNA (rRNA) is universally distributed, functionally constant, and extremely conserved.
[Prokaryote] ITS in funghi (Internal transcribed spacer)
2
Q
What is the role of two-components regulatory systems?
A
Cell mechanisms that allow for the detection of envioronmental changes through the use of sensor proteins that induce conformational changes.
3
Q
What are two-component regulatory systems composed of?
A
- Histidine Kinase - transmembrane protein that undergoes autophosphorylation in a histidine residue after the binding of a certain ligand or a specific envioronmental factor. This binding causes a conformational change in the HK that exposes the histidine residue to be phosphorylated.
- Response regulator (cytoplasmic) - phosphorylated by the histidine kinase on a conserved aspartate residues. Changes its conformation allowing to acts on DNA or proteins.
- Phosphatase - terminates the signal, desphorylating the RR. This can be the RR, the HK or another one.
4
Q
Describe Osmoregulation.
A
Envz protein (HK) when facing high osmotic pressure recruits OmpR (RR) that activates transcription of OmpC (short diameter) and represses OmpF (long diameter).
5
Q
Describe the pho regulon.
A
Pst system imports phophate and PhoU detects if this is happening. If not working PhoU activates PhoR (HK) and this phosphorylates PhoB (RR) which activates the transcription of the Pho regulon, that has genes responsible for using organic sources of phosphate.
6
Q
Describe the Arc system.
A
If quinones are present (oxydized form), means there’s a final aceptor in the electron transport chain. However, if there are only quinols (reduced form) the ArcB (HK) is activated and activates ArcA (RR) which triggers the Arc Modulon (genes associated with anaerobiosis), different promoters regulated by the same regulator; different operons under the same stimulus.
7
Q
What does the FNR protein do?
A
The formate nitrate regulation protein, in the absence of oxygen becomes activated and induces many anaerobically expressed genes and represses some aerobically expressed ones.
8
Q
Is the FNR protein a two-component regulatory system? What does it has to do with this?
A
It’s not an example of a two-system component. It will have an impact in the regulation of the Arc system and it is a switch between aerobic/anaerobic metabolism in the cell.
9
Q
What happens in the NarQ/NarP and NarX/NarL TCS?
A
These TCS are involved in anaerobic/aerobic transition. In low nitrate concentration NarQ and NarX will activate NarP and NarL and these will induce the expression of fumarate reductase and nitrate reductase allowing the acceptance of electrons by these compounds.
10
Q
How does OxyR work?
A
Regulates Oxidative Stress
OxyR detects hydrogen peroxide and activates genes for antioxidant enzymes, such as catalase and glutathione reductase, to protect against damage..
11
Q
How does SoxR work?
A
Regulates Oxidative Stress
SoxR senses ** superoxide radicals ** and activates SoxS, which induces genes involved in detoxification and repair.
12
Q
What are sigma factors in prokaryotic cells?
A
Regulatory proteins that bind to core RNA polymerase and provides with the ability to recognise specific promoter sequences on the DNA.
13
Q
How do sigma factors work, related with transcription?
A
Sigma factors recognize specific consensus sequences in promoter regions (-10 and -35 boxes).
- When the RNA polymerase holoenzyme (core enzyme + sigma factor) is fully assembled, it scans the DNA.
- Once the sigma factor binds the promoter, it helps unwind the DNA and initiates transcription.
- After initiation, the sigma factor is released, allowing RNA polymerase to proceed with elongation.
This ensures gene expression is regulated based on environmental cues.
14
Q
What types of sigma factors exist?
A
- sigma-70 (major one) - which drives the transcription of housekeeping genes
- sigma-N - responsible for the transcription of genes involved in nitrogen limitation
- sigma-S - associated with the stationary phase
- sigma-E - helps the cell respond to stress sensed at the periplasm level
15
Q
What controls alternative sigma factors availability?
A
- Anti-Sigma Factors – Proteins that bind sigma factors and prevent their interaction with RNA polymerase until specific signals release them (e.g., RsbW inhibits σ^B in Bacillus).
-
Proteolysis – Some sigma factors are degraded by proteases when they are no longer needed, regulating their levels dynamically.
