Midterm 2 Flashcards
True or false: the first tree of life, drawn by Darwin, was based on real data
False; it was simply a thought experiment
What was Haeckel’s tree of life based on? How were bacteria classified in this tree in general?
Based on the belief that evolution strives towards “higher” forms
- Protista (unicellular eukaryotes) are a Kingdom, but Moneres (bacteria) are only on the trunk
Based on later efforts to depict the universal tree of life, what was the order of kingdom evolution?
Monera -> protista -> plantae, fungi, animalia
Carl Woese impact on tree of life in general? (3)
- He decided that we should stop drawing the tree of life based on thought experiments or on “fuzzy” data like morphology. What was needed was a tree based on hard, numerical data.
- The best data would be DNA to quantitatively relate organisms
- He proposed that we use Sanger sequencing
What are four reasons why the 16S SSU rRNA gene is the ideal gene for making the tree of life?
- Universal to all organisms
- Does not undergo horizontal gene transfer between species
- Highly conserved (Functionally constant)
- Mutates at a constant rate (the number of mutations acts as a molecular clock)
Define phylogenetics and what it’s based on
Taxonomic classification based on evolutionary history (based on DNA changes, not metabolism or morphology)
What 5 things in particular did Carl Woese do?
- Made the tree of life quantitative
- Discovered Archaea and said that they’re “related to us, to the eukaryotes; they are descendants of the microorganisms that gave rise to the eukaryotic cell billions of years ago
- Proposed the 3-Domain system to replace the 5-Kingdom system
- Discovered that prokaryotes are not evolutionary artifacts of the earliest life forms. Evolution does not always progress towards more visibly “advanced” forms.
- Opens the door to studying diversity of uncultured bacteria
True or false: in the last 10 years, there has been an explosion of sequencing ability
True; we have much more efficient/cheaper sequencers today
True or false: Environmental rRNA genes lead to the discovery of new microbial species
True
True or false: There are only 4 major evolutionary groups (phyla)
False; there are at least 800 major evolutionary groups (phyla), and many of them are defined from environmental sequence alone (can’t be cultured)
More than 80% of characterized genera and species come from which 4 phyla? What is unique about these 4 phyla?
- Proteobacteria
- Actinobacteria (has G+ members)
- Fermicutes (Has G+ members)
- Bacteroidetes
These 4 phyla can be grown in the lab
What is the most abundant of cultured bacteria?
Proteobacteria
What is the most diverse group of cultured bacteria in nature?
Proteobacteria
True or false: proteobacteria can be divided into different classes, based on greek letters
True
What 4 species have ancestors that formed symbiotic relationships with eukaryotes to make modern-day organelles?
- Cyanobacteria
- Plastids
- Alphaproteobacteria
- Mitochondria
We use a _______ approach to describe a species
Polyphasic
A polyphasic approach to describe a species requires a description of which 4 things?
- Morphology: cell shape, visible structures, chemical composition (e.g. GC content), lipid composition, etc
- Metabolism: energy source, carbon source, aerobic/anaerobic, pH range, etc
- Genotype (most important key to naming a species)
- Evolution: Evolutionary tree
Describe the DNA-DNA hybridization (DDH) method for comparing the genomes of related species
Melt + combine DNA from 2 organisms. If there’s a high amount of hybridization between the genomes, then they’re probably the same species (and vice versa)
Describe the Average nucleotide identity (ANI) method for comparing the genomes of related species
Measure of nucleotide-level genomic similarity between the coding regions of two genomes
If the 16S rRNA gene sequence similarity is between 94-100, while the ANI is between 90-93…
Different species
If the 16S rRNA gene sequence similarity is between 94-97, while the ANI is between 90-96…
Different species
If the 16S rRNA gene sequence similarity is between 97-100, while the ANI is between 93-96…
Possibly same species
If the 16S rRNA gene sequence similarity is between 98.5-100, while the ANI is between 96-100…
Same species
If the 16S rRNA gene sequence similarity is between 94-96, while the ANI is between 96-100…
Not possible
16S rRNA similarity of at least 97% indicates…
Close evolutionary neighbours
16S rRNA similarity of less than 97% indicates…
Distant evolutionary neighbours
What 3 things does formal validation of a new prokaryotic species require?
- Detailed description of characteristics/traits (morpholigical, metabolic, etc)
- Deposition of viable cultures of the organism in at least two international culture collections (have to make DNA sequence publicly available)
- Proposal of a Latin name and publication in a very specific journal
What replaces kingdoms in the Linnean system of naming for prokaryotes?
Phyla
Species names are given in two parts. What are the generic name and species epithet?
Generic name: noun
Species epithet: adjective
True or false: the genome does not include plasmids
False; the genome is the entire complement of genetic information in an organism - chromosome + plasmids
- Includes genes, regulatory sequences, and noncoding DNA
Define genomics
The discipline of mapping, sequencing, analyzing and comparing genomes
What are the 4 basic -omic sciences?
Also explain what -ome means
-Ome= suffix implying global, collective, totality
1. DNA = genome
2. RNA = transcriptome
3. Proteins - proteome
4. Metabolism = metabolome
What does meta- mean and what’s an example of this?
meta- is a prefix implying beyond, more transcending - usually omics of a microbial community instead of a single strain
- e.g. metagenome = studies the genomes of at least 2 or more species
Define sequencing
Determining the precise order of nucleotides in a DNA or RNA molecule
Define genome assembly
Taking short fragments of sequenced DNA and puzzling them together into large contigs (ideally a complete chromosome)
Define genome annotation
Converting raw sequence data into a list of genes and other functional sequences present in the genome (assigning roles to sequences)
Define bioinformatics
Analyzing sequences and structures of nucleic acids and proteins computationally
Name the first widely used method of DNA sequencing and describe it (3 steps)
dideoxy method (Sanger sequencing)
1. Copy original ssDNA via PCR
2. Small amounts of ddNTPs are used with dNTPs. There is a different fluorescent dye for each ddNTP base
3. ddNTPs are chain terminators, preventing further elongation of DNA chain during PCR. They insert randomly, producing chains of varying length
Describe next-gen sequencing and the cost
Next-generation sequencing allows much greater throughput of sequencing at much lower cost than Sanger sequencing. The cost per base is about 100,000x lower.
- This has greatly accelerated genome sequencing
Closed vs draft genome
Closed: we know all of the nucleotides
Draft: 90% sequenced, but still hard to finish sequence
True or false: closing a genome often relies on extra work to fill in gaps, which is more expensive and requires more information
True
Define an open reading frame and how their functions are determined during gene annotation
Open reading frames are DNA regions between a start and a stop codon predicted to be “read” by ribosomes on mRNA. Identifying them requires searching 6 “reading frames” (bc DNA is double-stranded)
- The function of ORFs (and other genomic elements like tRNA and rRNA genes) is predicted by searching for similar sequences in DNA databases
What are the 7 steps of computer identification of possible ORFs?
- Computer finds possible start codons by scanning all 6 possible reading frames
- Computer finds possible stop codons
- Computer counts codons between start and stop
- Computer finds possible ribosome binding sites (RBS).
- If RBS is in front of start codon, this is a clue that the ORF is genuine - Computer calculates codon bias in ORF
- Computer decides if ORF is likely to be genuine
- List of probably ORFs is generated
Which of the following is not an important feature of a gene used for phylogenetics?
a) Universal to all organisms
b) Does not undergo horizontal gene transfer
c) Does not mutate
D) Highly conserved (functionally constant)
E) All of the above are important
C
- The gene mutates slowly
What is codon bias?
Different organisms have bias towards using certain codons for certain amino aids (recall that many codons code for a single amino acid)
If you’re comparing an unknown organism to a known organism, and you find that they have two different codon biases, what two things could this imply?
- If a gene in an organism has a codon usage pattern that does not match the typical codon bias of that species, it could indicate that the gene was acquired from another organism via horizontal gene transfer.
- Could be a clue that it’s not a true ORF (if an ORF has a highly unusual codon usage, it might be a false ORF)
True or false: the number of genes with roles that can be clearly identified (annotated genes) in each genome is greater than ~70% of total ORFs detected
False; the number of genes with roles that can be clearly identified (annotated genes) in each genome is ~70% or less of total ORFs detected
True or false: many genes in the annotation process are likely misidentified
True (the computer is often wrong)
What are hypothetical proteins, what % of the genome do they make up and what causes them?
Hypothetical proteins are uncharacterized ORFs (no match in the database) that likely exist but whose function is currently unknown, makes up the remaining 30% of the ORFs that aren’t annotated.
- Occurs due to lack sufficient amino acid sequence homology with known proteins for identification (don’t have enough homology to say that it’s the same as something else we know)
What might we do with hypothetical proteins during genome annotation? What organisms are hypothetical proteins found in?
