355: Bacteria Complete Your tRNA

This Week in Microbiology is brought to you by Microbe.tv and is supported by the American Society for Microbiology. To learn more about microbes, visit Microbe.tv and asm.org. this is twim this week in microbiology episode 355 recorded on april 30th 2026 the last day of april folks i'm vincent racaniello and you're listening to the podcast that explores unseen life on earth. Joining me today from Ann Arbor, Michigan, Michelle Swanson. Hello, everyone. I'm delighted to be with you. I can tell you're in Ann Arbor because you're in your office, right? Yes. Yes. All right. Also joining us from Charleston, South Carolina, Michael Schmidt. Hello, everyone. And if you hear a jet blast, it's only because the Blue Angels are practicing over our building. And so every now and then we get a jet blast. What are they practicing for? The air show that's going to be on Saturday in the center of Charleston on the river going back and forth. It was going to be at the Air Force Base, but due to the uptempo, they had to move it. And so it's going to be quite disruptive. I don't know. People ought to read a book. Stay home and read a book. or a research article or research article which we do for you here also joining us from Tacoma Washington Mark Martin always delighted to be TWIM adjacent with some of my favorite microbiologists thank you Mark yes we read papers for you so you don't have to read them you could listen to the results so you don't have to go watch an air show okay and be in the sun for hours and get skin cancer. Okay. Do the right thing, folks. If you enjoy these programs where we tell you about science, we'd love your support. Go to microbe.tv slash contribute. And we continue every two weeks having awesome papers. And today is no exception. And here's a paper that Alia would love, right, Michael? Absolutely. For our regular listeners, the snippet from our last episode, TWIM 354, spent quite a bit of time illustrating the importance of a microbe for which we're unable to grow. Well, in today's snippet, we're looking at an equally fascinating paper published in March of 2026 by the International Society for Microbial Ecology and their journal, ISME Communications. And the paper's titled, An Enhanced Domestication Method for uncultured bacteria, and it was authored by Andrew Morrison, Raffaella Jackson, Paul Fremont, and Harry Lowe, and they are in the Department of Infectious Diseases at Imperial College London. Their study offers us a clever approach that uses a variety of things, 3D printed technology, all sorts of peristaltic pumps, and traditional media in order to move the unculturable, if you will, to move them away from the nutritional shock of standard plating and towards a gradual transition that respects the organism's native environment and addresses one of the most persistent hurdles of our discipline that I affectionately refer to as the great plate count anomaly or microbial dark matter. Now, we've known for decades that the vast majority of microbes out there simply refuse to grow using standard laboratory techniques and media. And they're not unculturable. We just haven't figured out how, or dare I say, domesticate them properly. And while reading this paper, I had this tremendous sense of deja vu. It took me back to my days in graduate school and to the laboratory of Dr. Howard Guest at Indiana and Dr. Julian Wimpany of the University of Cardiff, who at the time was on sabbatical in Dr. Guest's lab. I recalled how Dr. Guest and Dr. Wimpany were trying to cultivate these very fastidious photosynthetic microbes. And it was here I was exposed to the gradostat and gradient plates. And then, as you might surmise, I fell through the licking glass, folks, and I went to see what's happened to this technology over the last 40 years, where I unfortunately found the obituary for Dr. Wimpany, who was lost to us in 2016. But in reading it, I reflected on what he might think of this paper. And in his obituary, I'm going to read a piece of it. It really sets the tone for this paper. Pure monocultures of microbes growing in liquid suspension never entirely satisfied him, and his application of his own novel techniques revolutionized and reinvigorated the subject. This innovative approach propelled him from the traditional era of shake flask and continuous culture microbiology to his newly invented gradient plates and gradistat devices, providing simultaneously multiple graded environments for selection and optimized growth of microbes. Microelectrode measurements on bacterial colonies on solid media led to analogies with growth of tissues. And using constant depth thin film fermenters led him to consider the problem of surface growth of mixed populations in biofilms in the real world of dental plaque and serious problems of microbial metal corrosion. And as a young graduate student, I just was fascinated by Dr. Whippany and the strange techniques he was pioneering in the cold room and warm rooms of IU. So now with that tribute to Dr. Wimpany and Dr. Gess, now back to the strengths of Eden. And Michael, if I could just put a sharper point on this, the authors point out that 99% of microbes remain uncultured. Absolutely, Michelle. So we have a lot of work to do. So back to the strengths of this Eden that they develop. It's simply a method for, as they