 Have to correct all right. Good morning. It is nine. We will try to start on time I will try to finish more or less on time that's Jacob asked me To respect that I'm scheduled today Very discipline guides or we'll try of course you still are Encouraged to ask question during the presentation. That's of course unavoidable but still we will try not to bother too much the speaker so that he can finish his Talk for time all right Okay, oh no We have some important issues to resolve based on our discussion from yesterday Probably the most important thing we talked about was this philosophical discussion of whether or not sleep was Fit the operational definition of dormancy which you may recall is the ability of an individual to enter a Reversible state of reduced metabolic activity I walked away feeling I didn't have a satisfactory answer for you So I went and did a little bit of research To find out what what what people in the field tend to think and so first I'll just briefly say that there are some people who think that that sleep is not a form of dormancy for a couple of reasons there's some There there's cognitive and informational processing going on. There's consolidation of memories and also We were using the example of of a parent who can instantaneously wake up when they hear a baby crying in the room so so it's not really a a Deep completely restful state And so the ability to respond quickly is one of the major arguments there But I still think that kind of fits the definition of dormancy in some ways and I was able to find this one opinion paper and nature reviews neuroscience it talks about the sleep viewed as a state of in a adaptive inactivity And I just highlighted this one phrase here that sleep could be best understood as a variant of dormant states even Throughout the plant and animal kingdoms and that is highly adapted because it optimizes timing in the duration of behavior So we could argue about the mechanisms and the the purpose of sleep But at least to some level I've I've hope I've provided some closure to this issue Not after 20 minutes of good conversations. I think we can say that sleep maybe fits some definitions of dormancy I ended yesterday Rather rapidly Trying to present that there's a bit of a conundrum at least with some forms of dormancy And I was specifically referring to endosporulation in groups of bacteria like bacillus and the clostridia These these organisms undergo this complicated pathway of transforming into a protein rich highly inert Cell they're very abundant in the natural environment Estimated somewhere on the you know order of 10 to the power 28 just in marine sediments alone But it struck us that this was a very energetically expensive and time-consuming Process to engage in which the conundrum is if you're a cell is this a responsive form of dormancy So you remember this is when an organism detects changes in its environment could be resources or other environmental conditions It senses these conditions and undergoes some responsive change to enter a quiescent or dormant state Endosporulation fits that bill, but it requires a lot of energy and I went through some calculations very quickly to show you how many ATPs that cost and how that compares rather to other cellular processes, so it is expensive So why would a starving cell choose to do something? That seems to be so expensive and we've kind of walked through some examples in our group about Bed hedging mechanisms not all cells do this some mother cells invest more in offspring end of sport offspring than others There's a time scale at which we can start to think about this where there's a break-even point where Other survival strategies that don't involve into sporulation need to be paid and those payments accrue instantaneously over time where Sporulation is a one-time investment So these are some potential explanations that could lead to the maintenance of endosporulation even though in many cases over The course of geologic timescales this trait has been evolutionarily lost So what I want to propose today is a segue a different way of thinking about this problem and that instead of just protecting From environmental stresses or energy energetic limitations that sporulation may Provide some additional benefits and we're going to view this through the lens of species interactions Okay, so can species interactions modify the benefits of investing in what otherwise seems to be an energetically costly decision process And so this figure that I have up here depicts this so yesterday We focused entirely on this dormancy module and the transitions and what I have on the left is kind of a classical food web module Which describes host parasite interactions There's resources that are being consumed by the host the host is the the focal group of organisms that can engage in dormancy And there is either a predator or parasite that can be feeding on those organisms And so as we described yesterday, this is a form of phenotypic switching where there could be preference for one type over another And we're interested. I'm interested in what degree that influences the process of co-evolutionary change between two species I'm not going to go into depth. I showed this really quickly. I've just described it We're going to focus today exclusively on one group of microorganisms These fall in this group this phylum of bacteria called the basalota They exhibit the responsive resuscitation under good conditions. They're dividing rapidly every 20 minutes typically under Good conditions, but when they sense specifically amino acid Alenine limitation this serves as a cue that Changes a lot of gene expression in the mother cell. It leads to development of a septum Which will create a new forest bore. There's a genome Replication, there's a translocation of that genome into the forest bore the the baby the forest bore consumes the mother And we're left with this highly resilient end of sport. So how does A phage that has the capacity to affect a genotype How is it affected by the ability of an individual to to spoil it? So we'll start first with the simple scenario where we can imagine that bacillus is not undergoing sporulation And we can imagine a classic litic reproductive cycle for it to be simple here so there are so over here We have a susceptible Active vegetative cell and there are phage particles in the environment that are moving around randomly via brownie in motion Contact is made and if that phage particle combined to the receptors on the surface of that active cell It will inject its DNA the DNA Takes over the cellular machinery of the host cell makes new virus particles that assemble They produce enzymes that will burst the cell and the viral progeny will be released back into the environment Okay, so this is a basic Bacteria phage litic life cycle if you will so we can imagine that happening but when we Think about what happens when that same virus interacts with an end of spore The dynamics are very different So we're showing you some data right here that we collected from what's called an absorption assay And the way you do it an absorption assay is you take a suspension of susceptible cells and you expose them to virus particles And then over time you measure the fraction or