We may assign it to a family or general function
Very common in genomes of uncultured environmental bacteria
Predicting the function of a gene is based on similarity to genes that encode characterized proteins. What could this lead to?
This can lead to errors via the annotation telephone
What does genome annotation result in, in terms of the unknown organism’s lifestyle?
Can lead to metabolic predictions
- Can piece together the lifestyle of the unknown organism
What does comparative genomics reveal in general? What is a specific discovery as a result of comparative genomics in bacteria?
Reveals details about the evolution of genes and bacteria
One discovery is that horizontal gene flow is very common in prokaryotes (before, it was thought to be rare)
What are 5 characteristics of genes originating from horizontal gene transfer?
- Presence of genes typically found only in distantly related species
- Presence of DNA with GC content or different codon bias that differs significantly from the remainder of the genome
- Presence of mobilome genes (transposons, insertion sequences, integrases, etc) that are likely pretty active in horizontal gene transfer
- Often encode resistance, virulence functions
- Often occur in clusters known as Genomic islands (different genes clustered in one spot on the bacterial chromosome)
What is vertical gene transfer?
Genome replication and cell division
What are the three mechanisms of horizontal gene transfer?
- Transformation
- Transduction
- Conjugation
What is transformation?
When bacteria takes up naked DNA from the environment
What is transduction?
Horizontal gene transfer facilitated by the use of bacteriophage
As the genome size increases, what happens to the total ORFs in the genome?
The total number of ORFs increases
How many genes are found in every megabase?
1000 genes
How many genes are found in every kilobase?
1 gene
How long is an ORF?
~1000 bp
Small organisms with small genomes usually have what type of lifestyle?
Endosymbiont (can’t survive independently of a host, need host enzymes for transcription and translation)
Intermediate-sized organisms with intermediate genome sizes usually have what type of lifestyle?
Parasite (have more genes and can live independently, but still need host for certain functions)
Large organisms with large genome sizes usually have what type of lifestyle?
Free-living
What 2 things are characterized by a complex metagenome?
- Lots of “repeats” in different genomes. Closely related strains are particularly problematic.
- Low coverage of rare genomes (many sequencing gaps)
What 5 things do cells need to grow?
- Water
- A carbon source
- Other building blocks (macro/micronutrients)
- An energy source (free energy)
- Reducing power (electrons
Heterotrophs vs. autotrophs
Heterotrophs obtain carbon from existing organic molecules, while autotrophs obtain carbon by generating their own organic molecules
Define catabolism
Energy-releasing metabolic reactions (breaking down complex molecules), which produces fundamental components (building blocks) for anabolism
Define anabolism
Energy-requiring metabolic reactions (building complex molecules), synthesizing new cell material from fundamental components
What is free energy, what are the units and how is it measured?
Energy available to do work (to change a system), measured in kJ/mol
- Measured as the change in free energy from before a reaction to after a reaction
ΔG°’ vs ΔG
ΔG°’= Standard free energy, which is the free energy for a reaction determined in the lab (25°C, 1 atm, 1M)
ΔG= Cellular free energy value
Exergonic processes have a (positive/negative) ΔG, and (require/release) free energy. These are associated with (anabolic/catabolic) reactions
Negative, release, catabolic
Endergonic processes have a (positive/negative) ΔG, and (require/release) free energy. These are associated with (anabolic/catabolic) reactions
Positive, require, anabolic
Define reducing power
The ability to donate electrons during a reaction
Redox reactions are comprised of how many half-reactions?
Two; electron donor and electron acceptor
Redox pairs are also written as what?
Redox couples and/or half-reactions
How are redox pairs written in the redox tower?
Oxidized/reduced forms
Define redox power and its symbol
Redox potential (E0’) is the tendency to donate electrons
The more negative the E0’…
The more likely it is to donate electrons to another redox pair
Formula for calculating the redox potential of a redox reaction?
E0’ acceptor - E0’ donor
Redox pairs written at the top of the redox tower are good electron ____, while those written at the bottom of the redox tower are good electron ____.
Donors, acceptors
When half-reactions are combined, what happens to the electron positions on the redox tower and what happens to the E0’ value?
The electrons will move down the tower to a less negative E0’
The greater the difference in redox potential, the greater the fall down the redox tower and…
The greater the energy yield (ΔG°’)
What are 2 examples of strong electron donors?
H2 and glucose
What are 2 examples of the strongest electron acceptors?
NO3-, O2
What do coenzyme electron carriers allow for? What is an example of one?
Allows us to take electrons from one spot to another
Ex: NAD+/NADH
What is a cofactor?
A metallic coenzyme
What are 3 energy conservation compounds (other than NAD+/NADH)?
- Phosphoenolpyruvate
- ATP
- Acetyl-CoA
What are 3 energy conservation mechanisms? Describe each
- Substrate-level phosphorylation (phosphate groups from organic molecules are transferred to ADP)
- Oxidative phosphorylation (electron flow generates proton motive force for chemiosmosis)
- Photophosphorylation (photons power the formation of proton motive force for chemiosmosis)
Organisms with a genome size of ~0-1 Mb typically have what lifestyle?
Endosymbiont
Organisms with a genome size of ~1-2 Mb typically have what lifestyle?
Parasitic
Organisms with a genome size of >2 Mb typically have what lifestyle?
Free-living
In respiration, an energy source (like glucose or H2, either organic or inorganic) is the (oxidant/reductant) in a redox reaction with a terminal electron acceptor (like O2 or NO3-) as the (oxidant/reductant)
Reductant (reducing agent), oxidant (oxidizing agent)
True or false: respiration involves the electron transport chain to generate proton motive force for chemiosmosis
True
The best terminal electron acceptors are those at the (top/bottom) of the redox table, with (positive/negative) E0’, and are usually (organic/inorganic) molecules
Bottom, positive, inorganic
True or false: The EMP pathway, also known as glycolysis, generates a little bit of reducing power
True (2 NADH)
What molecules produced by the EMP pathway (glycolysis) contain most of the free energy from the original glucose molecule?
The 2 pyruvate molecules
What 2 things can pyruvate undergo after glycolysis?
- Oxidation
- More electrons are stripped from pyruvate via the Krebs cycle (ultimately powering respiration) - Reduction
- Electrons are accepted from NADH via fermentation
NADH molecules produced from glycolysis can undergo which 3 things?
- Pass the electrons to an electron transport chain (respiration)
- Pass the electrons to pyruvate (fermentation)
- Use for cellular redox reactions
How many turns of the Krebs cycle completely oxidizes glucose into CO2?
2
True or false: anaerobic respiration does not involve electron acceptors and is only characterized by fermentation
False; when the electron acceptor of the ETC is inorganic, it is also considered to be anaerobic
What are 3 inorganic electron acceptors used by both chemoorganotrophs and chemolithotrophs
S^0, SO4^2-, NO3^-
What do organisms carry out if no electron acceptor is available? What happens to the electron donor in this process?
Fermentation, it gets reduced
Main difference between chemoorganotrophs and chemolithotrophs in terms of PMF generation?
Cheoorganotrophs use organic electron donors, while chemolithotrophs use inorganic electron donors (e.g. H2, H2S, Fe2+. NH4+, etc)
True or false: all bacteria can carry out both aerobic and anaerobic respiration
False
In fermentation, an energy source (like glucose) is the (oxidant/reductant) AND the terminal electron acceptor (like pyruvate) as the (oxidant/reductant)
Reductant, oxidant
Fermentation provides a mechanism to ____ NADH
Oxidize
- To maintain redox balance
True or false: In respiration, ATP is produced by both substrate-level phosphorylation and chemiosmosis, while in fermentation, ATP is produced mostly by substrate-level phosphorylation
True
What is a characteristic of fermentation products
These products are usually things that the organism can’t use
True or false: fermentation can be the primary source of ATP in some prokaryotes
- Explain the consequences
True; these organisms would grow extremely slowly
ATP yield from fermentation and aerobic respiration
Fermentation: 2 ATP
Aerobic respiration: 38 ATP (likely the highest number of ATP any organism could produce)
Describe homofermentative fermentation and which model organism carries it out
Glucose -> lactate + 2H+
Model organism: Lactobacillus spp.
Describe heterofermentative fermentation and which model organism carries it out
Glucose -> lactate + ethanol + CO2 + H+
(reduces 1 pyruvate to lactate and the other pyruvate to ethanol, so this organism can carry out both lactic acid fermentation and ethanol fermentation)
Model organism: Lactobacillus spp.
Describe mixed-acid fermentation and which model organism carries it out
Glucose -> lactate, formate, succinate, ethanol + CO2 + H2
Model organism: E.coli
What happens to the growth of E.coli carrying out mixed-acid fermentation
The pH drops so model organism E.coli stops growing
Describe butanediol fermentation and which model organism carries it out
2 pyruvate + NADH -> 2CO2 + butanediol
Model organism = E. aerogenes
What are the two types of fermentations that can be carried out by obligate anaerobes?