Environmental Signals – Certain sigma factors are activated in response to specific stress conditions (e.g., σ^E in response to envelope stress, σ^H during heat shock).
16
Q
How does the sigma-E work?
A
Sigma-E is an alternative sigma factor that helps the cell respond to stress sensed at the periplasm level.
- Protease DegS is activated and cleaves the periplasmic part of ResA (site-1 cleavage);
- Cytoplasmic domainis cleaved by protease RseP (site-2 cleavage);
- ClpXP degrades the ResA cytoplasmic domain, releasing sigma-E.
17
Q
How could we identify which are the newly synthesized proteins when a heat shock occurs?
A
Since all proteins have a methionine after being translated, we give M marked with radioactive sulfur; then, doing a 2D-Page (pH and molecular weight), we could use mass spectrometry to identify the proteins that weren’t present in the normal conditions.
18
Q
Explain the sigma-32 heat shock response.
A
Normally the sigma-70 transcribes rpoH gene that encodes sigma-32, but the transcript is degradaded at normal temperatures by RNases since it is unstable. At higher temperature sigma-32 can be formed, but it forms a complex with several chaperones. When in heat shock the chaperones are required due to the increase in misfolded proteins which bind to the chaperones, this frees sigma-32 that’s able to transcribe HSP genes. This HSP response can be triggered by other factors, not just temperature.
19
Q
Does a bacteria need a lot of autoinducer concentration to alter gene expression?
A
No, the detection of a minimal threshold stimulatory concentration of an autoinducer is sufficient.
20
Q
What are the general gram-negative bacteria autoinducers? And how are they secreted?
A
Acylated homoserine lactones (AHL/HSL). The membrane is permeable to them, no need for transporters.
21
Q
What are the general gram-positive bacteria autoinducers? And how are they secreted?
A
They employ secreted peptides as autoinducers. They are secreted via ABC transporter.
22
Q
How are autoinducers detected in gram-positive bacteria?
A
They use two-component sensor kinases that detect for the secreted peptide signals, and this interaction with the peptide ligand initiates a series of phosphoryl events that culminate in the phosphorylation of a cognate response regulator protein, allowing it to bind DNA and alter the transcription of the quorum-sensing controlled target gene(s).
23
Q
How are autoinducers detected in gram-negative bacteria? (Lux Operon)
A
They don’t have a two-component regulatory system, but two proteins. One that synthesises HSL (autoinducer) - LuxI-like proteins. And another one that binds HSL after a certain concentration level - LuxR-like proteins. The formed complex activates target gene transcription. It has a positive feedback loop where LuxR also makes more production of LuxI.
24
Q
Give an example of gram-negative quorum sensing mechanism (just the name).
A
The Vibrio fischeri LuxI/LuxR Bioluminescence System.
25
Explain the Trojan Horse in bacteria.
Due to a low cell density bacteria don't have enough quorum sensing molecules to turn on the virulence, this helps to escape the imune system in the beggining of the infection. Then once it reaches the threshold the infection can start. Virulence is like biofilm formation, toxin production, etc...
26
What quorum sensing systems are part of the Pseudomonas aeruginosa Virulance System and what do each of them do?
They are involved in motility, biofilm formation and toxin systhesis. LasI/LasRRhlI/RhlRPQS system (Pseudomonas quinolone signal)The first two systems act in tandem and are homologs to the LuxI/LuxR circuit (and also has postive feedback) - HSL. The LasI/LasR complex, besides induce several virulence factors, induces the expression of RhlR. The LasR induces PQS and PQS induces RhlI. It's an aditional link between the first two systems. These three control several genes together.
27
Explain biofilm formation.
**1. Attatchement -** Few motile cells contact with the surface
**2. Colonization -** Irreversible attachement to the surface, begins to divide and form colonies
**3. Maturation -** 3D structure with channels for nutrient exchange
**4. Active Dispersal -** In response to environment changes bacteria spread
28
What's quorum quenching?
The inhibition of QS using chemical or enzymatic means to counteract behaviors regulated by QS.
29
Say three different options to perform quorum quenching in gram-negative bacteria.