concentration of free virus particles over time Okay, so what you can see in the blue line is the absorption dynamics when phage particles are interacting with an active cell And you can see that they're decreasing exponentially over time You can measure that decay rate and that that's your that's the actual absorption rate Constant that you would include for example in a model And so what we did is the same experiment simple experiment with concentrated and purified endospores And you can see that the concentrations of free particles that we added at the beginning of the experiment stay constant over time There's no decrease that means that the that the virus is which are capable of infecting that genotype Cannot bind to the surface and start and initiate the first step of the infection process So this is a physiological form of resistance to those virus virus particles We refer to this as a refuge right the viruses can't get inside the host There's another aspect of this that I'm not going to talk about too much today But I think is really fascinating and I would love to I'm actively trying to work on this But there's some methodological problems some work from the 1970s and 80s where people have documented that Phages there the phage genomes can actually get inside of endospores and the way in which they do this is truly amazing so a Viper the process of a sporulation takes a long time eight hours So during that time a virus can get inside an actively growing cell as the process of dormancy and sporulation begins Many of these viruses contain what are called OA boxes that will bind to a sporulation a master regulatory genes bo zero a and that halts the infection process So viral infection cannot proceed if a virus contains one of these auxiliary genes. So so lytic reproduction is halted Then as dormancy proceeds There's this genome duplication of the mother and it needs to be translocated and transported into the endo spore The virus has stolen another type of gene a par gene, which is actually physically anchoring the genome and moving it into the endo spore So it seems that there are Genetic components within some viral genomes that allow viruses to actually be physically moved Into an endo spore and you can imagine that if the environment is not suitable for reproduction for the host It may is also not suitable for the reproduction and reproductive fitness of a virus But it would be advantageous for the virus a genome to be incorporated into an endo spore where it would benefit from the protection from the environment Importantly, it's not incorporated into the host genome It just rests with inside the the endo spore and this is something it fits a definition known as pseudo lysogeny Okay, so what fraction of the 10 to the power 28 endo spores in marine sediments and other ecosystems Have contained what are called Vero spores through this process of entrapment. I don't know any answers to that question But there's some problems in trying to study it, but I find it really interesting. Yeah. Yeah. Yeah, but but It cannot exit but upon germination Presumably and this is true. It has been documented that upon germination of the host the virus can resume its Lytic reproductive cycle. Yeah, that's a good question. So we talked a little bit about this at lunch. So Let's imagine we're talking about this situation the cell wakes up and then immediately the virus starts its reproductive cycle Lices the cell and maybe there's not a lot of other hosts. There can be stochastic components of germination So you would want to wake up if you're a virus You would want your host to wake up and where there are other hosts in the environment that you could subsequently infect so if that's not the case for some reason then Incorporating and replicating with the genome via lysogeny and profage would be perhaps a better strategy I don't know if I took it. We talked about this at lunch briefly. I'm not sure if that's really clearly Yeah, it might depend on the environment and the abundance of susceptible hosts there Anyway, this is sort of for me and when I learned about this My mind was kind of blown people do this for just as a practical purpose in their laboratory for height If you're if you're into Bacillus and you're into Bacillus phages People actually store their phages in endospores because it's a more stable way of maintaining cultures in your laboratory, which I think is just wild But to best my knowledge no one has ever documented this phenomena in the wild and there's a few studies from the 70s and 80s a Little bit more genomic work from roughly 2005. So I find this really curious But I don't have much more to say about it. How is the detection? No one no one has done any genomics on this So what so there's a feature that I this is a good time to talk about this these endospores are Heat resistant and so experimentally in the laboratory You can heat treat a sample and by doing so kill all the vegetative or active cells and you're left with a purified population spores And then if you polite those spores on media they'll germinate and if you do that then you can see that phage particles will emerge And you get plaques and so those plaques the viruses are also heat sensitive So the only way you could get a virus particle would be if they they came from inside the end of spore so these are the ways in which Sporulation might modify Interactions between bacteria and viruses some work I want to talk about now there's Maybe three things that we could get through today, and this is number one We wanted to ask the question about how Sporulation modifies the directional evolutionary process between a virus population and a bacterial population We used a virus in the lower left. It's bow zero one. This is a Cotto virales. It's a tailed phage It belongs to a family called the hell of iridae. It's very similar to t4 like phage and we used Bacillus subtlis on the right these organisms as I've described show endospores is a phase contrast image You can vary conveniently and beautifully see the white phase Particles that light does not transmit through because of the dense packing of materials in there along with some of the other vegetative cells So we can study these microscopically we can study them in a cultivation based approach using this heat treatment It's a really good genetic model system I'm going to talk about one of the genetic modifications that we made of a sporulation or dormancy trait And then we use a an experimental modification of an experimental evolution approach I assume many of you are probably familiar with these types of experiments, but they're pretty Straightforward you have replicate populations And you track individual populations or flasks over time doing what in our case serial transfers So we take 1% volume and transfer it every day into new media And we just population densities are fairly large in this case 10 to the 7 10 to the 8th So we have large effective population size which tends to favor selection for for adaptive mutations one rare So that this is a general approach and We have two treatments with regard to the seed bank. The first is really simple. So we Performed a genetic deletion of an essential gene called spo 2e which is required for the successful