So what I want to do today is recap a little bit what we talked about
last time, reiterate some of the important points,
and then show you how we can learn something about microorganisms in
the environment by talking about in-situ identification of
microorganisms as well as genomics. We'll first talk about genomics and
general and then talk about some applications of genomics to
environmental microbiology because I think there is some of the most
exciting new developments are in the area, actually.
So last time we talked about molecular evolution and ecology.
And just to recap, some of the main points were that we can actually use
genes or gene sequences for a couple of very important questions that we
want to explore. The first one was gene sequences act
as evolutionary chronometers.
Now, what do I mean by that? Basically what we said last time
was that each gene, each sequence in the genome
accumulates mutations with a certain probability. So what we mean is
that all genes accumulate mutations over time.
Now, these of course are the mutations that do not kill the
organisms, so not the deleterious mutations, but these are mutations
that are either slightly deleterious, or don't matter,
or are beneficial mutations. OK, and what this entails is that
each gene accumulates mutation with a certain probability over time.
It basically means that two organisms that come from species
that are relatively closely related to each other have gene sequences
that will be much more similar to each other than genes from an
organism that comes from a species that's much more distantly related.
So, in practical terms what this means is your genes are much,
much more similar to those of a monkey than they are to a crocodile,
for example. And we can take advantage of that by applying some
algorithms, some mathematical modeling essentially,
to constrain these relationships in those phylogenetic trees that we
talked about last time. And I also mentioned that the
ribosomal RNA genes are particularly important for that process.
In principle, you could do it with any protein coding machine or any
kind of gene in the genome, but we use the ribosomal RNA genes
in particular because all organisms have them. They're part of a
handful of genes that are what we called universally distributed
last time. And what this allows us to do is
then construct phylogenetic relationships for all living
organisms. And I just want to remind you of the tree of life that
I showed you last time where we can really explore the relationships
amongst all living organisms. And some of the important points
there that we made were, for example, that the tree of life
supports the endosymbiont theory, that when you actually look on the
tree where the mitochondria and the chloroplasts tree, they fall
into the bacteria. Now, there is a question where
somebody asked in the online survey, can the mitochondria and
chloroplasts still live outside of the eukaryotic cell?
And the answer is no, they can't anymore because over
evolutionary time the two organisms have become so integrated that the
mitochondria and chloroplasts both lost their ability to live outside
of the eukaryotic host cell. Another important point that we made
last time that I want to reiterate here is that gene sequences,
when we go into the environment, and obtain them directly from the
environment act as a proxy for microbial diversity in
the environment. So, the number of genes recovered
directly from the environment is a measure of diversity.
And we said that this actually plays a very, very important role in the
analysis of microbial communities, and I showed you the example here
where we went and took some ocean water and basically apply this
technique that outlined last time where we can actually amplify
ribosomal RNA genes from environmental samples,
clone them, determine the sequence, and then constructs phylogenetic
trees. And what you see here is a tree
where we summarize the major groups that we found in the sample have
been only for two of those groups where we show the entire set of
sequences that we actually obtained because there were so many of them
out there. And what we basically found was that over 1500 bacterial
16S ribosomal RNA gene sequences coexist in this environment.
And what we said also last time was that the analyses like these have
really taught us that microorganisms are the most diverse organisms
on the planet. So, most diversity is amongst the
microorganisms, and one of the big questions now is
what are all those microorganisms doing in the environment?
And so, today what I want to do with you is basically explore this
question of how we can actually figure out what those microorganisms
are all doing in environmental samples?
So we can say we are exploring the
function of microbes in the environment. At first,
I want to cover how we can actually identify them in the environment.
And I want to show you one specific example, and then I want to talk
about genomics in general, and then basically end with an
application of genomics to environmental questions.
So, let's first talk about the in-situ identification
of microorganisms.
And the basic problem that I alluded to already before is that most
microbes are only known [SOUND OFF/THEN ON]
-- from 16S ribosomal
RNA clone libraries.
