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This Week in Microbiology With Vincent Racaniello, PhD Episode #46: Spore! Recorded November 29, 2012 Released December 5, 2012 http://www.microbeworld.org/podcasts/this-­‐week-­‐in-­‐microbiology/archives/1313-­‐twim-­‐46-­‐spore Hosts: Vincent Racaniello, Michael Schmidt, Elio Schaechter and Jonathan Dworkin [0:06:38] Vincent Racaniello: I know that you work on aspects of sporulation. I thought we’d talk about that today. But you didn’t do sporulation in Peter Model’s? You did phage. Jonathan Dworkin: Right. I specifically did look at how phage infection turned on this sort of particular sets of gene expression in E. coli. Vincent: And in the Losick lab did you work on sporulation? Jonathan: I worked on sporulation. That’s where I fell in love with, sporulation as a discipline. Yeah, it’s great. Vincent: And that was in Bacillus subtilis which is a model organism used in many laboratories. Right? My wife did her thesis on B. subtilis. She did a phage phi 105. Yes, I could not marry anyone but a virologist. I remember in college a friend of mine said, “We have to find a good phage for Vinny”. I was just thinking of that. And we did. We found a good phage. But then she went on to parasites. Jonathan: That’s right. Bacillus subtilis has been a model system in microbiology since the very beginning when Robert Koch first reported it. It was in 1877. He talked in back-­‐to-­‐back papers in the Zeitschrift für Pflanzen Biologie because there were no microbiology journals in 1877. He described Bacillus anthracis and Ferdinand Cohn in the accompanying paper described Bacillus subtilis. So it’s been a subject of study for a very long time. The reason that Koch was really interested in spores and why they were interested in Bacillus was that in the middle of the 19th century, biologists were very interested in what is life. What makes living things different than nonliving things? Spores were very strange and troubling. Here you had these things that seemed very dormant, that could live outside without any nutrients or anything like that. They seemed like they were not living matter. Then you drop spores into broth and within 30 minutes you were seeing replicating bacteria. So it was a little philosophical conundrum I think which started the whole field going. Vincent: Are they living, spores? 1 Jonathan: Yes, probably, but very very slowly. Vincent: Because when we have this argument about whether viruses are living. I take the position that virions, the particles, are not living but the infected cell is living. And people say, “What about a spore or a seed?” I don’t know, ask them. Jonathan: The spores are certainly capable of regenerating and they regenerate quite quickly. We tend to think of them historically as dormant, as not metabolically active at all. But probably a more accurate description is that they’re metabolically active on time scales that are inconvenient for laboratory work. Enzymes, they don’t grow. Enzymatic reactions almost certainly occur but just on time scales of days and weeks, not in a matter of minutes or seconds or something like that. It’s very hard to actually do an experiment. Vincent: A spore has nucleic acid. Jonathan: It has a chromosome. Yes. Vincent: Can it make messenger RNA? Jonathan: The literature thinks that it can’t. There’s no report of it. The literature thinks that there’s nothing in a spore. They have such low ATP levels that it can’t do anything. It probably can’t do anything on any time scale that we can experimentally measure but there are things that go on. RNA seems to be degraded over a couple of days in the spores. So it could be that there are lots of processes going on just in sort of slow motion kind of thing. [0:10:16] Michael Schmidt: So what about C. diff.? I can understand Bacillus subtilis, but C. diff. which is the raging epidemic throughout healthcare facilities in the US. This is really an interesting concept because if C. diff., the spore, is metabolically active under aerobic conditions, it sort of rewrites our dogma about anaerobes if you will. Thinking about the reason C. diff. is an anaerobe is simply because it can’t deal with the toxicities of molecular oxygen. But if a spore is metabolically actable, albeit very very slowly, this really gives us an idea of why this damned spore is recalcitrant in the healthcare environment and could give us tremendous insight. Jonathan: Spores have this fantastic external structure. They have multiple layers of protective coats which we have some idea they exist, we don’t really know how they work but they are—the spore, and Clostridium has like this and Bacillus has these as well—they have a hundred proteins that serve as these surfaces. That in combination with the spore is largely dehydrated probably work together to keep it so protected. The Clostridium is very interesting because, of course, it is an anaerobe. So it can’t live out on surfaces but the spores can survive. There are reports in the literature that spores can survive hundreds of years. There are even articles published in Nature, I’m not sure that I believe them, that claim that spores last for millions of years. They’ve obtained spores from insects that were in amber. That was basically the idea of the experiment. It seemed interesting but it’s hard to actually do the careful controls in that case. Vincent: So Clostridia and Bacillus are the main spore formers. Jonathan: I think Elio thought or you were suggesting something else? 2 Michael: There are gram-­‐negatives that can sporulate. Elio Schaechter: Right, there’s a marine bacterium that somebody’s studying in Caltech. It’s a gram-­‐