production of an endosporb So we just deleted that gene and now we have bacillus mutant that cannot form sporulation and therefore Dormant individuals cannot accumulate and that's how we're defining as the seed bank, right? Okay, so that's one treatment and then You know, we passage these over time After generation one we introduced the viruses and we're able to track the dynamic the population dynamics phenotypic diversity and molecular evolutionary dynamics over time and this treatment And we're going to contrast this with a treatment that has a seed bank Figure looks a little bit complicated Maybe it is but I think I can explain it to you the first thing in this case is we don't use that Sporulation mutant. We're using the wild type a strain that can form an endospor and again that endospor confers Resistance phenotypic resistance to virus particles because it can't attach, right? So that's one way in which we can contrast things But what we noticed is that if you take like a chemist that we're just talking about before if you take a population that has Spores in it and you transfer it into fresh media. What happens? Ways up so quickly your seed bank from the previous time step just erodes like everything wakes up So you have no way of having a biological memory of Organisms from the past from dormant individuals because they just it erodes and they wake up from the previous time step And bearish and that took us a little bit of time to figure out and so what we said what we envisioned was and this is actually Biologically, I think reasonable is that there's an external seed bank out here In the Mediterranean sea there are organisms that are metabolically active that are in the pelagic the upper Suspended parts of the ocean there are a lot of dormant organisms that are living in the sediments below that get recruited from time due to changes in temperature and nutrient concentrations So the seed bank here in the Mediterranean is physically separated in some way from many of the active organisms that live within the water Com so we did something analogous to that we created an external seed bank and what that meant is that Every time step we took some of these dense cells we heat treated them killed off all the vegetative cells and we have this Falcon tube of of endospores purified endospores from time point T minus one and then we would keep this separately and then we would add back some of those at each time step But this whole external seed bank is a moving window of time And we transferred We wanted a relatively strong seed bank effect So we it was a four-to-one ratio of new endospores to old endospores You could imagine doing an experiment where you change the ratio of Individuals from previous time steps So there's there's a there's an introduction of new endospores from this collective external pool at each time step in the experiment so The heat treatment is to kill everything that is alive them all the active cells all the active cells because in and does the treatment also In use for relation in some of the new cells. No, we assume not. Okay. Okay. Thanks Yeah, I also have a question. So what causes this correlation in the end of the cycle it runs out of alanine as you described Yeah, a good question. So we I think we were transferring these Every day and during that time you you get there's enough time there for Resources we use the DSM medium to exhaust resources and induce Correlation, yeah, and I have some numbers somewhere on like what fraction of the population in this treatment. We're forming endospores So in the end of the cycle there is a mixture of the cells We just stopped growing because they run out of resources and spores, right? Yeah, I think that's right And what is the what makes alanine special why choose the alanine and not some other amino acid God knows I don't know why alanine is so important the media we use here wasn't necessary wasn't defined There's a medium you can order called DSM sporulation media which compared to LB Foster sporulation more in these in these populations of bacteria, but I don't know exactly what's in there There's a certain elements like calcium potassium things like this that are important for making endospore But it's not it's not to find media So we don't know for sure if it's alanine limitation, but there's some general resource limitation this environment that's inducing and I'm not sure I understood the parallel with Seed banks in the sitting in the sediments of the ocean and sort of like how do they get up? How do they wake up? I mean, it's not clear to me at all that they're able to cross this whatever minimum 100 meters of water and sort of like yeah, I Mean, I think yesterday I showed this one figure where there were proportion of inactive cells and different types of ecosystems So you could go into the surface water here and positive and you would find especially right now this time of year that there would be some Bacteria diatoms phytoplankton would have you that are metabolically inactive. So it's not True that these these pools are always distinct, but there's definitely a reservoir of inactive dormant organisms that reside in in sediments and in many other cases the Plants are another good example, right? Like if we go out and we look at the vegetation that's growing out of the ground those individuals are going to be largely Metabolically active and the seeds that are produced at the end of a growing seed get deposited in the soil so it's not it's not specific to to Lymnetic ecosystems in many cases the spores and seed bank is physically separated from actively growing individuals now So that's your question. I mean, I think there are things like if you're in the bottom of the ocean and you're asking what triggers It could be thermal activity Depending on how deep the water column is a penetration of solar radiation could be a cue I don't know a lot about what what leads to cyst Resuscitation For example, just wanted to add a comment on this. I'm no ocean oceanographer or anything But these know that there are Depending on where they're and stuff like that that can be like slow upwelling chorus. They basically go On on the bottom of the sea and then basically like mix everything very slowly and bring stuff from the bottom of the sea Great to the surface layer. So migration of dormant individuals right through passive mechanisms. Yeah, totally. Yeah That's question though More of as a comment than anything else, but I'm probably gonna confuse people So part of the population will also sporulate even when no starvation is present as a form of a bad hedging with quorum sensing Are you using the one six eight laboratory or some kind of undomesticated strain for? Bacillus subtilis in this experiment So right remember, there's two flavors or categories of transitioning. There's this responsive Transitions into and out of dormancy and there's stochastic forms of which can would not be dependent on things like resource limitation Bacillus subtilis and sporulation in this case. It seems to evidence seems to be Largely that this is an example of responsive transitioning where organisms are able to sense changes in resource availability But there have been Documentations of stochastic processes. I know I measured