College 1 – Microbial diversity
Microbes: micro-organisms such as fungi, bacteria, protist, virus and yeast
Microbes, what do they do?
-Driving biochemical cycles and recycling of nutrients
-Decomposition
-Agents of disease
-Antagonist against pathogens
-Purifying our water and cleaning environment
-Range of symbiotic interactions
-Maintaining soil structures
~1012 microbial species.
We do not know for sure how many microbial species there exists, because it is
difficult to know the difference between species. The diversity is so big because
the microbiome can tolerate a much bigger range of ecological ranges. Think of
PH, temperature, exogen levels, sunlight.
The most diversity on the planet is microbial, most eukaryotes diversity is also
microbial such as protists and yeast.
History
Antonie van Leeuwenhoek found out that microbes exists because he made the
first microscope, therefor we call him the father of microbiology. Because of this
microscope we were able to see a world we did not know existed.
After this we discovered that microbes are responsible for numerous activities.
After that we learnt how to cultivate them (grow them) and how to characterize
them (name them). We saw lots of different sizes and shapes but not enough to
distinguish 1012 species.
Later we found out that lots of them can not be cultivated so we made molecular
biological methods to characterize microbial populations without the need for
cultivation.
Whit “Genomics” and high-throughput approaches we now can determine “who’s
there and what they’re doing”
Who’s is there?
Great plate count anomaly: if you take a sample out of the environment than
you will count way less micro-organisms when you count the colonies on a agar
plate than count them straight under the microscope. This is because when
counting on an agar plate you will only count the colony forming units (CFU),
and not all microbiome will be able to grow on the agar plate. When counting the
cells directly under the microscope is doesn’t matter if you can grow on the agar
plate or not.
But remember both of these technics does not say a single thing about the
diversity of the environment you took your sample out of. Because it will count
how many cells there are not how many different species.
,Nowadays we use DNA to say something about the microbial diversity of a place.
Especially the 16S rRNA gene. It is chosen because it is the most established
marker.
-Homologous to all bacteria
-They have very conserved regions but also variable regions
-High copy number (more copies in one genome)
-Large and expanding database
-Works for uncultured taxa
Which of the following is not a reason that analysis of rRNA is useful for
interfering a Tree in Life? -> rRNA’s don’t vary between species. If everything
is the same than we cannot create a tree of life.
16S rRNA sequencing: take a sample, isolate DNA, PCR to amplify the 16S
gene, than we can sequence DNA and do DNA library construction.
We are looking at more and more complicated area like near a volcano or deep
down the ocean. Because of that the number of known microbial phyla increase.
But the majority of phyla have no known culture representatives. We only know
that there is a sequence and that we find them in a specific place, and that there
different enough that we have to put them in a different phyla. More than that we
do not know.
~only 9% of bacterial taxa are cultivated.
OUT (operational taxonomic unit) -> clusters of similar sequences, often
defined by a similarity threshold, usually 97%. This means sequences that are at
least 97% identical are grouped into the same OUT.
ASV (amplicon sequence variant)-> unique (vary by one nucleotide), error-
corrected sequences without clustering. ASVs are generated by algorithms that
model and remove sequencing errors, resulting in higher-resolution units that
distinguish true biological sequences from noise.
Defining a microbial species:
-Impossible by morphology of phenotype (and most can’t be cultured anyway)
-70% total DNA cross hybridization
-Molecular studies use arbitrary cutoffs such as 97% 16S RNA gene sequence
identity
- Genomic studies now allow the inspection of many genes, conservation of
organization and sequence identity across all conserved genes.
Because of the difficulties for defining a microbial specie we mostly just refer to
operational taxonomic units (OTU) and ASV’s.
Because technology is changing quickly and costs are going down we can use
metagenomics and genomics. In the past, we studied microbes primarily through
cultures. Later, through 16S rRNA sequencing. Now, through whole
genome/metagenomics sequencing.
Amplicon sequencing (bv. 16S): here you choose one specific gene (16S
rRNA), you do PCR and uses a sequence technique. Because of this you get an
idea which microbes are present in the sample.
,-Pro: cheap, easy
-Con: you can only see this gene, very little structural and functional information
Metagenomics sequencing: you sequence all of the DNA of the sample (all
microbes). Whit this we can make MAGs, metagenome assembled genomes.
These are almost whole genomes from the microbes. Less than 10% of MAGs
have cultured representatives, indicating that most microorganisms are known
only from their DNA.
-Con: complex
Alpha diversity: within sample. Questions as what is
there and how much is there?
Beta diversity: between sample. Questions as how
similar of different are samples from each other?
Other metrices:
-Alpha diversity and species richness: number of
species in a given sample
-Beta diversity: the diversity between samples. High beta
diversity means that two samples differ a lot from each other.
-Evenness: hoe even are species abundance distributed. For example you have
4 different species but one specie dominates, evenness is low
-Phylogenetic diversity: the phylogenetic distance of the observed sequences
-Functional diversity: the variance of functions of genes the species have
How are they doing it?
