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.