- Proteolytic fermentation
- Sacrolytic fermentation
Describe proteolytic fermentation and which model organism carries it out
Alanine + 2 glycine + 2H2O -> 3 acetate + CO2 + 3NH4+
- Oxidation steps use alanine, leucine, isolecune, valine or histidine
- Reduction steps use glycine, proline, tryptophan, or arginine
- Model organism = Clostridia
Describe sacrolytic fermentation and which model organism carries it out
1.5 glucose + H2O -> acetate + butyrate + 3CO2 +4H2 + H+
Model organism = Clostridia
During sacrolytic fermentation, the acetate and butyrate can result in a pH drop. What are the alternative products produced due to this pH drop?
The organism can switch to produce ethanol, acetone, isopropanol and butanol instead which are weaker acids
Describe secondary fermentation and which model organism carries it out
Fermentations products from one organism are the substrate for another
3 lactate -> 2 proprionate + acetate + CO2 + H2O
- Model organism = Propionibacterium
What type of fermentation gives emmental cheese its flavour
Secondary fermentation
What are the major inorganic electron acceptors in order?
- Nitrate (NO3-)
- Ferric iron (Fe3+)
- Sulfate (SO42-)
- CO2
(electron acceptors are switched in this order as each is used up, either by the same organisms or different ones)
How do the electron transport proteins differ in E.coli (cell membrane) compared to those found in eukaryotes (mitochondrial membrane)?
Only 3 complexes in cell membrane, cytochrome bo3 replaces complexes III and IV
In the absence of O2, what does E. coli use, and in place of what? What does this do?
In the absence of O2, E.coli uses nitrate reductase instead of cytochrome bo3 (complex III/IV)
- NO3- (nitrate) is reduced to NO2- (nitrite)
- Generates less PMF than using O2 (and generates less ATP because less H+ makes it across the cell membrane, since NO3- isn’t as good as O2)
Describe denitrification and which model organism carries it out
Model organism: P. stutzeri
In the absence of O2, Pseudomonads use nitrate reductase to reduce NO3- to NO2- and then an extended ETC to reduce NO2- to NO(g) to N2O(g) to N2(g)
- Gases float away
- Results in increased ATP production so P. stutzeri can outcompete E. coli
What is a benefit of denitrification?
An important part of sewage treatment (denitrifyers change our nitrogenous wastes into N2 gas)
What are 3 disadvantages of denitrification?
- Loss of fertilizer to atmosphere
- N2O is a greenhouse gas (N2O won’t always get reduced to N2)
- NO reacts with ozone and water to form nitric acid (acid rain) (NO won’t always get reduced to N2)
Describe iron-reducing bacteria and a model organism that carries this out
Geobacter uses acetate as an electron donor and ferric iron oxides as electron acceptors
- Electrons travel out through conductive pili (type IV pili) to reduce iron oxide minerals outside the cell
- Fe3+ (ferric iron) is reduced to Fe2+ (ferrous iron)
True or false: many species can also reduce other metals
True
- Bioremediation of metal contamination
- Uranium reduction
Which of the following is involved in anaerobic respiration but not fermentation?
a) Oxidation of an energy-source molecule
b) substrate-level phosphorylation
c) oxidation of NADH
d) reduction of an external electron acceptor
D
Which of the following statements is false?
a) Organotrophs conserve more energy than lithotrophs
b) Lithotrophic respiration can be aerobic or anaerobic
c) Both organotrophs and lithotrophs generate proton motive force
d) Phototrophs can use inorganic molecules as a source of electrons
A
- Energy conservation depends on the specific electron donors and acceptors involved (depends on redox potential differences)
- Lithotrophs can sometimes generate more energy, especially when using efficient inorganic electron donors like H2
D is incorrect because water (inorganic) is typically the electron donor in phototrophy
You study an organism that produces butanediol in the absence of oxygen. You are most likely studying:
a) A species of Clostridia
b) E. aerogenes
c) E. coli
d) A species of Lactobacilli
B
Which of the following is not a molecule you would expect to find when E. coli is growing in the absence of oxygen?
a) NO2-
b) Ethanol
c) N2O
d) CO2
C bc E. coli doesn’t carry out denitrification, only pseudomonads do
Describe sulfate reduction and what model organism it is seen in
- Also explain the difference between assimilative sulfate reduction and dissimilative sulfate reduction
Model organism: Desulfovibrio
Desulfovibrio uses organic compounds as electron donors and sulfates as electron acceptors
- SO42- (sulfate) is reduced to SO32- (sulfite_ and then to H2S (sulfide)
- If the reduced sulfur is used to generate organic sulfur compounds, it is assimilative sulfate reduction.
- If the reduced sulfur is excreted from the cell, it is dissimilative sulfate reduction
Which organisms are “scraping the bottom of the energy barrel”? Why are they said to do this?
Methanogens and acetogens
- The difference between CO2 and methane on a redox table is really small, so these organisms are barely getting any energy at all from this type of metabolism. But if it’s the best you can do, then this is an option
Describe methanogenesis and acetogenesis
These organisms carry out fermentation and generate H2 (the product of almost all the fermentations)
H2 acts as electron donor, and fix CO2 into methane and water or acetate (CH3CO2) and water
- Proton or sodium motive force (plus substrate-level phosphorylation for acetogens) yield ATP
Methanotrophs and acetotrophs are (acidic/alkaliphilic)
- Explain the consequences of this
Acidic
- Some of them use the sodium motive force for energy
What are the 4 major electron donors used by lithotrophs?
- Ammonia (NH4+)
- Ferrous iron (Fe2+)
- Sulfide (H2S)
- H2
True or false: the major electron donors of lithotrophs are typically the waste products of aerobic organisms
True
Most chemolithotrophs use __ as the final electron acceptor
O2 (aerobic lithotrophic respiration)
Most chemolithotrophs are (heterotrophs/autotrophs)
Autotrophs - generate reducing power to fix CO2 (carbon source, most use the Calvin cycle)
Describe H2 oxidation and model organism that uses this mechanism
Model organism: Ralstonia
H2 (dihydrogen) is oxidized by soluble hydrogenase to reduce NAD+ to NADH, with the subsequent formation of water
- NADH is generated for the Calvin cycle
- H2 is first split by membrane-bound hydrogenase
- O2 is the preferred electron acceptor
- Alternative to methanogenesis (using CO2 to get rid of H2)
- So the electron transport chain can use H2 to generate ATP, while soluble hydrogenase uses H2 to generate reducing power
Describe iron oxidation and the model organism uses this mechanism
Model organism: Acidithiobacillus ferrooxidans
Ferrous iron (Fe2+) is oxidized with the subsequent formation of ferric iron (Fe3+)
- O2 is the preferred electron acceptor (but this is an organism that can use alternates like nitrate)
- Uses reverse electron flow to generate NADH for Calvin cycle
Describe reverse electron flow and why it’s needed
Necessary because the energy difference on the redox table between most electron donors and O2 (electron acceptor) is not enough to reduce our NAD+
Through reverse electron flow, the electrons travel through quinones and complexes within the cell membrane to reduce NAD+ to NADH for the Calvin cycle
Why is reverse electron flow not necessary for NADH generation in H2 oxidation?
When we split H2, it has enough energy to reduce NAD+
Where are iron oxidizers prevalent?
In acidic aqueous environments (e.g. acid mine drainage and acidic volcanic springs)
How does iron-oxidizing bacteria look in water?
Fe3+ reacts with water and forms ferric oxides, resulting in a rust colour
- This is because these bacteria are converting Fe2+ to Fe3+
Describe the position of iron on the redox table and the consequences of its positioning
Fe2+ is near the middle, so sometimes it’s going to be used as the final electron acceptor in anaerobic respiration while at other times it can also be used as an electron donor with O2 as the final electron acceptor
Describe the first half of nitrification and the model organism that carries out this mechanism
Model organism: Nitrosomonas
- Oxidize ammonia to nitrite
- O2 is the final electron acceptor
- Electrons flow down ETC, generating PMF for chemiosmosis
- Use reverse electron flow to generate NADH for Calvin cycle
In general, what is nitrification important for?
Important in maintaining the global nitrogen cycle
What are nitrifiers prevalent in?
NH3-rich soils, wastewater, manure
Describe the second half of nitrification and the model organism that carries out this mechanism
Model organism: Nitrobacter
- Nitrite is oxidized with the subsequent formation of nitrate
- O2 is the final electron acceptor
- Nitrite stores less energy than nitrate, so we’re not getting as much PMF, and this results in less ATP formation
- Reverse electron flow generates NADH for the Calvin cycle
Describe sulfur oxidizers, and the model organism in which this mechanism is carried out
Model organism: Thiobacillus
Elemental sulfur (S0) and sulfide (H2S) are oxidized with the subsequent formation of sulfite and then sulfate
- O2 is the preferred electron acceptor (but nitrate can also be used)
- Uses reverse electron flow to generate NADH for the Calvin cycle
Thiobacillus (sulfur oxidizers) are also known as…
- also explain why they’re called this
Colourless sulfur bacteria
- Coloured bacteria are photosynthetic and have a lot of pigments
Where are sulfur oxidizers prevalent?
