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Hi, and welcome back to iBioSeminars. My name is Dianne Newman,
and I am a professor at the California Institute of Technology
in the divisions of Biology and Geological and Planetary Sciences.
I am also an investigator of the Howard Hughes Medical Institute.
This is part three of my three part series in microbial diversity and evolution.
And what I would like to give you an example of today
is ways that we can through laboratory experiments
today gain insight into metabolisms that were evolved in the remote past.
And this is a very difficult task and we are just at the beginning of our ability to do it, but I hope through this story
I am going to illustrate just how interesting this field of research is and where the important open questions still lie.
So the specific example that I am going to tell you about is how we have gained some insight into
an important class of biomarkers that are used to record the evolution of oxygenic photosynthesis,
one of the most important metabolisms ever to have been invented on the Earth.
And today we see examples of oxygenic photosynthesis at work on the globe,
as you can see in this beautiful image that is taken from satellite data averaged over three years.
And what it is showing you are chlorophyll profiles in the marine oceans.
Here you see blooms of phytoplankton that occur in not only the Atlantic, but also
here in the Eastern Equatorial Pacific off of South America, and extending far out into the Pacific Ocean.
Now these gyres occur when there are blooms of phytoplankton which today
are responsible for emitting into the atmosphere most of the oxygen that we breathe.
However, these organisms, these marine phytoplankton, are not the ancestors of photosynthesis.
They are the ones that are doing it en masse in the world's oceans today,
but the process of photosynthesis evolved many billions of years ago on the Earth.
And its story is one that we still don't fully understand.
One of the most basic questions we don't understand is when did it occur.
How did organisms figure out how to use water as a substrate for photosynthesis?
One of the things we do know is that this ability to utilize water
was evolved through a complex combination of photosystems
in different types of primitive photosynthetic organisms,
including anoxygenic bacteria like the type shown in these bottles that are called purple bacteria
by virtue of the beautiful purple pigments they contain within their membranes, that you can sometimes see
when you grow them on certain substrates.
And I spoke about these organisms in my introductory talk.
And as I said then, at some point the molecular machinery
in these organisms was combined with the molecular machinery
in other organisms, also of this anoxygenic general class
but nevertheless quite distinct organisms.
How that happened, we don't know, but an issue right now is that when
this did eventually happen, and these beautiful molecular machines came together
so as to enable these organisms to utilize water as the energy together of course the real energy source is the sun,
but water is the substrate that is then activated by sunlight
to a form that is biologically utilizable in the electron transport chain.
This is when oxygenic photosynthesis was invented,
and the perpetrators of this invention were the cyanobacteria,
shown here in this slide of beautiful bubbling tubes
where CO2 is being fed into these cultures and these beautiful green organisms
with their green chlorophyll pigments are converting that CO2 to
biomass coupled to the oxidation of water to molecular oxygen.
Now the chloroplast itself as I explained in the first lecture is what evolved into the plastids that today we find
in diatoms that you see an example here. These are marine phytoplankton important in the oceans.
And of course, terrestrial plants that we are all very well familiar with
due to their ability to take water and generate oxygen.
But when this group of organisms first arose is a subject of real debate.
And it had been thought that there were signs in old rocks,
either these banded iron formations, or other geochemical signatures
that indicated oxygen likely was rising up in the oceans, being produced by these
ancient cyanobacteria, reacting with iron and precipitating it from solution,
to form these deposits, and then eventually getting into the atmosphere so that
other types of geochemical signatures found in rocks such as mass independent fractionations
of sulfur isotopes that we find in different types of sulfur bearing minerals
giving a signature that we think is very diagnostic of having oxygen present in the atmosphere.
That all of this was occurring somewhere in the middle of this time span, and certainly by around 2.4 billion years ago.
But when the ability of organisms to first take water arose, before it had this profound global effect,
is something that has not been very well constrained.
So a dominant approach that has been used to try to date the rise of cyanobacteria in the rock record
has revolved around using these organic molecules shown here that are called hopanoids.
And these are very complex organic structures that resemble sterols in eukaryotic cells.
Now the story I want tell today is a reexamination of these structures as biomarkers
to ask whether or not they are appropriate to assigning the rise of this very crucial metabolism that transformed the Earth.
