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Micro Final Unit 1

QuestionAnswer
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
Created by: elliehall
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