MT2 Review Flashcards
How were bacteria originally classified on Haekel’s tree? Why?
Moneres (bacteria) were classified very low (near the “trunk” of the tree); are prokaryotic and thus less imporant
What were the three major Kingdoms of Haekel’s tree of life?
Plantae, Protista (unicellular eukaryotes), Animalia
Describe the tree of life that has Kingdom Monera at the bottom (LUCA)
Monera < Protista < Plantae, Fungi, Animalia (each diverged from Protista)
Describe Carl Woese’s contributions to the tree of life
Quantitatively related organisms based on numerical data (shared 16S SSU rRNA genes) instead of morphology
Why were the 16S SSU rRNA genes used to relate organisms?
- universal to all organisms
- does not undergo horizontal gene transfer (conjugation)
- highly conserved (functionally constant and low mutation rate)
- number of mutations acts as a molecular clock to determine divergence time between species
What is the “current” hypothesis for the 3 Domain phylogeny?
Divergence at LUCA into Bacteria and the common ancestor between Archaea and Eukarya, which diverged into those respective branches
Describe the relatedness between Bacteria, Archaea, and Eukarya
Bacteria is the oldest, Archaea and Eukarya are most closely related
What were the overall contributions of Carl Woese?
- quantitative approach to the tree of life
- discovered Archaea
- proposed the 3 Domain system instead of the 5 Kingdoms
- implied that prokaryotes are not current evolutionary artifacts; that have evolved alongside us
What is the trend in the number of bacteria species discovered versus time?
Has increased exponentially in part due to next gen sequencing techniques
How many phyla of bacteria have we discovered thus far?
~80, although 1500 is the predicted number
What are the four major phyla in Bacteria? What makes them “major”?
- Proteobacteria
- Actinobacteria
- Fermicutes
- Bacteroidetes
80% of characterized genera (cultured in lab) belong to these phyla
If more phyla could be comfortably cultured, how would our current major phyla change?
If more could be cultured, there would be more than 80 phyla and likely a greater makeup of “major” genera (our current ones would likely be diluted)
What is the most diverse and abundant phyla of cultured bacteria? Why?
Proteobacteria; can survive in culture, have many different metabolic strategies (anoxygenic, chemotrophs, autotrophs, lithotrophs, symbiotic, planktonic, etc.)
What are the classes of Proteobacteria?
Alpha, Beta, Gamma, Delta, Epsilon, and Zeta
Compare the divergence of plastids and mitochondria. Where did mitochondria come from?
Plastids diverged much sooner than mitochondria did; mitochondria diverged from Alphaproteobacteria
Describe the polyphasic approach to describe species
- morphology (cell shape, visible structures, chemical composition, etc.)
- metabolism (energy source [organo-, photo-, litho-, chemo-], carbon source [hetero-, auto-], oxygen requirements, etc.)
- genotype (use DDH or ANI to compare genomes of related species)
- evolution (tree constructed based on SSU rRNA)
How is DDH used to compare genomes of related species?
Known and unknown DNA is hybridized and the degree to which they hybridize determines their relatedness (more hybridized = closer relation)
What is the ANI % cutoff for different species?
Anything below 93% is considered a different species
What is the 16S rRNA sequence similarity % cutoff for different species?
Anything below 97% is a different species
What % of ANI and 16S rRNA must organisms share to be considered the same species?
ANI: above 96%
16S rRNA: above 98.5%
A high 16S rRNA gene sequence indicates what about the two organisms being compared?
They are close evolutionary neighbours (diverged recently)
What are the three requirements for the formal validation of a new prokaryotic species?
- Detailed description of characteristics/traits (morphology, metabolism, genotype, evolution)
- Deposition of viable cultures of the organism in at least two international culture collections
- Proposal of a Latin name and publication in the IJSEM (must be this journal)
How does the prokaryotic taxonomic hierarchy differ from the eukaryotic one?
Lacks Kingdoms, replaces them with phyla
What does the genome of a bacteria include?
The chromosomal DNA and plasmids
What is meant by the suffix -ome?
Implies global, collective, totality
What are the four basic -omic sciences?
DNA (genome), RNA (transcriptome), proteins (proteome), and metabolism (metabolome)
What is meant by the prefix meta-?
Implies beyond, more transcending, usually looks at omics of a microbial community rather than a single strain (analysis of at least two genomes
What is genome annotation?
Converting raw sequence data into a list of genes and other functional sequences present in the genome
What is bioinformatics?
Analyzing sequences and structures of nucleic acids and proteins
What are the steps for genomics?
- Sequencing
- Genome assembly
- Genome annotation
- Bioinformatics
Which step within genomics is considered the bottleneck of the process? Why?
Bioinformatics; slowest process because it takes the most work (analyze data)
What are the two sequencing techniques and how do they compare?
Sanger: more time consuming, less bases at a time
Next-gen: greater throughput at a much lower (100,000x) cost, faster
What is a closed vs. a draft genome?
Closed: every bp is known and sequenced, more expensive
Draft: most of the genome is sequenced except for repeats. Only know enough to distinguish between species
What is a contig?
A consensus sequence
What are ORFs?
DNA regions between a start and stop codon predicted to be read by ribosomes on mRNA. Identifying them requires searching 6 reading frames
How are the functions of ORFs predicted?
Searching similar sequences in DNA databases such as GenBank using BLAST
What are the steps for computer identification of possible ORFs?
- Computer finds possible start codons
- Computer finds possible stop codons
- Computer counts codons between start and stop (filters out shorter sequences)
- Computer finds possible RBS (upstream of start)
- Computer calculates codon bias in ORF
- Computer decides if ORF is likely to be genuine
- List of probable ORFs
What is codon bias?
The tendency of a on organism’s DNA sequence to use a degenerate codon for an amino acid (ex. E. coli prefers CGU, while fruit flies prefer CGC)
How is codon bias used to distinguish genomic regions?
Some species will have a preference for certain codons for an amino acid, so those codons are more likely to appear in ORFs
What percentage of total ORFs detected have a clearly identified function? Why?
70% or less, as many genes are misidentified
What are hypothetical proteins?
Uncharacterized ORFs that encode proteins that likely exist but whose function is currently unknown
What is a major cause of hypothetical proteins?
Lack sufficient amino acid sequence homology with known proteins for identification (cannot be compared to known proteins)
When are hypothetical proteins most common?
Common in genomes of uncultured environmental bacteria, as they cannot be intensively studied
Errors via the annotation telephone are caused by what?
Predicting the function of a gene based on similarity to genes that encode characterized enzymes
What evidence suggests horizontal gene flow is common amongst prokaryotes?
- presence of genes typically found in only distantly related species
- presence of a DNA with GC content or codon bias that differs significantly from the rest of the genome
- presence of mobilome genes (transposons, integrases, insertion sequences)
- often encode resistance, virulence functions (non-essential genes)
- often occur in clusters known as Genomic Islands within the genome
What are the three pathways of horizontal gene transfer?