it. Yeah, good So so we're gonna assume that this is mostly to answer your second question. We're using Delta One six eight. So this is a somewhat minimized cell of Bacillus subtilis that we've removed things like prophages Which would potentially be important for this? This system and the questions that we're asking So so we just track these, you know, this is just a plot showing the population dynamics of the bacteria on the viruses over time Remember, we have the seed bank treatment in place here so in this panel We've genetically removed the capacity of this organism to form endospores prediction would be that the cells there would all be Expressing receptors at least in the beginning of the experiment that would make them Infected by infectable by virus particles, right? So no No dormancy refuge here and in these populations they can form endospores But we also have this additional feature, which is the external seed bank And so statistically and even by eye you can see that these dynamics are somewhat different. There's a major Drop and host densities bacterial densities early on in the experiment reaching a minimum population size of 10 to the 6 at about 6 days You can see that this Minimum population densities with the seed bank is extended to much longer the population dynamics on average are more stable with the seed bank Than they are in the absence of that Another thing we did this figure is Looks simple and it's but but I could imagine somebody getting a little bit confused We are interested in the retention of phenotypic diversity in the population And what do we mean by phenotypic diversity when you challenge a bacterial population against a virus? It rapidly evolves Resistance in this case probably through a modification of a teacock acid Receptor on the cell surface and once that cellular modification has been made the virus particles typically can no longer attach And this is the mechanism by which a bacterium evolves resistance to a phage in this case Okay, so in the minus seed bank treatment You can see that we're starting the experiment at time zero where 100% of all the individuals are very close to 100% of all The individuals we sampled clones like 20 clones in this population and 100% of them were sensitive to the ancestral virus that we challenged them against But by transfer one two three and four we're seeing all of the 20 clones that we pull out of one of those Experimental units we challenge against the ancestral phage have are all resistant. They cannot in fact and create Viable virus particles, okay. I'm sorry this this one is one day or one hour One transfer and transfers don't equal day. So we we we sampled them every day, but we transferred every two days So that that ensures even more that's for relation the spores accumulate so two-day transfers We sampled every day. So there's a distinction between days and transfers and If you take E. Coli for example in stationary phase It's also known to be like particularly resistant to phage and actually people are struggle to find phases that are killing Stational phase E. Coli So would you expect that this is a property of spore nation or could you do the exactly the same way sort of like Keeping a stationary phase culture of E. Coli then you put them together with the sort of active E. Coli And they would be the one surviving but sort of this is not really You don't have to go through this expensive process of sporeation for that, right? Yeah. Yeah, great So there I think I am aware that Like organisms that are in some kind of quiescent state that might be a little bit different than what spore relation is Phages don't aren't as productive on those on Metabolically inactive host. I don't know but I assume that's just a function of the host metabolism, right? Ability of the the the productivity of the virus is determined to some degree by the productivity the host The question is What would be different is the mechanism of entry? So in this case with end of spores, we're not getting like actual physical binding So if you had an E. Coli cell that was starved, maybe it's receptive the the expression of receptors on the surface are modified And so in that way it would be similar, right? It's all it might be about initial contact and infection or could You know T4 or another E. Coli virus injected DNA and then it's just a rate-limited step due to metabolism the host you do you know Yeah, yeah So in the Seabank treatment If we look in the focal population our flasks the the black line looks qualitatively pretty similar we see a steep drop-off in the proportion of a Susceptible cells this is what we would expect just based on dilution Over time of what the concentrate of what the the sensitive cell types would be just due to dilution And what we see here as what's in the Seabank and you can see so remember there's some kind of protection being conferred Against virus attack that information can be stored in this in end of spores Which we purify and go into the into the spore bank and because of that be at least for some of these time-taps We're seeing the preservation or maintenance of sensitive phenotypes in the population for longer So there's some signal that that's phenotypic diversity is maintained through this seatbank effect. I Didn't explain it well so We know how many If the calculation that we're trying to basically I'm trying to remember what the first time point is or what we're measuring here But there are we know how many sensitive the frequency of sensitive Cell types are at the initial a time zero and if we expect that those are just being transferred And there's no introduction of new individuals with that sensitive phenotype That would be the expectation if there was just dilution or exponential big K due to dilution And what we see the point. I'm trying to make is is that the observe the observed gray diamond shapes Are greater than what you would expect just due to the effect of dilution based on what was in the population That's the very beginning which we documented So we knew the fraction of sensitive cells in the Seabank at time zero If there were no new inputs, then you would expect that those might just decay over time That's the point where we're trying to make the best of my knowledge right now I wanted to clarify one point about your Experimental setup when you do the experiment with the Seabank you also do dilution right by the same factor or You know when you do the heat treatment you kill the cells which didn't spoil it And then what do you do with the with the rest you just transfer all of it to the new flask or You somehow diluted There is a dilution that's made of the Seabank so that Every time step you would have one part old seed and four part new seed bank And then you would take a fraction of that 50 microliters of that cell suspension and in unoculated into the next time step of the transfer So in some sense all together if you combine the fact that the some of the bacterial cells die During they well during the heat treatment the ones which did not spoil it and this dilution the effective dilution in your experiment with Seabank is larger than the the dilution without it I'm just trying to compare apples with apples when you do the left side panel and right side panel