Hydrothermal vents, hot springs, etc.
Define phototrophy
The use of light energy to drive electron flow (instead of using organic or inorganic electron donors)
- Use electron transport chains to generate PMF for use in photophosphorylation (chemiosmosis)
True or false: Phototrophy is only oxygenic
False; it can be oxygenic or anoxygenic
Describe phototrophy when it’s oxygenic and what organisms this is seen in
- Explain generation of reducing power, carbon and energy
Split water to supply electrons with the subsequent generation of O2
- Seen in cyanobacteria (model organism), algae, protists, plants
Reducing power: H2O to 1/2 O2, electrons power ETC
Carbon: CO2 fixed by Calvin cycle
Energy: ADP -> ATP using light and ETC
Describe phototrophy when it’s anoxygenic and what organisms this is seen in
- Explain generation of reducing power, carbon and energy
Does not split water or form O2 in Calvin cycle, use molecules such as H2S instead
- Only carried out by prokaryotes
Reducing power: H2S to S0 which releases electrons, or S0 to SO42- which also releases electrons to power the ETC
Carbon: CO2 fixed by Calvin cycle
Energy: ADP->ATP using light and ETC
Most phototrophs are (heterotrophs/autotrophs)
Autotrophs - generate reducing power to fix CO2
True or false: both sulfur oxidizing lithotrophs and phototrophs are sulfate producers
True
What do purple sulfur bacteria tend to have a lot of in their membrane and why?
Tend to have a lot of carotenoids in their membranes that is going to allow them to absorb solar energy
Describe anoxygenic purple sulfur bacteria and the model organism for this bacteria
Model organism: Chromatium spp.
- Use a Q(quinone)-type photosystem (similar to photosystem II)
- Use cyclic electron flow to generate PMF
- The final electron acceptor is the original pigment
- Don’t require a source of electrons because these electrons cycle forever (so no water or anything else)
If anoxygenic purple sulfur bacteria don’t require a source of electrons, how do they generate reducing power for the Calvin cycle?
Occasionally, H2S gas will donate electrons to the cyclic electron transport chain, followed by reverse electron transport to generate reducing power (non-cyclic part of the transport chain)
- S0 accumulates in granules inside (periplasm) or outside of the cells
- S0 can be used when H2S is low
Purple sulfur bacteria are (heterotrophs/autotrophs)
Autotrophs
Describe anoxygenic green sulfur bacteria and the model organism for this bacteria
Model organism: Chlorobium
- Use chlorosomes so can respond to very low levels of light
- Use a FeS-type photosystem
- Electrons are used to reduce ferredoxin, so flow is non-cyclic
- Ferrodoxin carries electrons to reverse citric acid cycle for carbon-fixation into organic molecules
- Electrons are replaced by the oxidation of H2S to S0 to SO42-
- Maybe use electron flow based on arrangement? But this hasn’t been seen yet
True or false: anoxygenic green sulfur bacteria like Chlorobium spp. use the Calvin cycle for carbon fixation
False; they use the reverse citric acid cycle
Describe oxygenic cyanobacteria and the model organism for this bacteria
Model organism: Prochlorococcus
- Have both Q-type and FeS-type reaction centres, photosystem I and II (so cyanobacteria likely came into existence due to previous genetic exchange, likely between a purple and green sulfur bacterium)
- Use linear electron flow
- electrons are used to generate PMF and reduce NADP+
-Electrons are replaced by the oxidation of H2O to O2
- C-fixation via the Calvin cycle
Describe the 7 (and a half?) layers of the Winogradky column
- Cyanobacteria
- High water near top, low sulfide, lots of light
- Fix CO2 into organic molecules
- Water oxidized to O2 - Heterotrophic bacteria
- Organic molecules become CO2
- O2 becomes H2O in ETC - Iron oxidizing bacteria
- Fix CO2 into organic molecules
- Fe2+ oxidized to Fe3+ - Purple non-sulfur bacteria (didn’t talk about them)
- Organic compounds used to generate other carbon molecules
- H2S as electron donor, oxidized to S - Purple sulfur bacteria
- Fix CO2 into organic molecules
- H2S as electron donor, oxidized to S - Green sulfur bacteria
- CO2 fixed into organic molecules
- H2S as electron donor, oxidized to S
- Survive in low light conditions - Sulfate reducing bacteria
- Organic molecules become CO2
- SO4 reduced to H2S which feeds its way up the column
8 ish. Methanogens and acetogens
- Use CO2 and H2 to generate methane or acetyl groups
What would happen to the relative concentration of O2 if you put some oxic mud (soil+ water) and glucose in a closed bottle? The soil initially contains O2, NO3-, SO42-, Fe3_
Oxygen is going to be used up first. The aerobic respirers will use up the oxygen because its the best electron acceptor
Describe the difference between lithotrophic sulfide oxiation and phototrophic sulfide oxidation
In lithotrophs like thiobacillus (lithoautotrophs), they use O2 or another oxidant to oxidize H2S or S0 to SO42-, this produces energy and electrons that are needed to make reduced carbon from CO2
In phototrophs like purple sulfur bacterium chromatium, energy comes from light but electrons come from H2S
What type of organism dominates the fixation of carbon into organic matter? What method(s) do they use to fix carbon?
Autotrophs
- Use the Calvin cycle
What type of organism is the dominant photoautotrophs in terrestrial environments?
Plants
What type of organism is the dominant photoautotroph in aquatic environments? Is this the only type of metabolism present in aquatic environments?
Algae, bacteria and archaea
- Don’t forget about the chemolithotrophs
- Use the Calvin cycle, rTCA, etc
What organisms return carbon to abiotic reservoirs?
Organotrophs
- They respire and return CO2 to the environment
In oxic conditions, what process(es) converts organic matter into CO2?
Respiration
In oxic conditions, what process(es) converts CO2 into organic matter?
Oxygenic photosynthesis and chemolithotrophy
In anoxic conditions, what process(es) converts organic matter into CO2?
Anaerobic respiration and fermentation
In anoxic conditions, what process(es) converts CO2 into organic matter?
Acetogenesis and anoxygenic photosynthesis
What is nitrogen assimilation?
NH3 -> NH2 groups of protein (nitrogenous bases or amino acids)
What is ammonification?
NH2 groups of protein (organic sources) into NH3
Nitrification happens in (oxic/anoxic) environments
Oxic
Denitrification happens in (oxic/anoxic) environments
Anoxic
Do assimilation, ammonification and nitrogen fixation happen in oxic or anoxic environments?
Both
Describe annamox
- Stands for: Anaerobic ammonia oxidation
- Ammonia is used as an electron donor and nitrite as an electron acceptor to produce N2
- Organisms that complete annamox often have an anammoxosome
- Pseudo-organelle that generates PMF (happening internally and not in the cell membrane though, but can still produce ATP)
What is microbial symbiosis and what are the 4 types (in general)?
Prolonged and intimate relationship between organisms
1. Parasitic
2. Pathogenic
3. Commensal
4. Mutalistic
What is microbial parasitism?
The microbe benefits at some expense to the host (but still tries to keep the host alive for as long as possible)
What is pathogenic symbiosis in microbes?
The microbe causes a disease in the host (kills host)
What is commensal symbiosis in microbes?
The microbe has no discernable impact on the host
What is microbial mutualism?
The microbe is beneficial to the host
What is the general advantage of nitrogen-fixing bacteria and archaea?
They replenish organic nitrogen supplies lost due to denitrification
What is one of the most important symbioses known? And what is the most well-studied example?
The mutualistic association between plants and nitrogen-fixing bacteria
The most well-studied example is the relationship between leguminous plants and nitrogen-fixing rhizobia (legumes are any organisms with an internal pod, like soybeans, clover, peas, etc)
Describe the legume-root nodule symbiosis in general
Infection of legume roots by Rhizobia leads to the formation of root nodules that fix N2 - significant increases in soil nitrogen
- Biofertilizer
What is a cross-inoculation group?
What we call it when we have a good match between certain plant species and certain rhizobial species (the symbiosis between legumes and rhizobia is highly specific)
What is a flavonoid?
Compounds that plants secrete into the soil that promotes the growth of microbes around the root
- Chemoattractant
What is the rhizophere?
The microhabitat immediately surrounding the plant roots
Describe the steps for nodule formation on legume roots (3 steps)
- Recognition and attachment of rhizobia to root hairs using an adhesive molecule.