say, enhanced domestication of microbes that will gradually acclimatize the bacterium to culture media using a 3D printed continuous flow cultivation plate. Now, their method improves on taxonomic diversity from a polymicrobial population. It yields more unique and likely uncultured taxa and increases microcolony recovery. And they show in this paper, by using EDEN, they were able to isolate a novel bacterium with antimicrobial activity against multiple drug-resistant pathogens. What I found particularly elegant about this work is the marriage of these microphilitics using off-the-shelf materials. And one of the things that Mark loves to use are these inexpensive 3D printers. And they really make great use of things that, candidly, we did not have at our fingertips in the 1980s. So instead of throwing bacteria directly onto nutrient-rich agar, which often triggers a lethal response, this Eden approach relies on an exponential gradient to slowly transition the community from the sterile native pond water that they may have been growing on to the laboratory medium that was originally developed for the characterization of potable water. And that, of course, is Reasoner's 2A that many of us simply know as R2A agar or R2A medium. And they did this over a 28-day period. Now, their approach minimizes microbial exposure to abrupt environmental transition, which in turn leads to these potential nutrient shock, which often sends the entire community into a tailspin. And so what they did is they developed a 3D printed micro well plate, you know, the common 96 or 384 well plate that we all have seen, but they did it with off the shelf components, you know, the plastics that you can buy and they made them so that would work with the common 3D printers that are relatively inexpensive now that you can afford to purchase and incorporate into your laboratory. And they made sure that the materials they were going to use to fabricate this device were not toxic. Yes. They also did a trick where they had these peristaltic pumps with built-in continuous flow reservoirs. And I saw some of that when I was at IU over 40 years ago. And the use of a semi-permeable membrane then facilitate the diffusion of nutrients in and metabolic waste out to mimic the natural flow of the microbes' home habitat. at. And so the first questions that they concerned themselves with was demonstrating the utility of their system. And they measured the effectiveness of Eden to cultivate or, if you will, domesticate the bacterium over standard cultivation methods. And they did this by assessing population diversity. And I encourage you to read their method section. It's extremely detailed and it shows the rigor that the authors employed in order to demonstrate the value proposition that Eden could afford to the microbiologists who wish to effectively domesticate the population. And they coupled that rigorous approach to a very elegant design, which they show schematically in figure 1D. So you can see how they gradually pump in more of the optimal media, replacing the native water in this case. And their first figure could easily serve as a graphical abstract for this paper if this journal had a graphical abstracts. And they walk us through this Eden process with this elegantly simple diagram to digest and to support their narrative and their conclusions. Michael, I have a question for you. So they show you they're taking pond water here, some environmental sample, and they put it in the wells. And then in the end, they show one microbe type in each well. So how does it become clonal like that? It's how they're diluting it. It's the semi-permeable membrane. They're effectively putting it in the cereal dilution. The microbe is finding the right concentration of nutrients in which to grow. If you're familiar with the agar shake technique, where we suspend various concentrations of agar in a flask. This is old time technology. We're talking the 1920s instead of the 2020s. You could effectively grow bacteria in this gradient of agar with various nutrient concentrations. And in the old shake technology, they use mud at the beginning and they just effectively did a gradient that way. This technique is using the pumps and these magic 3D printed plates that they developed in order to facilitate the Goldilocks effect of the media. And the media that they selected or the medium that they selected was R2A, which is a complex medium. It's got peptone in it, case amino acids, yeast extract, dextrose, soluble starch. And it's that magic dilution where you're really having the Goldilocks effect. And that's how they get the isolated organism And so they evaluated Eden using three experimental setups They used Eden with the pond water and that started off with inocula of 10 to the 4 microbes And then they serially, gradiently, if you will, dilute it. And they have R2A as a control and they have pure pond water as a control. And it's this blended mix. So their first experiment asked, how did Eden do on the bacterial polyculture pond water with the slurry of 10 to the 4? They inoculated into the growth chamber and then it's effectively, if you will, pumped to extinction. So they have four setups and they did it in replicates of five. And after 28 days, the viable cells were counted across the three Eden cultivation conditions. You had the acclimatized