And we basically want to search and identify them in the environment.
OK, and I'll show you a specific example of that later on.
Now last time, we said that the ribosomal RNA sequences consist
really, like all gene sequences, in fact. We identified several
stretches of nucleotides, types of stretches, that can be
found. We said the A type stretches and B type stretches that are very
important for construction of phylogenetic relationships,
because we can align them and look for changes in the nucleotide
sequences because they are the same length and only differ in mutation
and single nucleotide base pair changes.
But then there's also those C type stretches, if you remember,
and those we said vary at much faster rates because they are not
functionally constrained in those genes.
OK, so they can actually also accumulate length changes.
And, it's these C type stretches that we can use sort of as
diagnostic sequence stretches for microorganisms.
So, what we can say is we identify organisms by the C type stretches,
C type sequence stretches. And we call those signature
sequences. OK, and they allow the differentiation
of closely related organisms, -- because they vary at very fast
rates between organisms. And the way we do this is that we
construct so-called phylogenetic probes. I should probably
write this over here. Now what are those phylogenetic
probes? They're basically short pieces of DNA that have a
fluorescent molecule attached to them.
-- DNA molecules that are roughly 20
nucleotides in length, and they carry a florescent molecule.
Now what the short, single-stranded stretches of DNA
basically are is they are complementary to those C type
sequence stretches --
-- in the ribosomal RNA. And so basically what we can do is
we can collect microbial cells from the environment --
-- make them permeable --
-- and then basically mix them with
those phylogenetic probes.
And these probes will then permeate into the cell and bind to their
complementary sequences.
Then we wash away the unbound probe --
-- and we can view it in a
microscope under UV light.
Let me show you an example of this. What you see here is basically a
light micrograph. So this is what you see basically
when you collect microbial cells from the environment under the
microscope. Most bacteria look the same, so you cannot actually
differentiate them all by just looking at them.
But then these cells were fixed and permeabilized and then basically
mixed with two different phylogenetic probes that identified
two different types of organisms. One was labeled with a red Fluor,
the other one with a green Fluor.
And what you see is that you can now differentiate those two organisms.
Now, why is this especially interesting? Well here's just a
specific example where people were looking for bacteria capable of
nitrogen oxidation. These are bacteria that are very
important in, for example, sewage treatment. And it was known
that there were two different types out there, one that oxidizes
ammonia to nitrite, -- and that a second one that
oxidizes nitrite to nitrate. And by doing this type of analysis
what people basically learned is that those two organisms live in
very, very close proximity at all times.
So the organisms that oxidized ammonia to nitrite are really
attached, and oftentimes even surround by the organisms that take
the nitrite to nitrate. So, where you have is a very close
cooperation between two different types of microorganisms,
and the transfer of one of the substrates that's a product of the
metabolism of one of the organisms to another one: so extremely
efficient process that really is very important to take into
consideration when you want to understand processes like sewer
treatment, but also nitrogen biogeochemistry and
the environment. Any questions?
OK, so for the remainder of the lecture I want to talk
about genomics, -- and then in particular also its
application to questions of environmental microbiology and
environmental science. So first, what I want to do is give
you a little bit of the definition of genomics, and then cover how it
is actually possible that we can sequence entire genomes,
and I want to give you some highlights of what we have found by
comparing different genomes to each other.
And then I want to talk about this field about environmental genomics
where we can use genomic techniques to actually learn something about
the function of different uncultured microorganisms in the environment.
So first, our definition, it's basically to interpret
or to sequence, -- interpret, and compare whole
genomes. And as you will see the comparison part actually plays an
increasingly important role because we have now actually genome
sequences available from almost all, or from at least some of the major
groups of life. So this, again,
is a different kind of representation of the tree of life.
You have bacteria, archaea, and eukarya again.
And as you can see, we have a lot of representatives.
In fact, this doesn't even come close to the diversity that we have
now sequenced as well over a hundred bacterial genome sequence now,
several archeael genomes, and increasingly also in
eukaryotic genomes. Now, genomes, so how is this done?