Transformation, Transduction, and Conjugation
How many genes can be located in 1Mb of DNA? 1 kb?
1000 genes, 1 gene
What is the trend in ORF number as genome size increases?
Positive linear
Why are metagenomes so complex?
Since they compare multiple organisms, there’s lots of repeats in different genomes. Closely related strains are particularly problematic. Also there is commonly low coverage of rare genomes (many sequencing gaps)
What do cells need to grow?
- water
- carbon source
- macro + micronutrients
- energy source
- reducing power
What are the two types of carbon sourcing? Explain them
- heterotrophs acquire carbon from existing organic molecules
- autotrophs acquire carbon from generating their own organic molecules out of CO2
What are the three types of energy sourcing? Explain them
- phototrophs acquire energy from light
- chemolithotrophs acquire energy from oxidizing inorganic molecules
- chemoorganotrophs acquire energy from oxidizing organic molecules
Catabolism
Energy-releasing metabolic functions that break down molecules
Anabolism
Energy-requiring metabolic functions that synthesize new molecules
Compare delta G to delta G knot
delta G describes cellular conditions where delta G knot describes the standardized conditions of a lab
Exergonic reactions
Negative G, release free energy, spontaneous
Endergonic reactions
Positive G, require free energy, non-spontaneous (require ATP)
What is reducing power?
The ability to donate electrons during a reaction. The more negative the redox potential, the better the molecule will lose electrons (become oxidized) and vice versa
What is the best reducing agent? Why?
Glucose. Has the most negative E
What is the best oxidizing agent? Why?
Oxygen. Has the most positive E
An electron donor is:
Oxidized, a reducing agent
An electron acceptor is:
Reduced, an oxidizing agent
How do you calculate the delta E of a redox reaction?
E (reducing agent) - E (oxidizing agent) = delta E
Compare coenzymes and cofactors
Coenzymes are organic molecules used to aid enzymes in their reactions. Cofactors are metallic ions used to aid enzymes in their reactions. They are not seen as reactants
What are three good energy conservation molecules? Why?
PEP (phosphoenolpyruvate), ATP, and Acetyl-CoA; they have the most negative delta G
What are the three energy conservation mechanisms?
- substrate-level phosphorylation
- oxidative phosphorylation
- photophosphorylation
What is substrate-level phosphorylation?
Phosphate groups from organic molecules are transferred to ADP
What is oxidative phosphorylation?
Electron flow generates PMF for chemiosmosis using inorganic phosphates to make ATP (ATP synthase)
What is photophosphorylation?
Photons (light) power the formation of PMF for chemiosmosis
Describe energy sources for respiration
May be organic or inorganic and acts as the reducing agent (gets oxidized)
Describe terminal electron acceptors in respiration
May be oxic or anoxic and acts as the oxidizing agent (gets reduced)
What separates fermentation from respiration?
Fermentation does not involve the ETC or generation of a PMF for chemiosmosis. Also, ATP is produced by both substrate level phosphorylation and chemiosmosis in respiration
Where are the best terminal electron acceptors found on the redox table? What are they usually?
Found at the bottom; usually inorganic, have a positive E, and are oxidizing agents
Glycolysis uses what kind of ATP production?
Substrate level phosphorylation
Where is the free energy from glucose stored following glycolysis?
Most of it is within 2 pyruvate, and a lot of it is in 2 NADH
How is the free energy in pyruvate released?
Oxidation via the Krebs cycle and reduction by NADH via fermentation
How is the free energy in NADH released?
Oxidation via ETC (respiration), fermentation, or other cellular redox reactions
What is NADPH?
A molecule similar in function to NADH that is used in some species of bacteria
Why is aerobic respiration the best way to generate PMF and ATP?
Because the redox potential from start to finish is the largest (1.14V) possible
What are the best electron acceptors and donors for respiration?
O2; glucose
True/False? Most species are restricted to one metabolic pathway
False. Many have more than one
What are the possible pathways for metabolism for chemoorganotrophs?
Fermentation, anaerobic respiration, and aerobic respiration
What metabolic strategies align with obligate anaerobes?
Fermentation or anaerobic respiration (both organo and litho)
What metabolic strategies align with obligate aerobes?
Aerobic respiration (both litho and organo)
What metabolic strategies align with facultative anaerobes?
Fermentation and aerobic respiration (both litho and organo)
Provide some examples of organic and inorganic electron donors
Organic: glucose, NADH, FADH2
Inorganic: H2, H2S, Fe2+, NH4+
Describe energy sources for fermentation
Organic and acts as the reducing agent
Describe the terminal electron acceptor for fermentation
Organic and acts as the oxidizing agent
How does fermentation maintain the balance of redox reactions within the cell?
Oxidizes NADH so it can be used for respiration if that mechanism is available
How is ATP generated in fermentation?
Substrate level phosphorylation
What kind of environment do some bacteria live in where they use fermentation as their primary ATP source?
Nutrient poor, they grow slowly
Compare the net ATP generated by fermentation vs. respiration
2 ATP vs. 34-38 ATP
What is homofermentative fermentation? What bacteria uses it?
Fermentation that results in one type of product; Lactobacillus spp. (lactate)
What is heterofermentative fermentation? What bacteria uses it?
Fermentation that results in 2 products; Lactobacillus spp. (lactate and ethanol)
What is mixed-acid fermentation? What bacteria uses it?
Similar to heterofermentative fermentation, except produces a wider range of products; E. coli (SELF = succinate, ethanol, lactate, formate)
What is butanediol fermentation? What bacteria uses it?
Conversion of 2 pyruvate into butanediol; E. aerogenes
What fermentation techniques does Clostridia use? What is Clostridia?
An obligate anaerobe; uses proteolytic and saccharolytic fermentation
Describe proteolytic fermentation. What are its products?
Use of amino acids to generate ATP via substrate level phosphorylation; acetate
Describe saccharolytic fermentation. What are its products?
Use of glucose to generate ATP beyond glycolysis via substate level phosphorylation; butyrate and acetate
What kind of fermentation (homofermentative or heterofermentative) is proteolytic fermentation? Saccharolytic fermentation?
Homo; hetero
What is secondary fermentation? What bacteria uses it?
Fermentation products from one organism are the substrate for another; Propionibacterium (turns 3 lactate into 2 propionate and acetate)
What are the major e- acceptors (in order) used for anaerobic respiration?
Nitrate (NO3-), ferric iron (Fe3-), sulfate (SO4 2-), CO2
No Fucking Shit Cunt
True/False? Anaerobic bacteria will always use ferric iron as a final electron acceptor if available
False. Will use nitrate first, if available
Why are energy yields in anaerobic respiration lower than aerobic respiration?
The difference in redox potentials is lower in magnitude
What fermentation method is used to make cheese?
Secondary fermentation via Propionibacterium
Describe the process E. coli uses to generate energy in the absence of O2. What metabolic and respiratory strategy is this?