Eden, which is the blending of the pond water and R2A. They have R2A by itself, and then they have the pond water by itself. And their second figure beautifully illustrates this. And you can look at the panels and it really shows you in the middle panel B that using Eden plates cultivates a greater diversity of bacterial communities than the standard methods. And the panel D of their second figure, if you will, really shows the power of the acclimatized. And acclimatized means the blending of the R2A. Gradual. Gradual. The gradual. Yes. And it's beautiful. And of course, we have all the standard modern techniques in here. We have applecon sequence variants. And their fourth figure in their manuscript, since this is a snippet, I'm skipping a bunch. Their fourth figure offers us that the acclimatized Eden polycultured yield significantly increased the likely uncultured applecon sequence variants. Again, this is well evident in panel A where their Venn diagram shows the proportion of likely uncultured amplicon sequence variants that are either unique to a cultivation condition or shared. And they didn't just show more things grew. They proved they were different things. And so the acclimatized Eden cultured recovered four times more likely uncultured taxa than their standard methods. And this include rarely cultured groups like holophagia and acidomicrobialia. Yeah. And in the word of microbiology, the proof is in the pudding, or in this case, the killing. What the authors finish us off with, and this takes us to figure eight, so I'm really jumping here. They finish us off with figure eight, which is the functional payoff for Eden, or that their 3D printed acclimatization system actually works. If you look at figure 8a, you'll see an agar overlay assay. And this is, if you go back to the old time religion to Julian Wimpany's technique of the gradient plate, they've taken isolate number 95, a novel microbe recovered from the pond water using the Eden method and challenged it against the lawn of MRSA, methicillin-resistant staph aureus. So what you see is isolate 95 has this beautifully clear halo. That's a zone of inhibition. It tells you that this previously uncultivated, now isolated and growing bacterium is pumping out something that MRSA simply can't handle. They ran it right next to Pantibacillus polymyxa, a known heavy hitter in the antibiotic producing world. And this isolate, number 95, isn't just participating, it's competing at a professional level. It's making an antimicrobial that knocks out MRSA. Now move your eyes over to the growth curve figures in 8B. This is where the data get real and should serve to warm the hearts of all our infectious disease colleagues out there concerned about where we're going to get our next generation of antibiotics. They just didn't stop at MRSA. They tested a crude extract of isolate 95 against a rogues gallery of pathogens. We're talking the nasties of the nasties. extended spectrum beta-lactamase producing E. coli's, we see 88% inhibition. Vancomycin-resistant enterococcus fecium, again, a solid 60% reduction in its growth. And then methicillin-sensitive staph aureus and methicillin-resistant staph aureus, both were hammered with a greater than 90% inhibition. So they literally took us from pond water all the way out to a potential isolate that can be producing an antimicrobial that we can effectively exploit. And they were able to pull out isolate 95 out of the pond water. And it was a previously undescribed species of an alpha proteobacterium that again produces this broad spectrum antimicrobial activity. And they just pulled it out of pond water. God knows what else is out there in environments that we haven't sampled. So it really demonstrates this functional success or, if you will, proof of concept of their Eden approach. It really helping us question, you know, effectively fulfilling or closing the loop on the experiments. Now, they admit. In particular, they show with their putting the pond water directly into the R2A media or into the pond water. They show that. It's not that the bacteria can't grow. They choose not to. Yes. But if you gradually introduce, gradually shift them over, the bacteria can do its thing and gradually adapt. Now, this is a resource-intensive technology. And 3D printing is, of course, coming down in price and accessibility, but it's really the automated controllers that they're using for their multi-pump peristaltic system that facilitates the control of this exponential gradient. And that's a significant step up in complexity and cost compared to the stack of Petri plates that I use to make gradient plates. And the other thing that should trouble microbiologists, because we're all type A personalities and you want the experiment done overnight, is the 28-day acclimatization period. This is a long wait and see for most microbiologists in this fast-paced world of research and also the five replicates. But again, it highlights the rigor to which these authors really attended to the detail. And it really advances the discipline or, if you will, the culturable frontier. It gives us a systematic way to tap into this dark matter of the microbial world. If we can grow them, we can study their physiology. And as we saw in episode 354 in Vincent's paper, where we had that one bacteroides that was responding to