Lecture 14 Flashcards
What does ubiquity mean?
they are present everywhere liquid water exists
What does metabolic power mean?
extremely chemically active in diverse ways
Microbial diversity in the oceans occur in which hierarchy?
Bacteria > Archaea > Eukarya
What do transmembrane ion gradients do?
gradients to generate ATP
What is syntropy?
Interactions where microbes feed together
What are Cultivation-Dependent Methods?
attempting to grow microbes present in the environment so they may be studied
oldest method of identification, different food sources, atmospheric conditions, temperatures, pH
What are Cultivation-Independent Methods?
assess microbial communities directly in sample through sequencing
Why is enrichment step is crucial for cultivation?
Enrichment means adjusting culture conditions to favor the growth of the desired microbes
What is culture bias?
You only cultivate microbes in the sample that grow best in the culturing condition —- There is a reason we know the most about aerobic chemoheterotrophs like E. coli bc they are easy to culture
Why can’t we culture so many microbes?
Many microbes flourish in nature because they are part of interdependent nutritional consortia
we don’t know they conditions they need to grow
Why is cultivation important?
is essential for hypothesis testing
The presence of a gene does not mean its being used when and how you think it may be, you must be able to show through controlled genetic experiments for which cultivation is a prerequisite
What are commonly used Cultivation-independent methods? (3)
1) Amplicon-based community profiling (most common)
2) Fluorescence in situ hybridization (FISH)
3) Shotgun metagenomic sequencing
What is genes are detected in Amplicon-based community profiling?
16S rRNA for prokaryotes
18S rRNA for eukaryotes
How does Amplicon-based community profiling work? (4)
1) Extract DNA from environmental sample
2) Amplify target sequences (16S rRNA) using PCR
3) Clone into bacteria or sequence directly (yeah Illumina!)
4) Followed by a bunch of data analysis….
Why is small subunit rRNA used in Amplicon sequencing?
ALL microbes within the community must have this gene
It must be possible to design universal PCR primers that can anneal to the gene in all microbes
There must also be variable regions of this gene that are different in all microbes
What are operational taxonomic units (OTUs)?
To classify the microbial community, its amplicon sequences are lumped into operational taxonomic units (OTUs)
What are the limits of amplicon sequencing? (5)
1) The “universal” primers are not completely universal and do not amplify target genes with equal efficiency in all species
2) The universal primers are also designed based on sequences available in databases and do not fully capture the diversity present in the environment
3) Least abundant microbes are often under-sampled and missed all together because PCR amplification is exponential
4) Can usually only reliably given genus-level resolution
5) Does not tell you anymore than WHO is there – no genomic information is gained
How does FISH work?
The same PCR primers used for amplicon-based community profiling can be used to make fluorescent DNA probes that hybridize with DNA and RNA in intact cells in a sample
Fluorescent FISH probes are hybridized with a sample and visualized with a fluorescent microscope
Provides spatial information
How does shotgun metagenomic sequencing work?
sequences ALL the DNA (Bacteria, Archaea, Eukaryotes and viruses) present within the sample
Eliminates culture bias and amplicon bias
very expensive compared to amplicon sequencing
What is Metatranscriptomics?
What genes are being expressed by a community? - sequence mRNA transcripts with similar method to metagenomics
What is Metaproteomics?
What proteins are being made by a community”? – Extract proteins and use mass spectrometry to determine which proteins are present and in what amounts
What is Metabolomics?
What kind of metabolism is occurring? – Extract metabolites and use advanced mass spectrometry to determine which metabolites are present and in what amounts