NITRATE REDUCTION
- reduces nitrate to nitrite with nitrate reductase (would regularly use complex III/IV with O2)
- generates less PMF than with O2
Anaerobic organotrophy
Describe the process Pseudomonas stutzeri uses to generate energy in the absence of O2. What metabolic and respiratory strategy is this?
DENITRIFICATION
- reduces nitrate to nitrite (nitrate reductase) to NO to N2O to N2
Anaerobic organotroph
Why is denitrification beneficial?
Sewage treatment requires denitrification filters
What is denitrification detrimental?
Loss of fertilizer to the atmosphere, N2O is a greenhouse gas, and NO reacts with ozone and water to form nitric acid (acid rain)
Why is N2O a problem if denitrifying bacteria can convert it into NO (and N2)?
Because it is gaseous, some may escape before it is used by denitrifying bacteria
Describe the process Geobacter uses to generate energy. What metabolic and respiratory strategy is this?
IRON REDUCING
- oxidizes acetate to reduce Fe3+ (final e- acceptor)
- other species may reduce other metals, like uranium
Anaerobic organotrophy
Why is Geobacter unique?
Energy production via iron-reducing is performed outside of the cell on electrically conductive pili to avoid iron build-up in the cell
Is metal-reducing as a form of energy restricted to just iron?
No, may also be used for other metals such as uranium, which allows it to be taken out of solution to be collected
Describe the process Desulfovibrio uses to generate energy. What metabolic and respiratory strategy is this?
SULFATE REDUCTION
- uses organic compounds as e- donors and sulfates as e- acceptors
- reduces sulfate to sulfite to sulfide (H2S)
- may use assimilative or dissimilative sulfate reduction
Anaerobic organotrophy
What is assimilative reduction?
When the reduced compound is used to generate organic compounds, like amino acids
What is dissimilative reduction?
When the reduced compound is excreted from the cell
True/False? Desulfovibrio requires an activation step to reduce sulfate
True
What is methanogenesis?
The reduction of CO2 to methane by anaerobic archaea. Goes hand-in-hand with acetogenesis (reduction of CO2 to acetate). Usually, the microorganisms are alkaliphiles
True/False? Methanogenesis is a commonly used form of metabolism, as CO2 is easily accessible
False. It generates a very very small amount of energy and thus is not favourable to rely on as a primary energy source
What do lithotrophs use to generate PMF?
Use inorganic electron donors (get oxidized)
What are the commonly used electron donors of lithotrophs? Which are used preferably?
Ammonium, Fe2+, H2S, and H2 in that order
What pattern do you notice in the list of lithotrophic e- donors?
They are the waste products of anaerobic respirators, so usually these bacteria are found together
What respiration and metabolic strategy do most lithotrophs use?
Aerobic lithoautotrophy
Describe the process Ralstonia uses to generate energy. What metabolic and respiratory strategy is this?
H2 OXIDATION
- oxidizes H2 into water, using O2 as the final electron acceptor
- may use CO2 in methanogenesis
- uses H2 to generate NADH for the Calvin cycle with hydrogenase
Aerobic lithoautotrophy
Describe the process Acidithiobacillus ferrooxidans uses to generate energy. What metabolic and respiratory strategy is this?
IRON OXIDATION
- oxidizes Fe2+ to Fe3+
- uses O2 as the final electron acceptor, although it may also use nitrate (nitrate reduction)
- uses reverse e- flow to generate NADH for Calvin cycle (insufficient energy like Ralstonia
- prevalent in acidic, aqueous environments (acidophiles)
Aerobic lithoautotrophy
Why is iron used both as an electron donor and an electron acceptor?
In Acidithiobacillus ferrooxidans, its final e- acceptor is O2 (lower E), so Fe2+ is oxidized to Fe3+. Whereas in Geobacter, its final e- acceptor is Fe3+, so acetate (higher E) is oxidized
Describe the process Nitrosomonas uses to generate energy. What metabolic and respiratory strategy is this?
NITRIFICATION
- oxidizes ammonia to nitrite, using O2 as the final e- acceptor
- uses reverse e- flow to generate NADH for Calvin cycle
- important for N cycle
- assimilative and prevalent in ammonia-rich soils, wastewater, and manure
Aerobic lithoautotrophy
Describe the process Nitrobacter uses to generate energy. What metabolic and respiratory strategy is this?
NITRIFICATION
- oxidizes nitrite to nitrate using O2 as the final e- acceptor
- uses reverse e- flow to generate NADH for the Calvin cycle
- important for N cycle
- prevalent in ammonia-rich soils, wastewater, and manure (located near ammonia-oxidizing bacteria like Nitrosomonas)
Aerobic lithoautotrophy
Describe the process Thiobacillus uses to generate energy. What metabolic and respiratory strategy is this?
SULFUR OXIDIZER
- oxidizes elemental sulfur and sulfide (H2S) into sulfite and sulfate
- uses O2 (preferred) or nitrate as the final e- acceptor
- uses reverse e- flow to generate NADH for the Calvin cycle
- prevalent in hot springs, hydrothermal vents
Aerobic lithoautotrophy
How is phototrophy different from chemotrophy?
Phototrophy uses energy collected from light to drive e- flow, while chemotrophs use compounds
What metabolic strategy do most phototrophs use?
Autotrophy (fix CO2 with reducing power)
True/False? Phototrophs are only known to be oxygenic (generate O2)
False. May also be anoxygenic
Which type of cell, prokaryotic or eukaryotic, is oxygenic? Anoxygenic?
Eukaryotic; prokaryotic
What do anoxygenic phototrophs use to reduce/fix CO2 and generate ATP?
Oxidize H2S to SO4 2- using solar energy
What do oxygenic phototrophs use to reduce/fix CO2 and generate ATP?
Oxidize H2O to O2 using solar energy. Uce Calvin cycle to fix CO2
Describe the process Chromatium spp. uses to generate energy. What metabolic and respiratory strategy is this?
ANOXYGENIC PURPLE SULFUR BACTERIA
- uses Q-type photosystem and cyclic e- flow to generate PMF
- oxidizes H2S to SO4 and use reverse e- transport to generate reducing power
- uses Calvin cycle to fix CO2
Anaerobic photoautotrophy
What may Chromatium spp. use in the event that H2S runs out? How do we know this?
Uses elemental sulfur; contains sulfur globules, which are visible under the microscope
Why does Chromatium spp. require an external electron source if it uses cyclic e- flow?
Requires electrons from H2S to power the Calvin cycle for carbon fixation
Describe the process Chlorobium spp. uses to generate energy. What metabolic and respiratory strategy is this?
ANOXYGENIC GREEN SULFUR BACTERIA
- oxidizes H2S to elemental sulfur to SO4
- uses ferredoxin as a final e- acceptor
- uses FeS-type photosystem
- carbon fixation by reverse citric acid cycle (do not use Calvin cycle)
- use chlorosomes (low levels of light)
Anaerobic photolithoautotrophy
Why is it hypothesized that Chlorobium spp. doesn’t use cyclic e- flow?
