Scientist Stories: Basem Al-Shayeb, Borgs/Extrachromosomal elements to augment methane oxidation

Hello everyone, I'm Kelly Eversole, the Executive Director of the International Alliance for Phytobiomes Research, and we are pleased today to have with us Bassem Al-Shaib from UC Berkeley who's going to be talking to us about Borgs, and not just the ones that are in Star Trek. So we really look forward to that, but before we get started, I'd like to give you a little bit of an overview of the Phytobiomes Alliance and what we're up to and what we're interested in. So we are an international nonprofit consortium of industry, academic, and governmental scientists, and we have a number of sponsors who make it possible for us to actually do this webinar and also to try to coordinate and research at the international and national levels. So a lot of people often wonder what we mean by phytobiomes. They are complex systems of plant-based agriculture. They are the plant microbiome, the soil microbiome, but they are also the plants themselves and the plants in a particular space. They include the soil and all of the other geophysical components like climate and weather that will actually interact with the plant in its particular space. And when we think of the phytobiome, we're really looking at this very complex system of interactions. And we're also, because we're interested in agriculture and food security, they are also all influenced by management practices. The Alliance vision is that by 2050, all farmers will have the ability to use predictive and prescriptive analytics based on geophysical and biological conditions for determining the best combination of crops, or not even com— crops might be the best variety, management practices, and inputs for a specific field in any given year. We will be hosting a phytobiomes conference in September of 2022, and this is a multidisciplinary conference that— where we will try to bring together experts in all disciplines that have some impact on the broad phytobiome. So you can get more information about that at the phytobiomes conference website. We are pleased already that we have USDA NIFA funding, and we look forward to seeing you at the conference. So our next webinar will be in December, and it's going to be about developing sustainable strategies to protect wheat roots from take-all disease. And before we move into our webinar today, just to remind you, the webinar is recorded and will be posted on the Phytobiomes Alliance YouTube channel in a couple of weeks. You can subscribe to the channel so that you never miss a new upload. The presentation itself will be followed by a Q&A session. You can submit your questions in the Q&A panel, but I would encourage you also to monitor the chat panel so that you can see messages from the organizers. So if there's a reference to a website or a particular document, you'll see a link to that in the chat. Already you can download both my presentation as well as the presentation on borgs in the handout panel of the dashboard. So without further ado, I want to introduce Bassam Al-Shaib from University of California in Berkeley. He's a PhD student, stud— and he's going to talk to us about borgs, giant extra-chromosomal elements with the potential to augment methane oxidation. So, Bassem, welcome to the Phytobiomes webinar, and I'll turn it over to you now.

