Archaea and the Tree of Life

Transcript of Part 2: Mysteries of the Methanogens

00:00:03:01	I've always had a deep curiosity for,
00:00:06:17	I guess, logic puzzles.
00:00:09:08	Trying to understand how things fit together
00:00:12:02	and why they do so.
00:00:15:23	At some point in high school,
00:00:17:10	I learned about the fact that microbes
00:00:19:20	can eat all kinds of things
00:00:21:15	that other groups of organisms cannot.
00:00:23:21	So that kind of drew me to environmental engineering,
00:00:26:08	as an undergrad.
00:00:27:11	Two years into grad school,
00:00:28:14	I took a summer course in microbiology
00:00:31:15	and that just changed everything instantly.
00:00:35:05	I knew that I had to stop, kind of,
00:00:37:00	thinking about the world from an engineer's perspective
00:00:40:07	and transition over to become a biologist.
00:00:43:10	I ended up joining the Department of Organismic
00:00:46:14	and Evolutionary Biology at Harvard.
00:00:49:05	And when I was doing my PhD,
00:00:51:10	one thing I recognized was that
00:00:53:24	methane is mostly produced biologically
00:00:56:00	by this one group of organisms.
00:00:59:00	(bright music continues)
00:01:02:08	They produce 80% of the methane that's being released,
00:01:07:03	emitted to the atmosphere annually,
00:01:09:14	and which has a significant impact on climate change.
00:01:12:06	So decided I wanted to delve more into the biochemistry
00:01:15:01	and the metabolism of these bugs.
00:01:21:07	So Methanosarcina acetivorans was isolated
00:01:24:20	from these dense kelp forests
00:01:27:09	off of the coast of La Jolla in California.
00:01:30:06	So these kelp forests are so dense
00:01:32:18	that the insides are completely deprived of oxygen.
00:01:36:14	And for these methanogens to grow, that is a necessity.
00:01:39:04	They cannot grow in the presence of oxygen.
00:01:42:10	So in that dense kelp forest
00:01:44:24	is a place where there is no oxygen.
00:01:47:06	And in those places, there's a lot of carbon available.
00:01:51:07	It gives the methanogens the right kind of environment
00:01:54:04	for them to grow and then as a result
00:01:56:13	they were extremely abundant there,
00:01:58:20	and it was easy to find them.
00:02:00:12	So when I started my post-doc,
00:02:02:13	the CRISPR-Cas revolution had taken off.
00:02:05:04	There had been many groups that had developed CRISPR
00:02:09:00	technology for eukaryotic systems, you know,
00:02:11:14	ranging from Arabidopsis to mice.
00:02:13:21	Similarly, there had been some attempts
00:02:15:19	to develop CRISPR technology for bacteria
00:02:18:02	to edit their genomes.
00:02:19:18	There had been no such efforts on the archaeal front.
00:02:22:20	No one had tried to develop these tools
00:02:25:05	to work in an archaeal system.
00:02:32:07	So one the first things that I did was
00:02:34:07	that I tried to adapt CRISPR-Cas9 genome editing
00:02:38:05	to an archaea, to a methanogen
00:02:40:18	that I knew how to grow in the lab,
00:02:44:15	luckily it worked.
00:02:47:02	And that really enabled us to do a lot
00:02:49:05	that happened afterwards.
00:02:52:00	(bright music)
00:02:54:20	One of the things that I subsequently worked on,
00:02:58:01	after developing CRISPR for this particular methanogen
00:03:01:23	was to look at the genes involved in forming methane,
00:03:05:11	specifically an enzyme that's involved in methane formation
00:03:09:13	called methyl-coenzyme M reductase or MCR.
00:03:13:01	MCR is one of the most abundant enzymes in our planet.
00:03:17:03	And about 80% of all the net methane emissions,
00:03:20:12	on our planet, come from this particular enzyme.
00:03:23:04	But very little is known about how this enzyme
00:03:25:22	actually makes methane.
00:03:27:15	So we thought we could use CRISPR technology
00:03:29:23	to understand more about how this enzyme makes methane.
00:03:34:05	MCR is unusual in many different ways,
00:03:37:07	but the one facet that appealed to us
00:03:39:21	was the fact that this enzyme
00:03:42:02	goes beyond the 20 canonical amino acids
00:03:45:04	that we typically see in most proteins.
