 So you see my screen now, or not? Yes, I can see it. But we can see your browser as well as your... Yeah, we are seeing all your computers. That's better. Right. Then, okay, so I'm going to, as Sam said on Monday, my mission today is to give a one-on-one course on Plasmid Biology. And which is, I had to select a few things to talk about, and I decided to talk about the conjugation and things like that. Plasmids and ICs, then replication incompatibility. So that means, more or less, some of the basic aspects of Plasmid Biology. So why doesn't it work? I don't know what happens. My screen is not... I don't know why. Anyway, so the first thing to know about what's the importance of Plasmids and so on is that, well, because antibiotic resistance is mostly carried by Plasmids. So it is already known that a reduction of antibiotic consumption will not be sufficient to control resistance because of the spread of resistance strains and resistance genes. And that's what my work, my group is working on. So probably the most famous Plasmid in all ages has been the Plasmid of Yersinia pestis, which carries the system for the control of the type III secretion system, which is essential for virulence of Yersinia pestis and therefore the plague, that isolated Europe for many centuries in the medieval ages and before and after. So what is interesting is that the Plasmid in Yersinia pestis is virulent because it increases the copy number when it's in the right environment and then it becomes virulent. So it's only because the Plasmid exists and increases the copy number that the Plasmid is virulent because carrying the Plasmid has a high metabolic cost to the host. And this idea of Plasmids being important for the spread of adaptive genes like antibiotic resistance or virulence and so on. And the cost of having the Plasmid is what drives most of many of the discussions in Plasmid biology. So what are the pros and cons of having a Plasmid or a given Plasmid? So I will talk first about Plasmid propagation. That is how Plasmids move from one string to another. So the first conceptual slide on what is the genetic structure of a Plasmid, of a conjugative Plasmid. A conjugative Plasmid has like for instance, R388, which is the Plasmid we work with, about half of its genome is 34 kb, of which half of it, about 15 genes, are dedicated to transfer to the conjugative machinery. Then replication, the region required for the Plasmid to replicate is just 1.5 kb. And that's the same for most Plasmids. Replication only requires a small segment of a Plasmid, I mean 1 kb or a little bit. And then there are a group of genes here in blue, which are involved in maintenance. And that means they are required for protection against other Plasmids for inducing when it's transferred to the recipient to accommodate to the new recipient and so on. And there are many functions which are starting to be known functionally, many genes which are required for these kind of things. So and then there is the adaptation module, which contains the genes which are not related to Plasmid physiology, if you want to call it like that, but they are completely external and they are the adaptation module, so that this module is involved in, for instance, in antibiotic resistance, like in R388, there is an integrand here, which contains several antibiotic resistances. So Plasmids are in fact natural devices for gene propagation. Establishment in new hosts and heterologous expression of genes, so they have to be prepared to do this. So the first thing I'm going to talk about is how we measure conjugation, how we measure the conjugation frequency, which is not the conjugation rate. A rate has a different formula than a frequency. A frequency is basically when you have cells with a Plasmid and cells without a Plasmid, which are called recipients, those are called donors, and then when you mix them, some Plasmids travel from the donor to the recipient, and in the recipient they are called transconjugants. So if you measure on a plate, let's say the number of T of transconjugants versus donors or recipients, you get the conjugation frequency. So you have that many transconjugants per donor or per recipient, and that's the way frequencies are measured. Rates are a bit more complicated, but there will be talks on this workshop specific about them, so I don't need to emphasize that anymore. So besides plating, we can do a high throughput conjugation assay, for instance, by using fluorescent protein like here. So you have donors which contain a green fluorescent protein under the control of a T7 promoter, a T7 promoter, which is not here, so therefore this gene here is not expressed at all. When you have a recipient like E. coli BL21, which contains the T7 polymerase in the chromosome, when the Plasmid goes to the recipient, you see light, and the light you see will be proportional to the conjugation frequency. That's why we can measure many, many conjugation assays at the same time by using this. So we do this in 96-well micro tighter plates, and we can see, for instance, assay like in this paper, many compounds to see if they are or they were inhibitors of conjugation. And we have done this also. We have analyzed conjugation, or you can analyze conjugation by calculating conjugation frequencies in natural environments, like here, when you can, you see that you have a fish here, you feed the fish with food containing donor and recipient bacteria, and you analyze the conjugation frequency here or here if there's no fish. And what you find is that in general, when there are fish around, the conjugation frequency is much higher for different Plasmids. We tested these two and several others, and the