negative. Anyhow, it’s an exception. It’s an outlier. So we don’t have to deal with that but most are gram-­‐positive alright. Jonathan: Most are members of the Firmicutes, which are also—things like Staph aureus and other bacteria in it that are not spore formers like Listeria. Elio: Most are rods but there’s even a coccus, Sporosarcina, right? Jonathan: Most are rods. Yes. There are coccal ones. Mostly we know of are rods but that doesn’t know—what’s out in the environment, there are a million spores per gram of soil. So there are a lot of spores out there. Vincent: So for Bacillus species the ability to make a spore is important because it lives in the soil. And that helps survive adverse conditions. Elio: Excuse me for interrupting. I found the paper that deals with the gram-­‐negative spore-­‐formers called Acetonema longum, it belongs to Veillonellaceae which otherwise are close to Clostridia. It’s very odd. It’s an outlier. There’s a beautiful paper which we may want to discuss someday by Elitza I Tocheva. Vincent: Sure, we’ll keep it in mind. Why does Clostridia have to form a spore? Jonathan: Why? I don’t know. Spores are a great survival strategy. Vincent: Clostridia is in the gut, right? Jonathan: Well, Clostridia is in the gut although it’s actually also in the soil. Vincent: Okay. Alright, so Clostridia are in the dirt so that makes sense too. But why don’t all the others make spores? Why not? Why doesn’t everybody make a spore? Who knows? Jonathan: It sounds like a religion discussion. Vincent: Ok. So we can’t answer that. Why don’t you tell us how sporulation works? Wait a minute. Before you do that, I wanted to ask you, why do you study sporulation? What appeals to you about it? Jonathan: I think it’s pretty amazing that you have a cell that can transform itself into this dormant cell form. It has a dramatically different morphology from the normal-­‐growing cell. That cell type can last for longer than human lifespans. Then when conditions change or improve, that spore can then exit that dormant stage and start growing again. I think that that’s pretty remarkable. It’s not the only way to survive bad times but it’s a very very good way to survive bad times and the natural world is mostly bad times. So it seems this is a pretty interesting model. [0:15:11] 3 Vincent: So spores are what was put in the envelopes in the Bacillus anthracis thing, right, after 9-­‐11. Did that effect you at all? Did people call you up and ask you your opinion on? Jonathan: Well, they didn’t call me up at that point but they did call up Losick and Peter’s people who were working on spores at a basic level. Because one of the issues with spores is that they’re actually very hard to get rid of. They’re so resistant that normal treatment, so this is why they’re a big problem in hospitals. Normal antiseptic treatments, basically, the spores don’t care. Actually, they didn’t even know how to decontaminate letters that were contaminated with spores because they said, “Well, could you stick it in a microwave?” There was sort of a quite a bit of interest in that and so there was subsequently quite a bit of funding in that area that’s fallen away. Vincent: So that’s why spores are a good weapon, especially for anthracis because it’s really stable. It floats around in the air. Jonathan: It’s pretty straightforward to aerosolize and the other thing is that you can put them in an envelope. There aren’t that many biological agents that you could—that are so robust that, as I say, wear on the bottom of your shoe all day. There’s limits with that but they’re remarkably robust hardy species. Vincent: So how hardy are they? You can dry them out and they’re fine, right? Jonathan: They’re fine. We routinely take spores in the lab and to prove that they’re spores, we treat them at 80 degrees Celsius, so not quite boiling water but pretty hot water for 20 minutes and they have no problem surviving that. Vincent: How long could they stay at that temperature? Jonathan: I don’t know exactly. It probably depends on the strain. I would imagine for at least an hour they’re probably safe at that temperature. There are some spores of other species that actually can go up to 95 degrees Celsius so just shy of boiling water and survive that. So they’re pretty impressive. Elio’s: What’s the name? “Desulfotomaculatum”? Jonathan: I think so. That may be the one that goes—there are a couple of species that seem they are even more temperature… Michael: Stearothermophilus is the one they typically put in the autoclave… Jonathan: …to test them. Right. Vincent: So how do you make Bacillus subtilis sporulate? What do you do? Jonathan: In the laboratory, basically, there are two ways of doing it. One is that you grow them in a somewhat of a rich media and they exhaust some components of that media. We think they’re probably carbon or nitrogen. Once they exhaust that they stop growing and then they start initiating sporulation. Over the next five hours, five or six hours, they make a spore. Other ways you can do in the lab is grow them in sort of a more minimal media and then basically shift them into a very minimal media. At that point, of shifting them into a very minimal media, then they begin to sporulate, and again over about 5 or 6 hours they make a spore. 