Thank you so much. All right. Hello, everyone. First, I'd like to thank Kelly and the Phytobiomes Alliance for extending the invitation to present today., and for the lovely introduction. I'm very excited to be talking to you about something that we found during my PhD studies to be totally nuts that we're still actually trying to fully understand that we call the borgs. So a lot of the work that I'll be talking about today is computational in nature, but it always starts in places like this. In this case, a plot of land that's a few miles from my advisor's property here in California. Where you wouldn't necessarily expect to find anything interesting, but as it turns out, everyone has a universe waiting to be explored in their backyard. So to give a brief primer of the process that generated these datasets, we collected many samples from the site that I mentioned that we were interested in, sediment and water as depicted here. And each cubic centimeter of these sediment samples that we collect has many billions of bacteria, archaea, eukaryotes, and viruses in them. So we usually take this sample and instead of sort of isolating those bacteria or archaea or viruses, we just extract all the DNA that's in the sample. And then we use next-generation sequencing to read through all of that DNA, creating terabytes of sequencing reads. After that, we computationally assemble all these sequencing reads to produce these longer, what we call DNA contigs, and then we bin these fragments of DNA into their separate genomes based on multiple different metrics, including GC content, tetranucleotide frequency, abundances in the sample, and more. And that would indicate that these different fragments belong together as part of one singular genome. And what I've focused on the most in my PhD was this world of extrachromosomal elements from our DNA samples that were essentially the misfits that didn't fit with the existing bacterial or archaeal genomes in the samples. And when I say extrachromosomal elements, I mean viruses, plasmids, and other pieces of DNA that aren't part of the main chromosome in a prokaryote but exist separately. And that led to— we found these giant bacterial viruses that far exceeded the average size of a bacteriophage, even surpassing the sizes of many bacterial genomes. Up to 735,000 letters of DNA, with many features that we typically think of as pertaining to life rather than viruses, including translational machinery, ribosomal proteins, CRISPR-Cas defense systems, and others. And we further dug in that same plot of land a year later to study these phages a bit more, and what we discovered were even bigger, more enigmatic elements that we call the borgs in the deeper samples in this environment. And these borgs, they replicate in archaea, which are arguably the most understudied domain of life after bacteria and eukaryotes. And these borgs, they tend to be linear in nature, double-stranded DNA elements, rather than circular, which has never been reported in archaeal exochromosomal elements. We named them after different colors that are shown on the left here, and what we found was in terms of the DNA structure, it had always two uneven replicores where the genes are encoded on two separate strands, one very long replicore and one very short one, and the genes are, as I said, on two different strands pointing in two different directions. With about 2,000 bases of long inverted repeats on either end of the chromosome. And they can go up to a million bases, base pairs of DNA, which is much larger than, you know, any of the bacterial viruses that we found. So this was very interesting to us to see what this could be, and they had very high read coverage, but in terms of mapping the sequencing reads to this genome, but they couldn't be further extended beyond the 2,000 bases of inverted terminal repeats, which tells us that these are 4 complete genomes that, that could not be further extended, for example, into another chromosome or additional size for the genome. And here I show the mapping, for example, for the black borg, where you can see that all the sequencing reads are mapping to our genome and they're all overlapping with each other. So you can think of it as sort of puzzle pieces or, you know, Tetris, for example, where you can fit all these reads together to form the complete genome. So we know that there aren't any gaps or any remaining areas in the genome to be further extended. And aligning these Borg genomes to each other showed their relatedness. So at the top here, we have the black Borg, for example, aligned with the purple Borg, where you can see similar blocks of genes that are rearranged differently in different borgs. Same here with comparing the lilac borg to the purple borg. We see this very similar region that's aligned in both borgs, with some additional genes in the purple borg expanded from the regions in the lilac borg. So we know that these mysterious elements are all related to each other. And they likely begin the replication at the genome ends, the terminal inverted repeats that I mentioned, and we know this from calculating the cumulative GC skew in these genomes. So for the GC skew, the— it tends to be positive in the leading strand and negative in the lagging strand, so you expect to see the switch in the GC skew just at the terminus or origin, with the peak corresponding to the terminus. So we can see that the origins are at the ends of the genome here, and this is something that we can see a lot in prokaryotes, where there is a richness of G over T in the leading strand, and that's how we calculate this GC skew and the cumulative skew plots that I show on this slide. So we analyzed the genome structure and looking further at the encoded genes, most of them had no hits to any database. So on the left here, I have the domain-level attributions of the different genes compared to NCBI and other databases. So you can see that a large portion of the genes had no domain annotations, whether bacteria, archaea, or eukaryotes. And then the majority of the genes after that were most similar to archaeal genes, followed by some genes very similar to bacterial ones, and some enzymes that were similar to eukaryotic viruses and eukaryotes. But looking at the species level of the genes, trying to see attributions for those encoded proteins. Most of them, as I mentioned, had no hits, but the first best hit after that was Methanoperidins nitroreducens, which is by far much, much more attributed than any of the other species that we analyzed, which was the first hint that maybe it is related to Methanoperidens archaea, or maybe replicates in