Once ferredoxin is reduced, it does not re-enter the cycle. Instead, H2S is used to replace the e- that ferredoxin took
Describe the process Prochlorococcus uses to generate energy. What metabolic and respiratory strategy is this?
OXYGENIC CYANOBACTERIA
- oxidizes H2O to O2
- uses both Q-type and FeS-type reaction centres and linear e- flow
- electrons used to generate PMF and reduce NADP+
- carbon fixation via Calvin cycle
Aerobic photolithoautotrophy
What is the order, from top to bottom, of bacteria you would expect to see in a Winograsky column?
- cyanobacteria (Prochlorococcus)
- heterotrophic bacteria (E. coli)
- iron-oxidizing bacteria (Acidithiobacillus ferrooxidans)
- purple sulfur bacteria (Chromatium spp.)
- green sulfur bacteria (Chlorobium spp.)
- sulfate-reducing bacteria (Desulfovibrio)
- methanogens
What are the pairs of bacteria that reverse each other’s metabolisms?
- E. coli and Pseudomonas stutzeri vs. Nitrobacter
- Geobacter vs. Acidithiobacillus ferrooxidans
- Desulfovibrio vs. Thiobacillus, Chromatium spp. and Chlorobium spp.
What is the main difference between lithotrophic and phototrophic sulfide oxidation?
Lithoautotrophs use O2 or another oxidant to oxidize H2S or elemental sulfur, producing energy and electrons needed to fix CO2. Phototrophs use light to generate energy and H2S to generate electrons (anoxygenic)
What metabolic strategy dominates the fixation of CO2 into organic matter?
Autotrophs
What are the dominant photoautotrophs in terrestrial environments? What do they use to fix carbon?
Plants; Calvin cycle
What are the dominant photoautotrophs in aquatic environments? What do they use to fix carbon?
Algae, bacteria, and archaea (also chemolithotrophs); Calvin cycle, rTCA, and many other processes (very versatile!)
Carbon is returned to abiotic reservoirs by:
Organotrophs
In oxic environments, CO2 must be ___________ to be fixed by ___________ and _________________.
Reduced; chemolithotrophs and oxygenic photosynthesis
In anoxic environments, CO2 must be ___________ to be fixed by ___________ and _________________.
Reduced; acetogenesis and anoxygenic photosynthesis
In oxic environments, organic carbon must be ___________ to be consumed as ____________ through ______________.
Oxidized; energy; respiration
In anoxic environments, organic carbon must be ___________ to be consumed as ____________ through ______________.
Oxidized; energy; anaerobic respiration and fermentation
The reduction of CO2 into CH4 is called _____________________, and the reverse reaction is called __________________.
Methanogenesis; methanotrophy
What is meant by comammox?
Complete ammonia oxidation (ammonia oxidized directly to nitrate without nitrification step)
What are the three products ammonia may be converted to?
Nitrite (oxidation, nitrification), nitrate (oxidation, comammox), N2 (oxidation)
What is anammox?
Ammonia is oxidized and nitrite is reduced to generate N2, requires an anammoxosome
What is an anammoxosome?
A pseudo-organelle that generates PMF to convert nitrite into N2 (denitrification)
What are the four types of microbial symbioses?
- parasitic (expense at host)
- pathogenic (causes disease in host)
- commensal (neutral on host)
- mutualistic (host benefits)
What are the fixed sources of nitrogen?
Ammonia and nitrate
Leguminous plants harbor _____________ in a _____________ process
Rhizobia; mutualistic
How is denitrification countered?
Using a biofertilizer with nitrogen-fixing bacteria
What is the rhizosphere?
The microenvironment surrounding the roots of legumes caused by the secretion of flavonoids
What are flavonoids?
Chemoattractants that legumes secrete to attract rhizobia and promote their growth
What is a cross-inoculation group?
When symbiosis is specific between certain plant species and certain rhizobial species (high specificity)
What does the cross-section of a newly-infected root look like?
The infection thread can be seen coming through the root hair and accumulating within the root (rhizobia). Major plant growth can be seen around this accumulation (lots of cells)
What is the symbiosome?
A compartment within the host cell that houses the endosymbiont (rhizobia)
What is a bacteroid?
A rhizobium within plant ells that has become swollen, misshapen, and branched. Loses its motility and independence
How do bacteroids start the nodule formation process?
Secrete an oligosaccharide (Nod factors) that:
- induce root hair curling
- trigger plant cell division
- trigger nodule formation
Describe the mutualistic relationship between legumes and rhizobia
Rhizobia (bacteroids) import organic acids from the plant (succinate, malate, fumarate, and pyruvate) for ATP production and to provide reducing power for N fixation via nitrogenase (N2 to NH3). The bacteroid exports NH3 to the plant
SMFP: Suck My Fucking Pussy (sorry vulgar)
What metabolic strategy do rhizobia use when they convert into bacteroids?
Aerobic Chemoorganoheterotrophy
Is the nitrogen fixation function of bacteroids assimilative or dissimilative? Why?
Dissimilative because it exports the product (NH3) away (to the plant)
Why is nitrogenase function complicated in root nodules? How is the issue solved?
Rhizobia require O2 to generate energy for N2 fixation, but nitrogenases (enzymes required for N fixation) are inactivated by O2. This is remedied by leghemoglobin, an O2 binding and carrier protein synthesized by the plant
What colour is leghemoglobin?
Red
Describe the function of leghemoglobin
It binds O2 and carries it to complex IV (ETC) for reduction to H2O, which allows nitrogenase to function without being inhibited by the presence of O2
True/False? Plant cells usually only carry a couple symbiosomes with a large volume of bacteroids
False. Carry a large number of symbiosomes, each harboring a couple bacteroids to increase SA
What is Chlorochromatium aggregatum?
A freshwater mutualistic microbial consorta consisting of epibionts (green sulfur bacteria, Chlorobium) and an unnamed flagellated rod-shaped bacterium. The Chlorobium piles up onto the unnamed flagellated rod-shaped bacterium, using it for motility (light, sulfide, oxygen) while it uses anoxygenic photosynthesis to fix CO2
How many epibionts can be associated in Chlorochromatium aggregatum?
13-69
The entire set of genetic elements in microbes include:
- chromosomal DNA
- plasmids
- transposable elements
What is the central dogma?
DNA -> mRNA -> protein
What is the typical genome size for microbial genomes?
4-8Mb for 4000-8000 genes (ORFs)
An endosymbiont has the genome size of:
0-1Mb (up to 1000 genes)
A typical parasite has the genome size of:
1-2Mb (1000-2000 genes)
A typical free-living organism has the genome size of:
> 2Mb (over 2000 genes)
What are the criteria of Chargaff’s rule?