4 Vincent: I remember you saying once it’s hard to get them to sporulate or hard to get a – do most of the population do sporulate, is that right? Jonathan: It’s a very good question. In the laboratory we’ve refined all of our methods so that they sporulate quite well. But whether or not in the real world that it’s as synchronized as what we get in the lab, it’s probably not. That’s one of the challenges of microbiology is to try to do something where you can actually do an experiment so that, for example, they all sporulate but also make something that is also physiologically relevant. Some of the work I actually did with Michael Elowitz was trying to understand bacteria are—when Bacillus is starving they have a number of strategies they can do. One is to become genetically competent, which is something they share with a number of other bacteria. One is to form spores. How they choose between those strategies is pretty interesting and not very well understood. Michael: A funny story. When I was a TA once upon a time in my graduate training, we had to prepare the reagents for the undergraduate lab in General Microbiology. One day we had to show the students what spores looked like in a vegetative cell. So we grew up a prep of Bacillus megaterium. The way graduate students do things, they inoculate a liter culture overnight and put it in the warm room and start it shaking. As I was walking it back to the lab, it obviously had stopped shaking and the oxygen tension must have been at a critical threshold. As I was walking back from the warm room to the lab to begin to dispense this material for the students the entire culture lysed. I had this beautiful preparation of spores and ghosts of where the vegetative cells came out. My advisor asked me, he said, “What did you use to do this? We’ve been trying to figure out how to make spores in synchrony and get them to autolyse on cue and you’ve just stumbled into something.” We never did figure out what I did. It was just one of those remarkable things that you remember of your training of the wonder. The cell actually lyses when it makes the spore. It releases this remarkable resting state. We thought for a long time it was manganese that was controlling the ability to sporulate. We tried all sorts of variations on the theme and never got that to do it again. [0:20:50] Vincent: Can you give us now an overview of how sporulation proceeds morphologically? Jonathan: What happens is most rod-­‐shaped bacteria, as you guys well know, the rods divide in the middle and they make two daughter cells that are identical. The first thing that happens in sporulation is they don’t divide in the middle. They actually divide asymmetrically towards one end of the cell so you get division results in having a large cell, which we call the “mother cell,” and a small cell called the “forespore.” After that division occurs, the first thing that happens is that one of the chromosomes gets pumped in through an active process and put in that small compartment. Kind of stuffed in. You can think about it as there’s a little machine sitting at the membrane that separates the two cells. It pumps in the chromosome. A little bit like what happens in phages when the DNA goes into the phage head. That’s the initial thing. So that happens. You have a small cell with a chromosome and a big cell with a chromosome. They’re identical chromosomes or, so far as we know identical chromosomes. Then there’s a very amazing thing that happens which is that the larger cell engulfs the smaller cell. About a period of about an hour the membranes of the larger cell migrate around the smaller compartment and totally enclose it. After about an hour of this process occurring called “engulfment,” you have a cell, by which I mean a chromosomal DNA, which is bound by actually two membranes at this point. It is in another cell, in this mother cell. So it’s a little bit like an endosymbiotic process. You have 5 this forespore now totally contained within the larger mother cell. The mother cell is responsible for synthesizing all of the surface structures of the spore. It kind of assembles over the next hour or so. As I said, about a 100 different proteins that are added to the surface. Once that’s complete, the mother cell autolyses and dissolves away leaving just the spore. Whether that’s a directed process, sort of suicide, or whether just because the mother cell exhausts itself and sort of basically lyses in a passive fashion, we don’t really understand. If you’re doing the cell, most mother cells lyse without having to do anything to the culture. Vincent: So do we understand how the reduced nutrients are sensed that starts this whole process? Jonathan: That’s a really good question. I’m somewhat embarrassed to know that we don’t understand very much what the proximal stimuli are. We know that limiting conditions of carbon and nitrogen sources probably contribute to this. There have been various reports that a fall in GTP, guanosine-­‐5'-­‐
triphosphate, levels in the cell are key. Those experiments are suggestive but certainly not conclusive. Elio: It is an interesting protein. Isn’t it called “Y”? Jonathan: Yes. Which senses those levels and that plays an important role. The way the cell senses is actually is what’s called the “phosphorelay” which is actually similar to what you see in eukaryotic cells where there’s a kinase that sits on the membrane typically and it senses something, although we don’t know what. When that kinase gets active, it proceeds through a series of proteins to activate a transcription factor which then gets sporulation going. There’s a master regulator of sporulation which is the DNA binding protein. Vincent: So that’s spo 0…? Jonathan: Spo 0A. Vincent: “Spo” standing for “sporulation”? Jonathan: Right. And then zero as in—so the original genetics of sporulation were done in the ‘60s and ‘70s where they classified mutants based on what stage of development they were blocked at. So there’s the Spo0 mutants which are blocked at the first stage, even before this asymmetric division I told you about. Then there’s spoI, spoIII, spoIV, spoV, spoVI. So there’s a whole set of mutants in each of those stages. [0:25:05] Vincent: But the first one, the spo0A, is a transcription factor and this turns on a lot of other genes that are all participating in the formation of the spore. Jonathan: So it turns on about 100 genes. It has a regulon that’s estimated to be about 100 genes to sort of get things rolling. It was identified starting in the ‘70s that there’s a series of transcription factors that are in each of these individual cells. So this is why sporulation is also a wonderful system. It’s sort of a model developmental system because of these two cells which have the same identical chromosomes but there are patterns of gene activation that are completely different and specific in each of those compartments. 