Methanoperidens archaea. And I'm showing here an example of the lilac borg, but you can see this plot for all borgs in the preprint. So we know they can't also be part of the Methanoperidens chromosome, because looking at the GC content of the borgs, it's very different from what we find in Methanoperidins. So if they were part of the same genome, they should have a somewhat consistent GC content, which we don't see here. They differ by about 10%. And looking further at the abundances in the samples, you'd expect, for example, if it was part of the same genome, then different regions of the genome would be sequenced at the same level. So the abundances of the reads would be similar between Methanoperidins and Borgs. But as you see from this plot of relative abundance, there are very little correlation with the Methanoperidins bins compared to the Borgs, which implies they're not part of the same genome. But also you can see that the Borgs don't necessarily have a consistent abundance relative to Methanoperidins, so it isn't a consistent copy number compared to the methanoperidins archaea. And looking further at the correlation of different methanoperidins bins from the same samples to the borugs that we recovered from the samples, we can see that groups of methanoperidins correlate very well, showing here the Pearson correlation with very high statistical significance shown in asterisks, groups of methanoperidins correlating well with groups of different borgs. So these borgs tend to be very abundant in the samples that also contain these methanoperidins bins, which gives some indication of which methanoperidins could be a host, if this is some kind of virus or plasmid or other extrachromosomal element. And looking at the concatenated ribosomal protein tree— so we took all the ribosomal proteins that are in the methanoperidins and we are able to concatenate those to build a phylogenetic tree and a representation of the evolution of the methanoperidins bins— and we can see that the methanoperidins bins or genomes that were previously reported, which are shown in gold, tend to cluster separately from the methanoperidins that we recovered in our samples, which was the first indication that maybe the methanoperidins that we're studying here are novel species compared to what was previously reported in this genus. And looking at comparisons of the average nucleotide identity as well supports that these methanoperidins co-occurring with the borugs, which are shown at the bottom here, compared to the previously reported methanoperidins shown at the top here, they seem to be very separate in terms of ANI. And this is a threshold that tends to be used a lot with comparing different species of microbes. So we can see that these methanoperidins that we recovered again are pretty different from the methanoperidins that were previously described in the literature. And looking at ribosomal proteins, I was thinking maybe this is a sister lineage to the methanoperidins that we recovered here, and that's why they tend to have very similar genes encoded. But what we found was that it lacked— all the borgs lacked most of ribosomal proteins, lacked most of what we call single copy genes that tend to exist in one copy in a single genome. So the fact that these borgs don't have the majority of ribosomal proteins implies that they couldn't be a separate lineage or an independent life form that sustains itself. They would have to rely on a host to translate their proteins. Somewhat like a virus, except looking at all the proteins that are encoded on the methanoperidins, we couldn't find any viral structural genes or any indication that this could be a virus. And peculiarly, all the borgs had this ribosomal protein L11, which currently in archaea at least has never been tested or known to be no tested function of in biochemical assay. And the ribosomal proteins that were found in the borgs, they tend to cluster with those identified in methanoperidins species, so they're pretty similar to but still distinct from the ones found in methanoperidins. So even though they are most similar to methanoperidins compared to Methanosarcina or other Archaea, they still form their separate subclade here. And next I compared these borgs, their protein families, everything that's encoded on the genome, to all archaeal viruses, all archaeal extrachromosomal elements, plasmids, and so on. And even compared them to all bacterial plasmids as well. And looking at all the encoded genes, they seem pretty distinct from all previously described extrachromosomal elements, and actually they seem to have more genes in common with their host than the other archaeal elements. But at the same time, This is sort of a zoomed-in view of the bottom of that graph. You can see that Methanoperidins has many, many protein families that are present in the archaea but not in the borgs, many protein families that are shared between borgs and Methanoperidins, but also many protein families that exist on borgs that are not found in Methanoperidins. And comparing them further to linear plasmids, for example, in bacteria, since no linear plasmids are currently known in archaea, they seem to also be pretty distinct. And looking specifically at protein families that might be shared between the borgs and other bacterial plasmids, these seem to mostly be methyltransferases and other enzymes that are not used in replication or with partitioning for plasmids, which is another indication that this might be a separate element that was not— was neither a virus nor a plasmid. Using, you know, different machine learning tools as well and classifiers, we were also not able to classify the methanoperidins, or the borghs, as a canonical plasmid. We've also seen evidence for acquisition of many genes from the host archaea to the borghs, and this is really what inspired the borgh name. So one of the gene clusters that we saw was transferred from the host to the borgh very recently is the mcr operon, and that's used for oxidation of methane to carbon dioxide. And that was very exciting for us because that also hasn't been shown to be horizontally transferred before. But also nitrogen fixation genes, this operon shown at the bottom here, and also multiheme cytochromes. And we can see this because there's a distinct separation of The GC, as I mentioned previously, the borghs tend to have a GC of about 33%, and these genes specifically were matching the GC content of the Methanoperidins host, so they were more about 42 to 46% GC, which is the red plot in the background here. So it really sticks out like a sore thumb that these genes were probably horizontally