So let's talk a minute about what it means to be a biomarker.
This is essentially acting as a molecular fossil.
That is a compound that is diagnostic of an ancient cell,
and a particular cell type if it is to be useful to identify organisms or their metabolisms.
So the idea is that a long time ago
cells hanging around in the environment, be they in the water column or in sediments
eventually through their sedimentation, and then through various diagenetic processes
where these sediments were transformed and over time compacted and converted
into solid structures that we see today uplifted in mountains and in various areas around the world
contain yet remnants of the original molecules from the original cells that once upon a time inhabited these environments.
Now these structures are not identical, but they are very, very similar, and so
what we are looking at today is what we call the biomarker
and this is the molecular fossil of what we infer to have been the original compound.
So here you can see something that we call a sedimentary hopane that is the diagenetic...
And diagenesis is just simply the process of transforming a sediment over time into a rock.
This compound is the diagenetic consequence of transforming the parent compound,
which we call the hopanepolyol, which has this core pentacyclic ring
here with a tail that has a variety of moieties on various carbon molecules,
and this is one where we are just showing hydroxyl compounds at these last positions, but they come in a variety of forms.
The bottom line is that some of these chemical decorations are lost,
but the backbone carbon skeleton remains.
And because it is so structurally similar to the original compound
it is very reasonable to infer that we are looking at a fossil of this molecule.
Now here is just another way of showing that. This is essentially a cartoon of diagenesis.
Where through the water column these cells ultimately sediment down, get incorporated into sediments,
and these sediments through geological processes
over billions and millions of years of Earth history wind up transforming,
and these molecules within them are converted into their most basic form.
Now why do people pay attention to these particular molecules?
These 2-methylated bacterial hopanepolyols.
The reason is because of an important study that came out about a decade ago
where it was claimed that 2-methylhopanoids were biomarkers for cyanobacterial
oxygenic photosynthesis. And the rationale for this was that at the time this work was done
all of the surveys of microbial cells for this particular molecule,
and when I say particular I mean the pentacyclic part
of the molecule and a methyl group at the second position of the first ring.
The other aspects of the molecule are not as crucial to the diagnosis,
but this initial part, and particularly the methylation at this second carbon atom
really was crucial to the whole story that I am going to tell.
So back when this initial study was published the investigators
reasonably were able to conclude that these molecules might be biomarkers for cyanobacteria because of the
fact that they were unable to detect their production in organisms other than cyanobacteria.
This wasn't entirely true because there were a few other strains that even in this initial study were reported
to produce these molecules, yet it was argued that in their sedimentary context
the most likely progenitors of these molecuels would have been cyanobacteria
because they were shallow marine enviroments in which the fossilized compounds were found
that were consistent with the growth of a phototrophic organism in this type of a habitat.
And also, the argument that the concentration of these molecules in a type that was believed to be
the most preservable was by far the highest in modern day cyanobacteria
lending credence to the notion that these types of molecules, when seen in the rock record
very well may be reflecting an ancient cyanobacterial community.
However, whether or not this was an absolutely valid assumption was debatable for a variety of reasons.
One of these reasons is that not all cyanobacteria can make these compounds,
so they are certainly not essential for the ability of oxygenic photosynthesis.
And so the argument that just because cyanobacteria make them means that they have
anything to do with oxygenic photosynthesis
is a whole other story in and of itself and is something that really needed to be looked into more for validation.
The second reason that this needed to be looked into more is that cyanobacteria
are capable of other types of metabolisms beyond oxygenic photosynthesis.
Some of them are called facultative oxygenic photosynthesis organisms
in that they are capable of using other substrates beyond water
in photosynthetic processes such as sulfide, for example.
And in addition, they are capable of fermentation.
And so just because cyanobacteria make them, doesn't a priori mean that they have anything to do with photosynthesis.
Although they might. At this point in the story, nobody knew, and so it merited looking into.
And so we got interested in this, and we thought that we would begin our studies
by coming up with just some rational criteria for what would constitute a robust biomarker.
The first of course is one I am not going to touch on because this really belongs to the province of geologists.
And that is that the biomarker must be indigenous to the rocks that it is meant to represent.