6 Vincent: So you said that when a chromosome is pumped from the mother cell into the spore through a portal of some kind. So DNA replication occurs just before the spore formation? Jonathan: Yes, it’s really a good question. We think that replication is already completed and there is a lag time between actual completion replication and when the chromosome is pumped in. But being certain that chromosome was at 99% percent complete vs. 100% complete, it is hard to differentiate those. Vincent: What else goes into the spore besides the chromosome? Anything else that we know of? Jonathan: We don’t think that there’s much that’s going in. Most of the DNA-­‐binding proteins that are on the chromosome seem like they’re getting stripped off the DNA as it gets transported. There’s this pore that is an ATP-­‐driven machine. It’s a multimeric, homomultimer, of a protein called spoIII which actually has homologs in the way plasmids get brought into bacterial cells. So this is a well-­‐conserved process. It seems like the pore would only allow a double-­‐stranded DNA through. It seems like specifically things aren’t included. I should say though that there is a pore at this stage. There actually is a port at a later stage in sporulation which we think, although the evidence is still coming in, that there is a pore between the two cells. That the mother cell not only produces or is responsible for making proteins that coat and structure the spore but actually provides nutrients to keep that spore alive in a true nursing fashion. Michael: So in addition to moving the chromosome into the spore because anthrax is virulent because of a plasmid. They also pump plasmids in too. Jonathan: That’s a really good question. They must. Michael: They have to because, otherwise, an anthrax spore wouldn’t be virulent. Jonathan: Right. And they are. They’re totally virulent. It’s a really interesting question how—and I should know this—but how the anthrax plasmids of which there are two plasmids that are important for virulence are appropriately transported. In subtilis, of course, we don’t have these plasmids. We don’t actually have any plasmids. Most actually spore formers have multiple plasmids. For example, megaterium has nine plasmids in natural form. Yeah, it’s an interesting question. Michael: If you think about it, it could really change the virulence profile. If you don’t get the right partition of the right complement—in the case of anthrax, you need the two. But in Megaterium, in order to confer the full genetic profile, Megaterium, if it doesn’t partition all nine of those plasmids properly, a fraction of the population of spores could be avirulent or aphenotypic. Jonathan: Absolutely. It’s a very good point. In fact, I may be speaking out of total ignorance. I’m not aware of this but there could be people who have investigated this and I just don’t know. It’s a good question. Vincent: So in this process of sporulation that you described, at what point, if you add nutrients, does it not reverse anymore? Jonathan: I’m laughing because this was actually something I worked on as a post doc in Rich’s lab so it’s a subject that’s pretty dear to my heart. When they made this polar septum, they will actually— now, if you take those cells and you give them rich media, like Luria broth, they will actually stop 7 sporulation and start growing. You get these actually fantastic looking and sort of weird looking cells where it’s almost like there’s heads growing out of this small compartment because they’ve actually started to grow again which they don’t normally do. Once they’ve started the turning-­‐on genes, specifically, in each of these compartments, in the forespore and the mother cell, then it doesn’t matter what you do. It’s a developmental commitment. Once they’ve really differentiated, by which I guess you mean, that the genes are differentially expressed, that’s it. It doesn’t matter what you do to them, they’re going to complete sporulation come hell or high water. Vincent: So you’ll get a spore coming out? Elio: In general, from a population point of view the story is a little bit complicated because sporulation is a great way to resist harsh chemicals and high temperature and so forth. It’s a lousy way to take advantage of good times. The spore is, unless it germinates, isn’t going to participate in the game. The bugs don’t take advantage of a good situation necessarily. Commitment to sporulation has to be done in a thoughtful way and I guess Subtilis does just that. It weighs the possibilities and not everybody sporulates. It’s a complicated story. Isn’t it, Jon? [0:30:45] Jonathan: I think you’re exactly right, Elio. I think what happens is they basically go some distance and have some flexibility but as I’m describing these morphological changes, the cells are very different. It’s a little bit like if you build a house and if you stop halfway through it and you haven’t put the roof on it doesn’t do much good to build the foundation. Right? Basically, at some point, they say, “Look, I know this may not be the right thing to do but I got to finish it because, otherwise, you know, you’re dead. It won’t do any good.” So there is sort of a point of commitment that I think is really important for the strategy. Basically, this is a long process. From a lifespan of a bacteria that can actually divide every 15 minutes, this process of sporulation is five or six hours. This point of commitment comes pretty early in this process. It come at approximately at