1. **Blockage of AHL synthesis –** Prevents the production of acyl-homoserine lactones (AHL), the signalling molecules used in quorum sensing, stopping the communication process before it begins.
2. **Interference with Signal Receptor –** Uses molecules that bind more strongly to the receptor than AHLs, blocking the receptor and preventing the bacteria from responding to the signals.
3. **AHL Inactivation –** Employs enzymes like lactonases to break open the lactone ring in AHL molecules, rendering them inactive. This is often done by gram-positive bacteria to disrupt the communication of gram-negative bacteria.
30
What is the major class of sRNAs in bacteria and what types exist?
Antisense sRNAs, actby base-pairing with mRNA to inhibit translation of their targets. Cis encoded: located in the antisense strand of the gene they will act on (they are 100% complementar). Trans encoded: located elsewhere in the genome far from the gene they will act on.
31
What does the RNA chaperone Hfq do?
It helps sRNAs exert their regulatory action. Hfq monomers assemble to form hexamers and dodecamers, which stabilize sRNAs and modulate base-pairing with target mRNAs. Besides that, Hfq is also a key player in the modulation of mRNA stability, it can protect transcripts against RNase E cleavage sites on mRNA (AU-rich single-strand regions).
32
What methods of repression can sRNAs do in translation (to mRNAs)?
**1. Non-nucleolytic repression -** the sRNA and Hfq chaperone bind to the ribosome binding site, causing it to be unable to connect to the target mRNA, therefore avoiding protein synthesis.
**2. Passive nucleolytic repression -** mRNAs may become more sensitive to RNase E attacks after base-pairing with sRNA, the previous bind of the sRNA exposes these sites to enzymatic action since no ribosome is covering those places.
**3. Active nucleolytic repression -** recruitment of RNase E on the target mRNA triggers formation of a sRNA/Hfq/RNase E complex that favors RNase E attacks, this complex becomes a specialized RNA decay machine.
33
What is the method of mRNA activation of translation, regarding sRNAs?
Initially the RBS is hidden in a hairpin in the mRNA; with the binding of the sRNA-Hfq complex, this site is exposed and is now able to ligate with a ribosome subunit, activating the translation.
34
What's a riboswitch?
It's a RNA structure located in the 5'UTR of mRNAs that regulate gene expression at the level of transcription, translation or splicing. By binding to an intracellular metabolite they can activate or deactivate depending on the concentration of the metabolite.
35
How does the Spot42 work?
When glucose levels are high, the cell doesn’t need to use galactose as the primary carbon source, hence Spot42 inhibits the metabolism of this sugar by preventing the translation of Gal K, which is responsible for the phosphorylation of galactose after it enters the cell (synthetase). When it is low it is not produced (sRNA) so galactose is metabolized.
36
What are the 3 structural genes in the gal operon?
galE (epimerase), galT (galactose transferase) and galK (galactokinase).
37
When is the Spot42 sRNA expressed? And what does it target.
When glucose levels are high. The gal operon (Gal K gene which is responsible for the phosphorylation of galactose after it enters the cell (it allow it to enter basically)).
38
What also controls Spot 42?
The complex cAMP-CRP (cAMP receptor protein) inhibits production of Spot42 when glucose levels are low. (Since cAMP is high).
39
How does the SgrS sRNA work?
It targets carbohydrate metabolism. PTsG a transporter specific for the influx of glucose and phosphorulates it when entering the cell. SgrS is activated when glucose levels are high and prevents PTsG expression.
40
What does the sRNA RybB do?
Targets mRNAs that encode for outer membrane proteins, such as Omp.
41
What does the sRNA InvR do?
It's involved in porin regulation. TCS activates HilD that activates InvR, this is involved in translation repression of OmpD mRNAs through sRNA-Hfq binding.
42
What does the RyhB sRNA do?
It supresses translation of iron-using and iron storage proteins by binding to the mRNA (increasing the degradation rate). Being negatively regulated by the ferric uptake regulator (Fur). This way, in iron limitation conditions, the Fur repression of RhyB is alleviated and it suppresses its targets, leaving more iron available for central metabolism
43
How does the The Pseudomonas GaCs/GacA system work?