How can we actually sequence genomes? Well,
on the face of it we use very large facilities where you have sequencing
machines present. There's one very important one at
MIT, actually at the Broad Institute, and here you see all those really
industrial scale production lines actually.
But the basic problem is that genomes are large.
E. coli, for example, has roughly 4.4 million base pairs,
and the human genome is even much, much larger.
It has about 3 billion base pairs. OK, so genomes are very, very large.
But a single sequencing reaction--
-- gives you only roughly 500-1, 00 nucleotides or base pairs. So
how is it that we can actually sequence entire genomes?
I'm going to walk you through this, and there is some variation on the
theme, but this is still a major approach that's still used in some
of the sequencing facilities. Now, you start out by extracting
genomic DNA from organisms, and then you use restriction enzymes
to cut the DNA into relatively large pieces of DNA, so about 160
kilobase pairs long. On average, this is shown here.
Kilo means a thousand, so 160,000 base pairs long.
These pieces are then cloned into specific cloning vectors that are
called BAC vectors.
So therefore, cloning large pieces of DNA, and BAC stands for Bacterial
Artificial Chromosome. And what they basically are,
are plasmids, very special plasmids that can carry large pieces of
genome, or large genome fragments. So, by cloning into those BAC
vectors, what you do is you basically divide up the genome,
and then the step number three is mostly done for eukaryotic genomes
because they are so much larger. You can actually map and analyze
the fragments, and map them onto genome maps where
you know the location of different restriction fragments and different
genes, actually. For bacteria, this step is mostly
skipped, actually. What you do with each one of those
BACs, is you cut them further up into 1 kilobase per fragment,
so much smaller fragments. And these are called,
and these are cloned then into normal plasmid vectors.
And so you generate what are called shotgun clones.
So, these are then cloned into E. coli, you go through the same type
of steps that we discussed before already with environmental clone
libraries. And you can actually determine the sequence of each one
of those pieces of DNA. And what you will then get,
is small fragments of overlapping DNA sequences.
That it shown here. You'll find overlaps,
basically, which piece together the whole genome. And so,
first to assemble, you piece together these genome fragments that
are present in the BACs, and then finally you piece together
the entire genome propose large sequence pieces,
and you get a so-called draft genome sequence.
The next step in this analysis, then, is that you do so-called
genome annotation is. And the first very important step
is that you translate the gene sequences into amino acids.
So, the nucleotide sequences into amino acids particularly in
prokaryotes. This step can be done right away --
-- and what this allows you to do,
is you can look for what we call open reading frames,
or ORFs. And what you look for is a start codon and a stop codon that
basically branches or frames a stretch of amino acids encoded by
the nucleotides. So you look for ORFs.
And these are your putative genes.
The next step that you can do,
then, is you can go to databases and now you compare your ORFs to
information that is present in the databases. So basically,
you inquire the database and ask, is a gene sequence that is similar
to the one that I have statistically significantly similar present that
allows me to say something about the function of this particular gene?
So function, can then be identified by comparison with databases.
Any questions?
OK, so that allows you, then, to basically say something
about the different genes that you have found in the genome,
but to give you an impression of how new this field really is and how
little we still know about the diversity of genes and organisms,
on average when we sequence a new bacterial genome we find about 30%
of the genes, or a third of the genes have no known functional
analog of the databases. OK, so there's a lot to learn about
the diversity of life and about the functional diversity of life.
In eukaryotes, there are some little twists,
as you all know. And basically, that is that genes
of course consist of introns and exons, right?
And so it's basically relatively difficult to directly identify those
open reading frames. And what you have to do is that you
have to actually oftentimes, so let's write this down.