There's actually great debate about this point, but let's just assume for the sake of argument that when we find these fossils
in ancient rocks that they really are as old as we think they are.
And we will let the geologists work out indeed whether that's true,
but I think that it is fair enough to say that many of these samples
now there isn't much of a question. These molecules really are for instance 2.4-2.7 billion years old.
So let's just grant that.
The second part which now becomes of relevance for biologists is that they must have a unique distribution
amongst modern organisms, or they must have a clear evolutionary history
where it is obvious from whence they originated.
What were the first type of organisms that were able to produce them and/or the third point,
which I think is actually the most crucial point,
that they have a specific and conserved biological function that's related to the process of interest.
And in our case for today's lecture, we are talking about oxygenic photosynthesis.
So let's begin now with this second point.
Do these molecules, these 2-methylhopanoids have a unique distribution?
So to begin to look into this we decided to go to the environment
and collect some diverse types of phototrophic organisms to work with.
And we began by working with various cyanobacteria
that we surveyed for their ability to produce these 2-methyl compounds.
As I said, not all of them do, so we picked ones that were abundant producers as our reference strains,
and then because we had also been doing other studies in my lab
looking at the ability of anoxygenic phototrophs to catalyze the oxidation of iron,
under anoxygenic conditions, we decided just for fun we would begin to test
whether or not they could produce these compounds as well,
thinking that this would be a negative control, because previous reports had shown that related strains were,
under the conditions these previous investigators used, not able to generate these compounds.
And so here is where we had one of the surprises that occur once in a while in science
that are very exciting and at first cause you great distress
because you are not sure whether or not it's an artifact.
And so this happened when my student Sky Rashby made the surprising discovery
that one of our "negative control strains", one that we called Rhodopseudomonas palustris
was capable of making these 2-methyl hopanepolyols, that's what this acronym here means,
2-MeBHP stands for 2-methylated bacterial hopanepolyol.
In concentrations as great as its cyanobacterial counterparts.
Now you can be assured that we did all of our proper controls to make sure we weren't contaminating the cultures,
and this was a very reproducible, very robust result.
And what was really quite significant is that this molecule here,
and here you can see liquid chromatographic spectrum, and now a mass spectrum
of these peaks, was identical between our cyanobacteria positive controls
and what we thought was going to be our negative control.
What was really crucial here is that the production of these molecules by our anoxygenic phototroph
was conditional upon the manner in which we grew them.
And I think this is a very important point that is worth emphasizing.
And that is, oftentimes in these interdisciplinary fields
they begin with a group of investigators who are excellent at a particular type of measurement
but not necessarily excellent at all, because who is? You know we are all specialists. We are all good at doing what we do.
And so the pioneering scientists who began this work were outstanding organic geochemists,
but were not people who routinely grew microorganisms in their own laboratories
so they were dependent upon getting strains from their colleagues
who would send them microbial samples grown up in whatever random growth condition
they happened to be doing at the time that they shipped the strain.
However, today, when we grow them in our laboratory, being microbiologists,
we are aware of the metabolic versatility of these organisms and are able to grow them under a variety of conditions.
And because of this we were able to find that it was under certain conditions
that these organisms made the molecule, and had we only looked at them in a more narrow window,
we would have missed their production entirely.
So there is an important lesson here, and this nice cartoon
of this filamentous cyanobacterium that is supposed to represent the cyanobacterium Nostoc punctiforme
that I'll show you later in the talk
is saying, "Do you make 2-methylhopanoids?" to our favorite model anoxygenic phototroph,
Rhodopseudomonas palustris, and she is answering, "Yes, I do."
This very nice cartoon was made by Sky, who is a very talented artist.
Now an actual image of this purple bacterium is shown here
in a transmission electron micrograph, taken by my postdoc Ryan Hunter,
that he overlaid upon sediments from the Sippewissett Salt Marsh in Woods Hole, Massachusetts.
And the reason we did this is because as I've been saying,
the geological record ultimately is emanating from sediments such as these,
where today we find purple bacteria and other types of phototrophs growing.
And one can infer that these types of environments also were good homes
for these organisms back billions of years ago.
And so what we are looking at when we are looking at the fossils
are ancient remnants of these types of habitats.