- As the bacteria starts to infect the root hairs, it starts to secrete Nod/nodulation factors (oligosaccharide) which induces the plant to start cell replication. The bacteria also starts to grow down the root hair into the main part of the root (forms an infection thread)
- The plant cells start to divide over and over again, resulting in the formation of a circular nodule that surrounds the bacteria coming inside
The rhizobia multiply in plant cells and become swollen, misshapen, and branched cells called _____ which are surrounded by plant membranes
bacteroids
What three things do Nod (nodulation) factors induce?
- Induce root curling (makes the root hairs more permeable to the bacterium)
- Trigger plant cell division
- Trigger nodule formation
The bacteroid takes in ______ from the plant for _____
Organic acids (succinate, malate, fumarate, pyruvate aka intermediates of the citric acid cycle) for ATP production and to provide reducing power for N fixation
Nitrogenase function
Fixes N2 into NH3
What does the bacteroid export to the plant? What do the plants use this for?
NH3
- Plant then uses NH3 to make nitrogenous bases and amino acids
What is an evolutionary conundrum in rhizobia and how is it resolved?
Nitrogen-fixing bacteria need O2 to generate energy for N2 fixation, but the enzymes that fix N2, nitrogenases, are inactivated by O2
- In the nodule, O2 is bound by the O2-binding protein leghemoglobin (red colour) that decreases [O2] so bacteria has an environment to fix N2
Define symbiosome
The plant membrane surrounding bacteroids where nutrient exchange occurs
Describe the structure of the bacteroid, and the 4 steps of N2 fixation
Bacteroid is surrounded by bacteroid membrane and symbiosome membrane (generated by plant)
1. Photosynthesis imports sugars and amino acids involved in citric acid cycle
2. Electrons are donated to electron transport chain from TCA in bacteroid membrane, but final O2 acceptor is delivered with leghemoglobin to complex IV (Lb acts as a carrier molecule)
3. ATP produced by PMF powers nitrogenase to fix N2 into NH3
4. NH3 is exported to produce amino acids and nitrogenous bases in the plant cytoplasm
What is chlorochromatium aggregatum?
A freshwater mutualistic microbial consortium between green sulfur bacteria (epibionts) and an unnamed flagellated rod-shaped bacterium
Describe the microbe-microbe symbiosis in chlorochromatium aggregatum
The green sulfur bacteria (Chlorobium) use light energy to fix CO2 using electrons from H2S (Anoxygenic photosynthesis)
- 13-69 ebibionts surround a single flagellataed bacterium. The motile bacterium move in the water column in response to light, oxygen and sulfide. Helps Chlorobium do photosynthesis and deliver these nutrients to unnamed flagellated rod-shaped bacterium.
What 3 things make up the entire set of genetic elements of any microbe?
Chromosomal DNA, plasmids and transposable elements
What is the typical genome size/ORF range for microbes?
4-8 Mb, 4000-8000 genes (ORFs)
True or false: All prokaryotes have one, circular dsDNA chromosome
False; some have more than one, such as cholera which has two circular chromosomes and the bacteria causing Lyme disease
Chromosomes are compacted into ____ supercoiled domains by which enzyme?
Negative, DNA gyrase
Thermophiles have ____ supercoiled domains by which enzyme?
Positive, reverse DNA gyrase
Chromosomes are (small/large), consisting of ____ bases while plasmids are (small/large), consisting of ___ bases
Large, 4-8 Mb
Small, 1-1000 kb
What types of genes do chromosomes code for? What about plasmids?
Chromosomes: Essential “housekeeping” genes
Plasmids: “Bonus” genes (essential in certain contexts, like presence of antibiotics but not for everyday life; bacteria with plasmids should be able to live without them
True or false: there are usually only one copy of the chromosome per cell, but there may be dozens to hundreds of different plasmids
True
True or false: Replication of plasmids are strongly regulated by the cell cycle, while replication of the chromosome is independent of the plasmid
False; Replication of chromosomes are strongly regulated by the cell cycle, while replication of plasmids is independent of the chromosome
Bacterial DNA replication is also referred to as…
Theta replication
Difference between origin of replication between prokaryotes and eukaryotes?
Prokaryotes only have one, while eukaryotes have multiple
DNA replication enzymes associate to form the…
Replisome
Prokaryotic transcription factors are called…
Sigma factors
σ70 is the transcription factor required for…
Transcription of “housekeeping genes” (proteins that are used on a daily basis)
Where does σ70 bind?
Upstream of transcription initiation sties, the -10 and -35 region
What controls transcription rates (specifically, rate of initiation)?
Variations in the consensus sequences at the -10 and -35 regions
- If the sequences differ significantly from the consensus regions then σ70 would bind with lower affinity
What is the RNA polymerase holoenzyme?
RNA polymerase + σ70 at the promoter before elongation
What happens to σ70 after transcription starts and what is this complex called?
After transcription starts, σ70 dissociates and becomes the RNA polymerase core enzyme which transcribes the DNA by reading the DNA template from 3’ to 5’
Describe transcription termination in prokaryotes
Inverted repeats in the DNA that are transcribed result in the formation of a termination stem-loop followed by a stretch of adenines on the template strand
- This causes RNA polymerase to stall and “fall off” the DNA
Genes for enzymes of related function are sometimes organized into an…
Operon
What is an operon? What type of mRNA is produced when operons are transcribed?
Multiple genes ordered sequentially under the control of a single promoter
- Genes are co-transcribed to produce a single transcript called polycistrionic mRNA
What are the two types of transcription factors?
- Activator proteins
- Repressor proteins
Describe activator proteins
Turn transcription “on” by binding to an activator binding site (upstream of promoter)
Describe repressor proteins
Turn transcription “off” by binding to an operator (downstream of promoter)
What are effector molecules in general and what are the two types?
Molecules that allow (or prevent) transcription factors to bind DNA by interacting with transcription factors
1. Inducers
2. Corepressors
What is an inducer?
An effector molecule that binds to activator proteins to help activate transcription
What is a corepressor?
An effector molecule that binds to repressor proteins to help stop transcription from occurring
Describe enzyme repression in general and provide an example
Some genes are only expressed when the end-product of a biochemical pathway is absent
- The gene(s) are normally “on” with nothing bound to the operator
Ex: Arg acts as a corepressor and binds to a repressor protein. The corepressor-bound repressor protein binds DNA at the operator region of the arginine-synthesis gene(s). With a repressor attached to the operator, RNA polymerase can’t transcribe the gene - the cell stops making arginine. If the cell runs out of arginine, Arg falls off the repressor, which falls off the operator, transcription starts and the cell will make the enzymes necessary to produce arginine.
Describe enzyme induction (derepression) in general and provide an example
Some genes are only expressed when a substrate molecule is present
The gene(s) are normally “off” with a repressor bound to the operator
Ex: Lactose acts as an inducer and binds to the repressor protein that is bound to the operator already. The inducer-bound repressor protein falls off the operator region of the lactose-metabolizing gene(s). With the repressor off the operator, RNA polymerase carries out transcription - the cell metabolizes the lactose. If the cell runs out of lactose (e.g. all the lactose is metabolized), lactose falls off the repressor, which binds to the operator, and transcription stops.
Describe enzyme activation in general and provide an example
Some genes are expressed rarely or in very small amounts. These genes are normally “off” because they have weak promoters.
- Ex: Maltose acts as an inducer and binds to an activator protein. The inducer-bound activator protein binds DNA at an activator binding site upstream of the promoter. The DNA-bound activators improve sigma factor/RNA polymerase binding, transcription occurs, and the cell metabolizes maltose. If the cell runs out of maltose, maltose falls off the activator, which falls off the activator binding site, and transcription stops
True or false: prokaryotic translation is virtually identical to eukaryotic translation
- Explain why or why not
True
- The ribosome binds mRNA to RBS (Shine-Dalgarno sequence) and initiates translation at a start codon
- Elongation proceeds as tRNAs deliver amino acids based on complementary codon-anticodon interactions
- Translation ends at a stop codon
What are 4 mRNA differences seen in prokaryotes compared to eukaryotes?
- Polycystronic mRNA (multiple RBS) in prokaryotes
- No 5’ cap
- No poly-A tail
- No introns
True or false: prokaryotic mRNA doesn’t require processing
True; they lack a 5’ cap, poly A tail and introns
True or false: prokaryotic mRNA has no protection from degradation
True; they lack a 5’ cap and a poly A tail
What are 5 differences in translation in prokaryotes compared to eukaryotes?
In prokaryotes:
1. 70S ribosomes
2. First amino acid is fMet
3. Codon bias differs among species
4. Not all ORFs in polycistronic mRNA are in the same reading frame
5. Some species use “stop codons” for selenocysteine (Sec) and pyrrolysine (Pyl)
Prokaryotes don’t have a nucleus. What is a result of this?
mRNAs don’t have to be exported before translation begins (transcription and translation tend to happen at the same time
What is a prokaryotic polysome?