- A = T, G = C
- A + G = T + C = 50%
- A + T ≠ G + C
Typical prokaryotic chromosomes are ________________ supercoiled by (enzyme)
Negatively; DNA gyrase
List the key characteristics of prokaryotic chromosomal DNA
- large (4-8Mb)
- carries essential housekeeping genes
- one copy per cell
- replication strongly regulated
List the key characteristics of prokaryotic plasmid DNA
- small (1-1000kb, 1-100 genes)
- carries bonus genes (antibiotic resistance, sugar-metabolizing, etc) not needed for life in normal conditions
- 1-100 copies (copy number)
- dozens-100s of different types of plasmids
- independent replication
What are insertion sequences?
Small transposable elements located within plasmids
What is theta replication?
Semi-conservative replication of a bacterial chromosome
How many ori are possible for each plasmid and chromosome?
1
What is the replisome?
Collection of DNA replicative machinery
Describe the importance of σ70
It is a transcription factor responsible for the recognition and binding of the consensus sequence and subsequent recruitment of RNA Pol for housekeeping genes
What are the promoter sequences of importance?
-10 (Pribnow) and -35
Describe the importance of the consensus sequences and σ70 recognition
σ70 binds with different affinities for each variation of the consensus sequence, thus has some regulatory control over transcription
RNA Pol holoenzyme
Includes RNA Pol and σ70 during promoter recognition and transcription initation
RNA Pol core enzyme
After the start of transcription, σ70 dissociates, leaving the core enzyme to transcribe the rest of the DNA
Describe transcription termination
Inverted repeats in the DNA result in the formation of a termination stem loop (complimentary base pairing of daughter strand with itself) followed by the polyA region. This causes RNA Pol to stall and fall off the DNA
What is an operon?
Genes for enzymes of related functions occur sequentially within the genome under the control of a single promoter
Polycistronic mRNA
mRNA that includes more than one ORF, used for operons
Activator proteins
Positive regulation of transcription, bind upstream of promoter at activator binding site
Repressor proteins
Negative regulation of transcription, bind between promoter and transcription start site (operator) to prevent function of RNA Pol
Effector molecules
Molecules that allow or prevent TFs binding DNA, inducers and corepressors
Inducers
Induce transcription
Corepressors
Prevent transcription
Describe the function of the Arg synthesis pathway
When the end product (Arg) is present, it acts as a corepressor and binds to a repressor. Together, they form the corepressor-bound repressor protein that binds to the operator for Arg-synthesis genes. RNA Pol can’t transcribe the gene, so the cell stops making arginine.
In the absence of Arg, the repressor cannot bind to the operator, allowing for transcription
What kind of enzyme control is used in the Arg synthesis pathway?
Enzyme repression
Describe the function of the lactose metabolization pathway
In the presence of lactose, it acts as an inducer and binds to the repressor protein, reducing its affinity for the operator. With the repressor off the operator, RNA Pol may carry out transcription and produce the products needed to metabolize lactose. Repressor binds operator in absence of lactose
What kind of enzyme control is used in the lactose metabolization pathway?
Enzyme induction
Describe the function of the maltose metabolization pathway
In the presence of maltose, it acts as an inducer and binds to an activator. The induced activator binds upstream of the promoter and improve σ70 and RNA Pol binding. This leads to the transcription of maltose-metabolizing genes and thus the metabolism of maltose. In maltose’s absence, the activator cannot bind to the activator binding site
What kind of enzyme control is used in the maltose metabolization pathway?
Enzyme activation
What is the RBS for prokaryotes?
Shine-Delgarno sequence
What are the differences between prokaryotic and eukaryotic translation?
Prokaryotic:
- polycistronic mRNA (multiple RBS due to multiple ORFs for added level of regulation)
- doesn’t require processing (no cap or tail (shorter life span), no introns)
- 70S ribosomes (euk is 80S)
- uses fMet
- codon bias differs among species
- not all ORFs are in the same reading frame
- some species use “stop codons” for selenocysteine and pyrrolysine
Why are transcription and translation coupled in prokaryotes?
Because they don’t have a nucleus, so ribosomes are free to bind to newly synthesized RNA
What is a polysome?
Many ribosomes translating the same transcript at once
If the transcription start site is on the left side of a photo, describe the positions of the ribosomes and why
Longer chains of ribosomes will be found on the right side of the photo, as the longest transcript will be there. The shortest transcripts will be nearer to the transcription start site and thus the polysomes will be smaller too
Why do most prokaryotic proteins not require special transport to targeted locations?
Prokaryotes lack organelles, so most proteins (except membrane or periplasm) are in the cytoplasm
What post-translational modifications may prokaryotic proteins need?
Chaperones may assist in folding, adding cofactors, quaternary structures
At what state (on/off) is the Arg operon until cellular conditions change?
Constitutively on until [Arg] increases (addition of repressor)
What is an inverted repeat?
A DNA sequence that has the identical sequence to another part of the DNA a few bases up/downstream. During transcriptional termination, they base pair, forming a stem loop
At what state (on/off) is the lactose operon until cellular conditions change?
Constitutively off until lactose present (removal of repressor)
At what state (on/off) is the MalT operon until cellular conditions change?
Constitutively off until maltose present (addition of activator)
Describe how sRNA can turn translation off (2 ways)
- can bind RBS, blocking access for ribosome
- can recruit ribonuclease to degrade mRNA
Describe how sRNA can turn translation on (2 ways)
- can bind the 5’ end of the mRNA, preventing it from binding to itself and making the RBS inaccessible
- can bind 3’ end of the mRNA preventing ribonuclease digestion
What is sRNA?
Small RNA (40-400bp) that base pairs with mRNA, allowing for the regulation of translation
What is a riboswitch?
A level of translational regulation in which the RBS may be made inaccessible to ribosomes upon aptamer binding
Explain how a riboswitch works
Upstream of the RBS, a stem loop is formed by regions 1 and 2. Region 3 (RBS) is unbound and thus translation may begin. When the metabolite (serine) binds within the loop, it displaces the stem loop, which now forms between regions 2 and 3, known as the expression platform. This disables ribosome binding to region 3, thus no translation occurs
Describe attenuation when trp is present
Coupled transcription and translation allows the ribosome to translate the leader sequence, which requires 2 trp. In high trp abundance, the leader sequence is translated quickly, which causes the ribosome to block regions 1 and 2 on the mRNA. This causes regions 3 and 4 to form a hairpin that terminates the transcription of the rest of the gene
Describe attenuation when trp is absent
Coupled transcription and translation allows the ribosome to translate the leader sequence, which requires 2 trp. In low trp abundance, the leader sequence is translated slowly, which causes the ribosome to stall and block region 1 on the mRNA. This causes regions 2 and 3 to form a hairpin that allows the transcription of the rest of the gene
Why is the trp operon only found in prokaryotes?
It requires coupled transcription and translation
What are the three possible pathways foreign DNA may be utilized after horizontal gene transfer has occurred?
- may be degraded by nucleases to provide the recipient with nucleic acids
- may exist and replicate autonomously (plasmids)
- may recombine with the recipient chromosome
How can recombination of horizontally-transferred genes occur?