So what we thought we would begin to do would be to study Rhodopseudomonas palustris
to ask whether or not we could gain insight into the molecular biological basis for the production of these compounds,
and have this help us parse whether or not they were accurate biomarkers
for cyanobacteria or, and/or, oxygenic photosynthesis.
Now R. palustris is a terrific organism to use for many reasons. It grows rapidly.
It is metabolically versatile. It's genome is sequenced.
And it is amenable to genetic manipulation. And so it's a system primed for discovery
in terms of the biosynthesis and the cellular function of 2-methyl BHPs.
So one of the first things that we wanted to answer was whether or not
there was a specific enzyme responsible for methylating hopanoids
at the C2 position because given the fact that these molecules were produced very conditionally
upon specific quirks of how one were to grow them, what would be desirable
in terms of asking the question which organisms writ large are able to make these molecules
would be to bypass this growth requirement and simply be able to go to an organism's genome
to see if there were some gene that were diagnostic, that could predict the ability to make these molecules.
So Paula Welander, a postdoc in my laboratory,
sat down with the genome of Rhodopseudomonas palustris
and through some very clever bioinformatic work identified a whole part of the chromosome that encoded genes
that she suspected might have something to do with hopanoid biosynthesis.
And that's because in the middle of this cluster there is a gene annotated as shc that had previously been shown
to encode an enzyme responsible for the first step in hopanoid biosynthesis,
the cyclization of squalene into the first pentacyclic ring.
Now what we wanted was the enzyme responsible for a later part in the biosynthetic pathway
and that is the methylation at C2 because a wide variety of bacteria are capable of making hopanoids in general,
but what had been thought, as I told you, to be the diagnostic feature was this methyl group at the second position.
So if we could find a methylase that was specifically responsible for putting this methyl group on that position
we might be in a position to ask whether or not different organisms beyond cyanobacteria and this one freak
discovery we made, were capable of also making 2-methylhopanoids.
So to do that Paula looked at this genomic region, and she identified a gene over here
that seemed to have attributes that might be consistent with its product being
capable of catalyzing this methylation reaction.
And so to test that she made a clean in frame deletion of this gene,
and then she asked whether or not the mutant
strain that she called delta 4269 was capable of making methylated hopanoids in comparison to the wildtype.
And so here what you are looking at is a liquid chromatogram showing peaks of elution
of various types of hopanoid structures.
These over here are called...this one is diploptene, and this one
has a methyl group at the 2 position, so it is called 2-methyldiploptene
and this is a different type of hopanoid, with a slightly different R group on the end.
And again here, this peak that elutes first is the 2-methylated version of the molecule.
And as you can see when you compare it to the mutant that Paula made,
there is no peak here in the foreground showing that the 2-methylated versions
of these hopanoids are not being made in this mutant background.
Now when she complements and adds back to this mutant strain the wildtype
gene, then again she sees, indeed, just where she should, the peaks
that indicate the production of the methylated versions of those hopanoids.
So this was a very nice demonstration that indeed this methylase was responsible for
producing these methylated hopanoids.
Now in collaboration with another postdoc in the lab, Maureen Coleman,
Paula and Maureen went on to show that this methylase gene that they dubbed hpnP,
was much more broadly distributed than we had suspected.
And so what matters here is not the individual names of these organisms,
by any means, but simply the fact that what you are looking at is a complicated tree
that's drawn using the 16S ribosomal DNA sequence for a whole variety of organisms.
And what you are looking at now is in blue those organisms on this tree that are capable of producing hopanoids in general.
And there are many, as I said.
But the really key piece is that here, which is this outer ring
in which you can see in three different groups, in the cyanobacteria, in the acidobacteria,
and down here in the proteobacteria, evidence for the hpnP gene from the genome.
And this evidence is based on a very stringent cutoff where we are asking for a very high degree of similarity
in sequence identity between putative hpnP sequences
and our sequence of hpnP that we have shown is required for the methylation at the C2 position.
Now the other thing to note is that in every case that these sequences have been found in the genome
where the measurements of the actual ability to make the compound have been made, we have found
a 100 percent correspondence. And inversely, we have never found an instance
for an organism that is capable of making 2-methylated BHPs where they don't contain this gene.