the right side panel Not in this but maybe in a previous slide The the left panel would you you just do the dilution regular way because there is no, you know There are no spores or anything. So and this is a dilution by factor hundred if I remember correctly, right? You do you take 1% and then on the right you do Heat treatment which kills. I don't know how much but let's say 50% of and plus do this dilution of the seed bank Still right by a factor hundred So if I just trace the the life history of one bacterial cell The chances of it to be transferred to the next generation are Lower on the right than on the left I'm not sure I followed all that but I know I think I know where you're going But the amount of the volume that we're putting in from the seed bank is on the order of 50 microliters It's a relatively small volume to the volume that's being transferred over from the from the experimental unit in the previous time step I'm not sure if that's what oh, well, maybe maybe then I I'm I'm now confused So you I thought you only transferred the the seeds. No, no, I see okay that that that's my fault So you you you still do the regular dilution just like and then yes, you add to it the the seed back Okay, okay, I didn't catch a relatively small volume Yeah, and what what happens you it looks like you have some sort of a stable Coexistence even though the jury is still out. Maybe the phages on the way out How does it they coexist they coexist but of course the resistance evolved right so you have the resistant So what is what is the coexistence? So there is a mix of resistant and susceptible Just on the left If you if I look at day 28 or something like this So I see coexistence between phage and the host and yeah, it is a Fage susceptible and resistant all three coexistent together. Yeah, so I should say that The evolution of phage resistance is costly. You're modifying these receptors often those are used for transport or metabolites or whatever So so there's a cost to being resistant and there's a cost to making a spore And what we see here is your your documenting surrogates that there is coexistence They stay they coexist together and both of them are undergoing this co-evolutionary arms race The the take-home story is that the the rate and the magnitude and the intensity of co-evolution is dampened by the seed bank So we still see these processes taking place. It's just they're modified Presumably due to the external seed bank and the physical refuge of the cell not being able to be spores being able to be infected Okay, got it. Okay Yeah, so the last thing we did this was some work we did where we we did pool population sequencing Will who's not here Helped and contributed to this and so what we did is just measured the mutations and the host populations at the end of the experiment This measure of gene multiplicity is basically just talking about the the occurrences or abundances of mutations and replicate populations corrected for things like a gene size because larger genes have a higher probability of getting hit in a population and so We're looking at is a distribution of the the abundances of mutations At the end and so there are some mutations that are really abundant in common We found across replicate populations and then there's these long tails of rare mutations Seed bank theory would would predict that dormancy can retain that genetic variation That might otherwise be eliminated due to selection or random genetic drift And these observations show that we're getting about two to two and a half times more rare mutants that are being retained in these Populations when there's a seed bank versus when there's not So so what I've showed you is that that seed banks I'm going to transition so I'm wrapping up So they this experimental evolution approach shows that we we know that there's a Benefit of forming an end of spore in terms of contact and infection rates Through absorption that that when we create this external seed bank we can see modified population dynamics We see the retention of phenotypic diversity in terms of sensitive and resistant cell types and we're seeing the the retention of Rare genetic mutations in the population that otherwise would go extinct which perhaps could be beneficial In the future, right? Can you maybe explain a bit more what exactly is being? Plotted here you sort of sequenced some sort of and then yeah Genes I'm just trying to understand so you could start with a reference genome for Delta 168 And we know what all those mutations are in their ancestral state and then at the end of this experiment only at the end of this We actually did sample through time But we we extract DNA We sequenced those the that DNA and we map reads back on to the reference genome and we use a program that allows us to Identify insertions deletions and single nucleotide mutations and so you could imagine that there might be a gene here And you can find that in multiple populations and we can assess and quantify how many times a Sample that would have a gene that would have a high multiplicity score would be hit in multiple populations over time and Rare ones would be a gene where you only find a mutation occurring one so it's a way of standardizing mutations across populations and Accounting for differences in gene size because In a no expectation you would expect that larger genes should be hit more times than not I'm just trying to understand why that the rank stops at half for the minus c5 So we're ranking the mutations based on their frequency in the population and so on this end of the Distribution you're seeing lots of mutations that are commonly found in populations and as you move out These are ones that are rare. So we're finding a lot more rare mutations in the populations that have a seed bank and fewer mutations But you just have twice as many mutations That's right But then I mean shouldn't you then rescale to get the frequency distribution the other one? I would they then look like almost exactly the same. I Just don't understand this comparison. How can you have twice as many mutations? How can you have twice as many mutations in a population? Is that yeah, I thought you wanted to compare the frequency distribution of the mutations These are the this is the recovery of mutations in populations And so in the seed bank your observations right there's more mutations Being recovered in the seed bank about two and a half fold and most of those are rare and this is their abundance and The population I will really like to understand this so you have yesterday We saw a figure where we saw these rank abundance distributions, which are pretty common for looking at frequencies Yeah, I understand what is the rank abundance? Okay, I'm just trying to understand the rank abundance of what is being plotted here So you have two populations of cells and and then you sequence them and you map them to the reference genome So are you starting from two identical sized populations of cells? Sure Are the population sizes equal? Is that what you're asking? Population sizes. Yeah, we're standardizing by DNA concentration as well Maybe just to continue because I'm also intrigued by this plot But could it be the fact that the one without the seed back went went in through