 y los Fernando. No, you didn't lose. Here you are. Hello. So, eh, now we continue as expected. Eh, we are late already. Okay, anyway, eh, with the Dal Dagan, we will talk about the Darwinian fitness of extra chromosomal genetic. You have 20 minutes. Yeah. Thank you very much. Good. See the presentation fine. Yes. Awesome. Okay. So, right. So, thank you again Fernando de la Cruz also for the introduction for plasmids. Also saying that plasmids are quite complex. So what I would like to maybe try to convince you that they are also not only complex but could be considered as real Darwinian entities. So, the view on the determinants of plasmid persistence in the population is that we have an interplay between the effect of plasmid effect on the host fitness and this might depend on the environmental conditions. We take here the example of antibiotic resistance plasmids if we start, if a plasmid is found in, we start usually in the lab for example with 100% hosts of the plasmid. In the absence of selection we would expect the plasmid to decrease in frequency due to the negative fitness effect on the host. But with the selection for example for the plasmid presence in here antibiotics we would see the plasmid frequency in the population going up. But according to that kind of view if the plasmid always has a very negative effect on the host fitness then you would expect the plasmid to decrease in frequency and at the end to go extinct from the population. Yet, also as noted here in the previous talk, I think by Konis Mala, this might not always be the case very often you might have conditions in the environment where the plasmids have a less, a smaller effect on the host fitness, which we even might consider as neutral plasmids or even neutral conditions for the plasmid presence in the host. Right, so what happens under non selective conditions. In order to try and answer that question. A couple of years ago we performed an experiment of plasmid evolution and the non selective conditions. Here we evolved a model plasmid that we call the ancestor plasmid. This is a PBBR1 derivative, so it's relatively low copy number of about five. If you take it into the naive host, which is in our case, Escherichia coli and we made it very, very small so that the fitness effect on the host was almost negligible. So you might say it's a semi neutral plasmid. We evolved it in under non selective conditions for the plasmid presence. Nonetheless, the plasmid was unstable so the frequency of the plasmid in the population was decreasing. Then when the PG student back then, Tana Wain, exposed the population to antibiotics so selecting for the presence of the plasmid and then went on with the experiment under non selective conditions. We could see several plasmid variants that evolved stability so they were present in the population continuously in 100% of the hosts and not, yeah, were not lost anymore. When we sequenced the plasmid, we observed a small duplication in the plasmid genome segment, a duplication that apparently made this plasmid stable. So we show here evidence for evolution of plasmid stability or stable plasmid inheritance under non selective conditions. Now if you think of what happened during this experiment, during sometime during our experiment in the populations, there was, yeah, occurred the segmental duplication that we observed in the plasmid. So the way we would picture it is that we would have heterogenic host that include both plasmid types, the ancestral one, and the, and the evolve on with the segmental duplications. So our next question was then, is this genetic variation in the plasmid genome indeed associated with variation in fitness of the plasmid we have from this experiment or the preliminary answer, but we could ask this in a much more direct fashion. So what is important under non selective conditions. The most important is that the plasmid will be able to complete the plasmid life cycle in a way that it can replicate, resolve the plasmid copies and then divide or segregate into the, into the daughter cells. Right, so failure to undergo any of these steps would lead to the plasmid loss and over time also to the plasmid extinction. So we hypothesize the plasmid persistence under non selective conditions would depend on the plasmid Darwinian fitness. In other words, you could call it reproductive success. So the success of the plasmid to complete the plasmid life cycle. So what is competition experiments and here I show for the benefit of those to don't do it every day what does it mean for cells right if we complete cells. We mix them in. So we have two genotypes here the blue and the red. We mix them in a ratio of 5050 or one to one. Then we transfer them over time and ask in each generation, how many do we have of the red or the blue