transferred from somewhere else. And also looking at the phylogenetics of these genes, we saw a similar pattern to the ribosomal proteins, where the MCR genes were clustering with those from Ethnoparidens but separately in a distinct subclade. And the nitrogen fixation genes were also very interesting because of the relevance of this to agriculture, for example, and how these were found in sort of a foresty area. But we also saw many different transfers between the borgs, but the horizontal transfer of the MCR operon specifically was of most interest to us because the methane cycle is arguably the most tightly linked to climate change. So methane is a, is a very significant greenhouse gas that's roughly 30 times more potent than carbon dioxide, and approximately a gigaton is produced every year by methanogenic, or methane-producing, archaea that sort of inhabit these anoxic environments that are devoid of oxygen. And global emissions tend to be essentially regulated by these specialized methane-consuming microorganisms that could oxidize the methane either to methanol or to carbon dioxide. Because we, as, you know, the human species, we don't really reduce— we only know how to reduce methane emissions but not as much how to sequester or consume the methane emissions. So this is primarily done by these microbes that are found in anoxic environments and in the deep ocean. But these microbial lineages like methanoperidins, they can gain energy exclusively from the anaerobic oxidation of methane. So these microbes essentially is where methane would go to die. And in part, these archaea help to regulate the Earth's climate by mitigating the efflux of methane from the natural sediments into the atmosphere, as I mentioned. But despite the importance of this, the biological processes and archaea in general are very poorly understood. And this demonstration was the first time that we were able to show that the genes for the MCR pathway, for example, can be horizontally transferred, possibly between lineages. And even beyond the horizontal transfer from the host to the borgs, as I mentioned, there are also blocks of identical regions between different borgs that indicate recombination between different borgs. So not only can they acquire genes, for example, from their host, but they can also acquire genes through recombination with different borugs. And this was also another indication that several borugs may have coexisted in the same host cell at some point that enabled them to exchange this DNA. We've also seen evidence for many tandem direct repeats that were very puzzling across Borg genomes. So everywhere that you see here a gold line is where we found these tandem direct repeats. So these are repeats that occur, you know, anywhere from 3 to 15 times, mostly enterogenic in nature, but in some cases also found within proteins. And these were also not found in the Methanoperidens host. And the function of these repeats is still currently unknown to us. And looking at the repeats that were found, for example, within the proteins, a lot of times they are possibly corresponding to amino acids since they're divisible by 3. And in, for example, the N-terminus, you can see that these repeated regions are not present in homologs that are found in either Methanoperidens or other archaea. And same result that we saw in the C-terminus and in most of the proteins that had these repeats. And looking at the sequence level similarity of the repeats across a single borg and across the different borgs. We don't see any sequence similarity or conservation between these repeats across a singular borg or compared to other borgs. And speaking of repeats, we also found CRISPR-Cas systems in the borgs. So these borgs have their own CRISPR array that are these interspaced repeats with spacers between them.. And similarly to what we saw in phages, these CRISPR-Cas systems in the borghs tend to lack the acquisition machinery, so they don't have the Cas1 or Cas2 genes that are usually used to integrate new spacers into the array. And typically in a bacterium or archaeon, these arrays tend to have spacers that match different viruses that infect them. So it's a record of previous infections within, for example, a Methanoperidin's genome. But what we found, for example, in phages was that these phage CRISPR arrays tend to target other viruses that are competing for the same host. And in this case, we saw a similar situation where the CRISPR array in the Borg tends to target a region in the Methanoperidens genome that is a mobile region that is likely a mobile element in itself, where this CRISPR array, it's an RNA targeting system, and we saw that it targets specifically the sense strand of a protein in this mobile region. Which was also very interesting to us, seeing this possibly warfare between the borgs and other mobile elements that are in the methanoperidins. And looking at what other functions are enriched on borgs, the majority of genes are, as I mentioned previously, still a mystery to us with unknown functions, but there are quite a lot of genes for DNA or RNA manipulation, energy metabolism, transmembrane proteins, and many other functions, which to us is, you know, a very interesting field to start to explore. And looking at specifically the genes that have annotations, credible annotations, even beyond energy metabolism and electron transfer machinery, that are shown at the top left here that must be augmenting the Borg's ability to do this, or at least influencing it in some way. There are also many genes involved in the central carbon pathway. They have genes for polyhydroxybutyrate synthesis, also known as biodegradable plastic in the synthetic biology community. They also have many genes for stress response, for responding to the environment, and as I mentioned, CRISPR-Cas systems that target a mobile region in Methanoperidins. So clearly this megabase of DNA is highly influencing the host, whether positively or negatively, while it exists in the host cell. And we've since found over 20 examples of these extrachromosomal elements in many different environments from the East River in Colorado and rifle, as well as different mercury mine discharge in California. And we still continue to study these borgs to identify what exactly they're doing and what the 80% of unknown genes on these genomes might be encoding. And with that, I'd like to thank all the colleagues, mentors, mentees, friends, and collaborators who made this work possible, as well as, you know, the rest of my thesis and helped me grow as a scientist during the past 5 years. Thank you, and I'll take any questions.