-Omics approaches allow us to assess microbial
communities at multiple molecular levels, revealing
their potential (genomics), actual gene expression
(transcriptomics), protein activity (proteomics), and
functional output (metabolomics)
Which microbes are there-> you measure
nucleic acids (DNA/RNA). You use methods like
amplicon sequencing (16S) ore SSU rRNA
approaches.
What are microbes doing? -> you measure RNA, proteins, metabolites. You
use metatranscriptomics, metaproteomic ore metabolomics.
What is the genomic/genetical can do? -> you measure DNA. We use
metagenomics
Look back this part!
Summary:
-Microbial diversity is exceedingly high
- Culturing fails to capture this diversity (great plate anomaly)
- Sequencing of amplicons (e.g. 16s) and metagenomes helps
- Alpha diversity measures diversity within communities
- Beta diversity measures diversity between communities
- OTU and ASV are ways of partitioning taxonomic groups
, - Omics approaches can help to determine what microbes can do
(metagenomics) or are doing (transcripts/proteome/metabolome)
What is competition?
Use or defense of any resource that limits the availability of that resource to
other individual.
A resource is any substance or factor that can lead to increased growth rates as
its availability in the environment increases, and which is consumed by an
organism.
This definition is limited to consumed items that are utilized for maintenance and
growth. What about other unconsumed resources such as sunlight ore oxygen?
Resource competition (-/-): limited resource, no direct interaction necessary.
Costs may be symmetrical
Interference competition/ allelopathy (+/-): units interact directly for access
to a common (but not necessarily limited) resource. Costs are asymmetrical.
Competitive exclusion: complete competitors cannot coexist because they
both want the exact same resource. But if complete competitors can’t coexist,
which species wins in competition? Best competitor for a resource wins. A best
competitor grows fastest, reaches highest density and persist at the lowest
resource concentrations.
Note: if species DO coexist, it implies a mechanism for coexistence
Experiment example:
When alone the Saccharomyces has both a higher growth rate and a higher
carrying capacity, so it grows faster and can
reach a larger population than
Schizosaccharomyces.
But when we put them together in one
environment we see that Saccharomyces
and the Schizosaccharomyces grows less in
the presence of the other than alone.
Both species experience negative effects
from competition – even the weaker species (Schizosaccharomyces) reduces the
growth of the stronger species (Saccharomyces) slightly.
Tilman model of competition: shows how species
compete for a shared resource, and predicts who will
win based on their minimum resource requirements.
Curve A shows how a species' growth rate increases
with increasing resource (R).
The horizontal line MA represents the mortality rate ant
it is constant, regardless of resource. The intersection of the growth curve and
mortality is R*: the minimum resource level required to maintain the population.
Microbes: micro-organisms such as fungi, bacteria, protist, virus and yeast
Microbes, what do they do?
-Driving biochemical cycles and recycling of nutrients
-Decomposition
-Agents of disease
-Antagonist against pathogens
-Purifying our water and cleaning environment
-Range of symbiotic interactions
-Maintaining soil structures
~1012 microbial species.
We do not know for sure how many microbial species there exists, because it is
difficult to know the difference between species. The diversity is so big because
the microbiome can tolerate a much bigger range of ecological ranges. Think of
PH, temperature, exogen levels, sunlight.
The most diversity on the planet is microbial, most eukaryotes diversity is also
microbial such as protists and yeast.
History
Antonie van Leeuwenhoek found out that microbes exists because he made the
first microscope, therefor we call him the father of microbiology. Because of this
microscope we were able to see a world we did not know existed.
After this we discovered that microbes are responsible for numerous activities.
After that we learnt how to cultivate them (grow them) and how to characterize
them (name them). We saw lots of different sizes and shapes but not enough to
distinguish 1012 species.
Later we found out that lots of them can not be cultivated so we made molecular
biological methods to characterize microbial populations without the need for
cultivation.
Whit “Genomics” and high-throughput approaches we now can determine “who’s
there and what they’re doing”
Who’s is there?
Great plate count anomaly: if you take a sample out of the environment than
you will count way less micro-organisms when you count the colonies on a agar
plate than count them straight under the microscope. This is because when
counting on an agar plate you will only count the colony forming units (CFU),
and not all microbiome will be able to grow on the agar plate. When counting the
cells directly under the microscope is doesn’t matter if you can grow on the agar
plate or not.
But remember both of these technics does not say a single thing about the
diversity of the environment you took your sample out of. Because it will count
how many cells there are not how many different species.
,Nowadays we use DNA to say something about the microbial diversity of a place.
Especially the 16S rRNA gene. It is chosen because it is the most established
marker.
-Homologous to all bacteria
-They have very conserved regions but also variable regions
-High copy number (more copies in one genome)
-Large and expanding database
-Works for uncultured taxa
Which of the following is not a reason that analysis of rRNA is useful for
interfering a Tree in Life? -> rRNA’s don’t vary between species. If everything
is the same than we cannot create a tree of life.
16S rRNA sequencing: take a sample, isolate DNA, PCR to amplify the 16S
gene, than we can sequence DNA and do DNA library construction.
We are looking at more and more complicated area like near a volcano or deep
down the ocean. Because of that the number of known microbial phyla increase.