Multiple ribosomes attached to mRNA at the same time as transcription (ribosomes bind and begin translation as soon as there’s enough room)
True or false: in prokaryotes, proteins may need to be targeted to specific locations but the vast majority of them don’t need to be targeted
- Explain why or why not
True, due to the lack of organelles
What type of genes require repressors?
Those that are transcribed a lot (constitutively expressed, e.g. enzymes required for everyday function and essential end-product molecules like amino acids, lipids, etc)
What types of genes require inducers?
Those that are only transcribed when needed (e.g. enzymes required less often, metabolize substrates when present in the environment, like sugars)
What types of genes require activation?
Those that are rarely transcribed (e.g. enzymes rarely required, rare sugars like maltose or other energy molecules)
True or false: sRNA (small RNA) is solely responsible for decreasing gene expression
False; it also works to increase gene expression, it just depends on the context
Describe how translation is inhibited through sRNA regulation at the 5’ end of the mRNA
When sRNA is complementary and parallel to the RBS and blocks it, no translation occurs and ribonucleases are free to degrade the mRNA while the sRNA is bound
Describe how translation is stimulated through sRNA regulation at the 5’ end of the mRNA
Sometimes, the 5’ end of the mRNA can be complementary to the RBS, naturally blocking the binding of the ribosome to the mRNA. sRNA can base pair with the 5’ end of the mRNA to expose the RBS, allowing translation to occur
Describe how translation can be inhibited through sRNA regulation at the 3’ end of the mRNA
sRNA can recruit ribonucleases to the mRNA and bind to the DNA to stimulate degradation
Describe how translation can be stimulated through sRNA regulation at the 3’ end of the mRNA
sRNA can base pair with the 3’ end of the mRNA and prevent ribonucleases from binding and degrading the mRNA
In an mRNA molecule with an aptamer, describe the aptamer’s structure when the protein that it codes for is at a low concentration
Regions 1 and 2 form a stem loop, and the RBS is exposed, and the ribosome binds to increase transcription of the gene
In an mRNA molecule with an aptamer, describe the aptamer’s structure when the protein that it codes for is in high concentration
The signal metabolite binds, resulting in the step loop formation between regions 2 and 3 (3 contains the RBS so the ribosome can’t bind and translation does not occur)
True or false: riboswitches are only present in mRNA to regulate translation
False; they’re also present in DNA to regulate transcription
Attenuation as a form of transcriptional regulation is unique to (eukaryotes/prokaryotes)
- Explain why
Prokaryotes, because it requires the coupling of transcription and translation
Describe attenuation if the amino acid product is in high concentration in the cell
The 5’ end of the mRNA is translated quickly because the amino acid can be incorporated into the leader peptide (blocking regions 1 and 2), resulting in a hairpin forming between regions 3 and 4
- This stem loop terminates transcription (causes RNA polymerase to stall and fall off the DNA)
Describe attenuation if the gene product is in low concentration in the cell
The 5’ end of the mRNA is translated slowly because the amino acid cannot be incorporated into the leader peptide, blocking region 1 only, resulting in a hairpin that allows transcription (and translation) to continue
DNA transferred by any of the horizontal gene transfer mechanisms can do which 3 things?
- Degraded by endo/exonucleases
- Exist and replicate autonomously (independent of the host chromosome, as a plasmid)
- Incorporate DNA into host chromosome through recombination
What 2 ways can recombination occur between the DNA transferred by horizontal gene transfer and the chromosome?
- RecA-mediated homologous recombination
- Site-specific (non-homologous) recombination (new DNA inserted into chromosome and chromosome gets longer)
Transformation usually happens when the donor cell…
Dies (cell lysis releases DNA that breaks into small (10kb) dsDNA fragments)
Cells that can uptake naked DNA from the environment (undergo transformation) are said to be…
Competent
Cells that can uptake naked DNA from the environment (undergo transformation) take up what type of DNA?
ssDNA
What is an example of a competent cell?
Streptococcus
True or false: competence only happens in nature
False; competence can be induced in the lab
What are the 3 steps for inducing competence in the lab?
- Negate charge differences
- Rupture membrane (cold/heat shock, electroporation)
- Take up dsDNA
Describe the 3 general steps for transformation of DNA into the cytosol
- Donor dsDNA binds to the top of the extended pilus of a competent living cell
- Pilus retracts and brings DNA into the periplasm of G- cells or through the cell wall of G+ cells
- Rec2/Com (competence) transporter brings ssDNA into cytosol
Describe the 3 steps for how donor dsDNA is is brought into the cytosol as ssDNA and incorporated into the bacterial chromosome
- Donor dsDNA binds to DNA-binding proteins (Rec2/Com) on recipient inner membrane
- Nuclease degrades one strand as the other is moved into the cytosol
- Rec-A mediated recombination if sufficient homology exists between donor DNA and host chromosome
What happens during the transformation process if sufficient homology doesn’t exist between donor DNA and host chromosome?
The ssDNA is degraded
Can transformation occur with plasmids?
Yes but only in the lab
What is conjugation and what does it require?
Transfer of PLASMID DNA from a donor cell (F+) to a recipient cell (F-)
- Requires cell-cell contact
Where do the cells that undergo conjugation get the genes required for conjugation?
Conjugative plasmids contain the genes required for conjugation
True or false: not all plasmids are conjugative, but non-conjugative plasmids can move between cells during conjugation
True
What was the first/best studied system of F plasmids?
E. coli
What is the general function of the tra region in the F plasmid?
Makes the plasmid conjugative
What 2 things does the tra region in the F plasmid code for?
The F pilus (conjugative pili) and the type IV secretion system (not the same as type IV pilus)
oriV function on F plasmid?
Plasmid DNA replication
oriT function on F plasmid?
Origin of transfer, where transfer of the plasmid starts from the donor to the recipient
What else is contained within an F plasmid other than the tra region and the oriV/oriT
Numerous insertion sequences (IS) and a transposon (Tn)
Upon attachment of the recipient cell with an F pilus, what happens next?
The pilus retracts and the cells come close together
Function of type IV secretion system
Creates bridges that connect the cytoplasms of cells that have come close together for conjugation
What are the 5 steps for rolling circle replication?
- Tra operon indued by mating pair formation
- One strand is nicked at oriT
- As the nicked strand is “rolled” off the plasmid, it is replaced by DNA polymerase
- The donated strand is moved into the recipient, circularizes, and a complementary strand is synthesized
- The recipient cell is now a donor cell (F+)
What is the end result of rolling circle replication during conjugation?
Produces 2 F+ cells
In Hfr cells (high frequency of recombination), the F plasmid is an…
Episome
What is meant by the F plasmid being an “episome” in Hfr cells?
It can integrate into the host chromosome and the genes on the plasmid are still expressed within the host chromosome
What specifically allows for F plasmid to integrate into the host chromosome in Hfr cells?
If the plasmid has an integration sequence that matches that of the chromosome, recombination can occur
True or false: When the F plasmid integrates its genes into the host chromosome, it is still called an F+ cell
False; it is now called an Hfr cell
What are the 4 steps for Hfr conjugation?
- Following mating pair formation with an F- cell, the DNA is nicked at oriT within the plasmid region of the new host chromosome
- Rolling circle replication starts moving chromosomal DNA into the recipient cell
- The chromosome is very long, so the connection between the two cells breaks during the transfer and only 1/2 of the plasmid DNA and part of the donor’s chromosome is transferred to the donor cell
- The linear stretch of DNA transferred into the recipient may be degraded OR the donor DNA can recombine with the recipient chromosome at high frequencies
True or false: After Hfr conjugation, the recipient can radically change its phenotype due to high recombination of transferred genes
True
After Hfr conjugation, the donor cell is a __ cell while the recipient cell is a ___ cell
Hfr, F-
What is an F’ plasmid?
Chromosomally integrated F plasmids can be excised and occasionally, chromosomal genes can be taken with the plasmid DNA (because there’s many insertion sequences on the host chromosome)
During conjugation, what happens to F’ plasmids?
The donor chromosomal genes are completely transferred to the recipient
True or false: regardless of what happens to a plasmid, transposons can still act independently
True
True or false: All phages are transducing and all prokaryotes are transducible
False
Describe how transduction is important medically and industrially
Medically: It serves as a mechanism of transferring virulence factors and antibiotic resistance among organisms, especially in the GI tract
Industrially: Serves as a mechanism of moving genes from one organism to another
Describe generalized transduction
Occurs during lytic infection by virulent phage. During assembly, host DNA is randomly packaged inside a viral capsid instead of viral DNA. This is called a defective phage. Following release, these detective phage can still adsorb to host cells. After penetration, the donor DNA can either be deraded or undergo homologous reecombination
What is a defective phage also known as?