cells. Right and if you see that the red cells are increasing over time we would say that they have a higher reproductive success, or therefore higher fitness. Together with the news we adopted this idea in order to quantify the fitness of placements using that concert but this time in a pair wise in cellular competitions between placements. The idea is we can bring the placements into the same cell right so we call it the head to head competitions we then evolve them under non selective conditions. And then we ask for the evolving evolved population. What's the frequency of the competing placements. We do it like head to head competition as we plotted here and in addition, you can also ask what happens if we have a preemptive competition, when the plasma is invading a cell that is already populated by a competing plasma, we call it here the endemic plasma. The question is that if plus mid stability is a determinant of the plus mid fitness, then the stable plus mid in our case the one with the segmental duplication is expected to out compete the unstable plus mid. The model placements we used for our competitions were exactly the placements I already told you about but without too much details we have here the unstable plus mid which was our back then ancestral plus mid. And we have the stable plus mid which is the evolved one with the small segmental duplication this is this extra piece that we have in that plus mid button here. And having two different antibiotic resistance genes on no market genes on the two placements enables us to quantify them in the population after the experiment. We also replace the markers so that we can also check the check the effect of their marker but I will not show that one here. At any rate we competed placements, so we were able to bring them together into into the hosting cells and then let them evolve over a very short time and ask at the end, which of the placements is more frequent in the population so we have here the in blue the unstable plus mid. And then the stable plus mid. And what we see in this graph. This is the head to head competition. We have here 36 replicates of the competitions. And each of these bars are stacked bar plots that shows the frequency of the placements in the population. In the replicate population, what we see here is mostly that the stable plus mid was winning in most of the cases in the head to head competitions. And when we repeated the competitions with the unstable plus mid here as the endemic one, of course also the stable plus mid is the endemic one. We have here some cases of coexistence of the plus mid. Yeah, and also some cases here of the unstable plus mid winning, of course there are some auditors in this work you can see it in the, in that publication. The conclusion here is that the stable plus mid wins in such a competition, therefore, if we take the lesson from competition experiments between cells, we can say that the stable plus mids have a higher fitness in comparison to the unstable ones. Now that's kind of a proof of concept to say that we can quantify plus mid fitness and plus mids can evolve stability in the lab and also win over unstable plus mids. And that enables us to go into the next question, which is about the origins of essential plus mids or plus mids that encode essential genes. We were interested to know whether such plus mids might first evolved a stable inheritance or stability first, or first essential to the host and then evolve a stable inheritance. So what is an essential gene just for the formal definition this is a gene that is required for the reproduction of an organism. Typically, we would find such genes on bacterial chromosomes but not always. We do have examples of plus mids encoding essential genes. And one example here is vitamin B, biosynthesis pathway in Rizia, there's an endosymbion of humanoid lice, or Luzin and tryptophan biosynthesis pathways in Buchenera, an endosymbion of aphids, or the very extreme example, the ribosomal RNA operon in Aurimonas, which was isolated from soybeans. Such examples, indeed, occur in nature. And again, we are interested them to test different scenarios for the evolution of such essential plus mids in the essentiality first, the acquired essential plus mid evolves a stable inheritance, and the stability first, and initially stable plus mid becomes essential. That's by gene gain. And, yeah, mathematical models support this scenario, this is a Patsyman and Al paper from an MBE several years ago. Right, so, yes, Fernando said bioinformatics would be easier than going to the lab so we did both but we started with bioinformatics and one here first asked how often do we see essential genes in plus mids. He analyzed the data set of about 600 ashericia genomes, and tested the presence of about 500 