the bottleneck Or it really dropped to you know orders of magnitude below what it was in the beginning and during the bottleneck Maybe some of the mutations got fixed So what what is behind it in other words there were no mutator strains. Why why you have this factor two Do you have any any kind of yeah, it's that how to understand it? So I think what we would expect Independent of the methods being used here is that if you had populations with and without a seed bank that you have more genetic and phenotypic Functional information that's retained in a population that otherwise would be at risk of being lost because Organisms aren't dormant and can have higher rates of mortality So in this experiment where transferring things things are getting diluted out That would be an example of about a potential body. That's bottlenecking, right? We see here that there's bottlenecking at around time seven, right? So yeah, so those processes of individuals being randomly lost over time would contribute to Selection and a component of drift that would lead to differences in diversity We see that with the seed bank you're getting more genetic diversity preserved in the population Mutations that are arising during the experiment are not lost either due to selection or drift and they're being retained at about Two-and-a-half fold and many of those mutations are rare. That's that's the that's the that's the conclusion or interpretation of the figure Did you try to classify those mutations because again the other Interesting possibilities that there are two types of mutations the ones which are good for you know being passed through the Seed bank channel and the other ones which are just good through, you know, evolving the phage resistance So is it possible that this factor two-and-a-half is due to the? Basically two transmission channels each one puts a different selective pressure So what you'll see is that there are some different line types And those are for the the populations that evolved in the presence of phage and minus phage So that that general relationship is not affected by phages at all the mutations that we do Find over on this right-hand side of we have classified those and there's nothing really That seems obvious. There's no specific Family of proteins or functions that seem to be retained, which again is something that we might expect if there's just random There's this retention of genetic information In the absence of selection Okay, that's interesting So in other words you would if you were to repeat this experiment without phages at all you would expect Well, you did right and you found the same profiles. This is that what we're seeing here is Independent of any phage effect. This is a pure seed bank affected We don't see any statistical effect of the virus in the population on the retention of this genetic information So it seems to be solely due to the influence of the seed bank So I think this is a naive question, but did you look at how? how this kind of Gene multiplicity rank looks at the beginning of a trans or like Early on after a transfer and then late after it transfer. So kind of how does it change over the time of a transfer? Yeah, I mean so this question about bottlenecking would beg that question. We didn't We didn't choose to sample at that frequency resolution to address that question. But yeah, you might imagine that Yeah, you could look at this for each at the end of each transfer Yeah, and I guess my expectation would be is that the initially early on in the experiment Maybe the the retention of diversity in the seed bank would be minimal and you would see this effect Grow over time would be a off the top of my head Yeah Yeah Reset it. I see and how quickly does this set up? Yeah. Yeah. Yeah. Thank you Can I ask you the last question? Yeah Now I was trying to understand Where does the seed bank come from in terms of how do you generate the seed bank population in this experiment? Yeah Through first step one by having a strain that can produce endospores But that's not enough. The second thing is to create this external seed bank where we preserve and Purify endospores and put them in the external seed bank And do you do it while the experiment is running? Okay, so basically you have a population that is Growing and producing endospores. Yeah, so does the seed bank reflect so the diversity of the seed bank reflects the Diversity of that population. Yes. Yeah, there's a seed bank for each replicate population It's not a global seed bank across Experimental units, and I think there's a lot that could be done with this by expanding the the the ratios and the amount of time You could imagine you could purify a seed bank from different environments and have migration between seed banks Okay, so Yeah, maybe it's like ten minutes that we have is that right? I Got two things to talk about and I don't think we can get through two of them I'm just going to give you a preview on on this work, which is actually not quite baked yet It's some work that we did and so The general question is is can dormancy prevent the spread of viruses? And this is kind of the way we're setting this up is this is sort of a spatial question And the previous experiment we have well-mixed flasks where phage particles are coming into contact freely in a well-mixed system but there are many cases where Viruses are kind of isolated and local populations and patches And so what motivated this were a couple papers that came out Over the past four or five years The first one is from Joshua whites who's collaborating on all this work. They have been studying sulfolobus And what they there's an Archean and what they found is that they could Deactivate phage particles with UV radiation and if they came into contact with the host just the physical contact would initiate Dormancy in this in this population. So just purely physical contact was enough to allow Sulfolobus Icelandicus to enter a dormant state There's been some other work in Listeria and in CRISPR cast out of the Miska group in Washington showing that CRISPR can be used to down regulate RNA in the entire Cell but it also kills off virus infections and so through this way this mechanism of CRISPR induced dormancy You can actually prevent phage infections from spreading in population. So these types of papers were coming out They were interesting to us and the thing that we're going to focus on here are The size of plaques that form on a lawn and this is a pretty People have been doing this for hundreds of years. We can use this to enumerate them the number of Fage particles in a population but the morphology the shape the size of those those Clearings on a lawn of susceptible hosts may also convey some information that can be of value I Want to get through the last thing But I just want to kind of show that what one of the things we did is we infected hosts that could Spoilate and those that couldn't so a wild type strain that can form endospores Plate them out on a lawn put down infectious phage particles and characterize the plaque size We did the same thing for a host where there had been a mutation in Spoe To e which is an essential gene that's involved in sporulation and when we did this you can see that the the plaques that form