A transducing particle
True or false: generalized transduction is often succesful
False; success is rare because most progeny phage are virulent, and recombination is not guaranteed
Describe specialized transduction
Occurs during lysogenic infection by temperate phage. During lysogeny, viral DNA is inserted at a specific (att) site. During induction of the lytic cycle, host chromosomal DNA - adjacent to the att site - can be excised with the viral DNA. During the next round of lysogeny, those prokaryotic genes can be inserted into the next host genome.
During specialized transduction, when is it said that “phage conversion” has occured?
If in the next round of lysogeny, the phenotype of the next host changes since the previous host’s genes could be inserted into the new host’s genome.
Define virus
A subcellular particle (not actually considered “living”) that can only replicate within a living host cell
What type of parasite are viruses classified as and what does this mean?
Obligate intracellular parasite (absolutely must have a host)
True or false: viruses have been found for organisms in all 3 domains
True; for every cell type, there’s about 2-3 viruses that can infect it
What is the most abundant form of genetic info?
Viruses
What 2 forms/states do viruses exist in?
- Virion
- Replicating genome
What is a virion? Is it infectious?
Virus existing extracellularly, it is infectious
What is a replicating genome? Is it infectious?
Intracellular virus
- non-infectious…ish (is only infectious if you insert genetic info into another host cell)
True or false: virions have a very specific and constant size range
False; virions have a large size range
- e.g. polivirus (the size of a ribosome) vs pandoravirus (larger than most bacterial cocci)
What are the three possible virion ordered symmetrical structures?
- Helical
- Icosahedral
- Complex
What is a virus capsid?
Protein “coat” that currounds the genomic material
Capsid subunits
Capsomeres
Describe the viral envelope
Some viruses have a envelope made of a phospholipid bilayer that often contain glycoprotein “spikes”
What are non-enveloped viruses called?
Naked
True or false: some virions contain viral enzyme(s) required for infection
True
True or false: viruses are made of linear dsDNA
False; viral genomes can be DNA or RNA, single-stranded or double-stranded, linear or circular, and virions have a large genome size range
True or false: bacteriophages are often just called phages now
- Explain why or why not
True, because they can attack Archaea just as well
What is an example of a complex virion?
Bacteriophage
What are plaque assays? What can they be used to estimate?
A bacterial lawn is prepared on an agar plate. A viral solution is then spread on top. Plaques (clear areas) form where the bacteria have been killed.
- Counting plaque-forming units (PFU) gives estimates of titer (concentration)
What is the eclipse stage of a viral one-step growth curve?
Upon viruses being added into a bacterial culture, the extracellular virus concentration quickly depletes because the virions are getting inside the host cell
- There is then a plateau phage characterized by viral replication, where the virus is taking over the host and typically won’t get new infections in this stage
What is the maturation stage of the viral one-step growth curve?
Virus coats start assembling
What is the burst size of the viral one-step growth curve?
Difference between initial and final virus concentrations
What is the latent period of the viral one-step growth curve?
eclipse + maturation stages
Describe phage receptors
Host cell phage receptors perform normal functions for the host cell, viral evolution has made these cell components targets for attachment.
What type of infection do virulent phages cause?
Lytic infection
What is an example of a virulent phage
bacteriophage T4 and its E. coli host
How do T4 bacteriophages attach to their E. coli host?
Tail fibers adsorb to LPS carbohydrates on host outer membrane
How do T4 bacteriophages penetrate their host? (4 steps)
- T4 tail fibers retract, bringing tail pins into contact with the lipid bilayer.
- The tail sheath contracts, pushing a tail tube through the outer membrane.
- T4 lysozyme forms a pore through the cell wall by degrading the peptidoglycan in the cell wall
- T4 dsDNA moves into cytosol
What is “early” T4 protein synthesis after the T4 genome is injected into E. coli? (3)
- Host RNA polymerase transcribes a series of viral genes that are only expressed at the start of infection (designed to take over the host)
- Includes anti-sigma factor proteins that inhibit host sigma factors, which stop host transcription and translation (to take over the host)
- Includes a phage-specific replisome (helps ensure that the viral DNA is replicated, not the host)
What is “middle” and “late” T4 protein synthesis? (4)
- Includes capsomere, tail, and tail fiber proteins
- Includes enzymes to facilitate assembly and release
- “Late” T4 genes encode a packaging motor complex, which fills the viral capsids with copies of the viral dsDNA
- “Late”T4 genes encode enzymes that compromise the host cell membrane and cell wall.
What happens to the linear copies of T4 dsDNA during replication?
The linear copies are joined to generate a long concatemer through recombination
What is the “headful” packaging mechanism? Because more than a genome-length of DNA fits into each capsid, what happens?
During assembly, the phage DNA is moved into each capsid until it is full from the packaging motor, and then cut from the rest of the molecule.
Because more than a genome-length of DNA fits into each capsid, the DNA is cut at different sites, and each progeny virion has a different gene order (different gene termini in each capsid)
The T4 genome is terminally redundant. What does this mean?
Genes at one end of the molecule are repeated at the other end.
True or false: T4 progeny viruses all have the same genotype, but the gene order is switched
True
What type of infection do temperate phages cause?
Lysogenic infection
What is an example of a temperate phage?
Bacteriophage λ (linear dsDNA) and its E. coli host
What is a prophage? How is it different from a lysogen?
Prophage: integrated viral DNA
Lysogen: host with integrated viral DNA
Describe the λ adsorption/penetration to E.coli (2)
- The λ tail attaches to a host maltose transport protein
- Penetration like T4
What happens to the λ genome after entry into the host cell?
The 5’ ends of the λ DNA contain short, single-stranded, complementary, “cohesive ends”, so following entry into the host cytosol, λ DNA circularizes, forming a cos site (bc overhangs are now complementary and parallel)
True or false: most λ infections result in a lytic infection
True
What happens if the λ repressor (cI) gene is expressed upon penetration?
If the λ repressor (cI) gene is expressed upon penetration, cI accumulates, and most λ genes are repressed (λ DNA becomes a prophage)
- high cI, Cro is repressed, lysogeny
What happens if the cro repressor (cro) gene is expressed upon penetration?
if the cro repressor (cro) gene is expressed upon penetration, cro accumulates, and the cI gene is repressed (λ enters the lytic cycle)
- low cI, Cro not repressed, cell lysis
How are host stress levels “monitored” upon λ infection? What happens if conditions deteriorate?
A few λ genes are expressed during lysogeny to “monitor” host stress levels and induce a switch to the lytic cycle if conditions deteriorate
λ integrase function
λ integrase nicks the DNA at the att sites (which is found in both λ and host DNA), and the viral DNA enters the host chromosome (λ integrase is only expressed if the cell is undergoing lysogeny)
λ excision enzyme function
Cuts the prophage out of the host chromosome
Describe λ synthesis (2)
- If expressed, “early” through “late” λ protein synthesis occurs like in a T4 infection
- The circular λ DNA is copied by rolling circle replication. The λ DNA is copied continuously, generating long concatemers. Individual λ genomes are cut from the concatemer at each cos site and packaged into assembling phage capsids
How do λ phage concatemers and T4 concatemers mainly differ?
No terminal redundancy, instead have the exact same molecule going into the λ capsids
True or false: many eukaryotic viruses are specific to certain tissues
True; they’re specific to organisms and tissues
Eukaryotic viruses follow a similar infectious pathway as phages, but what are the 3 main differences?
- Capsid enters the host
- Host cells have a nucleus
- Viral genome (sometimes) “hides” in a membrane-bound viral factory named the viroplasm (some viruses replicate in the ER)
What are the 4 possible outcomes of eukaryotic infection? What is the most common?
- Virulent infection (most common)
- Latent infection
- Persistent infection
- Transformation
What is the virulent infection pathway of eukaryotic viruses?
Cell lysis, releasing the progeny viruses
What is the latent infection pathway of eukaryotic viruses?
Viral genome becomes integrated into host chromosome, instead of prophage is called “provirus”
What is the persistent infection pathway of eukaryotic viruses?
Virion leaves the host cell by budding out of the cell membrane (no host lysing)
What is the transformation infection pathway of eukaryotic viruses?
Infection results in cancer because the virus is either going to be producing some enzymes that affect our DNA or cell cycle, or the virus inserts its genetic info into our genome.
What are the 4 differences of plant viruses compared to animal viruses?
- Plant viruses have a broad host range (less plant or tissue-specific) because…
- Most plant viruses are not enveloped
- Plant cells are harder to infect - insects with viruses on mandibles usually bit and damage the plant cell wall allowing for infection
- Viruses can infect adjacent cells through plasmodesmata (viruses can’t really burst out of a plant cell due to its cell wall)
What is the Baltimore classification scheme in general?