genes identified by others in lab experiments, knockout experiments as essential genes. What you see out of the results in here we see here the genes and here the each dot is basically a plus mid. And our results show that out of those essential genes only 17 of those could be found in plus mids. We have a large number of plus mids, but this is a bit biased, because the SSB the single standard binding protein that was mentioned in the context of conjugation is also essential with the many tears of the chromosome. But otherwise you see here that this is actually a very case that an essential gene would be on a plus mid. Many of those plus mids encoding essential genes are conjugative. And these genes that we see in here is essential and appearing on plus mids are very often duplicated in the isolate so present in two copies in the chromosome and the plus mid, except for one case of magic in a single isolate. So we were very curious them to to try and go forward in and ask the same question in the lab essentiality first versus the stability first and we picked as our test case here the Goyal Goyas the chaperonin. So these two genes are translated, co translated very often, and they are part of the chaperonin complex which is a, yeah, it's a chaperonin of protein folding, which is known as obligatory in Asher Asia. So, right, the evolution of that the Goyal Goyas on plus mids is actually in quite an interesting case of these essential genes being on plus mids. One recently performed this analysis asking what is the origin of these chaperonins on the plus mids. And they actually found out that they are part of transposon TN one to five, where they are present in two different types of this transposable element, also accompanied by antibiotic resistant genes we see here the two types so this is one, this is two. This is a phylogenetic tree of the Goyal part of of this transposable element. We see here the chromosomal part you see here in black, sorry in blue. This is all of the chromosomal encoded Goyal. This is here the plus mid encoded Goyal. So this is the blue and the pink. And on the outer ring we see the organisms in this analysis of the chromosomal encoded Goyal occurs here in the data set that we work with CLABSILA, some monela and Asher Asia. And here in the placement we can see that this type of the transposable element is mostly present in Asher Asia and some monela so this blue and yellow. This type of the transposable element is mostly present in Asher Asia and CLABSILA. We actually identified here only one putative case of transfer of this Goyal between plus mid and chromosomes. So this one is grouping with the chromosomes and occurs on a plus mid. In that context you might want to look at this publication showed up in Nature Communication three weeks ago by Ackman et al with much more data on this transposable element. Right, so we said that we will go to the lab and indeed turn it around back then a PG student in the group tested this wanted to test this essentiality first versus stability first. In the lab what she did, she used the two model plus mids that we had the unstable and stable plus mids. Right, so the stable one always have this small segment and duplication here that makes it stable. And this time she cloned into those plus mids the chaperonin Groy exactly as we found it in the transposable element. Now, these two plus mids, the stable and unstable plus mids encoding the chaperonin were introduced in two types of hosts. One is our model organism asher ishakoli mg 1655 the one we the usual one we work in the lab. And when you introduce this, these two plus mids into that host, the Groyal is found in two copies the chromosome and the plus mid. So it is not essential in this host background. The other one is mg 1 mg m 100. So that was a cloned in a time that Groyal was a highlight subject for research in asher ishakoli. And what this constellation or this background does we can silence the expression of the Groyal in the chromosome. And in that background, when we silence the chromosomal copy the plus mid copy is essential. Right, so the essential essential yes no is determined by the host background. And the plus mid stability is determined by the plus mid type. Right, so we started from with both plus mids and both host backgrounds and serial transfers experimental evolution. And what we see in here this is the non essential host. And we have here the line showing the proportional force in the population over time for the stable and unstable classmates. So the unstable is green, the stable is pink. And we can see that when the plus mid is not essential for the host, right, because the Groyal is also encoded in the chromosome. The plus mid is lost, regardless of the plus mid stability. The most stable and unstable plus