on the wild type are About two two and a half times smaller than the plaques that result when you plate them on a lawn of the Bacterium that cannot form endospores So that was sort of interesting it suggests that dormancy might be preventing the spatial spread of Viruses in these structured Petri dishes So that's macro scale observation one I'm unfamiliar with these I say but like what in the absence of sporulation what limits does the spread of the plaque What is sort of like what determines the its limits? Okay, that's a really interesting question. What did what what what? What determines the size of a plaque and then that was sort of the question that that's a general question Usually I think it's resource depletion of the the host right and so that we're just what we're talking about as the lawn grows And it gets really dense and the virus is moving there's resource depletion around the perimeter And there's not enough energy for the host to sustain reproduction of the virus part That would be a resource-based explanation for what ultimately limits the size of plaque from taking over the entire plate It's two times the same phase right You say it's the same phage on the left and right. Yes, same phase. Yeah, but it's two different back Drive from the same wild type strain So we have the wild type or Delta's 168 and we have a single gene mutation and spoke to me It's just the same bacterial population that we used in the previous experiment We don't see any fitness consequences associated at least in liquid and I mean by eye You can see the lawns form so I don't that wouldn't be what I would would first suspect So we see some differences in cell size the other thing so this is the macro scale observation. There's a micro scale observation We grew we made little mini plaques on auger plates and looked at them underneath the microscope And there was a micro scale phenomenon that stuck out to us that initially seemed really Interesting and could potentially explain why the plaques don't get bigger You can see in the bright field on the left that there's this zone of clearing here and There's cells on the lawn around it and we had a the one strain that we were able to use is a GFP reporter which is fused to Caught Z which is a protein that's involved in the end of sport Protein proteinaceous component of the of the sport And what you can see is that there are a lot of mature spores forming a ring a Rampard or a wall that you might imagine physically could prevent the the movement and diffusion of virus particles in these environments So my first reaction was like oh, that's really cool. We also have to start to think about well. Why would that ring form? And on the right panel what you're seeing is a A distance profile if you will where we measured the amount of GFP expression From the center at zero of the of the plaque and outwards and so you can see this big ring of GFP There's still we're still seeing spores being produced elsewhere on the plate presumably just due to pure resource depletion But but those concentrations go down So there's seems to be at the at the leading front of a plaque you're seeing enrichment of spores Question is how how and how wide might that happen? I'm sorry. Maybe they could lost what is in the center clearing that there's no cells So in this bright field you can see that there's Cells out here. No cells in the middle because they've been killed by the viruses Here you see preferential enrichment of endospores around the perimeter of the of the plaque clearing And here you can still see that there are endospores out in this region being produced But not to the same density as here Wouldn't it be sort of expected if indeed the boundary of the ring is due to starvation and If if the besattlist runs out of resources, we know that it forms spores So could it be just a byproduct of the fact that The ring stopped where it did because bacteria ran out of nutrients Yeah, so your your one hypothesis could be is that there's just more resource depletion at this point. Yeah, okay? We explored this I'm not gonna have a chance to go throughout all these but that's one hypothesis another one is this contact mediated due to virus particles Accelerate or initiate the the process of sporulation And then the last hypothesis and I'm gonna try to move this along so we know is we create we use some Partial differential equations to kind of model this and come up with some predictions We can easily get the macro scale differences in plaque size That's not hard to do the thing that's a little bit more challenging is to get this ring And the thing that we needed to do to get the ring was include a A defusible metabolite a danger signal if you will something that sells Here so if there's lysis occurring here can imagine that there's some kind of defusible compound We don't know what it could be That accelerates the onset of of sporulation. That's the working hypothesis the models Need that We don't have any Experimental evidence we're working on it to back that up, but there must be some kind of Chemical it could be something like a lysate it could be peptidoglycan, which is known to be a signaling molecule We don't know yet So I think this is really cool. This this reminds me a lot of Hypersensitive response in plants. Have you heard of this concept? Explain it. Yeah, I think basically when on a plant leave, you know, there's a I guess maybe a fungal pathogen that hits the plant at some point that you know You will see a ring of death cells basically programmed cell death to contain this Yeah, yeah sort of as a Type one immune response of the plant or is it more of an adaptive immunity? It's I guess yeah, I guess this is a very general immunity Yeah, but you would you would see in this case, you know, for example the viral particle and you'd be like Oh, this is signal to to shut down so to contain this and so maybe you can find inspiration and yeah Yeah, thank you. Thank you Okay, so I don't I don't want to I Think I gave away the punchline. We created some models We needed to update those models and we needed to include this diffusible metabolite in order to get the Effects that we see here where we can recreate plaque size variation and this ring structure still a work in progress and I think these experiments beg that we Think a little bit more about chemistry that might be involved in these danger signals and the mechanisms of What's leading to that protective putatively protective ring? I just want to end really quickly on the last side I think so one of the things we know about studying viruses and other systems as in the context of co-evolution Parasites usually find ways to get around the defense mechanisms of the host and And this is true in the case here as well You would think that maybe creating an endosport that has no receptors that a virus could potentially recognize and has no way of getting inside of a cell that is Biologically and energetically inert would be a dead end for the virus But it seems that there are ways in which they've co-opted dormancy to their advantage the first sort of not entirely chronological way in which we approached this was thinking about Documenting the existence of auxiliary genes so some of you may know that viruses have