00:03:48:02	In MCR, many amino acids are modified
00:03:50:18	so that they look like something completely different now.
00:03:53:17	And what I wanted to understand
00:03:55:09	was why are there so many amino acids,
00:03:57:17	these building blocks for proteins modified in MCR
00:04:01:04	and are those modifications important for its function?
00:04:04:18	For instance, there is a very simple amino acid
00:04:07:05	called Glycine.
00:04:08:23	In one particular place in the protein
00:04:11:06	that Glycine had been modified to form
00:04:13:13	what we call Thioglycine.
00:04:15:17	And in principle, the difference between Glycine
00:04:17:12	and Thioglycine is the replacement of an oxygen atom
00:04:21:02	with a sulfur atom.
00:04:22:18	With Thioglycine it's extremely interesting in the fact that
00:04:26:06	we don't know of any other protein
00:04:29:03	or enzyme out there in nature
00:04:31:04	that has a Thioglycine in the protein.
00:04:35:09	So MCR up until now, it seems to be the only protein
00:04:38:19	that has Thioglycine instead of a regular Glycine
00:04:41:21	at that particular location.
00:04:43:23	So that was odd and unusual and extremely rare.
00:04:48:08	For the longest time researchers in the field,
00:04:50:06	who've noted this have considered
00:04:51:23	that Thioglycine kind of going beyond the Glycine
00:04:55:08	and modifying it must be really important for this enzyme,
00:04:59:11	possibly important for the formation of methane.
00:05:03:14	And so when we developed our CRISPR tools,
00:05:05:16	we thought, okay here's an interesting question.
00:05:07:15	Here's kind of this, you know, dogma in the field.
00:05:10:10	Why don't we go and look at what that Thioglycine does?
00:05:14:00	So to look at it, we identified the genes
00:05:16:12	that make those modifications,
00:05:18:01	that take a Glycine and make it into a Thioglycine.
00:05:21:11	And we modified those genes,
00:05:22:23	we deleted them.
00:05:24:06	And then we converted the enzyme from the form
00:05:26:16	where it has this unusual Thioglycine
00:05:29:15	back into a form that just has a regular Glycine
00:05:32:04	and looked at the differences between them
00:05:34:08	and try to ask the cell,
00:05:35:14	okay, how well are you growing?
00:05:38:00	How much methane are you producing?
00:05:40:09	And once we deleted those genes,
00:05:42:02	we essentially had cells that were making MCR.
00:05:45:20	But now that Glycine was no longer
00:05:48:14	getting modified to Thioglycine
00:05:50:21	and the cells were growing just fine
00:05:52:14	under most conditions that we tested and making methane.
00:05:57:04	And what that revealed to us was that
00:05:58:19	this Thioglycine is not that important
00:06:01:02	for the cells to make methane.
00:06:03:04	To some extent, we were kind of maybe disputing,
00:06:08:01	you know, a hypothesis that was well accepted in the field.
00:06:11:09	What we'd shown was the fact that
00:06:12:20	this enzyme just works fine with the regular Glycine,
00:06:15:24	which made us scratch our heads a little bit,
00:06:17:24	because what does it do?
00:06:19:07	Why go through the bother of making this,
00:06:22:00	you know, modification if it's not that important?
00:06:24:18	And to figure that out, we tried subjecting the cells
00:06:28:14	to a bunch of different conditions that we thought
00:06:30:12	were environmentally relevant.
00:06:32:12	And here I'm showing you one parameter
00:06:34:16	that often changes out in the natural environment,
00:06:37:05	which is temperature.
00:06:38:18	So we grew the cells up
00:06:39:19	at a bunch of different temperatures,
00:06:41:13	starting at 29 degrees Celsius,
00:06:43:14	which is on the lower end for these groups of organisms.
00:06:47:05	At these low temperatures,
00:06:48:11	we essentially detected no difference
00:06:50:06	between the wild type strain.
00:06:52:14	So the strain that has the modified Glycine,
00:06:54:19	the Thioglycine,
00:06:56:06	and the strain that has now just a regular Glycine
00:06:58:22	in its place, in green.