two hours into this process so they still have two and a half to three hours where they can’t do anything except finish building the structure. So, obviously, if conditions change at that point, they’re pretty much vulnerable. Michael: In the case of the Clostridia, like perfringens, it has a generation time of nine minutes, it really drives home the… Elio: It does? Hey, wait a minute. Michael: Nine minutes. Perfringens has a generation time of nine minutes. It cheats. It has multiple copies of everything. Vincent: It sounds like Elio doesn’t believe you. Michael: I think he doesn’t. Elio: There’s a Vibrio natriegens, which in the literature that I know of, is supposed to be the world champion fastest grower. Its generation time is 13 minutes. So this is news to me. You probably entirely right. I don’t doubt it for a minute, Michael, but I didn’t know this. 8 Michael: I just asked Dr. Google and I went off to Health Protection Agency of the UK which came up first. Under optimal growth conditions the organism has a generation time of 10 to 12 minutes. That’s the C perfringens so my neurons are functioning again. Elio: They sure are. Thanks for enlightening us. Vincent: You mentioned that at the end of the whole process the spore which is developed inside at one end of the Bacillus then ruptures the host. Do we know how that happens? There must be some enzymes that digest away. Jonathan: There seem to be enzymes that digest the cell wall of this mother cell, of the larger cell. It’s basically like an antibiotic. It’s the same kind of mechanism that penicillin would do and that seems to be the process. There is some question as to whether or not this is really an active process or it could be it’s a cell that essentially sort of gives up the ghost and there are holes that happen in the cell wall just by normal sort of things and then they just fade away. It seems unlikely though that—I’m not a firm believer that things just happen by chance. I think there’s a directed mechanism. Vincent: So what aspect of sporulation are you focused on? You mentioned it earlier. Tell us again. Jonathan: We’re interested in dormancy. We’re interested in what the cell does to begin to prepare itself to become dormant, the spore. We’re also very interested in what causes the dormant spores to germinate, to start to grow again. I think this is a really fascinating, fascinating issue that not much is known about in microbes. This is such an important decision because if they decide too early to start growing again, they can be vulnerable and can be killed. But, of course, if they’re too conservative with that decision, then their neighbors are going to eat up all the nutrients and take over the population. So it’s clearly a very important decision to be made. Vincent: And how is that made? [0:34:46] Jonathan: One of the things in my lab is where—I’ll talk about the germination issue. People for a long time, starting with Koch and Cohn in the 19th Century, people have considered the role of nutrients as being detected by spores to cause them to germinate. In the laboratory, for sure, you can get spores to germinate with high levels of certain amino acids, like alanine, sort of millimolar levels of alanine. I’m not clear where in the environment or in a host that you would see millimolar levels of an amino acid but those work very well. They germinate 100% of the spores. Elio: Or “germinants,” aren’t they? Jonathan: They are called “germinants” because they induce germination, although it’s not exactly clear what the mechanism is. We took a somewhat different perspective which said, “Well, “Why would spores ever want to germinate?” Nutrients are one possibility but maybe they’re also detecting other bacteria in the environment and specifically that there are other bacteria that are growing. Then that begs the question: how can they detect that? It’s actually a little known, but very interesting observation, that both gram-­‐positives and gram-­‐negatives to a lesser extent release a substantial quantity of cell wall material as they grow. It’s a little bit like molting that snakes would do. As they grow, they can’t totally fix their peptidoglycan and conserve it. So some of the peptidoglycan gets released into the milieu and this happens during growth. It’s a natural consequence of this assembly. 9 We said, “Well, maybe they’re detecting. Maybe spores detect the growth of other bacteria in the environment by this peptidoglycan.” We actually found, in fact, that spores do detect peptidoglycan. They detect chemically synthesized parts of peptidoglycan or peptidoglycan that we purify from growing cells. So we think that that’s an important thing. It’s sort of a quorum sensing signal, in the sense that they’re detecting bacteria in the environment but they’re most importantly detecting that they’re active and growing. Vincent: So if you add peptidoglycan that’s enough to get them out of dormancy? Jonathan: That will be sufficient to get them to exit dormancy. Elio: I should say that this was an amazing phenomenon and it was your discovery, Jonathan. Jonathan: Well, thank you, but yes. Elio: Really a beautiful piece of work. Michael: Have you ever thought of using ground up peptidoglycan as a spray to get C. diff to germinate in the hospital to die? Jonathan: I haven’t actually with C. diff but I have actually have been contacted by members of the US government, shall we say, who are interested. Of course, that is the problem with things like anthracis. No one knows how to get rid of spores. If you can germinate things like anthracis they’re just as vulnerable. They’re vulnerable to all sorts of things. You can wipe them off with a wet tissue paper until you do that. There is some interest in figuring out ways to do that. The peptidoglycan is a very interesting molecule and it’s actually fairly labile