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Micro Final Unit 1
Question | Answer |
---|---|
serial endosymbiosis hypothesis | nucleus is formed, is later engulfed Based on mitochondrial 16s falling in with protobacteria and chloroplasts with cyanobacteria |
symbiogenesis hypothesis | "hydrogen hypothesis" bacteria is engulfed by archaea, one produces hydrogen and one needing hydrogen The nucleus forms later |
How does diversity originate within microbial populations? | HGT, mutations |
What roles do selection and genetic drift play in microbial evolution? | Bottleneck events change the allele rations in a population, mechanisms of evolution select for traits associated with higher fitness (reproductive success) |
What does rapid trait change plus slow speciation mean for microbial genomes within a species | Lots of diversity within a species |
What is a core genome? | select genes that are all shared within a species |
pan genome | the totality of the genes present in the different strains of a species |
What is the current definition for a microbial species? | Group of strains sharing certain characteristics with a relatively recent common ancestor |
where is HGT most common | HGT is common within a group, greater similarities |
What two methods are commonly used to characterize a new species? | SSU rRNA gene sequencing and genomic similarity |
What cut-offs (to groups strains into a species) are used for 16S sequencing | 97% |
DNA-DNA hybridization cut off | 70% |
Are there exceptions to these cut offs | B subtilis and B anthrax have 99% similarity but VERY different |
what are the taxonomic methods that were discussed | gene, multigene, genome sequencing, MLST, Phenotypic analysis |
Gene, multigene, and genome sequencing | Whole strand, 16s with recA and gyrB, ANI (average nucleic identity) |
MLST (multilocus sequence typing) | Good for strains, sequencing several portions of several genes, label alleles with a number and compare allele profiles |
phenotypic analysis (FAME) | Extract fatty acids, form methyl esters, then gas chromatography |
If a new organism is discovered, what must happen in order for it to be classified as a new type of microbes (a new taxon)? | Must be cultured in a pure culture + deposited into culture collections, characteristics need to be described, need proposed name, and published information |
vertical transmission | generation to generation |
horizontal transmission | from one member of a species to another |
mechanisms of HGT | transformation, transduction, conjugation, vesicle, nanotubes, GTA |
GTA is | not fully functional phage, gene transfers but no infection |
nanotubes | use cell-cell contact with straw |
How are HGT events detected? | Difficult to detect, look for unusual proteins/genes, DNA with different GC content, phylogenetic discordance |
How often does HGT occur between related organisms? | not sure |
Do rates of HGT vary across organisms? | We think there is more HGT between close relatives, the more similar → more HGT? |
We think there is more HGT between close relatives, the more similar → more HGT? | Rates are impacted by how much DNA is in the environment, sequence homology, genetic drift, benefits of a gene, how frequently DNA is introduced into an environment |
What is the overall impact of HGT on phylogeny? | We do not know!! Likely contributes to speciation events and MAY have a larger role in evolutionary history |
What is the great plate count anomaly? | discrepancy between the number of microbial cells observed by microscopic examination and the number of colonies that can be cultivated from the same natural sample |
What are some proposed reasons for this discrepancy? | 90-99% of bacteria cannot be grown independently Cross feeding organisms exist, organisms need to consume a byproduct of another organism Slow growth for some organisms |
Why is cultivation of microbes important? | Best way to understand physiology and look for evolutionary tracking, comparison is crucial |
Explain the idea behind enrichment cultures | Adding targets to make specific organisms grow exponentially better (CO2 for autotrophs) |
Why is GFP not a useful tool for natural populations? (green flourescent protein) | It cannot be found naturally |
What about populations of anaerobes? | It is oxygen dependent, anaerobes cannot be in the presence of oxygen so it would not work |
Describe how FISH and CARD-FISH work | FISH: providing hybridizes with target RNA (there is fluorescent dye on the probe) CARD FISH: enzyme joins to help with glowing (greater intensity), the signals are amplified and tyramide is added |