But the majority of phyla have no known culture representatives. We only know
that there is a sequence and that we find them in a specific place, and that there
different enough that we have to put them in a different phyla. More than that we
do not know.
~only 9% of bacterial taxa are cultivated.
OUT (operational taxonomic unit) -> clusters of similar sequences, often
defined by a similarity threshold, usually 97%. This means sequences that are at
least 97% identical are grouped into the same OUT.
ASV (amplicon sequence variant)-> unique (vary by one nucleotide), error-
corrected sequences without clustering. ASVs are generated by algorithms that
model and remove sequencing errors, resulting in higher-resolution units that
distinguish true biological sequences from noise.
Defining a microbial species:
-Impossible by morphology of phenotype (and most can’t be cultured anyway)
-70% total DNA cross hybridization
-Molecular studies use arbitrary cutoffs such as 97% 16S RNA gene sequence
identity
- Genomic studies now allow the inspection of many genes, conservation of
organization and sequence identity across all conserved genes.
Because of the difficulties for defining a microbial specie we mostly just refer to
operational taxonomic units (OTU) and ASV’s.
Because technology is changing quickly and costs are going down we can use
metagenomics and genomics. In the past, we studied microbes primarily through
cultures. Later, through 16S rRNA sequencing. Now, through whole
genome/metagenomics sequencing.
Amplicon sequencing (bv. 16S): here you choose one specific gene (16S
rRNA), you do PCR and uses a sequence technique. Because of this you get an
idea which microbes are present in the sample.
,-Pro: cheap, easy
-Con: you can only see this gene, very little structural and functional information
Metagenomics sequencing: you sequence all of the DNA of the sample (all
microbes). Whit this we can make MAGs, metagenome assembled genomes.
These are almost whole genomes from the microbes. Less than 10% of MAGs
have cultured representatives, indicating that most microorganisms are known
only from their DNA.
-Con: complex
Alpha diversity: within sample. Questions as what is
there and how much is there?
Beta diversity: between sample. Questions as how
similar of different are samples from each other?
Other metrices:
-Alpha diversity and species richness: number of
species in a given sample
-Beta diversity: the diversity between samples. High beta
diversity means that two samples differ a lot from each other.
-Evenness: hoe even are species abundance distributed. For example you have
4 different species but one specie dominates, evenness is low
-Phylogenetic diversity: the phylogenetic distance of the observed sequences
-Functional diversity: the variance of functions of genes the species have
How are they doing it?
-Omics approaches allow us to assess microbial
communities at multiple molecular levels, revealing
their potential (genomics), actual gene expression
(transcriptomics), protein activity (proteomics), and
functional output (metabolomics)
Which microbes are there-> you measure
nucleic acids (DNA/RNA). You use methods like
amplicon sequencing (16S) ore SSU rRNA
approaches.
What are microbes doing? -> you measure RNA, proteins, metabolites. You
use metatranscriptomics, metaproteomic ore metabolomics.
What is the genomic/genetical can do? -> you measure DNA. We use
metagenomics
Look back this part!
Summary:
-Microbial diversity is exceedingly high
- Culturing fails to capture this diversity (great plate anomaly)
- Sequencing of amplicons (e.g. 16s) and metagenomes helps
- Alpha diversity measures diversity within communities
- Beta diversity measures diversity between communities
- OTU and ASV are ways of partitioning taxonomic groups
, - Omics approaches can help to determine what microbes can do
(metagenomics) or are doing (transcripts/proteome/metabolome)
What is competition?
Use or defense of any resource that limits the availability of that resource to
other individual.
A resource is any substance or factor that can lead to increased growth rates as
its availability in the environment increases, and which is consumed by an
organism.
This definition is limited to consumed items that are utilized for maintenance and
growth. What about other unconsumed resources such as sunlight ore oxygen?
Resource competition (-/-): limited resource, no direct interaction necessary.
Costs may be symmetrical
Interference competition/ allelopathy (+/-): units interact directly for access
to a common (but not necessarily limited) resource. Costs are asymmetrical.
Competitive exclusion: complete competitors cannot coexist because they
both want the exact same resource. But if complete competitors can’t coexist,
which species wins in competition? Best competitor for a resource wins. A best
competitor grows fastest, reaches highest density and persist at the lowest
resource concentrations.
Note: if species DO coexist, it implies a mechanism for coexistence
Experiment example:
When alone the Saccharomyces has both a higher growth rate and a higher
carrying capacity, so it grows faster and can
reach a larger population than
Schizosaccharomyces.
But when we put them together in one
environment we see that Saccharomyces
and the Schizosaccharomyces grows less in
the presence of the other than alone.
Both species experience negative effects
from competition – even the weaker species (Schizosaccharomyces) reduces the
growth of the stronger species (Saccharomyces) slightly.
Tilman model of competition: shows how species
compete for a shared resource, and predicts who will
win based on their minimum resource requirements.
Curve A shows how a species' growth rate increases
with increasing resource (R).
The horizontal line MA represents the mortality rate ant
it is constant, regardless of resource. The intersection of the growth curve and
mortality is R*: the minimum resource level required to maintain the population.