in ways we don’t totally understand, to be honest. So it’s not a good thing but if you could figure out a mimetic that was chemically stable that would still stimulate it. I can tell you some people who would be very interested in that. Elio: You’re something of an expert on chemical modifications of peptidoglycan. Aren’t you? Jonathan: Well I’m not an expert but we have worked on it. There are a lot of very interesting modifications. Yes Vincent: What senses the peptidoglycan in the spore? Jonathan: That’s really interesting. It turns out that what senses the peptidoglycan in the spore is a membrane kinase which has, at its extracellular domain, something called “PASTA repeats” which are seen in some other bacterial proteins. These bind peptidoglycan. The intracellular domain of this protein is a kinase. It’s a very interesting kinase and that’s actually been subject to a lot of work in my lab in the last couple of years. It’s a serine/threonine kinase that looks just like eukaryotic serine/threonine kinases. Bacteria have signal transduction pathways. They have serine/threonine kinase and serine/threonine phosphatases that look structurally basically identical to eukaryotic kinases and phosphatases. Elio: Here’s the big question. How can this poor kinase sense the outside when it’s surrounded by a cortex and this and that, all kinds of layers around it? How can it go out that far? 10 Jonathan: It’s a great question. Actually, what turns out is that, I think—well, there’s two things. One is that the muropeptides that we think it senses, that it can certainly bind to in vitro, are small enough that they probably penetrate this. In the textbooks, the spores are written as having this impenetrable coat but actually it turns out that if you actually look at the literature, molecules smaller than 5 kilodaltons can probably cross these layers. So it’s very good for keeping out proteins. That’s actually why spores are resistant to lysozyme because the lysozyme can’t gain access to the peptidoglycan that protects them because it’s held off by this coat. The other thing is that, which we don’t totally understand, but I think is very exciting, is that the spore actually is not a static structure. It seems to be a somewhat dynamic structure. It almost looks as though it’s breathing in and out. This is difficult to visualize because these spores are a micron in size and so our methodologies for seeing this are really cutting-­‐
edge. I think there is some good evidence that there is actually dynamics in the structure. Vincent: So when this kinase recognizes peptidoglycan, it starts a phosphorylation cascade? [0:40:35] Jonathan: It starts a phosphorylation cascade which we, to be honest, we thought at one point we understood. We think it directly regulates translation in the bacteria. We have some evidence that it regulates the activity of elongation factor TU. Spores, like many dormant cells, have actually pretty large storehouses of RNA. That’s interestingly not very limiting. We think that the spores are set with basically, they have RNA and they need to start translation. We think that this signal that comes on through peptidoglycan starts translation of the RNAs in the spores and that’s where the whole thing starts. We’re not quite there in establishing the whole signal transduction cascade but it seems like it’s going to work out something like that. Vincent: So they’re translationally dormant and then you turn on somehow, EFTU, and that gets them going back into vegetative phase? Jonathan: It’s hard to work with spores because, well, they’re designed not to be messed with. Their whole raison d'être is to like be robust. So that makes them not great things to use for biochemical assays. They’re not easy to break open. So we’ve been studying a lot with how the—the converse question which is how do spores become dormant. What causes them to become dormant? We’ve had more success with that because those cells are actually much more tractable. Until they reach this final stage of dormancy, they’re more like regular spores. What happens, and this is work that we haven’t published but we’re very excited about, is that actually during sporulation the spore turns off translation. It does that by regulating the phosphorylation state of this protein EF-­‐Tu. When EF-­‐Tu gets phosphorylated, it gets inactive. If you look in spores, they become translationally inactive. As they proceed through sporulation we think that that eventually comes to a stop when they’re finished. At some point, they just completely stop translation all together. Vincent: Remind us what EF-­‐Tu does in translation. Jonathan: EF-­‐Tu is a key protein. It’s the most abundant protein in bacteria so it’s probably one of the most abundant proteins on the planet. It’s responsible for delivering the tRNA to the ribosomes so it performs an essential function. Every time there is a new codon, EF-­‐Tu has to be engaged. Vincent: Can you delete the gene for this kinase, the sensor kinase, and are those bacteria viable and then will they go and make a spore but not be able to be resuscitated from the spore? 