So the HK GaCs after a stimulus activates the RR GacA which will activate the sRNAs RsmZ and RsmY which will target the mRNA encoding for RsmA inhibitting its translation. This RsmA activates swarming motility and inhibitts biofilm formation. So its inactivation will establish biofilm formation.
44
Describe rhizobia bacteria.
Gram-negative bacteria form nodules mainly on roots
45
Define Quorum Sensing.
Quorum-sensing can be defined as a mechanism of gene expression regulation mediated by cell population density. Quorum-sensing bacteria produce and release chemical signal molecules called autoinducers that increase in concentration as a function of cell density. The detection of a minimal threshold stimulatory concentration of an autoinducer leads to an alteration in gene expression. Bacteria use quorum sensing communication circuits to regulate a diverse array of physiologic activities such as symbiosis, virulence, competence, conjugation, antibiotic production, motility, sporulation, and biofilm formation.
46
Describe the general characteristics of sRNAs.
Pos-transcriptional gene regulation by small regulatory RNAs (sRNAs), between 50-500 nucleotides long. Small non-coding RNAs are usually located in the intergenic regions (trans) or containing the 5’-UTR and the 3’-UTR (cis), which lack start and stop codons. Most sRNAs are transiently expressed (only expressed in stress conditions) and can act as repressors or activators of target genes by decreasing translation and/or increasing mRNA turnover.
47
Why are eukaryotes better for fine-tuning of gene expression?
Because they have one promoter per gene, so we don't have to transcribe all the genes, unlike prokaryotes which have one promoter per operon.
48
General order of binding of factors and proteins in the DNA in eukaryotes transcription.
First and the more upstream factors bind, the specific transcription factors (they are the ones that modulate the activity of the RNA polymerase).The mediator complex is recruited (it's sort of a support unit) and joins the TATA box the STF.The general transcription factors and the TATA binding protein are recruited and bind. GTF bind to a GC-rich and CCAT boxes.The RNA polymerase is recruited to the TATA box and DNA transcription starts.
49
Why do Specific transcription factors can have multiple random binding occurrences?
Since their DNA binding domain recognizes sequences with 5 to 8 bp (not so specific).
50
What's a good starting point to study TF-DNA interactions but doesn't gives us the full information?
Indirect methods: by mutating a TF and doing RNAseq and analyzing with motif discovery tools to find downregulated genes, since the TF in question can regulate another TF that regulates our gene in study (indirect regulation).
51
Explain the in vivo direct method for TF-DNA interactions.
Chromatin Immunoprecipitation (ChIP) If "it doesn't work" we can't say that it doesn't bind since it can need diferent conditions:
First a condition of study is selected (it's a full condition-dependent method).Use formaldehyde to perform crosslinking, this promotes interactions and fixes them.Lyse the cells and do sonication (around 200 bp fragments of DNA).Using beads covered with an antibody for the TF in study (it can be a tag inserted in the TF like a GFP) we immunoprecipitate the TF bound with the DNA.Reverse crosslinking using heat.If we know which promoter we are looking for we can do a PCR with the corresponding primers if not we can do ChIP-on-ChIP (microarray with all the promoter regions of the organism as probes)
52
Explain the EMSA.
The in vitro technique of electrophoresis-mobility Shift Assay is to see if the interaction is possible and can sometimes indicate if more than one protein molecule is involved in the binding complex and also the affinity of the interaction: Starts with the target DNA being amplified through PCR, the fragments are marked.One sample is just the fragments themselves running in the gel, the other has nuclear protein extracts (antibodies against the TF in study can also be used).The DNA isolate will migrate more, the shift will appear if the TF binds to the DNA, also if the antibody is present it will create a supershift.
53
Exlain the DNase footprinting assay.
This is an in vitro technique to see where the TF binds in the DNA since we already know that it binds. Amplify the target DNA and mark it with radioactive nucleotide in the 5' terminus.Join the purified TF.Join DNases that will cleave randomnly in every place but not if the TF is bound (several sizes of fragments will appear).Run one sample with TF and another without TF to compare.Since we are using a gel that distinguishes from 1 nucleotide we can see where there's a fragment missing using autoradiography (that's where the TF binds).We can have several binding places.