mids are being lost. When we perform this experiment in the begon of the MGM 100 where the plus mid is essential for the host. We see, right, that the plus mid proportion in the population is always 100%. In other words, whoever is losing the plus mid is immediately dead. So the plus mid is stable in the population or has a stable persistence in the population regardless of the inheritance stability of the plus mid. Okay, the next was to ask, right, what's the effect of the plus mid on the host fitness and here we have the ancestral results for the ancestral hosts. So the stable plus mid is pink and the unstable is green. And we can see that when we look at the host MG 165 where the plus mid is not essential, the plus mid introduction reduces the host fitness. And also in the essential begon MG 100, the plus mid introduction reduces the host fitness. Then when we look here at the evolved host, what we see in here is that the MG 165 when the plus mid is not essential. Even after the evolution experiment here, the plus mid still has an effect, a negative effect on the host fitness. But if you look here at the essential begon MG 100, we see that the host and plus mid are now well adopted. And nonetheless, it doesn't really matter if the plus mid was initially stable or unstable. We see here that the plus mid now could persist in the population or be fixed in the population. Right, so taken together. Our result showed that redundant chromosome a gene in a plus mid is disadvantageous to the host cell. And we think that this is very often due to those effect, even for the shaperonin and that plus mid essentiality can lead to a long term persistence of the plus mid regardless of the initial stability of that plus mid. In the population. Right, so to summarize evolution of plus mid stability can lead to the persistence of neutral plus mids in the population or plus mids in the population under neutral conditions. We find that there's a direct link between plus mid fitness and the plus mid genetics, just like we know for living organisms. Yes, yet unstable placements can be maintained in the population if they are essential for the host. And our research suggests that natural selection can operate at two different levels on the host population to maintain the plus mid or on the plus mid to evolve stability. Overall, we can now say that principles that apply for the evolution of cellular entities may be applied to understand the evolution of plus mids as well. Right, with that I'm done for today I acknowledge here the funding agencies and the pictures of the group that I didn't show on the way with the leading authors. Right, I thank you very much for your attention and I'm happy to take your questions. Questions for Paul. I've got a question. Oh, Stephanie, you can go first. Okay, you hear me. Yeah. Yes. I had a question about the first part of your talk so when you showed the competition between placements within cells. In the majority of the cases it's the stable glass, mid that worn the competition, but you still had some cases where it was the other one or you had coexistence and it was actually replicates. So really identical conditions initially, as much as could control them so you have. I mean if we do competitions between two genotype of cells, usually the winner of the competition is always the same. ¿Crees que es debido a la procesación de la procesación de la plasmid o cómo te explica esta solución? Sí, sí, sí, así que pensamos que hay un montón de procesos de plasmid aquí, así que lo que tenemos en la publicación también es el test estratégico de eso, así que cuando el plasmid venga, lo venga significativamente. Por lo menos, como en el año, por ejemplo, tenemos algunos casos en los que el plasmid venga. Y esto puede ser, por ejemplo, si no sabemos exactamente por qué, podríamos ir y verlo en detalles, pero, por ejemplo, para empezar con eso, el plasmid fue, de alguna manera, logrado tener el número de copies más alto en el principio. Esto podría tener afecto. Pero esto no puedo decir, sé que es más estochastico. Los casos que nos notamos aquí en la existencia, fuimos adelante y chequearon qué son ellos. Y esos fueron, en realidad, plasmid fusiones. Entonces, resulta que, porque los plasmid fueron muy similares en el backbone, se confundan y producen no solo dimensas, sino también timers y más. Y, por lo menos, esos multimers pueden segregar monomers. Entonces, una de las conclusiones que tuvimos en esa publicación era que una de las fusiones plasmid puede mantener una variante de plasmid incendios en la población, en un tiempo bastante largo, especialmente si tendrían condiciones que seleccionan para la presencia de los dos plasmid. Gracias. Sí, hay un par de preguntas en el chat. Así que, solo responde. Entonces, el inicio dice que la