the lactic acid that was a consequence of the heart attack, it really opens the potential for doing the science that we all love in being able to fulfill and demonstrate equivocally that it's just not causality, but we are not coincident, but we do indeed have causality. Michael, so can we take the entire gut microbiome now and grow it? We probably will be able to do this if we can figure out what's in your stomach, Vincent. that unfortunately you don't eat r2a but r2a is is stand the test has stood the test of time for growing microbes out of potable water maybe r2a will be the answer but i think we have to work on what the ideal diluent is if you will they got lucky with principles hold there's no reason to think we couldn't apply the same approaches. Michael, just a couple of things, if you don't mind. This is the elegant grandchild of the work that Kim Lewis and Slava Epstein were doing at BU, you know, with the buried Petri dishes in the muck. Oh, yes. Right. So this is like much more elegant than that. I do have a concern about adaptation because in the microbial world, that's often evolution. And the problem is, is you don't have the original isolate to compare to because it has to evolve to that. Now, I am not faulting finding really cool things, you know, and I've played with R2A a lot. And there are a lot of things that'll grow on R2A that won't grow anywhere else. I've found those. But what are your thoughts on that? We don't know if it's evolution, which implies a genetic change, or whether it's adaptation, signal transduction and turning on and off different but how could you tell the difference if you don't have the original organism but what you do is you have the original pond water right and you can do total genome sequencing of the original pond water okay yeah and you can do total genome sequencing of the isolate and then with the metagenome you can compare and contrast And you can look for epistatic variants, which is your evolution argument. Or as Michelle said, you can figure out if it's some magic Goldilocks effect of adaptation. And so I think it's, in short, Eden reminds us that if we want to see what's really living in our world, We have to stop treating microbes like they belong in the lab and start treating like they're still at home. And it gets to your adaptation. And, you know, it goes back to is that E. coli that is causing the urinary tract infection in your bladder, the same one that's effectively growing in your gut, providing you vitamins. Yeah. And because it was a snippet, Michael couldn't go through all the detail, but they also set up the experiment so that they were looking at what they called polycultures. So not just monocultures, but appreciating that bacteria live in communities. So they set it up so that each micro well could have a different combination of bacteria from the pond water. Yes. Thank you, Michael. You're welcome. All right. Now, this paper has been in the queue for a long time, right, Mark? Ha ha ha ha ha. Oh, you got it. I'm very impressed. Mark generally gets those kind of jokes. He gets corny jokes. Thank you. Thank you. So this is, I have to tell you, I put, let me see if I did this right. Here we go. This whole article is a flashback to my whole background in eukaryotic biology and biochemistry. and I shiver the minute I see enzyme kinetics. And this is well before I learned the one true microbial faith. And the title of this paper is the Oncogene SLC F35F2, that's a name, is a high specificity transporter for the micronutrients cuene and cuicine. Now, I have to tell you, there are 17 authors on this paper, and they're primarily from Florida State and the University of Dublin. The first author is Leboni Brutniak and the corresponding authors are Vincent Kelly of Trinity College in Dublin and Valérie de Cretilagard at the University of Florida in Gainesville. And this appeared in the Proceedings of the National Academy of Sciences June 17th of last year, 2025. I have to tell you, there's a couple of wonderful introductory overviews of the article, and I put one of them up in the show notes. And I have to tell you, a lot of people are going to be interested in this paper because it lends itself to the whole idea of how we interact with our gut microbiota. The value of the article is quite broad, and let me quote from the paper. The identification of the SLC transporter links cuycine availability directly to several critical physiological processes including brain health memory formation and cancer suppression That's a lot. Yeah. And that's why you'll see a lot of a few places. In fact, there's a whole entry in Wikipedia on this. Yeah. And while you've emphasized the human impact, I love that these micronutrients are only synthesized by bacteria. Exactly so. And that's so interesting to me in a lot of different ways. There's a lot of stuff in this paper that we don't have time to cover. It's got so much to it. And so my apologies if I oversimplify for things. What's especially impressive is the breadth of approaches. Still a lot to know. And the abstract and the significance section of the paper give you a great roadmap of what's to be covered. And so the very first thing, if you want to have a look at this article, is to read the abstract after you read the significance section. You're going to be really, really