And what people oftentimes do,
then, is that they search for matching sequences in so-called cDNA
libraries. Now what are cDNA libraries? Let me just show you
this on the next slide. Skip this. Basically what you can do is you can
isolate messenger RNA from cells and that translate the messenger RNA by
a process called reverse transcription that the viral enzyme
that translates RNA into DNA, so you can translate it into DNA
fragments. And you can then clone those DNA fragments into plasmids,
sequence those, and then basically see what are the pieces that are
actually, what are the introns in the genes?
What are the pieces that are excised when the messenger RNA is actually
created from the genome?
And so, let me just cover now a few of the major insights that people
have come up with. Of course, it's a very growing
field and a lot of excitement is coming out.
And I first want to talk about
bacteria and archaea --
-- and then say a few words also about eukaryotes or eukaryote.
First of all, what we learned about, bacteria and archaea,
is that their genomes are very compact.
Whenever they have pieces of DNA
that are not frequently used, they're actually lost from the
genome. OK, so they lose genes, I should say, relatively easily, and
we can see this that the genome size is correlated to metabolic
diversity.
So, for example, we have Mycoplasma genetalium and
Streptomyces --
coelicor are two very different bacteria. The first one is an
obligate intracellular parasite.
OK, so, which means it's actually bathed in a nutrient solution in the
eukaryotic cells that it invades. It doesn't have to make amino acids.
It gets it just from the host cell. And it turns out it has a very
small genome, so only 0. 8-based mega-base pairs, so 580,
00 base pairs, and only 517 genes. And interestingly,
actually people are now using this organism to try and ask,
well, what's the minimum number of genes that organism can actually
will live with? And so, they are deleting in a
stepwise fashion the different genes in this organism,
and it turns out that you need about two to 300 genes minimum in order to
make the things survive. On the other hand,
streptomyces is a soil bacterium --
-- has a very complex lifestyle, can degrade a lot of environmental
substrates, and it has a very big genome, one of the biggest bacterial
genomes. And so, those two organisms basically span
pretty much the range of bacterial genome sizes.
And so, it's thought that it has about 7,846 genes.
Now, we also have a very large
genetic diversity --
-- between species. And typically what you find is that
roughly 15 to 30% of genes are unique to a specific species.
And that's really because bacteria and archaea have the capability to
affect a lot of chemical reactions that eukaryotes,
for example, cannot. There's about 20 million known
organic substances, organic chemicals, and almost all of
them are biodegradable by bacteria. Even the minutest compounds if it
were not biodegradable bacteria, would build up in the environment,
OK? So, if it just where a cofactor that some organism produces because
we have such a long period of time of evolution on this planet and
evolutionary history, you probably would be able to dig it
up in your backyard. One of the other very important and
interesting insights that has come out with comparing genomes for
microorganisms is that lateral gene transfer is a very important process
amongst microorganisms.
Now what do I mean by lateral gene transfer? It basically means that
we find evidence among bacterial genomes that they have actually
taken genes from completely unrelated organisms.
And I just want to show you one example here from that of
thermotoga maritima --
-- which lives in hot springs. This is a very interesting
bacterium that lives in hot water of around 80∞C and thrives only in
those kinds of environments. And they coexist there with many
archaea. And when people sequenced the genome of thermotoga maritima
what they found was that about 25% of the genes have their closest
relatives in archaeal genomes. So roughly 25% of genes in
thermotoga are of archaeal origin. And how can we actually figure
something like that out? Well, the most important technique
is, again, phylogenetic tree construction. And so when you have,
for example, gene A, well let me draw this, actually,
on a new board.
So you're comparing, say, three organisms,
organism A, B, and C and you compare gene one with gene two.
And you notice that most genes adhere to this pattern,
but that every now and then there's a gene that gives you this
type of pattern. What you can then conclude is that
this gene, C, has not coevolved with the other genes in the genome of
these organisms but was actually transferred into it from another
source. And I don't have time to go actually into the mechanisms.
If you're interested, I teach a graduate class that undergraduates
actually take in our department, environmental microbiology, where we
discussed a lot of the mechanisms. It's basically a lot of viruses can
affect gene transfer but also plasmids and transposons.