00:07:00:14	But as we increase the temperature from 29,
00:07:03:19	all the way to 45 degrees Celsius,
00:07:06:03	we noticed something interesting.
00:07:07:24	As the temperature increased,
00:07:09:15	we noticed that there was more and more of a growth defect
00:07:12:24	for the cells that did not have that Thioglycine
00:07:15:17	in their MCR anymore.
00:07:17:15	And what that indicates to us is that
00:07:20:09	as the temperature increases,
00:07:22:01	if you ever experience a high temperature stress
00:07:24:12	in the environment,
00:07:25:16	having that Thioglycine is really important
00:07:27:19	to keep your MCR happy and making methane.
00:07:31:07	And if you don't have it,
00:07:32:07	you probably don't do it as well.
00:07:34:10	So now our new hypothesis for this
00:07:36:12	Thioglycine modification in MCR
00:07:39:07	is that it actually helps the enzyme
00:07:41:17	stay stable and stay active under stressful conditions,
00:07:45:02	such as the one shown here,
00:07:46:17	that is high temperature stress.
00:07:48:20	So we started out with Thioglycine
00:07:50:17	because it was so unusual and so rare,
00:07:53:01	but when you look at MCR,
00:07:56:08	we find that in addition to Thioglycine,
00:07:59:00	it often has other amino acids
00:08:00:24	that also undergo these changes.
00:08:02:17	That also go beyond the 20 canonical ones
00:08:05:13	that we typically see.
00:08:07:06	And after this work, we followed up
00:08:09:10	on some of the other ones
00:08:10:16	and we're starting to see very similar patterns.
00:08:13:23	Most of the ones that we've studied so far,
00:08:15:20	and we've studied three so far,
00:08:17:19	are not crucial for this enzyme to make methane,
00:08:21:05	contrary to what we kind of believed for a while,
00:08:24:10	but they play important roles
00:08:25:20	under very specific environmental conditions.
00:08:28:17	Temperature is often one that we see
00:08:30:12	some interesting responses to that make us think
00:08:34:10	that maybe we were thinking of this incorrectly before,
00:08:37:23	you know, when an organism's growing out in the wild.
00:08:40:12	That organism experiences, extremely fluctuating conditions,
00:08:44:17	and especially if you're a unicellular archaeon,
00:08:47:18	you kind of have to have your defenses up
00:08:49:12	to make sure that you can make your way
00:08:51:02	through those conditions.
00:08:52:02	So if the temperature suddenly shifts,
00:08:53:21	you'd still need to grow and divide.
00:08:55:22	And we think now we're kind of changing our paradigm
00:08:58:22	and thinking now that these modifications to MCR
00:09:02:04	are more to adapt the enzyme to different environmental cues
00:09:07:02	like temperature, than for making methane itself.
00:09:14:06	So I spent my post-doc studying this one particular
00:09:17:05	methanogenic archaeon and developing tools for it,
00:09:21:04	but I'm extremely cognizant of the fact
00:09:22:21	that this is one particular strain, one organism,
00:09:27:23	and it's one amongst many that are found in the environment.
00:09:30:23	And what I'm trying to do in my lab
00:09:33:05	is trying to kind of, is being cognizant
00:09:36:00	of the diversity that's out there
00:09:37:15	and trying to see if we can leverage that diversity
00:09:41:00	in our understanding of methanogens and archaea.
00:09:44:03	It's an interesting time to be an archaeobiologist
00:09:46:23	because we're kind of in this, you know,
00:09:49:21	major transition in thinking about archaea
00:09:52:17	from an evolutionary standpoint.
00:09:54:22	And so that leads to interesting discussions
00:09:57:16	and conversations,
00:09:58:20	kind of at a big picture level about
00:10:00:20	what archaea mean and how we should study them
00:10:03:06	and what systems should we look at
00:10:05:09	to try to understand archaea?
00:10:07:05	I wish I could pinpoint one thing about archaeal research
00:10:10:15	that I find the most interesting.
00:10:12:01	I think what I find the most interesting about archaea
00:10:15:04	is the fact that there's so much.
00:10:17:03	They're such an understudied group of organisms
00:10:19:11	that I don't think there's one right question to ask.
00:10:22:12	I think there's so many things that we can look at.