excited. It's a really great example, as Michelle just said, of the role that gut microbes have on how our own cells work. I wanted, and I know you all agree with this, I have to tell you before I do that, I have a couple of quick things we have to say. nightmares from graduate school. What does KM mean? I'm still scarred from the experience. And what does it mean? It's just the concentration of substrate of half of the maximal velocity of a reaction, in this case, to transport of a substrate. Secondly, and this is something I have to ask you all about because I've taught about this kind of thing quite a bit and the nomenclature might have changed. The term oncogene, I was always taught to use the word proto-oncogene for the normal cellular version of such a gene that undergoes changes to promote carcinogenesis. We then call those oncogenes. Has this changed somehow while I wasn't looking? No, that's what Howard Temin originally said. It's okay. The problem is not every proto-oncogene changes. It's sometimes It's a matter of overproduction and that's enough. Exactly so. Exactly so. And what's really interesting to me, and I used to – I'm sorry. I don't teach introductory biology anymore. It's such a big deal to me to point out that these proto-oncogenes are absolutely necessary for signal transduction and cell division. And that's the – and when you just see oncogene, your mind goes to cancer. That's my concern. Well, that's – it's actually a misnomer, right? Yes. Because they're not there to cause cancer, yet that's what the name implies. They are there to regulate cell growth. And the first ones described by Harold Varmus and Mike Bishop, they called them oncogenes and oncoproteins, and we're stuck with it, unfortunately. Yeah. But it's not right. Language can be a prison. Yes. Finally, what a wonderful opportunity to really think about transfer RNAs, which most people, when they're teaching about it or learning about it, kind of gloss over. I've shown you all there's a little model of one that my undergraduate students made me out of beads. And so I really enjoyed some of the information here, how marvelous and strange they are, the modified bases that we're going to come back to, and how anticodons recognize codons, leading also to this whole issue of degeneracy of the code. And one of the things that I used to have issues with when I would teach about this is why are there so many codons for a particular amino acid, say histidine, but you don't find the same concentration of all of those transfer RNAs. And so that's something that I really enjoyed teaching about. So I was really ready to talk about this. Now, transfer RNAs have modified bases, and some of these modified bases are based on Q-ene and the nucleoside version of a Q-acene. Now, I have to tell you that in this article, they call Q-acene capital Q and Q-ene small Q. And because I'm trained as a geneticist, you can imagine what fun I had with that. It became structures and chromosomes, which don't let it throw you. So if you go to figure one, great place to start. And it's easy to forget the relationships here as you go through this very, very detailed paper. Now, queuing is being used to modify that transfer RNA using QTRT type genes, as you can see in the figure. Q-acene, on the other hand, needs to be made into queuing, and that's by gene products QNG1 type genes. Now, just as we said at the beginning, and I want to thank Michelle again, we need these compounds in our cells and we do not make them. So they must come either from our diet or from our microbiota providing them to us. And this is a very, very big deal because we knew we needed them and we didn't know how they were getting into our cells. And we do have part of the answer now, not all of it, but part of it. These weren't just nice to have to give you a little more energy. You actually need them for your tRNAs to work and for DNA to go to RNA to go to protein. So what's interesting about a codon versus an anticodon is usually when you talk about the codon, it's that third position that we like to call the blobble one. It's responsible for most degeneracy you see in the code. But when you think about what the anticodon is, that's going to be a position one. And that is where you often find cuing. And the anticodons for histidine, tyrosine, aspartate, and asparagine. So you can say that's involved with this whole wobble position if you feel like it. The other thing to remember, and I emphasize it again, because I have it in my notes. Most eukaryotes don't even make the stuff. They get it from diet and gut microbes. I just am amazed at people who measure small amounts of stuff. So we know that humans have circulating levels of QE of about eight nanomolar. And that's amazing to me. And no one knew the nature of the transporters for QE or QE. So if you label molecules like this, if you label the two Qs, for lack of a better term, with tritium, what you find out is there are two different transporters for Q-ing. One is fast with a low KM and the other is slow with a high KM, high affinity, low affinity. And competitive experiments showed the mechanisms were specific to Q-ing. Now, I'm going to spoil it a little bit here by getting ahead of myself, but it's really important. Some pathways involving cell division, mitogenic pathways, actually seem to modulate cueing