But for bacteria, again, you should remember that often new function is
actually oftentimes arises by lateral gene transfer.
And one of the interesting things is that lateral gene transfer is
actually very important in the evolution of pathogenic bacteria.
So, the so-called virulence genes,
which are the genes that basically affect pathogenesis. Do
you have a question? Among pathogenic bacteria,
often arise by lateral gene transfer. OK. Any questions?
OK, now for eukarya, I just want to make the point that
their genomes are generally orders of magnitudes larger --
OK, and that the exons,
so the stretches that really encode the protein that make up the
organism, the exons are only typically a few percent
of the genome. That's particularly in higher
eukaryotes. Yeasts, for example, have a much more
compact genome also. We, for example, are full of DNA
that people still have a very hard time figuring out what that actually
does. But it seems that the majority of the genome,
so-called repeated sequences -- -- many of which seems to be ancient
retroviruses that have inserted themselves into the genome and have
since then lost actually their function. OK,
so the remaining time I want to just give you an example of how we can
now use these techniques that I outlined before to learn something
about microorganisms in the environment.
It's called environmental.
Basically, the way this all started
was by going into the environment and extracting nuclear gases and
treating them exactly the same way as if you had a single genome.
But, again, remember, we have a very large mixture of microorganisms
present in the environment. And where this is mostly done was
in the ocean, actually. And what people did, was they
constructed those BAC clones directly from the environment and
then looked amongst those BAC clones for specific 16S ribosomal
RNA genes. Remember, this is the marker that we
have for microorganisms in the environment. We know the diversity
of microorganisms through those types of genes,
and we have a lot of the data available. And so,
in order to link a specific function of such an organism that we only
know from the 16S ribosomal RNA genes.
So, to ask the question of what much of this organism might be carrying
out in the environment, it's very useful to sequence BAC
clones that have 16S ribosomal RNA genes on them,
and determine what kinds of protein coding genes are on there that might
reveal some of the function of the organism in the environment.
And one example that I want to show you is that of the proteorhodopsin.
So basically, the initial task was to sequence BAC clones containing
ribosomal RNA genes, and look for other genes that might
reveal some of the function.
So, you don't want to look for all the genes that encode proteins that
are important to the cell cycle and things like that,
but really sort of metabolic genes that might tell you something about
the type of metabolism that this organism carries out
in the environment. And so, what the first example that
turned out to be really, really important is that people
found rhodopsin genes on one of those BAC fragments,
and it turns out this rhodopsin catalyzes or these rhodopsin genes
produce a protein that inserts itself into the bacterial membrane,
and it's a photoreceptor that when it's hit by light,
it actually becomes a proton pump. So, it expels protons from the cell
interior to the outside, and you already know that this is
important in energy generation in all living cells.
So proton gradient across membranes basically give the cells sort of a
battery status that can be exploited by ATPase molecules or ATPase
proteins that equalize the proton gradient and affect ATP
synthesis in doing so. Now, why is this so important?
Well, it turned out that this type of protein is present in almost all
microbial cells that were previously thought to be heterotrophs alone in
the ocean in the parts of the ocean that receive enough life.
And what this means is that our estimates of the global carbon
budget of the ocean were basically wrong because most microorganisms in
the ocean have this.
So most prokaryotes in the ocean have a light-driven proton pump
which is called proteorhodopsin. And it basically allows them to gain
energy from sunlight. And there's an increasing number of
such examples now where we are learning to interpret environmental
communities, and the function of environmental microbial communities
through those genomic approaches. And it reveals basically an
enormous diversity of organisms out there. And what we also are
learning to do now is to assemble entire genomes from those samples by
applying genomic techniques. And this is an example here where
you see, this was published last year, where people went out and
basically were able to piece together from pieces of genes
obtained from the environment, entire genomes or fragments of
entire genomes. And that's shown here.
Those are contiguous sequences. OK, so if you have any questions
let me know by e-mail, or if you're interested in pursuing
this further I also teach another class in civil and environmental
engineering.