Thank you very much, Bassam. It was a very interesting presentation. And one that I think in general extrachromosomal elements are very significant and probably play some interesting roles, not only in the fields and the environments in which you extracted them, but perhaps in directly in agricultural environments. So I just want to remind everyone to please put any questions you may have into the Q&A panel. We do have one question regarding then-if genes. Can you be more specific as to which ones?

The NIF genes? Yeah. Yes, I can go back here. So we see NIFK, NIFD, NIFH, as well as different regulatory genes for the NIF operon. We also did a phylogenetic tree of NIFHs, and as well as NIFD and NIFK, which are found in the BioRxiv that is— the citation for is down here. Where you can see, you know, conserved residues, for example, and how the NIF genes are likely acting for nitrogen fixation.

Yeah, all right. So in particular, when the question had been NIF, but it was really the NIT genes, N-I-T genes.

Yeah.

So sorry for asking that question the wrong way, but— so one of the reasons I'm very interested in— well, one among many reasons I'm interested in borgs is because of the potential for carbon sequester— for methane sequestration, in fact, and its relation to the potential for actually having, you know, an impact on agricultural emissions that may occur and actually getting us to net emissions in agriculture. Can you talk a little bit about that? You haven't looked at any environmental, agricultural environments yet, but is that planned, or what do you think about the potential for that?

Um, yeah, definitely, and that's something that we're actively working on. We have, you know, several new samples from agricultural sites that we're currently studying borgs in. So it's definitely on our radar, and there's a lot of potential for both methanoperidins and borgs in influencing agriculture.

Right. So with one of the things I thought was interesting is You actually showed that the methanoperidins and the BORGs are different from a structural standpoint, yet they seem to associate with each other. Have you identified any kind of dependence? I mean, do you think BORGs can exist without the methanoperidins, or do you think methanoperidins can exist without BORGs? Have you looked at that particular aspect?