- RecA-mediated homologous combination (new DNA swapped in for “old”)
- site-specific (non-homologous) recombination (insertion into the chromosome)
What is transformation? Explain its mechanism
Naked DNA is uptaken from the environment due to the death of a donor cell. Competent cells extend their type IV pilus to grab dsDNA, which then contracts and brings it into the periplasm (GN) or through the cell wall (GP). Rec2/Com transporter degrades one strand as it moved through the membrane, making ssDNA. Then, RecA-mediated recombination may occur if sufficient homology exists, otherwise, degradation
What makes a cell competent? What are some species that do this?
The presence of the Rec2/Com transporter determines competence. Streptococcus, Bacillus, and Vibrio
How may dsDNA be absorbed into the cytoplasm, bypassing the Rec2/Com transporter?
If the cell membrane is punctured, then dsDNA may make its way through. This only occurs in lab
Are all cells with a type IV pilus competent? Why or why not?
No, because although they have the necessary structure, they’re unable to bind dsDNA with the tip of their pilus. Plus, they must also have a Rec2/Com transporter
What is conjugation?
The transfer of a plasmid from a donor cell (F+) to a recipient cell (F-). It requires cell-cell contact through the pilus
True/False? All plasmids that move through the pilus during conjugation are conjugative
False. Conjugative plasmids contain the genes required for conjugation, but non-conjugative plasmids may also move between cells during this process (piggy-back on conjugative plasmids)
If RecA-mediated recombination is utilized, what happens to the genotype of the chromosome?
Genotype changes (genotype is not stationary)
What level of regulation does attenuation work at?
Translational control of transcription (transcriptional regulation)
Which organism has the best studied conjugation system?
E. coli
Which region of the plasmid is necessary for the distinction between conjugative and non-conjugative plasmids?
tra (transfer) region
What are the regions of an F plasmid and their respective functions?
- tra region: encodes F pilus and type IV secretion system
- oriV: plasmid DNA replication
- oriT: where one strand of the plasmid is broken, allowing for the transfer of the plasmid into the recipient cell
- IS and Tn sequences: recombination
What stops F+ cells from receiving more plasmids from other F+ cells?
F+ cells lack the F pilus receptor, so they do not attach to the F pilus of other bacteria
What is type IV secretion?
Once the F pilus attaches to a bacteria and contracts, the bacteria form a bridge between them to pass the F plasmid through
Describe the steps of rolling circle replication
- induced by mating pair formation
- one strand is nicked at oriT
- as the nicked strand is rolled off the plasmid, it is replaced by DNA pol
- donated strand is moved into the recipient, circularizes, and a complementary strand is synthesized
What is an Hfr cell?
A donor cell in which the F plasmid has been integrated into the host chromosome as an episome via recombination at an IS sequence
What must the plasmid and chromosome have in common to become an Hfr cell?
They both must have homology at an IS site to allow for recombination
True/False? The genes on the F plasmid are no longer expressed in the form of an episome. The episome must be excised to allow for F pilus formation and type IV secretion
False. Genes can be expressed in episomal form
True/False? After mating with an Hfr cell, the recipient cell is now F+
False. Still F-
Describe the difference between Hfr rolling circle replication and F plasmid rolling circle replication
F plasmid RCR only involves the plasmid, while Hfr RCR involves the plasmid and the chromosome
Why is the recipient of an Hfr conjugation not F+?
The oriT is in the middle of the episome, so usually only half of the plasmid is conjugated. The tra region is the last to be transferred because its on the other side of the oriT
The type IV secretion bridge is unstable during conjugation. Explain why this is important for Hfr conjugation
Because the bridge is unstable, not all of the chromosome may be passed through the bridge before it breaks, so it’s unlikely that the entire donor chromosome is transferred
What may happen to the donor DNA following Hfr conjugation?
- degraded for extra nucleotides
- can recombine with recipient chromosome at high frequencies due to homology
What is an F’ plasmid?
An F plasmid that has previously been integrated into the chromosome, then excised, sometimes taking part of the chromosome with it. This allows for the transfer of chromosomal DNA with an F’ plasmid, creating a larger plasmid and smaller chromosome
How does an F’ plasmid happen?
When an IS sequence is located near the original IS sequence in which the plasmid was inserted, allowing for the chromosomal gene between these two IS sequences to be excised with the episome
What is the potential origin of plasmids?
Transposons
What is transduction?
The transfer of chromosomal DNA from a donor cell to a recipient via a phage vector
True/False? Not all phages are transducing and not all prokaryotes are transducible
True
Why is transduction medically and industrially important?
- mechanism of transferring virulence factors and antibiotic resistance among organisms
- mechanism of moving genes from one organism to another
Explain generalized transduction
Occurs during the lytic cycle where host DNA is randomly packaged inside a viral capsid instead of viral DNA. These defective phages may still adsorb to host cells, injecting bacterial DNA into the new host
What two pathways may bacterial DNA undergo after generalized transduction?
Degradation or recombination
Explain specialized transduction
Occurs during lysogenic cycle in which viral DNA is inserted into the host DNA at the att site. Once the lytic cycle is induced, some chromosomal DNA adjacent to the att site can be excised with the viral DNA, which has the chance of being inserted into the next host genome due to homology
What kind of phage is required for generalized transduction? Specialized transduction?
Virulent; temperate
What is phage conversion?
If the host phenotype is changed via specialized transduction
True/False? During specialized transduction, all of the viral genome is excised along with a partial host chromosome
False. Some viral DNA is left behind
What two forms may viruses exist in?
Virion and replicating genome
True/False? Viruses are found in all domains
True
What is a replicating genome?
The intracellular version of a virion that is only composed of genetic material. Not infectious unless directly injected
What is a virion?
An extracellular subcellular particle that is infectious due to its adsorption structures
What are the structures virions can be found in?
Helical, icosahedral, and complex (non-symmetrical)
What are the structural components of virions? Functions?
- capsid (stores genetic material as a protein coat made up of capsomeres)
- envelope (PPL bilayer, often contains glycoprotein spikes)
- sometimes viral enzymes (required for infection, packed within the capsid)
What is a naked virus?
A virion lacking an envelope, only has a capsid
List the characteristics of viral genomes
- DNA or RNA
- single or double stranded
- linear or circular
- large size range (3-2500 genes)
True/False? Viral DNA is smaller than the DNA of the bacteria they infect
False. While most are smaller, there are some viruses with a larger genome size than their targets
Provide an example of a complex virion
Bacteriophage (phage is more general)
What is a plaque assay?
A bacterial lawn is prepared on an agar plate, then a viral solution is poured over top. The cleared areas (plaques) are where bacteria have been killed by viruses
How may viral concentration be measured? What is viral concentration called?
Counting plaque-forming units (PFU); titer
Describe each stage of the viral growth curve
- virus count decreases at first (infecting bacteria)
- titer remains constant (eclipse phase)
- titer increases drastically (maturation phase)
When does cell lysis happen on the viral growth curve?