54
Explain transcription factories.
Transcription occurs at discrete sites in the nucleus, where multiple active RNA polymerases are concentrated and anchored to a nuclear substructure. Hence, instead of having the machinery in tracks, they are in specific positions in the nucleus, and DNA is drowned to thereby folding. The presence or absence of certain TFs can increase the potentiality of genes to get transcribed by modulating their access to the transcription factory.
55
How is transcription favoured related with histones?
By acetylating the lysine tails in the histones the positive charge is removed which decreases its affinity for the DNA promoting transcription.
56
In which regions is the nucleosome occupancy lower by default?
Intergenic and promoter regions.
57
Which types of mutations acelerate MDR mutations?
Mutations in DNA repair systems accelerate the formation of mutations that together confer multidrug resistance
58
Explain the P-gp transporter. Why is the P-glycoprotein a good MDR mechanism?
It's an ABC transporter with two cytoplasmic nucleotide binding domains (NBDs). Since it has two sites for drug binding and transport with different specificity, that interact in a positive cooperative manner. This may help P-gp expand the range of substrates it transports. High local substrate concentrations may allow for broad substrate recognition by one or more of P-gp’s relatively low affinity binding site(s) within the lipid bilayer. The more lipophilic a drug is, the easier the export mediated by P-gp is since it will be more in the membrane, independently of its affinity to the transporter.
59
Explain the hydrophobic vaccum cleaner model.
It's an ABC transporter. Acts as a phase separator: drugs detected in the lipid bilayer are excluded and injected into the aqueous environment surrounding the cell.
60
Explain the flipase model
It's an ABC transporter. Drugs are flipped from the inner leaflet to the outer leaflet by P-gp where they diffuse into the aqueous environment surrounding the cell.
61
Say a method to inhibit the P-gp.
Using a drug that is not a pump substrate or is a rapidly fungicidalUse the drug in combination with a pump inhibitor;Use a multi-functional inhibitor that affects target, efflux pump activity and efflux pump transcriptionUse the drug in combination with a proton pump inhibitor that reduces the concentration of ATP and dissipates de pH and electrochemical gradients;Uptake of drug increased in order to exceed the efflux
62
What do MFS use to transport?
The Major Facilitador Superfamily are secondary transporters (indirectly coupled with ATP) using the proton gradient to transport the several stuff.
63
Explain the polyene antifungal drug, and its possible resistance development.
It binds to ergosterol and forms membrane pores. It can be toxic since it also binds to cholesterol in mammalian cells. Some cells can develop resistance but they will be less viable, by controlling more the ergosterol synthesis, either through the TFs or the biosynthetic genes.
64
Explain Fluoropirimidines antifungal drugs and its possible resistance.
It's a pro-drug, being converted to a drug in the cell, where it's primary target is DNA and protein synthesis. It's not toxic the pro-drug, since it only enter pathogens cells. However if the cells die and release the drug it has a toxic effect, it can cross BBB. Since it uses some specific transporters, the cell only has to mutate those transporters. (FCY2), or the enzyme that converts it (FCY1).
65
Explain the echinocandins antifungal drug and its resistance.
They target the cell wall biosynthesis (Fsk1 the production of the glucan layer), not needing transporters, nor being toxic for us. The resistance can come from the overexpression of this enzyme or the mutation of it "hot-spot" regions.
66
Explain the azoles antifungal drug and its resistance.
They target ergosterol biosynthesis through Erg11. There can also be toxic sterol intermeadiates that disrupt the membrane. Imidazole - more toxic and effective Triazole - less toxic and effective The resistance can come from overepression, mutation of Erg11. Mutation of the transporter. Increase sphingolipids. Increase the lenght of phospho or it's saturation.
67
What converts more rigidity to the cell membrane?
The more saturated lipids exist and the longer the lipids. Since this increases the number of interactions. Unsaturated are trans bonds
68
Explain the general stress response.