Yeah, so we definitely looked at, you know, all the methanoperidins enrichments that we could find, for example, and a lot of the previously published methanoperidins that were enriched, we couldn't find Borgs in those bioreactors, which implies that, you know, maybe there is some specific species of methanoperidins that harbor Borgs or specific, you know, environments that where the methanoperidins could have borgs. But the borgs specifically, I don't think they can exist without the host because just of the dependence, or very highly likely dependence, for protein production since they only have one or two ribosomal proteins in any given genome. Despite the sort of megabase in size. So they likely depend on a host for protein production, that they can't exist separately.

Yeah. So do you think there's enough energy-related machinery in the borgs that would be required for NIF or carbon or whatever functions that you may find down the road?

Yeah, they have a lot of ATPases as well as the different, you know, energy machinery that I mentioned. And like I said, that 80% of the genome is, you know, essentially dark matter at this point. So there's a lot still that we don't understand in how exactly they influence the host. We just know definitively that they must be influencing it with this many genes.

All right. So have you tried to image the borgs in order to locate them inside the microbial cells?

That's also something that is still not achievable currently, just because we still don't have enrichments of methanoperidins that have borgs in them. So all the enrichments that we've looked at from you know, collaborators and published data don't have orgs in them.

So having, having the inability to culture these have been a major drawback. Do you see any progress being made in that realm? Because it could have a— it could help us really get to that next level of understanding.

Yeah, definitely. I mean, the knowledge for culturing these microbes is growing every day, and there are practices for how to enrich Formothenoperidins, so it should be possible to enrich the ones that— from the samples that have Borax in them. It will just be a matter of time.

How will you actually disassociate them in that process?

Well, if they replicate within their host, then it would have to be imaged, for example, specifically, you know, in the host, or, you know, harvested, you know, the DNA specifically from the host. But I don't think it would be possible to separate it completely with the current methods that we have.

Yeah, so you've spent your PhD working on extrachromosomal elements, and so I'd like to talk a little bit about those. You know, they often talk about non-essential, that they are filled with non-essential but useful genes, and by that I'm assuming that means they're not essential for the element itself but useful to their host. And in the case— mostly these are circular, and yet in Borgs you found it to be very linear. Can you talk about that a little bit?

What makes them so unique? Um, yeah, so the genome structure specifically was something that really piqued my interest in, in this element, because as you said, most exochromosomal elements tend to be circular. There are a few linear plasmids in bacteria that were previously reported, specifically in Streptomyces, which I showed some comparisons to here, that it's still pretty rare compared to the circular ones, and that also indicates sort of different methods of DNA replication and different, you know, structures for the genome itself, that there's a lot of, I think, interest in trying to understand how that works differently, especially with something like the Borgs. They have the, you know, 2KB inverted terminal repeats that somewhat reminiscent to a transposon, for example, except no transposon anywhere is going to be a megabase in size with inverted repeats that long. So it was really interesting, for example, to see if it integrates into the host genome, but as I mentioned, trying to extend the genome into maybe the host genome led to no evidence for integration. So it's definitely something that we're still trying to understand how and why they're so different from other reported elements. And for me, you know, coming into the PhD, extrachromosomal elements was my, you know, passion and interest, whether it's phages or BORGs. So these are all the questions that, you know, I hope to answer.

Yeah. So you mentioned dark matter, and we no longer call it junk DNA because we know it's not necessarily junk, but 80% of the Borg is comprised of dark matter and repetitive elements. And, you know, it's for, you know, I've spent a lot of time working with plants, and that's a number that's very similar in plant genomes. Is there any relationship, you think, to the kind of plants that might have been in that region? I mean, is it possible there's been horizontal gene transfer in that, or is that just completely out of the realm of possibilities?

I mean, I never say never. Like, I know there are maybe some papers that will describe sort of cross-kingdom gene transfer. I haven't seen any evidence of that here, but I always keep an open mind.