Transition between eclipse and maturation phases
What is the latent period?
The combination of the eclipse and maturation stages of a viral growth curve where no bacteria are being actively infected
How do virions target and attach to their hosts?
Use extracellular components such as receptors, channels, glycoproteins, pili, flagellum, etc
The lytic cycle can begin with infection via the ________ phage
Virulent and temperate
Provide an example of a virulent phage and its host
T4, infects E. coli
How does T4 attach to its host?
Tail fibers adsorb to LPS carbohydrates
How does T4 penetrate its host?
- tail fibers retract, bringing tail pins into contact with the PPL bilayer
- tail sheath contracts, pushing tail tube through outer membrane
- T4 lysozyme degrades peptidoglycan to form a pore in the cell wall
- dsDNA moves into the cytosol, passing the inner membrane
Explain the steps for T4 synthesis, assembly, and release
Early protein synthesis
1. Host RNA Pol transcribes a series of viral genes only expressed at the start of infection (anti-sigma factor proteins and phage-specific replisome)
Late protein synthesis
2. Synthesis of capsomeres, tail tube, and tail fiber proteins, as well as enzymes to facilitate assembly
3. Synthesis of packaging motor complex, filling capsids with DNA
4. Remaining virion components assemble
5. Synthesize enzymes that compromise host cell membrane and wall
6. Cell lysis
What is the function of the anti-sigma factor protein?
It inhibits the host sigma factors to stop host transcription and translation
Why does host RNA Pol transcribe the viral genes?
They have stronger promoters than the host, so RNA Pol has greater affinity for them
True/False? DNA packaging into capsids is GTP-dependent
False. ATP-dependent
What is a concatemer?
A very long piece of genetic information composed of viral genomes attached end-to-end, creating a very long linear strand
Describe headful packaging
During assembly, the phage DNA is moved into each capsid until it is full and the cut from the rest of the molecule
Why do phages that use headful packaging have different gene orders?
Because more than a genome-length of DNA fits into each capsid, the DNA is cut off at different sites, and each progeny virion has a different gene order
What does it mean that the T4 genome is terminally redundant?
Genes at one end of the molecule are repeated at the other end
The lysogenic cycle can begin with infection via the ________ phage
Temperate
What is a latent infection?
The viral genome is replicated as the host continues to grow and divide
What is an example of a temperate phage and the host it infects?
λ page, E. coli
What is a lysogen?
A bacterial cell with a prophage
Describe the steps for λ adsorption
- λ tail attaches to a host maltose transport protein
- penetration similar to T4
- 5’ ends of λ DNA contain cohesive ends, that are complimentary, so they bind and circularize the viral genome within host cytosol
What is a cos site?
The location where the cohesive ends have joined, the sequence is palindromic
True/False? Temperate bacteria usually result in host cell lysis
True. While they may allow entry into the lysogenic cycle, they usually just enter the lytic phase
When does λ enter the lysogenic cycle?
If the λ repressor (cI) gene is expressed upon penetration, cI accumulates and the λ genes are repressed. This causes λ DNA to become a prophage
When does λ enter the lytic cycle?
If the cro repressor gene is expressed upon penetration, cro accumulates, and the cI gene is repressed. This causes λ to enter the lytic cycle
How does λ switch from lysogenic to lytic?
A few λ genes are expressed during lysogeny to monitor the host stress levels and induce a switch to the lytic cycle if conditions deteriorate. This allows the phage the replicate before it dies with the host
How is λ integrated into host DNA?
λ integrase nicks the viral and host DNA at att sites, causing sticky ends that bind to each other, allowing for the prophage’s insertion
How is λ excised from host DNA?
λ excision enzyme cuts the prophage out of the host chromosome at the att sites
True/False? Bacterial DNA requires an att site for the λ DNA to integrate
True. Both the viral and host DNA have the att site so sufficient homology exists to allow for recombination
How does λ synthesis happen during the lytic phase?
Similar to T4 synthesis, except it does not use headful packaging
What is the difference between T4 and λ concatemers?
T4 is linear, while λ is circular, so it requires rolling circle replication (no headful mechanism, 1 genome/capsid, sticky ends)
Describe rolling circle replication in λ. What does this remind you of?
The λ DNA is copied continuously, generating long concatemers. Individual λ genomes are cut from the concatemer at each cos site and packages into assembling phage capsids; Hfr and F+ plasmid replication during conjugation
If a respiratory virus finds its way onto the skin of its host, does it cause an infection? Why or why not?
No. Viruses are specific to the tissues they infect, so a respiratory virus must be breathed in for it to cause an infection
What are the three differentiating features of eukaryotic vs. prokaryotic viruses?
Eukaryotic:
- capsid enters host
- host cells have a nucleus
- occasional presence of a viroplasm
What is a viroplasm?
A membranous cellular component in which viruses replicate. Sometimes called a virus factory
What are the four possible outcomes of a eukaryotic viral infection?
- Virulent infection (cell lysis)
- Latent infection (provirus)
- Persistent infection
- Transformation
Describe a persistent infection
Virions replicate inside the host and leave by budding out of the cell membrane, leaving the cell alive for more replication of the virus
Describe transformation in relation to eukaryotic viruses
The infection results in cancer either because:
- virus produces enzymes that interfere with regular cell division
- viral genome inserts within a tumor-suppressor gene, causing it to be dysfunctional
What are the four differences of plant viruses vs. animal viruses?
Plant viruses:
- broad host range
- naked (no envelope)
- cell wall makes them harder to infect, so damage is needed for infection (via bugs)
- can infect adjacent cells through plasmodesmata
Why do plant viruses have a broad host range?
They lack spike proteins (specificity) since they have no envelope
True/False? Plant viruses may still induce cell lysis despite being different from animal viruses
False. The cell wall is too strong for the cells to lyse
What is the Baltimore Classification Scheme?
A widely used method of organizing viruses based on what the virus must do to produce mRNA
What is the mRNA produced for viral infection labelled as?
The plus, sense, or configuration strand
Describe the genome type of Class I viruses and the representative examples covered in lecture
- dsDNA genomes
- T4 and λ phages (prokaryotic)
- variola major: smallpox (eukaryotic)
What type of DNA replication (conservative/semi-conservative) do Class I viruses use?
Semi-conservative
Describe the steps for mRNA(+) synthesis in Class I viruses
The DNA(-) strand is used as a template to make mRNA(+)
Describe the genome type of Class II viruses and the representative examples covered in lecture
- ssDNA(+) genome
- ϕX174 (prokaryotic)
- parvovirus (eukaryotic)
Describe the steps for mRNA(+) synthesis in Class II viruses
First, the complimentary DNA(-) strand is synthesized via rolling circle replication. Then, the mRNA(+) is synthesized from the DNA(-) strand
In rolling circle replication, which end of the DNA (3’/5’) is inserted into the recipient?