Widely used method of organizing viruses based on what the virus must do to produce mRNA
In the Baltimore classification scheme, what is mRNA labelled as?
“positive” or “plus” strand (or sense or configuration strand)
In the Baltimore classification scheme, what is the negative/minus strand?
The template strand used to make positive strands
Class I virus genome
dsDNA
What 3 viruses are class I viruses?
- T4 phage
- lambda phage
- Variola major (smallpox)
How is mRNA synthesized in class I viruses?
mRNA is synthesized from the DNA - strand
What type of DNA replication happens in class I viruses?
Semi-conservative DNA replication
Class II virus genome
ssDNA (+)
What 2 viruses are class II?
- ΦX174 phage
- Parvovirus (eukaryotic)
What first happens to the class II virus genome upon infection?
Complimentary DNA (-) strand is synthesized first to form a template for mRNA synthesis. This forms a dsDNA replicative form.
How is mRNA synthesized in class II virsues?
mRNA is synthesized from the DNA (-) strand
How are genome copies made for progeny viruses in class II viruses?
Genome copies are made by rolling circle replication
In class II viruses, what is the result of rolling circle replication?
One dsDNA replicative form, and one ssDNA positive strand copy
ΦX174 (class II virus) structure
Structure: Naked icosahedral virion
Describe ΦX174 (class II virus) genome
small ssDNA positive circular genome, many of its 11 genes are overlapping so parts of the genome are transcribed in more than one reading frame.
Describe ΦX174 (class II virus) adsorption and infection mechanism
Viral particles adsorb to LPS on E. coli outer membrane (like lambda and T4 phages). Host polymerases generate a dsDNA replicative form. Capsids form spontaneously and exit by cell lysis
Parvovirus is also known as ____ disease
Fifth (or slapped cheek disease)
Parvovirus genome
ssDNA positive genome
Parvovirus host and transmission mechanism
Host: humans and animals
Transmission: aerosols/contact
Parvovirus symptoms
Mild rash and swollen joints
Class III virus genome
dsRNA
What 2 viruses are class III?
- Φ6 phage
- Rotavirus (“stomach flu”)
What happens to dsRNA in class III viruses?
ssRNA positive strand is made using dsRNA in the capsid by RNA replicase (RNA-dependent RNA polymerase) for more protection from the host immune system
- RNA replicase is packaged into the capsid
What happens to the ssRNA derived from the dsRNA in class III viruses? What about the dsRNA?
ssRNA (+) is exported for translation and loaded into capsids with replicase (so that the next generation of virus has the enzyme) . The dsRNA is synthesized afterwards in capsid and the virion is released
Rotavirus host and mechanism of transmission?
Host: vertebrates
Transmission: oral-fecal
Describe rotavirus adsorption and symptoms
Adsorbs to cell surface glycans on gut cells. Infection increases electrolyte concentrations in intestinal human (released as the viruses lyse)and water follows the [] gradient, causing diarrhea
- Causes gastroenteritis
Class IV virus genome
ssRNA positive genome
What virus is class IV?
Poliovirus
How are proteins produced in class IV viruses?
Genome is used directly as mRNA
How does the class IV genome replicate?
RNA replicase synthesizes RNA - strands, then uses the RNA - strands as a template for RNA + to synthesize mRNA and the genome (some used for translation, some used as progeny genome)
Poliovirus is also called what?
Poliomyelitis
Poliovirus structure and genome
Structure: naked icosahedral virion
Genome: ssRNA + genome with a polyA tail (to protect genome in the cytosol long enough to start making copies)
How is the poliovirus genome specifically translated and replicated? (what protein helps…)
VPg protein facilitates ribosome binding to translate the mRNA
VPg also acts as a primer for RNA replicase to replicate the + sense genome into - sense to make more + sense copies
Poliovirus host and transmission mechanism
Host: human only
Transmission: oral-fecal
Poliovirus severity?
Most cases mild, but 1:200 develop neurological damage when virus moves to CNS (meningitis, encephalitis, paralysis)
What environmental conditions does the Chlorochromatium aggregatum consortium move toward?
Low oxygen, high light, and high sulfide
Class V virus genome
ssRNA (-) genome
What viruses are class V?
Rabies and influenza
How are proteins produced in class V viruses?
RNA replicase synthesizes RNA (+) from ssRNA (-)
How is the class V virus genome replicated?
RNA replicase uses RNA (+) as a template for RNA (-) to synthesize the genome
Describe the structure of the flu virus
Pleomorphic (all viruses look slightly different)
Describe the influenza genome
linear ssRNA (-) genome, 8 gene segments
Influenza spike proteins?
HA: hemagglutinin binds cell receptors
NA: neuraminidase facilitates virion release
What causes influenza to be pleomorphic?
The virion buds from the cell membrane. Everytime the capsid does this, it takes a different amount of phospholipid bilayer, resulting in pleomorphic viruses.
Where is HA/NA ORIGINALLY embedded?
In the host cell membrane; influenza gets the HA/NA during the budding process
Influenza host and transmission
Host: mammals and birds
Transmission: aerosols
What 3 diseases can be caused when influenza gets serious?
Meningitis, encephalitis, pneumonia
How are yearly flu shots designed every year?
WHO monitors and predicts upcoming strains, and the vaccine is based on viral spike proteins (HA)
Class VI genome
ssRNA (+) RETROVIRUSES (viruses that use reverse transcriptase)
What virus is class VI?
HIV
How are proteins produced in class VI viruses?
Reverse transcriptase (RT) synthesizes dsDNA, which is then used to synthesize mRNA.
order is: ssDNA (-) made, ssRNA + that it’s attached to is degraded and dsDNA is made.
How is the class VI virus genome replicated?
Reverse transcriptase (RT) synthesizes dsDNA. ssDNA (-) is used as a template to synthesize the genome.
HIV structure and genome
Structure: Enveloped segmented spherical virion
Genome: linear ssRNA (+) genome, 2 COPIES (advantage to virus bc mutations can happen in one copy which don’t affect the other copy)
What 4 enzymes are included in HIV capsid?
- Reverse transcriptase
- Integrase
- Viral tRNA
- HIV protease
HIV spike proteins? What do they bind to? What happens after they bind?
gp120 and gp41 bind CCR5/CD4 on host macrophages and T cells, and then membranes fuse
Describe how HIV generates dsDNA and what happens next
RT generates dsDNA in the capsid. dsDNA is released and migrates into the nucleus where integrase inserts the genome into the host chromosome forever (now virus is referred to as provirus)
How are HIV proteins produced?
Host RNA polymerase generates viral mRNA for translation of viral proteins, produces a polyprotein cleaved by HIV protease
How are HIV genome copies produced?
Host RNA polymerase generates RNA (+) genome copies
How are new HIV virions released?
New virion spread by budding (similar to influenza, but a lot slower. HIV keeps infected cells alive for longer)
HIV host and transmission
Host: primates
Transmission: contact
What type of cells does HIV adsorb to?
Macrophages and T cells
True or false: HIV infections can be latent for years before initiating AIDS
True
Why is HIV normally fatal?
Due to secondary opportunistic infections (because immune cells are dying)
What are 4 HIV “treatments”?
HAART (Highly Active Anti-Retroviral Therapy):
1. Fusion inhibitors (synthetic antibodies that block gp41 so viruses can’t bind to host cells)
2. RT inhibitors (Azidothymidine - nucleoside analog, blocks DNA synthesis of RT)
3. Integrase inhibitors (prevents provirus formation)
4. Protease inhibitors (prevents post-translational modification of viral proteins)
What could a potential HIV vaccine target?
gp41/gp120
Class VII virus genome
dsDNA (but different from class I because it’s partially dsDNA)
What virus is class VII?
Hepatitis B
How are proteins translated in class VII viruses?
mRNA synthesized from DNA (-) strand
How are class VII viruses replicated?
mRNA is used as a template by RT to synthesize dsDNA. RT changes mRNA into DNA (-) strand, which is then used to make a partial DNA + strand
Hepatitis B structure and genome
Structure: Enveloped icosahedral virion
Genome: Partially dsDNA circular genome, all genes overlap (multiple genes transcribed from the same stretch of DNA by initiating transcription in a different reading frame)
What happens following adsorption of hepatitis B?
The virus migrates to the nucleus and RT synthesizes the complete dsDNA genome
What does transcription of the hepatitis B genome produce? What happens after this is produced?
A ssRNA (+) the size of the genome
The ssRNA (+) enters a capsid where RT synthesizes a complete DNA (-) strand and then a partial DNA (+) strand
Hepatitis B is also known as…
Hepadnavirus
Hepatitis B and host and transmission
Host: Animals
Transmission: contact (blood)
What is a longterm effect of hepatitis B?
Acute liver disease (chronic infections can cause cirrhosis and cancer of the liver)