transport, but that's not all that goes on. But again, they didn't know the nature of what was transporting it. So in this paper, we'll talk about how they do that in a moment. That's how they identified SLC35F2. That's quite a name. I'm used to drosophila biologists to come up with names like Sonic Hedgehog for things. Right? And so SLC F35F2 appears to act as a transporter for cuisine and cuing in yeast, trypanosomes, and humans. Microbes. Right. And just to help our listeners, cuisine, remember, has the sugar part on it. And queuing doesn't have the sugar part. It's so much fun to look at it because you can tell they're related to guanine and you might think that from the name. And by the way, that's why Vincent's been throwing queue jokes at me for a week, right? Yes. Get the queue. Now, HeLa cells, which are kind of a workhorse for a lot of cell biology, they were able to show that this particular gene, SLC35F2 product, is localized to Golgi and plasma membrane. Again, they suspect and show that it's the primary high affinity transporter for cueing and a sole transporter for cueing. And we'll come back to those things. Again, when you look at the paper, they basically go large Q and little Q. So I want to talk about the results section because that's interesting to me. And we've got some wonderful things to talk about. Now, if you go to figure one, what you're going to see is some stuff here that's really interesting. What we're talking about is QTRT gene, and that encodes this transfer RNA in guanine transglycosylate that exchanges guanine for QE at position 34 in the transfer RNA. And you can see that in figure one. So that's one thing. We've got the transporter, and we've got the stuff that processes it. So how are they going to find it if they don't know what they look like? and they did something that really impressed me. I'm so used to folks just looking for sequence similarity, which doesn't help you if things aren't terrifically related on the sequence level. So they looked at characteristics that had to be true about that transporter, the number of transmembrane motifs, that kind of thing. And as I read this, because I'm currently involved in retirement issues. When I read SSN, it comes up social security number to me, but that's not what they mean. Sequence similarity network. And they want to look for things that are similar to QTRT1, which is what they think is the transporter, right? And they do this by prediction. And that's what you see in figure two. And I have to tell you, that's graph theory, which is what my wife does for a living. So I have hidden this from her. So she doesn't, she doesn't look at it. Right. So one thing they thought is how am I going to show this to be the case? And what's funny is there are some eukaryotes among the fungi in particular that do have QTRT and many that don't. So they basically look for things that are related, that they would be present in 184 out of 189 QTRT positive fungi that are transporting and absent in all the ones that not. Yeah. So these became candidates, suspects. Yes. And this is how they got this journey. And they go into quite a bit of detail in the journey. And that's how they got to SLC 35F2. And I was exhausted when they got there because it was very exciting to me. And then it's time to do what I love, where you make mutants. And this takes us to very busy figure number three. Now, I have to tell you there was a type of a northern blot they talk about called APB. And what that can be used for is identifying modified transfer RNAs. That's what they're talking about here. So what they started to do is make knockouts of the gene candidates, render them homozygous. They're using CRISPR and they have a puramycin selection technique to make sure that they got the homozygous ones. And if you look at part A of figure three, they're doing these northern blots, these APB northern blots to see that transfer RNA modification. And you can see what effects when they're using little cueing versus guanosine. And you can see that the four SLC F35 F2 homozygous knockouts aren't modifying the transfer RNA when they're giving cuisine, but they make a little of it when giving cuing. Now, in part B, they focus on knockout number one. They give it cuisine, no modification. giving more and more of the, of the QE and you can get some modification, but not terribly efficiently, efficiently. And that's where they come up with the idea that SLC F35 F2 is the sole transporter for QE and also a high affinity transporter for QE. And because they are related in their structure, I'm okay with that. You're not a believer? No, I agree with you because Because they're probably looking at the aromatic rings and they're grabbing onto the rings. Yeah. Now, what's interesting to me is that you can see where, and this is part C of that figure, where you can see where SLC35F2 is localized. And this is just amazing to me how far we've come. They make a GFP labeled ectopic copy of that gene that they put in and they able to show with fluorescence microscopy where it localized and surprise huzzah to the Golgi and plasma membrane There's some beautiful stuff about lipid rafts and where in the Golgi things are being localized. Now you know why they had so many authors. Yes. So again, why should we care? Yes, it's delightful. we are getting these