11 Jonathan: That’s a great question. We can delete the kinase and what happens is that, it seems to be at least, in the laboratory, that the spores are more or less okay but they are no longer responsive to peptidoglycan as a germinant but they still respond to alanine, which is maybe not surprising. This is an important enough decision that the cells probably have a number of signal transduction cascades and there’s a number of signals that are going on. Vincent: So it’s not just peptidoglycan. That’s great. That makes perfect sense. But the vegetative bacterial doesn’t need this kinase to survive. Right? Jonathan: It doesn’t need this kinase but I should say—and this is maybe something that touches on other things you guys have talked about in this series—but we study bacteria in a very strange way. We study mostly what’s going on in log-­‐phase: in laboratory conditions, 37 degrees, all the nutrients. It’s clear that most microbes don’t live that kind of life. They are very living in a very nutrient limited in most conditions. They sort of manage to squeak by. Under laboratory conditions this kinase is not essential. What’s interesting is that if you delete the kinase the bacteria grow fine in log phase but they die in stationary phase. I don’t want to get all metaphysical but the question of what’s essential. We think about, “Oh, yes. It’s in log phase then it’s not essential.” But stationary phase is what most microbes exist in. So if things die in stationary phase that’s a pretty essential phenotype. [0:45:00] Vincent: Is this kinase present in all bacteria or just those that form spores? Jonathan: The particular form of this kinase which binds peptidoglycan is present in all gram positive bacteria. There are potential homologs in gram-­‐negatives but they are sufficiently different in how they bind peptidoglycan that we don’t know. There are kinases like this in all bacteria that have been examined. We’re hopeful that this is a general mechanism by which bacteria can slow down their growth and maybe in cases become essentially quiescent and in subsequent times start growing again. Michael: Do you think that this kinase system, if you think about the wholly grail of microbes that are always in maximum stationary phase even when they’re in exponential growth, which I always view as mycobacteria, do you think that if you can noodle on this sensor kinase regulon or the associated proteins we may have a new target for an antibiotic for TB? Jonathan: No, it’s a great questions. In fact, one that we got very excited about early on, and it’s been slowed because we’re not experts in TB, and as you know it’s a very hard organism to work on. One of the key observations with TB is that there are proteins called “resuscitation-­‐promoting factors” in TB. You may have heard about them. These are proteins that are essential for sort of viability of TB in various host models. These proteins are very interesting because it was not known what they were until they were crystalized. They were seen to be essentially lytic transglycosylases. In other words, proteins that could digest peptidoglycan. So one of the things we were very excited about was whether or not the RPFs, these resuscitation promoting factors were working to cause MTB to start growing again by digesting peptidoglycan and making the signal that would then go back and start them growing again. It doesn’t seem like maybe it’s that simple but it’s a very hard organism to work with. It takes three weeks to get a colony on plates, which for someone as impatient as I am, a frustratingly long time. Elio: By the way, some years ago, somebody came up with the tantalizing idea that mycobacteria makes spores and it was your ex-­‐mentor, Rich Losick, who didn’t like the idea. Is there any new development on that? 12 Jonathan: The evidence was not great to begin with. It was certainly an alluring possibility. What’s interesting is that the evidence they had was some pictures, which are always sort of interesting but it’s hard to know how to evaluate them. They also identified a bunch of genes that they thought were homologs in MTB to the sporulation specific genes of Bacillus subtilis. But it turns out that most of the genes they saw as homologs had homologs in other bacteria as well and probably were doing different kinds of functions. I think it’s pretty accepted that MTB is not making spores. On the other hand, I should say that even though they aren’t making spores, there’s pretty good evidence that they can exist as dormant species for a long time. One thing that’s classic with TB is that people can have these granulomas where there is not a lot of bacterial growth for their lifetime. They can basically get infected when they are a child and these things don’t reactivate for 50 or 60 years. They’re not spores but they are pretty impressive dormant or quiescent species. Elio: They resist drying. They can be found in dust. If somebody coughs up TB they stay in the room for a long time because they don’t die on drying. Jonathan: That actually is the case. It’s a great point, Elio. A lot of bacteria, it turns out, have states where they actually are pretty—you know, they’re desiccation-­‐resistant. Legionella does this. There are a number of bacteria people have looked at where if you do a proper drying them down and stuff like that, they will resuscitate quite well. So this seems that it’s a pretty, maybe a more general phenomena of microbes than we sort of care to appreciate. Michael: So this resuscitation promoting factor is really—it may be a universal function that bacteria have to get them to move out of stationary phase back into a logarithmic phase of growth. Jonathan: Those kind of proteins, in MTB they’re called RPFs, but Bacillus subtilis and all other gram-­‐
