Lecture 14

Question 1

What does ubiquity mean?

Answer 1

they are present everywhere liquid water exists

Question 2

What does metabolic power mean?

Answer 2

extremely chemically active in diverse ways

Question 3

Microbial diversity in the oceans occur in which hierarchy?

Answer 3

Bacteria > Archaea > Eukarya

Question 4

What do transmembrane ion gradients do?

Answer 4

gradients to generate ATP

Question 5

What is syntropy?

Answer 5

Interactions where microbes feed together

Question 6

What are Cultivation-Dependent Methods?

Answer 6

attempting to grow microbes present in the environment so they may be studied

oldest method of identification, different food sources, atmospheric conditions, temperatures, pH

Question 7

What are Cultivation-Independent Methods?

Answer 7

assess microbial communities directly in sample through sequencing

Question 8

Why is enrichment step is crucial for cultivation?

Answer 8

Enrichment means adjusting culture conditions to favor the growth of the desired microbes

Question 9

What is culture bias?

Answer 9

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

Question 10

Why can’t we culture so many microbes?

Answer 10

Many microbes flourish in nature because they are part of interdependent nutritional consortia

we don’t know they conditions they need to grow

Question 11

Why is cultivation important?

Answer 11

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

Question 12

What are commonly used Cultivation-independent methods? (3)

Answer 12

1) Amplicon-based community profiling (most common)
2) Fluorescence in situ hybridization (FISH)
3) Shotgun metagenomic sequencing

Question 13

What is genes are detected in Amplicon-based community profiling?

Answer 13

16S rRNA for prokaryotes
18S rRNA for eukaryotes

Question 14

How does Amplicon-based community profiling work? (4)

Answer 14

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….

Question 15

Why is small subunit rRNA used in Amplicon sequencing?

Answer 15

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

Question 16

What are operational taxonomic units (OTUs)?

Answer 16

To classify the microbial community, its amplicon sequences are lumped into operational taxonomic units (OTUs)

Question 17

What are the limits of amplicon sequencing? (5)

Answer 17

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

Question 18

How does FISH work?

Answer 18

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

Question 19

How does shotgun metagenomic sequencing work?

Answer 19

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

Question 20

What is Metatranscriptomics?

Answer 20

What genes are being expressed by a community? - sequence mRNA transcripts with similar method to metagenomics

Question 21

What is Metaproteomics?

Answer 21

What proteins are being made by a community”? – Extract proteins and use mass spectrometry to determine which proteins are present and in what amounts

Question 22

What is Metabolomics?

Answer 22

What kind of metabolism is occurring? – Extract metabolites and use advanced mass spectrometry to determine which metabolites are present and in what amounts