So while of course it is possible that in the future another type of methylase will be discovered,
right now there is a 100% correspondence between position of this gene in the genome
and the ability to make the compound as measured directly.
So it appears as though it is a fairly robust indicator.
All right so, there are a few things to note here, as I said before, not all cyanobacteria make this.
So a priori, it is certainly not required for oxygenic photosynthesis.
The question is whether...and number two, more than cyanobacteria make it. It is not just a case of one random organism
being a flier. No. We find that there are many other types of bacteria here in this proteome,
alpha-proteobacteria family, that are capable of producing these compounds.
Some of these actually have even been shown in that very original paper to produce the methylated hopanoids.
It just had been claimed that they weren't producing them in abundance that was relevant for diagenesis.
And so that deserves a bit of re-examination.
But here is this whole other group, the acidobacteria, that hadn't previously been appreciated to make these molecules.
And I should say that this is just the tip of the iceberg, because these are only the genomes
of microbes that currently have been sequenced, which represent a minute fraction of the microbial diversity
in nature, and so now we have the tools where we can go out into the environment and ask more broadly,
where do we see these sequences? What type of organisms are likely containing them?
And we'll get a much better picture of the phylogenetic diversity of the organisms that make them.
However, to get back to the central point in terms of whether or not this is a robust biomarker,
even though today we see that these molecules are distributed amongst many organisms,
they still might be a valid biomarker of cyanobacteria
provided we can have some good evidence suggesting
that their root evolutionarily was within the cyanobacterial group.
So to get at that question of what is the root of this enzyme,
where was it first invented, Maureen Coleman decided to look at the phylogenetic history of the enzyme family.
So she made a tree where she was able to show, and now this is an unrooted tree,
the different branches of the organisms that we know can make this.
All right, so these are two different groups of proteobacteria. Here we have the cyanobacteria.
And here we have the acidobacteria.
And what we wanted to know was which of these groups was the first to invent this enzyme.
Now here is where it became complicated, and also where it became very important to do a rigorous
statistical analysis to align these sequences in various ways, use different algorithms,
to infer the most parsimonious evolutionary path to their divergence.
And when Maureen did this, quite literally by doing an in silico experiment, by varying a variety of parameters,
none of which was clear what was the absolute correct assumption to make,
but all of which would affect the topology of the trees that ultimately would result.
What she found was that the root, at least at present, is ambiguous,
that statistically there was as good support for these three different topologies,
that led to very different answers.
The first where the ancestral root of this methylase would be in the cyanobacteria.
The second where it was quite simply unresolved,
where there was no clear branching pattern,
of which subset would have been the earliest one to make this enzyme,
or the opposite result where the ancestor would be inferred to be an alpha-proteobacterium.
We hope that in the future as more sequences are gained we will be able to,
with greater confidence, resolve between these three options, but what we can confidently say
now is that we cannot with confidence interpret that cyanobacteria were even the evolutionary inventors of this.
And so the case remains open whether or not in the future they will be proven to be so,
but at present it is invalid to argue that there is an absolute certainty
that these molecules are biomarkers even for the cyanobacteria themselves,
saying nothing about oxygenic photosynthesis.
So let's cut to the chase and talk about oxygenic photosynthesis
because that is really what motivated this all along.
Could we find a biomarker that would give us insight into the process of oxygenic photosynthesis?
And of course, just because an organism makes a molecule, doesn't necessarily
mean that it has anything to do with a particular function.
And so what we wanted to ask was whether or not there was a connection between
the ability to produce this molecule and photosynthesis directly.
So, as I told you, hopanoids resemble sterols.
And if you take a look at the biosynthetic pathway
for cholesterol over here and compare it to bacteria hopanoids over here,
you see that they go through very similar structures.
Of course the rings are different between them, but they are reminiscent.
And so it would be reasonable when thinking about the universal functions for these molecules
that we would turn to sterols for some inspiration.
And this is a very fascinating field in cell biology in eukaryotes,
and I am sure there are some interesting commentaries on this in other iBioSeminar series that you can refer to.
Very briefly what I will just say now is that it has been demonstrated
that sterols in eukaryotic membranes have roles in both membrane fluidity and integrity.