5’ end (3’ is used for replication)
Describe the ϕX174 phage
- naked icosahedral virion
- ssDNA(+) circular genome (Class II)
- targets E. coli through adsorption of lipopolysaccharides
- capsids form spontaneously and exit via lysis
- has overlapping genes due to its small genome size (11 genes)
How can genes overlap, such as those in ϕX174?
The genes are transcribed in more than one reading frame
Describe parvovirus
- ssDNA(+) genome (Class II)
- humans and animals
- transmissible through aerosols and contact
- “slapped cheek disease”
What are two other names for parvovirus?
- Fifth Disease
- Slapped Cheek Disease
Describe the genome type of Class III viruses and the representative examples covered in lecture
- dsRNA genome
- ϕ6 (prokaryotic)
- Rotavirus (eukaryotic)
Describe the steps for mRNA(+) synthesis in Class III viruses
The mRNA(+) is made in the capsid by RNA replicase (from ssRNA(-)), then exported into the cytosol for translation. It’s also loaded into capsids with replicase, so that it may generate the ssRNA(-) within the capsid to restore the dsRNA
Why does the Class III genome remain in the capsid?
Because it’s mRNA, it gets degraded very quickly in the cytosol, so it must stay protected
What is unique about RNA replicase?
It is an RNA-dependent RNA polymerase, unlike the traditional RNA Pol we see (DNA-dependent)
Describe rotavirus
- dsRNA genome (Class III)
- naked and segmented icosahedral
- outer capsid is degraded in the lysosome, releasing the inner capsid into the cytosol
- translation and assembly occur in a viroplasm (ER) as an extra layer of protection
- causes diarrhea by increasing [electrolyte] in the intestine, water follows
- adsorbs to cell surface glycans on gut cells
What type of DNA replication (conservative/semi-conservative) do Class III viruses use?
Conservative
Describe the genome type of Class IV viruses and the representative examples covered in lecture
- ssRNA(+) genome
- poliovirus: poliomyelitis (eukaryotic)
What is unique about Class IV viruses?
Their genome can be directly translated into the viral proteins
Describe poliovirus
- naked icosahedral
- linear ssRNA(+) genome (Class IV) with polyA tail
- VPg protein facilitates ribosome binding, and the translation yields VPg, RNA replicase, protease, and capsid proteins
- VPg also acts as a primer for RNA replicase
- whole genome is translated at once and is cut up by proteases into the various viral proteins
- causes meningitis, encephalitis, paralysis
Describe the genome type of Class V viruses and the representative examples covered in lecture
- ssRNA(-) genome
- rabies and influenza
Describe influenza
- Class V virus (ssRNA(-))
- enveloped segmented pleomorphic virion (8 segments)
- HA and NA spike proteins
- uses budding
What is meant by pleomorphic?
The buds of a virus come in many different shapes due to “messy” process of wrapping the genome in the host cell membrane
What are HA and NA?
HA: hemagglutinin binds cell receptors (adsorption)
NA: neuraminidase facilitates virion release (budding)
How often are pandemics caused by influenza? What is used for vaccines?
Every 10-50 years, vaccine uses attenuated virus and mRNA based on spike proteins (HA). General vaccines are hard to make as the strains are constantly changing
Describe the genome type of Class VI viruses and the representative examples covered in lecture
- ssRNA(+) retrovirus
- HIV
What three modes does reverse transcriptase have, in order of use?
- reverse transcriptase (RNA to DNA)
- exoribonuclease (degrades ssRNA)
- DNA Pol III
Describe HIV
- enveloped segmented spherical virion
- 2 copies of linear ssRNA(+) genome (Class VI)
- virion carries reverse transcriptase, integrase, viral tRNA, and HIV protease
- gp120 and gp41 spike proteins bind CCR5/CD4 on macrophages and Th cells
- nucleocapsid released into cytosol following membrane fusion, releasing RNA close to the nucleus
Describe how HIV infects its host
- Virus adsorbs
- reverse transcriptase generates dsDNA in the capsid
- dsDNA is released and migrates into the nucleus
- integrase inserts genome into host chromosome permanently
- host RNA Pol generates viral mRNA for translation of viral polyprotein and for the genome of progeny
- polyprotein cleaved by HIV protease
- new virion spread by budding once ssRNA(+) strands loaded into capsids
Compare retroviruses and lysogenic viruses
Similarities:
- integrate into host genome (site specific)
- latent phase
Differences:
- provirus vs. prophage
- provirus is permanent
- lysogenic eventually switches to lytic, retrovirus buds
What is targeted by HIV? How does it manifest as AIDS? Does it kill the host itself?
Primate macrophages and Th cells targeted. Latent until virus reaches threshold titer, where symptoms are more severe. Fatal due to secondary infection (targets host immune system)
What are the possible treatments for HIV?
HAART:
- fusion inhibitors (blocks spike protein gp41)
- reverse transcriptase inhibitor (uses nucleoside analog Azidothymidine that cannot be added onto)
- integrase inhibitors (prevents provirus formation)
- protease inhibitors (prevents cleavage of polyprotein)
Currently no vaccine, but it may target the spike proteins
Describe the genome type of Class VII viruses and the representative examples covered in lecture
- partial dsDNA genome
- Hepatitis B
Describe Hepatitis B
- enveloped icosahedral virion
- partially dsDNA circular genome
- all genes overlap
- transcription produces ssRNA(+) the size of the genome
Which strand, + or -, is the partial strand in Class VII viruses?
DNA(+) is partial, -ve strand needed for full transcription of mRNA(+)
What is the advantage of having overlapping genes?
Multiple genes can be transcribed from the same stretch of DNA by initiating transcription in a different reading frame
True/False? The genome for Class VII viruses is assembled within the capsid, not packed into the capsid before its completion
True. ssRNA(+) is packed in, then reverse transcriptase synthesizes the partial dsDNA genome
Chronic infections of the liver caused by Hepatitis B can cause:
Cirrhosis and cancer of the liver
Which viral classes require RNA replicase?
Classes III, IV, and V
Which viral classes require reverse transcriptase?
Classes VI and VII
Describe the steps for mRNA(+) synthesis in Class VII viruses
- mRNA(+) synthesized from DNA(-) strand, then used as a template by reverse transcriptase to synthesize dsDNA
- DNA(-) used as template for mRNA(+) and genome
Describe the steps for mRNA(+) synthesis in Class VI viruses
- reverse transcriptase synthesizes dsDNA, by:
1. synthesizing ssDNA(-)
2. degrading ssRNA(+)
3. synthesizing dsDNA - integrase integrates viral dsDNA into host genome and DNA(-) used as a template for mRNA(+)
Describe the steps for mRNA(+) synthesis in Class V viruses
- RNA replicase synthesizes mRNA(+), then uses it as a template for mRNA(-) for the genome
Describe the steps for mRNA(+) synthesis in Class IV viruses
- genome is used as mRNA(+)
- RNA replicase synthesizes ssRNA(-), and then uses it as a template for mRNA(+) production