compounds from the microbes in our gut. We need them so that we can optimize translation in our own cells, something I wouldn't have thought was a big deal in the sense that it would be a microbial product, which is amazing to me. So, quote, a number of recent studies have highlighted the contribution of SLC-F35F2 overexpression to the progression of a variety of cancers, including non-small cell lung cancer, papillary thyroid cancer, and bladder cancer. And this is what Vincent and I were talking about. When you get multiple copies or overexpression of a particular gene, something that has a normal function can suddenly promote overgrowth, and in some cases, cancer. They didn't have a good figure for this, but they describe it in the supplementary information. If this is the case, what they just said, then you would expect that the HeLa cells that have been rendered homozygous, SLC, F35, F2 deficient should grow more slowly. And they do, which is interesting. So this may be an issue of growth. Or fitness? Yes. And actually, in my opinion, I'm not alone in this one. A lot of cancer is really evolutionary in terms of adaptation and changes over time. But what I found really interesting, and when you talk about eukaryotic papers, they always find a way to bring it back to cancer. the anti-cancer drug, which had been used for many years, YNM155, and they knew it was transported by this particular gene, SLCF35F2. It's product, I should say. So this is part of the whole idea, is that if you get changes in that particular gene, then the cancer therapeutic agent would be less efficient. So this is pretty amazing stuff, but that takes us to figure four. Now, figure four is busier still. And I say this with a big smile on my face because I loved looking at these things and trying to puzzle it out. And remember, I can't talk about everything in this paper. What they're interested here is uptake and let's shiver together kinetics of QE and QE uptake in human cells. So if you go to figure four part A, you're looking at Q is seen uptake with uptake with QNG1 knockout kinetics. Let's have a look at that. I'll go down to figure four so I can do this nicely. Oh, look at you. So what's happening here is you can't convert Q Q is seen into QA, which is what you need to make the modification. So if you look in the first part a figure for, I have a little red X knocking out that function, right? And you can see they're looking at transport there. Transport's pretty awesome. Okay. Then they wanted to say, let's knock out the ability to move queuing completely because we think, remember, we think that that SLC 35F2 is transporting both big Q and little Q. So the first thing they, and you can see this in part B. They use a QTRT1 knockout, so it can't transport the cue as seen and what amount of cueing it can normally bring through. But you can see in the figure, now you've got something where they're bringing it through from this unknown cueing transporter. So that brings you to panel C, where they say, okay, let's not use that knockout now. Let's do something different. because YM-155, that's the anti-cancer drug that I mentioned, is a competitive inhibitor of SLC-35F2 transport. They just put a boatload of that in there. So neither big Q or little Q is entering. So what they got is just the little Q. I want to point out to you, they don't know what this secondary transporter is, which is really interesting to me. And I've seen this kind of thing many times where there's more than one transporter for things. So it's really delightful to see the kinetics that they're getting from this. And again, there must be a mechanism that eliminates excess cuisine from cell once the transfer RNA is finally Q-modified, as it's called. And you can see that in D&E. What they're showing you in D&E is competitive stuff. And I want to draw your attention in all cases to when you use YM-155. You notice they're using different kinds of nucleosides or nucleobases in D&E. And they're able to show that it's not competing. So this is very specific for queuing and queicene, depending on what you're talking about, right? So compare on the left-hand side, well-type HeLa cells, and then the knockout HeLa cells. And once again, when you use YM-155, that is a competitive inhibitor of the major transporter. So it's just lovely to look at. And again, gave me nightmares. oh no because i had i had to do a bunch of enzyme kinetics in graduate school of transport that's that's why it's it's a nightmare to me and i bless the memory of robert simoni at stanford university who got me through this because i kept trying to repeat experiments to like make the differences and the significance more and more it was driving me crazy we're not really talking about what most interests me how does this stuff get from our gut into our cells So it turns out a lot of folks have looked at SLC35F2 expression because of potential interest in cancer in all kinds of conditions, not knowing that it had this function of transporting big Q and little Q. So actually, you can show that it appears to be expressed in the human alimentary canal. So this means this is very probably how stuff's getting from the gut into our cells. What this means is our microbial friends are contributing Q-A-C-E-N and Q-E-N through diet and that SLC-35F2 imports big Q and little Q into cells