They can play roles in membrane curvature. They are also important
in creating what are called lipid rafts, that involve the recruitment
of specific lipids and proteins to particular places within the membrane.
All of these are very active areas of research in eukaryotic cell biology,
and one can reasonably ask whether or not in bacterial and in archeal,
or even microbial- eukaryo systems these hopanoid molecules
that resemble sterols might play similar functions.
And one can go further to ask whether or not in any way these functions are related specifically to oxygenic photosynthesis.
So, one thing that we can do to begin to address this is simply to figure out in different types of microbial cells
where hopanoids reside. And the point here is that it is not obvious,
because microbial membranes are actually very complex,
particularly in phototrophs where we have these
beautiful inner cytoplasmic membrane lamellar invaginations.
We can have other types of invaginations
that are either vesicular or tubular, there are many different types of
internal membrane structures that can form,
and as I said this is a wonderful opportunity for future research in microbial cell biology.
But getting back to the focus here, the question is, and in this cell,
what I am showing you is a thin section of my favorite organism Rhodopseudomonas palustris,
where do the hopanoids reside? Are they in the outer membrane or are they in the cytoplasmic membrane?
Are they in this membrane called the inner cytoplasmic membrane,
which is where the photosynthetic machinery is housed?
And the same question can be asked of cyanobacteria, where you see these inner cytoplasmic membranes,
which also host the photosynthetic machinery in cyanobacteria.
And this is an organism that isn't even photosynthetic,
but does indeed produce 2-methylated hopanoids
and as you can see, it has complex membrane systems as well.
So where are the hopanoids, in which of these membrane systems?
And just for the remainder of this time, I am only going to focus on showing you work
we've done using a representative cyanobacterium called Nostoc punctiforme.
So Nostoc punctiforme is a terrific microbial system for studying cellular differentiation.
And the reason for this is that there are three states it can exist in, as the vegetative growing cells,
some of which can differentiate to form specialize structures called heterocysts,
and this is where nitrogen fixation occurs in this organism.
But these organisms are also capable of creating a spore like state,
that is known as an akinete, and this is a very stress resistant cell type
that forms under stressful conditions that is not metabolically active. It is metabolically quiescent.
So Dave Doughty, a postdoc in my laboratory decided to take Nostoc
as a beginning entry way into understanding
where hopanoids would partition in cyanobacteria.
And what he decided to focus on were the different membranes that I just described:
the outer membrane, the inner membrane, and the inner cytoplasmic membrane
in the vegetative, heterocyst, and the akinete cell types.
And what you can see when you do these thin sections of the organisms
and look at them under electron microscopy,
is that the cell types change quite dramatically.
For example, there are very few, what he is labeling here as thylakoids,
this is just borrowing a term from eukaryotic cell biology
but what this is also really talking about here is the inner cytoplasmic membrane
that these cyanobacteria have that houses the photosynthetic apparati.
They have very few when they are in the akinete state, which makes sense, because these are resting cell types.
The heterocysts have a much thickened cell envelope, and these are the cells in which nitrogen fixation occurs.
And this is just a normal view of a vegetative cell,
where you have what Dave calls a thylakoid membrane, and what I have been calling the inner cytoplasmic membrane.
As well as an outer membrane and an inner membrane in the overall cell envelope.
Now what we wanted to ask was depending upon the way in which the cell was grown,
would we find that hopanoid content and also hopanoid localization to these three membranes change?
And the answer was dramatically yes.
And so what Dave showed was when he was able to grow them as vegetative cells,
as heterocysts. Isolate the heterocysts, and then grow them so that they went into the akinete cell type
and then fractionate each of those three different cell types with respect to the cytoplasmic membrane,
the thylakoid membrane, and outer membrane,
that for these cell types the concentration of hopanoids, micrograms of hopanoids per milligram of total lipid,
varied quite dramatically.
And what I'll have you note here is that for the cytoplasmic membrane and the thylakoid membrane
the scale is much smaller than for the outer membrane.
Here this is only going up to ten, and here it goes up to sixty.
And so while indeed we do find some of these hopanoids in akinetes
and in vegetative cells being generated in the thylakoid membrane
by far the greatest concentrations are in the outer membrane of the akinetes, which are the resting cell types.
So as I told you, akinetes can be very resistant. They are survival structures.