positives have homologs of those. In fact, Bacillus subtilis has one of them that we’ve studied in the lab that auto-­‐regulates itself via this kinase that I’ve been telling you about that is responsible for germination. So you ask what this kinase is doing in log phase. It’s not essential but it serves as a signal transduction pathway where it induces the expression of this hydrolase, this thing that destroys peptidoglycan in response to there being free peptidoglycan. [0:50:17] Vincent: I wanted to just ask you a few questions about another paper that you’ve published. Before we do that, any other questions before we move on? Elio: I think this was a wonderful explanation. I’m really so delighted for this. Thank you, Jonathan. Vincent: You have a paper in collaboration with Michael Elowitz’s lab called Pulse Feedback Prefers Cellular Differentiation. I just wonder if you can explain—the general idea here is that when you nutrient starve Bacillus subtilis, it takes a while before it sporulates. What’s going on? Jonathan: This a textbook model of what’s going on. The cells, basically, decide, in one-­‐cell cycle, to start this developmental pathway. This transcription factor that I’m talking about, Spo0A, which is the master regulator for kicking off sporulation, the idea was that basically that thing got phosphorylated and then that was it. That was the key thing. In part, with this paper and there’s observations from other labs as well that have come out in the last couple years, it’s suggesting that it’s not that simple. The idea is that if you look at some of the experiments that are in this paper and, as I said, from other labs, show that this phosphorylation actually occurs much before this starvation event occurs. The cells 13 are—maybe, you can say sort of sampling the environment. They are going through cycles where they go up, they increase phosphorylation, then they decrease phosphorylation, they go up. One argument for that is there isn’t such a dramatic thing as like this switch gets turned on or turned off. It gets turned on a little bit then maybe if things get really bad it goes up more and if things get a little better it goes down. I think this idea of this black-­‐and-­‐white transitions between growth and something like sporulation or something like competence and other response to starvation conditions is clearer but that’s not the simple model going on. If you look earlier on, now that we have these very nice microscopy techniques that Michael and his colleagues have pioneered where you can actually follow single cells. You can see, in fact, that these transcription factors are getting turned on or getting phosphorylated in cells that are like five cycles away from ever becoming a spore. These things are happening but you wouldn’t see that because, of course, when we’re thinking about sporulation, I told you in the earlier part of our discussion, we only can see this because they divide asymmetrically. But that’s already an hour and a half into sporulation. They’ve committed themselves to going, or more or less committed themselves to going, at that point. But what we’re seeing now is we can look at individual cells and I think this is a very exciting part of microbiology. You can see that there are things going on in these cells where there’s no obviously gross morphological changes that you can observe. But there are changes in the levels of these transcription factors that are indicative that there actually is a lot going on. They’re not simply doing it all at once. Vincent: You can look at individual cells because you hook up reporter fluorescent reporters to promoters that are controlled by certain transcription factors. Is that how it works? Jonathan: Yes. Before we had these kind of approaches you could look at a population of cells and say they were doing this. There are very nice things about a population but it’s really an average. If not all cells are doing that it’s not easy to tell that. But with a single cell thing, I view it as it’s making microbiology into an organismal biology science as well because we’re looking at what’s going on in this cell versus another cell. These are not different bacteria. They’ve been growing under the same conditions. They’re on the same microscope slide. What’s really striking is they do very different things. It’s so obvious that we didn’t’ think that was going on. I couldn’t even imagine until you see these bacteria that are responding very differently to what you think of as the same conditions. Vincent: The figures in this paper are beautiful. These red and green fluorescent proteins. I particularly like this picture, A Time Course from Zero to 24 Hours and the bacteria start out black then they turn green and then you can see the spores forming beautifully and they’re all different shades of green. Jonathan: It was about 10 years ago that Michael and I started making movies of subtilis sporulating. One of the first things we were struck by was how heterogeneous the population was. As microbiologists, in these bulk cultures who were used to thinking about, “Oh, yeah, they’re all doing this. At two hours after some treatment they’re all doing this.” But in fact, they’re not. What’s really interesting is not just that they aren’t but the biological implications of that that there really is heterogeneity which is good. Some of them may not make an accurate assessment and some of them may be very aggressive in making the assessment. But in the end what you want is some of them to survive. That’s the goal of the population. So this fits in with this thinking about them as real cells as individuals. 14 Vincent: I think the same happens in other systems, say, virus infections as well. We study populations of virus-­‐infected cells. We never look or rarely look at individual cells. If we did, we would probably find some very different stories. But we don’t do that. Probably worth doing. Anything else gentlemen? Are you all satisfied? Michael: I tell you. The first time I heard about sporulation back in the middle ‘80s was from Rich Losick and the Cascading Sigma Factor story impressed me and showed me the elegance of bacterial gene expression. In the subsequent 25, almost 30, years now watching it evolve into this elegant system. It even further reinforces how truly amazing bacteria are. Vincent: They are. That’s why we’re all here, right? Michael: Yes. Without them, we’d have a dull life. [0:56:48] -­‐ End -­‐ Content on This Week in Microbiology (microbeworld.org/twim) is licensed under a Creative Commons Attribution 3.0 License. Transcribed by: Frank Shinneman 15