pretty streamlined genomes Most of their gene content is made for infecting cells Taking over host machinery and the assembly of new virus particles and releasing them into the environment very small genomes But if you go out here, and you were to look at viruses in the ocean You will find that they contain Metabolism genes phosphorus genes nitrogen genes fermentation genes Mone oxidation genes sulfur metabolism genes. These are referred to as auxiliary metabolic genes And in some cases people have been able to show or infer through Transcriptomics and such that that this may be advantageous to the virus by by using those genes and during an infection a host can Allocate more energy to reproduction and you will get more viral infections. This is the idea This led us to ask the question. Okay, there are a lot of viral genomes available to us now And we know what the list of 500 sporulation genes that could be potentially Obtained by viruses and perhaps they could use those in ways it would modify the decision-making behavior of an endospore forming cell And so we had we collaborated with Kelly right in a Colorado State University who does a lot of work She had a lot of data on viral metal genomes from various different ecosystems And so what I'm showing here is a list of some Sporulation genes essential sporulation genes on the x-axis and we have a heat map here showing the recovery of Sporulation homologs from Metagenomically assembled virus metagenomes in different ecosystems We focused mostly on the human gut and found that there were a lot of sporulation genes that were inside of genomes And that was an interesting pattern So then the last slide I want to show is I did some work with Daniel Schwartz who's now moved on and is working in the Netherlands and we looked in actual Culture collections of viruses and we knew what their genomes were and we found another room that had Transcription factors or what are called sigma factors that are involved specifically in the formation of endospores So we had an isolate with a sporulation homologue that had presumably been obtained through horizontal means Some of those were very very closely related to Sporulation genes and bacillus subtlas and so what we did the first step was to to use CRISPR-Cas editing to remove those those Sporulation homologs from the virus and see if they were essential or not if it's an auxiliary gene You should be able to delete it and it should have no effect on the ability of that virus to infect its host And that's what we found so then we took there's some limitations to what you can do in the system we took that Sporulation gene it was inside a virus we cut it out with CRISPR and we inserted it into Bacillus and we at topically expressed that protein inside of the host cell We saw that when we expressed that gene that it led to Changes preferentially in genes that are involved in transcripts and sporulation pathway and in this case over here You can see that it disrupts the ability of the host to form endospores by 99 percent So it this is favoring the reproductive fitness component of the Virus by preventing it from entering that refuge You could also imagine that perhaps different types of genes might increase the probability of those cells Going forming an endospore and if viruses can get in and form zero spores then that would Perhaps be beneficial in terms of its survivorship component of fitness So the full story is that we got into this really super naively So yeah, this cell type probably can't get infected as much and we've shown through experimental co-evolutionary means that That's true. It affects population dynamics retention of phenotypic diversity and genotypic diversity We've been able to kind of ask some questions about how and to what degree Sporulation may prevent the spread of viruses and spatially structured populations and then maybe we need to think a little bit more about Metabolites and things that are diffusing in this environment, whether they're actively produced or if this is something that's just in a lysate of a dead cell and Finally, we were seeing some evidence using metagenomics that Viruses have the ability to tap in co-opt hijacked and potentially continue this co-evolutionary arms race Via means that manipulate the transition probabilities of cells into and out of dormancy Yeah, so only three minutes late Okay, thanks That is how that there is time for one or two quick questions Going back to the gene rank abundance graph. Did you look by any chance what these rare mutations are? Yeah, we did and we thought there might be some kind of unique Functional signature associated with them and there's really it seems like there. It's it's there's nothing special going on with those genes And so, you know it suggests that Yeah, maybe you're just retaining Neutral variation in certain genes in the population So there's nothing that really distinguishes those genes from the common mutations that we saw in the population But they are different and another one was I was thinking about the sporulation the other graph you showed and Would there be any chance of cyclic amp in Dicty When they start forming spores, there's cyclic amp waves going on And it's a key factor in sport forming in Dicty. So you're talking about the plaques or no When when they there's no food and they start to starve there's cyclic amp going on so they form spores In order to survive starvation, okay, I just want to make sure I understand what figure you're talking about This is early on and it's the other page Like I think it's one Yeah, that's the one this one, okay Yeah, yeah, so the question is is could cyclic amp specifically be I don't know. I mean, I don't tend to think about I mean, this is how science works, right? You see a pattern and you got to try to figure out what's going on So I've been trying to think broadly about what could be going on. I have a little bit of I've thought a little bit about peptidoglycan and how those I mean those are like PAMP molecules, right? That induce inflammatory responses. So there's really good evidence that things like LPS or Peptidoglycan conserved signaling molecules, maybe cyclic amp I you know, I don't know and I'd be I'd be happy if anyone had some suggestions and then the question is like how do you go about You know, what would you identify a potential chemical Canada that could be a dangerous signal? How do you go about studying that? I think the first thing for us right now? We're working on some experiments in the lab where we can take whole cell lysates generated by by virus infections and seeing whether or not we can increase the kinetic onset of Sporulation can we because if that if you could do that just at an aggregate level without identifying even what the chemical is you could say that there's something in the lysates that is is Accelerating sporulation and that would be one explanation for the ring formation Because you can see that there cells on the outside that are undergoing sporulation Right, so the conditions are suitable for spore formation. It's just that There's higher densities which would suggest that maybe they can enter dormancy earlier due to the processing of Signals on the environment normal questions So if they are not the things