When would you use CARD-FISH instead of FISH? | CARD FISH is good when looking at gene expression of slow growing microbes |
What are some common problems with PCR-based techniques? (DNA extraction issues) | variation based on: method of extraction, lysing, sample you are processing (some inhibit certain extraction methods), soil samples have compounds that cause inhibition at certain steps , is it DNA from active organisms? they could be dead |
PCR issues | even if DNA extractions are equivalent, certain pieces of DNA will not amplify with PCR to the same degree secondary structure, GC content |
How would you use PCR and sequencing to determine the active portion of a community? | amplify with PCR, look at total ssu rDNA genes *whole community ( RNA is usually only produced by active organisms and it doesn't stick around for a long time this gives an idea of the active portion in the community) |
How do dNTPs and ddNTPs differ: dNTP focus | dNTPs have an OH on the 3’ which allows for elongation, they are DNA building blocks |
How do dNTPs and ddNTPs differ: ddNTP focus | ddNTPs are used for sanger sequencing, they are missing an OH on the 3’ so they terminate synthesis |
basic process of Sanger sequencing | Make DNA fragments, terminate each with ddNPT, then separate fragments based on size via electrophoresis Fluorescent ddNTP allow for identification of A,T,G, or C |
differences between a PCR reaction and a Sanger sequencing reaction: Sanger focus | Sanger uses 1 primer, dNTPs and ddNTP and does one gene at a time to determine the order of nucleotides, it builds the complement |
differences between a PCR reaction and a Sanger sequencing reaction: PCR focus | PCR uses 2 primers and amplifies a specific DNA sequence, creating multiple copies of ds |
similarities between PCR and Sanger | BOTH rely on thermocycling, BOTH require DNA polymerase |
What are the advantages to next generation sequencing? | It is cheaper and faster PER piece of DNA, it can sequence 1000s of genes in a single run |
Define metagenomics | Next gene sequence and analyze microbial genomes from an environment -Targeting any and all genes -Huge amounts of data (high-throughput sequencing is necessary) Look at potential of a community |
Define metatranscriptomics | Investigating gene expression of the entire community - RNA-seq **no dead or dormant organisms Can use the same basic methods as metagenomics - Use a reverse transcriptase to convert mRNA into cDNA |
Define metaproteomics | Uses all the proteins to tell what is happening, functions and interactions |
How do metagenomic studies link function and diversity? | Use smaller pieces to assemble larger pieces. They study all the genes in a species and give the possibilities, not only what is happening Display what is different/similar within a species, links genes to phylotypes |
What challenges are there to study the metatranscriptome of a sample/environment? | mRNA is used, but it is in fewer numbers than DNA or rRNA,, there is less activity and it is NOT stable, it has a short half life (degrades quickly!!) |
Why would one want to study the metatranscriptome or metaproteome rather than the metagenome? | You can determine what genes are active, while a metagenome may tell you genes that are dead/dormant |
issues with metagenome | too much data and tells how much potential the entire community has and has ever had (some of those organisms could be dead) |
Explain the basic procedure for microautoradiography (MAR). | Incubate cells with substrate containing radioactive isotope, Fix cells to a slide, Dip slide in photographic emulsion and incubate in dark, developed and areas with silver deposits (dark spots) indicate active cells location |
Why would combining this technique with FISH be useful? | FISH identifies what/who is active (phylogenetic group), MAR tells you what is occurring **link diversity and function |
How would stable isotope probing allow you to identify which organisms in a particular environment are utilizing a substrate of interest | Heavy DNA produced as a substrate is incorporated to cellular components (DNA, RNA, lipids) then centrifuged to separate by density so the organisms that incorporated the heavy isotope can be identified |
What are some potential issues with these techniques? (Why should you interpret results with caution?) | Only tells what an organism is capable of. (NOT preference), Strain variation that we are missing, Complex metabolic webs, cross feeding may be occurring |