But what I want to add to that description is the fact that
they can develop when cells are grown completely in the absence of light.
And so while in this experiment Dave did he'd originally grown them up photosynthetically,
and then subjected them to starvation conditions so that they went into the akinete type.
They do not need to ever go through the photosynthetic phase in order to become akinetes.
Therefore, since the majority of the methylated hopanoids in this organism
are localized to the outer membrane of the akinetes, one can infer that
the most likely source of 2-methylated hopanoid deposition into sediments, at least from this organism,
would be from cells where photosynthesis is not occurring.
And because of this we can go back and interpret the rock record and ask the question,
when we find both these molecular fossils of 2-methylated hopanoids
as well as these morphological fossils that paleontologists who are very clever and able to look at
these images and actually tell you when it is an akinete versus some other type of cell
and date them to very old times in the rock record.
Here we are talking about billions of years, so 1.5, 1.6, and 2.1 billion year old samples
where the akinete cell types as diagnosed by these paleontologists
are known to have been produced, we can ask whether or not
the 2-methylated hopanoid record that we find at similar ages
might have been produced through the shedding of the akinete envelope
into the sediments and may be reflecting a stress response
as opposed to anything to do with oxygenic photosynthesis per se.
That said, there is still more work to be done in examining whether there is some type of indirect connection
with oxygenic photosynthesis. There may be.
And we have a lot more to discover, so this is a very exciting field to be in
and will remain a very fruitful area of research for many years.
So to summarize what I hope I have been able to show you through an example
is how we can re-examine molecules that have been used as biomarkers for ancient metabolisms,
and ascertain whether or not these are valid biomarkers.
In this particular case, we have shown that these compounds
2-methylated bacterial hopanepolyols, or hopanes, as they are called in the rock record,
are likely to have been generated by many diverse bacteria.
And we can not yet say which type of organism invented the ability to make them.
So the jury is still out whether they can be used as biomarkers of cyanobacteria.
That said, even if cyanobacteria did invent them first,
it is not at all clear that they have anything to do with oxygenic photosynthesis
and in fact, all of the available evidence really would most reasonably lead you to conclude otherwise.
And that is because cellular localization studies show that they are mainly in membranes
that do not have anything to do with active cells,
nor do they necessarily have to reflect going through a photosynthetic process.
And in experiments which I don't have time to show you here,
we've been able in Rhodopseudomonas palustris to actually knockout these methylated compounds,
and find that even for the process of anoxygenic photosynthesis
they are not required and so, it doesn't appear that they play a direct role in metabolism.
But what it does seem to be important is that they are involved in stress response,
and likely this is manifesting through effects in membrane permeability and membrane fluidity.
Potentially there are other roles, and we await future discoveries to eliminate the full menu of options
that these molecules are serving in terms of their biological function in modern cells.
All right, so to summarize what I hope I have been able to show you from this story
is that we can use studies of modern bacteria to gain insight into the genesis of ancient metabolisms.
And while there is still a lot more work to do, we have a very exciting
future ahead of us where we have these remarkably geostable
compounds that hang around in rocks that are incredibly old, going back over two billion years,
and we need to be able to interpret them rigorously. And so in the case of these 2-methylated bacterial hopanes,
it appears that they really are no longer the best indicators of oxygenic photosynthesis,
however, they might be good indicators of microbial stress responses and other things.
And so in the future we hope to understand what their biological functions
are in both cyanobacteria and the other organisms that make them,
and be able to utilize these molecules as biosignatures of wonderful inventions in the history of cell biology.
So with this, I would like to acknowledge the people in my lab
that I have been referring to who have done this work.
It's been a collaboration over many years, and it began at Caltech with the student Sky Rashby,
co-advised by my colleague Alex Sessions, and in collaboration with my colleague Roger Summons at MIT.
And then continued by two postdocs, primarily Paula Welander and Dave Doughty.
Paula doing the work in Rhodopseudomonas palustris, and Dave with Nostoc,
and now continuing with Rhodopseudomonas, helped out by phylogenetic work with Maureen Coleman
and microscopy by Ryan Hunter.
And the Howard Hughes Medical Institute, NASA, and the NSF have contributed to supporting our research.