It's first cascade response that is trigged, and only afterwards a specific response is generated. Aims to protect critical features of cell physiology, that confer short term protections until a specific mechanism is activated. It are involved in several biological functions. Usually upregulated genes include genes related with oxidative stress and chaperone production. And down regulated are like cell cycle, translation and transcription. The transcription of the genes activated during general response is mainly regulated by two specific transcription factors: Msn2 and Msn4.
69
What is lipid peroxidation and what its consequences?
It's a non-enzymatic reaction and results in hydrophobicity lost and ROS production which target DNA and proteins (thiolation - reversible - and carbonylation - irreversible).
70
Say two antioxidant enzymes.
Superoxide dismutase (SOD1 and 2 in cytosol and mithocondria) and catalase (CTT1 and CTA1 in cytosol and peroxisome).
71
How does the redox buffering system work?
Glutathione peroxidase removes the oxidized state in proteins and lipids, transferring these electrons to two GSH molecules and forming GSSG The reduction of the enzyme is mediated by GSH reductase, with the help of the NADPH cofactor (2GDH per 1 cofactor)The regeneration of the cofactor comes from a metabolic shift
72
How does the metabolic shift occur?
When NADPH is needed, the cell shifts its focus from ATP production to reducing power production, through metabolic pathways such as pentose phosphate cycle. (No ATP just 2 NADPH).
73
What other proteins also work as redox buffers?
Thioredoxin and glutaredoxin with the difference of not needing two molecules to form a thiol group, as each protein as two cysteines in their composition. Uses NADPH.
74
Say all the chaperone groups and their differences.
**Small HSPs: ** Prevent aggregation, protect cells under stress.
**Intermediate HSPs: ** Actively assist in protein folding and stabilization.
**Large HSPs: ** Disaggregate misfolded proteins and facilitate recovery or degradation.
75
How are proteins degraded in the cell?
80% by proteosomes (ubiquinated proteins) 20% lysosomes (endocytosis)
76
What happens when the cell faces high osmotic stress (in the case of glycerol)?
Either through Hog1 (TF) phosphorylation that activates genes related with glycerol biosynthesis. Or with FPS1 (aquaglyceroporin) phosphorylation that closes this transporters.
77
What makes these species pathogenic? Typical characteristics of pathogens (Candida albicans). Other examples of other organisms.
Persistence within the host.phenotypic switching (differences in cell wall, adaptation, etc...)morphological dimorphism both are needed for full virulence (hyphae and yeast)adherence to the host (biofilms) invasion of the host cells injury of the host cells Trojan horses, use macrophages Prevent maturation of the phagosome Titan cells
78
Distinguish between multidrug resistance and drug resistance.
Normal drug resistance is the acquisition of resistance to a specific or a family of xenobiotics, it can happen by a mutation or overexpression of the drug target in case it is a protein, usually occuring upon exposure to it. MDR is the acquisition of resistance to chemically different xenobiotics, happen by mutations in DNA repair mechanisms which will acelerate mutations that together acquire MDR. The mutation in multidrug transportes is also another example by increasing the efflux of these drugs.
79
Explain the formation of mature nitrogen-fixing bacteroids.
Bacteria secret NF signals that are specific to the plant they are infecting. These factor are recognized by the plant and in response release a specific flavonoid to the bacteria. These activate the nod genes in bacteria that stimulate more production of NF.This is a specific interaction since flavonois from non-host plants inhibit nod gene transcription.After the (correct) NOD factors are recognized by bacteria: Strong calcium oscilations and change in the cytoskeleton which makes curling of the root hairs, trapping bacteria. Also simultaneously, Nod factors stimulate root cortex cells to reinitiate mitosis and these cells will form the nodule primordium, and give rise to the cells that will receive the invading bacteria.Bacteria have to produce a simbiotically active exopolysaccharide Reorganization of cellular polarity causes the inversion of the tip growth and the formation of the infection thread and bacteria penetrates the tip of the plant cells. When the infection thread reaches the cell, it will result in endocytosis of the bacteria.To differentiate to bacteroids endoduplication they will increase in size without dividing. They now can start to produce nitrogen by the enzymatic complex nitrogenase and cannot reproduce.In the absence of O2 it is fixated by plants, and plants give it energy (malate) TCA componentes creating a symbiosis association.