Yeah, well, that— one of the other questions that has been is that, is there something about the soils or the climates that result in the potential for the borers to be there? Because it looked like, at least just glancing at the pictures of those 3 environments, they are similar. They're not, you know, they're isolated. They may have some commonality. Have you looked at that at all? Any kind of comparison of where, when, soil type, etc.?

So we had some chemical profiles that we published in the supplement of the preprint. The one thing that we can say maybe more definitively is that we found these more in, you know, really deep soils and anoxic environments that might be devoid of oxygen. I would still expect them to be found in other places where you could find methanoperidins, whether that's, you know, in the deep ocean, for example, or other places. But that still remains to be explored.

All right. What do you think about the evolutionary significance of borgs compared to to other extrachromosomal elements or other microbes for that matter?

Yeah, it's a really interesting question for us to try and maybe speculate how these borgs came to be to begin with. So they have a lot of genes, as I mentioned, that sort of cluster with but separately from the ones from Methanoperidins. And so that raised the question, for example, like with the ribosomal protein, was this a sort of sister lineage of Methanoperidins that got reduced, or is this maybe, you know, a plasmid-like element that got really expanded with more gene insertions? There are, you know, many different speculations and things that we could suggest might have happened that led to the borgs. But right now we can't say anything with certainty how they evolved to be the way that they are.

Do they currently replicate, or have you seen evidence of that?

So we haven't calculated replication rates, but we were able to see how it would replicate, which was indicative from the GC skew plots that I showed. But anything further than that would still require more sampling and maybe bioreactor studies.

Have you done anything in terms of calculating the numbers, you know, perhaps in the ones, the numbers in the deep soils, compared to the oceans, etc.?

So that sort of large, larger-scale analysis of prevalence would still need to be in, you know, further work. But looking at abundance in the soil specifically, we didn't see a consistent abundance. So there isn't, you know, like a 2-to-1 ratio, for example, compared to the Methanoperidins, it seems to be anywhere from, you know, 2 to 8 times the Methanoperidins genome. So it didn't seem very consistent, and it seems to be about a third of the Methanoperidins genome size.

So you mentioned the nitrogen fixation genes that you found. Can you talk a little bit more about that? At least if I recall in looking at the chart, it was not— there were not a great number of those genes. But so can you talk about what potentially might we might be able to do with those?

So we found the, you know, this specific NIF operon that was found in many different borugs as well as in many different methanoperidins. So it's likely that they function in nitrogen fixation, but right now we don't have any experimental evidence to show that, you know, they could be deployed in some way for nitrogen fixation.

So were there plants in— when you— at least for the ones in the— where you went to the deep soil, were there plants there, and did they Were they plants with deep roots or any common characteristic there that might have benefited from those genes?

There were definitely roots in the samples that we recovered. So, you know, we had to specifically remove those in order to be able to extract the DNA more efficiently. So it's possible, it's definitely possible. And yeah, I look forward to, you know, having more evidence from the specifically agricultural sites that we're looking at.

So if you actually tried to separate the plants from the borgs, how did you— or is it possible— well, let me say it another way. Is it possible that they aren't— the genes aren't from the borgs, but in fact are from the plants, that there is some possibility of contamination? Contamination in the process, or which you would maybe see in one or two sites but not across the board?

So when I mentioned, you know, removing the roots, that was purely like physical process before the DNA extraction. In terms of contamination in the genome, that's something that we look after with every study that we do. And when I showed, for example, the sequencing reads all aligning to the Borg genome, it's very clear that these genes all, you know, align one after the other within the same genome. So even though it has the GC content similar to the Methanoperidens host, it fits in the Borg genome using the overlapping and paired-end reads from the sequencing. So it's highly unlikely that it is from contamination. But all the reads are publicly available, and people can do that analysis themselves.

Right. So is there any signature sequence of DNA found in Borgs? For example, like what is used in 16S sequence and bacteria?

So we haven't seen specific genes that are shared between borgs that are highly unique to borgs that we've used for something like that. But looking at the L11 ribosomal protein, which is the ribosomal protein that I showed was present in all borgs, that we reported, that tends to be a pretty good indicator of the Borg genome because it's very similar to the Methenoperidins but a separate sort of subclade. And so looking at the L11 from any given sequence, if it clusters with the L11 from the Borgs, that's likely going to be a Borg genome.

