ESPM 131 Study Guide - Midterm Guide: Enrichment Culture, Secondary Metabolite, Anaerobic Respiration
14 May 2018
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ESPM 131: Soil Microbial Ecology - Review Questions for Exam 2
Covers lectures from Cultivation (2/26) through Phosphorous (3/23).
What does the “uncultivated dark matter” refer to?
●Organisms that we know are there but can’t culture in a lab
Why is it so hard to grow soil bacteria in the lab? – give 3 reasons.
●Soil is very heterogeneous
●ey’re picky
●A lot of them are symbionts w/ plants
●Microbe-microbe interactions afect culturing outcomes
Explain the concept of enrichment culture.
●It is when you make a culture that is nutritive to the organisms you want to grow but not to
others
●Allows selection for specific metabolisms
●Can be sequential
Explain 3 “improved” isolation strategies.
●Pour plates : add inoculum to agar in order for subsurface colonies to occur
●Roll tubes : O2 concentration gradient, helps things grow that are obligate anaerobes
●Gradient plates : can see what organisms prefer and is more heterogeneous
Explain what in situ cultivation entails.
●Cultivating organisms in their natural environments
What characteristics make an efective biomarker?
●Of biological origin
●Doesn’t exist outside the organism very long
●Can be detected in small quantities
●Occurs in all organisms to be detected
●Unique to each organism
What are the characteristics of nucleic acids that make them good biomarkers?
●Unique to each organism
●Degraded easily
●Can be detected in small amounts/amplified
In relation to DNA (PCR amplification) explain what a chimera is? Why is it important to identify and
remove chimeras from DNA sequence data?
●From more than 1 organism, annealed together
●Distorts est. of diversity and ruins tree
●Can also happen during DNA assembly
Briely explain how “DNA sequencing by synthesis” works.
●Re-assembling base-by-base, instruments used have chemistry that allows to add (dideoxy) one b
at a time
●Modifications to bases being added, luorescent ( illumina )
●en add enzyme that chops of dideoxy, repeat cycle to build complementary DNA molecule
Why has 16S rRNA been such a popular biomarker for characterizing bacterial/archaeal communities,
and what are the limitations of using it?
●ere are conserved and variable regions to check for variation btw organisms/communities
●It has been sequence in many taxa, known in general, have databases for it
●Housekeeping genes like rRNA are rarely horizontally transferred
●Good for phylogeny and metabolism, but not for function
What is the ITS region and why would one use it?
●Spacer btw rRNA subunit sequences
●Fungi have 18S which isn’t as variable
●Shows high variance in size/seq
●Used for fungi
What is Fluorescence In Situ Hybridization (FISH) and how is it useful for detecting and counting soil
bacteria?
●It uses luorescent probes that attach to parts of the chromosomes
●Can be used to find specific sequences/types of bacteria
●Can be used to calculate actual abundance
How predictive is 16S rRNA gene sequence similarity of the function of bacteria?
●Only really useful for phylo analysis b/c ribosomal structure doesn’t say much about
ecological/bio function
What is meant by an “Open” or “Closed” format for sequence-based analysis of a microbial community?
Closed : know what you’re looking for, microarrays, have primers, i.e. 16S
Open : open to discovering new things, i.e. shotgun sequencing
Define the terms: metagenomics; metatranscriptomics, metaproteomics
Metagenomics : application of modern techniques to study communities and bypass the need for
isolation
Metatranscriptomics : sequencing the combined expressed genes of a community, good for phylog but
not function
Metaproteomics : “sequencing” the combined proteins of a community, use MS, need DNA sequences to
interpret
Contrast ‘gene-centric” and “genome-centric” metagenomic approaches to analyzing the functions of
microbial communities.
Gene-centric : focuses on genes themselves and function, process all reads, annotate function,
count/sum rel abund
Genome-centric : try to piece whole genomes together, compare function btw genomes and try to match
to organism, infer metab/predictive models
Does the detection of expression of a gene (mRNA) prove that an organism is performing a specific
function in soil?
●No, because it could be in small amounts
●May be inhibited/multiple metab capabilities
●Consequential evidence
What is stable-isotope probing? How can it be used to associate soil microbes with a specific function?
●Using atoms w/ 1 extra neutron, makes higher-density DNA
●Can use problems/amplicon sequencing to find functional genes
●Can test for metabolism
●Use stable isotope imaging
Give two examples of techniques (each) used to measure relative and absolute abundance of soil
microbes.
●Relative : microarrays, shotgun metagenomics
●Absolute : FISH/microscopy, qPCR
Can you prove microbial activity in situ using ‘omic data? Explain why.
●No, you can only predict function- you need to see how they work in the ecosystem (in situ)
●May not be using them
What roles would you expect methane oxidizers to play in a wetland soil with a foot of water overlying an
anaerobic sediment? Explain.
●Net source, can’t consume all
●At oxic/anoxic interface using the methane produced by methanogens in anaerobic sediment
What roles would you expect methane oxidizers to play in a well-drained upland soil? Explain.
●Net sink
●Need CH4 from atmosphere b/c no methanogens
How does nitrification cause soil acidification?
●NH4 + O2 → NO2 + 4H+
●e reaction causes a net production of protons, which acidify the soil
ere is one central metabolic pathway through which nitrogen is mineralized (converted from organic N
to NH4+). What is that pathway? What is the dominant controller of the occurrence and rate of
N-mineralization?
●GDH - deamination
●If plant material has low C/N
●If organism needs to metabolize C skeletons
Whether net N-mineralization occurs during decomposition depends on each of the three following
controllers; Briely explain each.
C/N of the decomposer: their growth requirement for N relative to C
Eficiency of the decomposer.: how much C is converted to body mass vs how much is released as
CO2
How long decomposition has been going on: the relative C/N ratio decreases over time bc
microbial bodies decompose
Document Summary
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