proporción de la plasmid, el T0, cuando empezamos el experimento, es siempre 100%. Porque los plasmid que usamos aquí en el centro tienen la encoda para una resistencia antibotícola. Gena, y podemos seleccionar para todos los hostes que tienen ellos en el principio antes de retirar la selección. Gracias. Sí, y luego la pregunta sobre dónde los plasmid y el plasmid son los fitos, que es nuestra conclusión sobre la fórmula más alta de la reproducción de la plasmid incendios es una estatística. Así que tenemos aquí 36 replicaciones. Y tenemos, sí, usamos aquí un test binomial para preguntar a cuál plasmid ganó significativamente más que lo expectó por la oportunidad, por ejemplo, si hubieras dicho que fuera de 1 a 1. Sí, Jamie, tienes la pregunta. Gracias, El. Así que te ha mostrado muy bien que en una competición directa entre la esencialidad y la estabilidad, la esencialidad ganó. Pero esto es solo mirar la parte de la selección de la evolución, ¿no? Y no estoy pensando que un plasmid es esencial más probable evolver bajo un backgrounds estable porque es solo un gran tamaño de populación. No estoy segura sobre el tamaño de populación. Desde este experimento, nuestro take es que un plasmid es esencial es menos probable evolver en un background estable, un background estable de un plasmid, porque es muy fácil llevarse a ese efecto, ¿no? Y así que, en realidad, la presencia o la duplicación de un gen esencial en un plasmid esencial podría crear, digamos, un conflicto entre el plasmid y el plasmid. Así que lo que piensamos es que puede encontrar un plasmid esencial con un plasmid gen y posiblemente un replacimiento o que debería divergir un poco. En realidad, para este caso, sí, solo hemos encontrado esta publicación hoy, pero es también muy fresco de tres semanas atrás. Ellos predictan o inferen que la origen de este trasposable elemento es de Arcineto Bacta. Y, interesante enough, hemos encontrado un par de meses de un papel desde el último año en el bio, demostrando la compatibilidad entre el agoriel, el chaperón, entre el Arcineto Bacta y el Arcineto Bacta. Así que es un tipo de fitz, pero eso significa que esto es realmente un evento, este chaperón en un plasmid, en el Arcineto Bacta es realmente un transferimiento gen y no una duplicación de la goma goma. Así que sí, pensarías como en la goma que tenemos aquí, es un ejemplo muy inusual, tiene que ser para ir a varios pasos si se encuentra en un esencial o, digamos, un homologo de un esencial gen en el plasmid, ni siquiera no fue esencial antes de que el plasmid llegue, en la esencialidad de después, ni siquiera hay varios pasos de replazamiento de la goma goma. Gracias. Sí, Sonia. Sí, gracias mucho por este really interesting talk. I was wondering if you could say a little more about the concept of essentiality here. So, because I guess in some ways you could say that any gene that is selected for is in a sense essential because you'd expect the strains with that gene to compete the other ones. So do you expect the results that you present to hold for any gene under selection or for genes that are under very strong selection? Yes. So first of all on the data that we have here, it's all coming from experiments usually of knockouts. At the most new addition to this is a very good publication from Francois Rousseau et al in Nature Communications, Nature Microbiology, two years ago. They compare the essentiality of genes in different Asherisha strains in three different media conditions and indeed they show that essentiality, real essentiality, depends also on the conditions which is of course expected, right? And many of those were tested in maybe two media types, like the very rich media and relatively poor media. So, yes, I think, right? If we talk about selection, coefficient or pressure, then this is probably the highest one we can have, right? Maybe you will go into that into your talk. Yes. So I guess that if you would live all of your lives in antibiotics, then antibiotic and this gene will become essential. Yeah. Maybe you can explain more on that. Excellent. Liam. Thanks, Tal. That's really interesting. I just wanted to say on that because I'm on that ACVAN 2022 paper. So, yeah, very much the focus there is on the antibiotic resistance gene, which is this NDNB dilactamase. So it's really interesting that there, I don't think we even clocked that we wrote, grow airs, grow out. Hadn't clocked that it was a chaperonin like and as you explain that it's this essential thing. So that's really interesting pairing of how success of a plasmid can be linked to those things. Thank you. Yeah. Very nice paper. Yeah. Gates. Okay. If there are no more questions, we can go to the next speaker. We, Sonia Letinen, will talk about why do some bacterial genes reside on the chromosome and others on plasmids.