to modulate the transcriptional efficiency. We still don't know very much about the secondary transporter of QE, but it would say as a take-home lesson, because I'm watching our time, bless your microbiota and feed them well because they're feeding you. What did you folks think? Well, the one question I had, Mark, was in notobiotic or germ-free animals, do the microbes that we use to effectively colonize the notobiotic animal so that it will grow, do those lactobacilli actually make sufficient concentrations of these molecules to allow the mouse to grow? Or is that why it's so problematic to grow notobiotic mice? I would love to have a laundry list of what microbes are secreting these things. Yes. And I don't know how you could do that off the top of my head, other than probably leveraging guanine metabolism. Yeah. That may be it with knockouts and other things. And can I also say when someone says within half of a sentence they made homozygous knockouts, that was probably six months of work, just to be clear. Oh, yeah. Oh, yeah. Yeah, well, this is what happens with technology, you know? Yeah. I was thinking about the oncogenicity aspect of this. So this is not clear mechanistically, but what they say is, so, all right, so SLC35F2 has oncogenic behavior. If you put it in cells, it makes them divide. And so they say maybe this works because when you overproduce the F2 protein, you get more QQ in cells, more modification of tRNAs. And maybe that makes the cells divide faster. And that could be. I would love to see that actually done. Now, as you said, in HeLa cells, if you knock out F2, the cells grow slower. But if you add QQ micronutrients, it doesn't fix the slow growth defect, which tells us that F2 is doing something else besides QQ. Yeah. And this is what, okay, I'm getting too excited. Excuse me. Yes, calm down, professor. Yes. But what I find so interesting is that this gene had been studied by many people not knowing what it really did other than being a target for chemotherapy. And so I'm really hesitant to say that all it does is transport the cues. I think it's doing something else. Of course, it must. If adding Q doesn't help the HeLa cells, well, anyway. Well, you not only have to have a supply, but it has to get to the right part of the cell. Yes. The Golgi seems to be involved. So it might be that that's why just you can't just supplement. Trafficking is the issue inside the cell. And it's so interesting to think about – you would think that this effect would be like a very general thing in us. Well, in eukaryotes, just making sure that things are being expressed at different levels. And this is why their yeast system might be very useful because there are a lot of experiments you can do with yeast that you can't do in HeLa cells. And that's how we learned a lot about oncogenes originally is by studying them in yeast. Yeah, I also think about the evolutionary implications of this, right? So how far back in our phylogeny do you have to go and not see this dependency? Because you can imagine at one point some ancestor was doing okay, and then some bacterium colonized them, and then they got better. So I would love to be able to go back and see how far back you can go, because obviously the gut microbiome becomes simpler and simpler the farther back that you go. I mean, some organisms have just one species of bacteria, right? And the yeast, Pombi, and the parasite, T. brucii, both are also scavenging cues from the environment. So they've got the transporter. But not all yeast. That's the interesting part, right? Not C. brucii. Yeah. And so it just itches at the base of my brain because it means something that I'm not getting. And I know it's important, but that's the advantage of microbial sciences. We learn more every day. And here we're looking at something, you know, they did this wonderful work. My mind goes to just exactly as several of you have said, who's making this stuff that our cells are taking up? Is it being transported through the bloodstream? Well, we know what the concentrations are in the bloodstream, don't we? And what foods do I need to eat to encourage those microbes that are giving me cues? To make the stuff. So, oh, I know. I don't want to hear about supplements. We're going to go there in a second, I'm sure. But Michelle, that's why some of the stuff that you're going to read from the University of Florida is really good. Because it not just says the microbiota are important, but it talks about feeding our microbiome. Right. By eating more high fiber, et cetera. Thank you, Mark. You're very welcome. All right. That's TWIM 355. You can find show notes at microbe.tv slash TWIM. If you like these programs, we'd love your support. Microbe.tv slash contribute. Michelle Swanson is at the University of Michigan. Thank you, Michelle. My pleasure. Thank you all. Mark Martin is at the University of Puget Sound. Thank you, Mark. You're very welcome. And Michael Schmidt is at the Medical University of South Carolina. Thank you, Michael. Thanks, everyone. I'm Vincent Racaniello. You can find me at microbe.tv. I'd like to thank the American Society for Microbiology for their support of TWIM and Ronald Jenkins for the music. This episode of TWIM was edited by Ray Ortega. Thanks for listening, everyone. And we'll see you next time on This Week in Microbiology.