So somewhat of a unique identity. Yeah. Yeah. So is there a sequence similarity for any other specific bacteria or archaea?

No, the most of the similarity has been to the methanoperidins. There are some genes, for example, that are involved in extracellular electron transfer. So, you know, flavoproteins and other machinery that are used for that are very similar, for example, to the ones found in Geobacter sulfurireducens. So that's, you know, another bacterium that also uses extracellular electron transfer for their respiration. Using different metals. So that's something that was very similar to the ones in Geobacter, which— and also similar to the ones in methanoperidins. So it's very clear that it likely influences the respiration of the methanoperidins using possibly different metals as electron acceptors.

All right, thank you. I'm going to kind of step back from your particular paper and talk a little bit about the work that's going on in Dr. Banfield's lab regarding carbon cycling and the role of microorganisms in that. And not only from its standpoint of production and degradation of methane, but very generally for what we're starting to see more and more is that, in fact, microbes do sequester carbon. They're heavily involved in the carbon cycle, more so than what we thought in the past. And just curious what you see as kind of the future research opportunities in that particular area.

Um, yeah, I mean, for me, sort of environmental microbiology has always been a deep interest, and I think there's a lot of potential in studying how different microbes impact the carbon cycle and how, you know, things like borgs, you know, the more we recover and study, the better understanding that we'd get from that. And there's a lot of research, for example, on chloroflexii that tend to fix carbon somewhat differently. That was a preprint that we put out earlier this year as well. So if there's interest, you could check that out too, by my colleague Jacob West-Roberts. So yeah, there's a lot of interest in that area and in carbon sequestration. And I think it's just going to continue to grow. Right.

So you are a PhD student who has published a preprint. Yet you were all over the media. I mean, not just you personally, but this paper has generated so much interest. How have you handled that as a PhD student? I mean, you've been— it's been in Nature, Science, it's been discussed in a number of different venues. So how have you dealt with that as a PhD student?

Yeah, it was definitely a bit overwhelming. We had some media attention from the phage work that we did that we published about a year earlier and sort of the CRISPR-Cas work that we did from that as well. So I learned to handle that a bit better than maybe I would have if I hadn't had that previous exposure. But yeah, it's definitely a bit overwhelming when my focus is on the science and advancing the work. I've learned to communicate my research with people maybe more clearly than you know, with other scientists. So it's definitely been a valuable experience.

Yeah, not everybody gets a chance to interact with media and journalists, you know, so it is, you know, a unique opportunity. And I suppose just in listening to your statement that you would think that It's important for PhD students or students and individuals involved in science generally to very clearly articulate their science in a manner that is approachable to general public.

Yes, absolutely.

All right. Do you have any other last words of wisdom for the other PhD students that are out there?

Um, just I would say it's maybe not impossible, but very difficult to sort of set out to, you know, solve a giant problem and then do that. A lot of times it's, you know, discoveries made by very curiosity-driven research. So this plot of land that we found the borgs in, this was very much a, you know, a hike past that we decided, oh, what if we collected samples from here and looked to see what kinds of microbes are there? And we ended up finding, you know, the huge phages and the borings in samples like that that were, you know, essentially in Jill's backyard. So you never know what you're going to find. Something like CRISPR also came from very curiosity-driven research. So I think, yeah, just follow the research that interests you and you'll eventually find very interesting things from that too.

You have to be open to opportunities. I think once you see something that pops up just out of the blue, you know, you can then widen your horizons in that regard. Well, thank you very much, Bassam. It's been a pleasure to have you in the webinar today, and I really look forward to seeing this work published in a peer-reviewed journal and wish you the best of luck in your PhD and in your career. So thank you again for being with us, and we look forward to seeing everyone else on our next webinar in December. So thank you.

Thank you so much.