conjugation frequency goes up like one to two locks when there is a fish. That meaning that a conjugation within the gut of the fish makes more efficient conjugation than if it's dispersed here in the liquid of the fish bowl. And we then can test the inhibitors we tested before to see if they work in this by diminishing the conjugation frequency. And we find here that when you have the water ecosystem, which is this one, the conjugation frequency is reduced when you have the inhibitor. And the same happens in another experiment in which we used mice, and we did the experiments of conjugation in mice. So what this means is that we can measure conjugation frequencies by several methods, and this will give us some conjugation frequencies that are under different conditions. So with this, I pass to the next idea and is how is the expression of the genes in a Plasmid. So here we have Plasmid R388, and it contains the 15 promoters. So we took each promoter here and we cloned in the same expression vector to check what was the strength of the promoter. And you see here in black for each promoter, in black is the let's say the constitutive expression of this promoter. But then when you add R388 in trans to the same, so you have the clone Plasmid containing the promoter and some reporter gene. And then you add Plasmid R388 on top on that cell. And you see that in all cases, if you start here, one, two, three, you see all of them are completely reduced. So that means they are repressed. So instead of having activators and repressors, Plasmids usually have always repressors, at least R388 here. The only gene which is expressed is from this promoter, and this promoter, not casually, is the antibiotic resistance genes. So that means we have a Plasmid, which is a 34KB, which has like 35 or so genes, and it's only expressing one, which is the antibiotic resistance, because the others are shut down all the time. All the time when the Plasmid is growing with the bacteria. But what happens during... So, well, that's the same idea here. But this is just to show that we have identified what is the repressor for each of these promoters. It's not important in this discussion. So here, this is a slide. You don't need to understand, but you just need to listen to what I'm saying. When a Plasmid, which has only repressors, shutting down the expression of all the promoters, goes to a recipient cell, something happens, because when the DNA is there at the beginning, there are no repressors. So then there is what is called an overshoot of expression. So everything is expressed, but when the repressors build up, then expression is already down. So people that study conjugation frequencies or conjugation rates and so on, so they should realize conjugation is usually at low level. But when a donor goes into a recipient, there is a transient state which lasts a few generations and all that is explained in this paper. So there are a few generations in which the expression of all the machinery for conjugation is high. So the Plasmid is what is called transiently did repressed. And so it goes, so when one Plasmid conjugates, a number of Plasmids conjugates around there, and then everything is shut down again. And that should be for modelers and so on, it should be taken into account. Okay, so how does conjugation happen? We need to realize or to think of three important protein or protein complexes. One is the relaxase protein, which is the protein that cleaves the DNA at the origin of transfer, and it is going to start the function, right, to start the action. Then we have the coupling protein, which is the green protein here, which is bound to the channel, which is the blue thing, the blue cylinder, which is the channel by which the DNA will pass through the recipient. So the mechanism by which conjugation occurs is like this. First, the relaxase cleaves the donor DNA, and well, donor and recipient come together because they are put together by the conjugation channel. So here, the relaxase is cleaving the DNA on the donor. And then one of the copies, one of the strands of the DNA binds the coupling protein, which is an ATPase, and it starts pumping the well, the type 4 secretion system, the channel, is in fact a protein export channel. So it exports, or it moves. Sorry, I have a question about the previous slides. The previous. Yes, yes. The question is, you said before that maybe 40% of the genes were functionally due to maintenance and some of it to replication of the plasmid itself. So isn't it strange that they're all shut down outside of conjugation, that only the antibiotic resistance gene was active? Yes, only the, so when you have a plasmid and under vegetative growth, everything is repressed. Repressed doesn't mean there is no protein at all. Repressed means is 100 times less protein than when the promoter is activated, basically. Okay, so these are just low expression, all the maintenance and replication genes. Are expressed to low level. Yes, okay. So when conjugation happens, when this DNA enters the recipient, what happens is that here, the repressors, for a time, they are not there because the repressors were expressed here and only the DNA passes to the recipient. So therefore here, everything will be expressed in a burst for a few generations, maybe two to four generations, which is what takes for the, it depends on the growth rate, for the repressors to gain the same concentration that they had in the donor cell. Okay. Yes, thanks. Okay, so I continue. So we are here, then the relaxase links to the coupling protein and is exported by the typhus regression system like this, right? So you see the DNA is entering the recipient, five prime to three prime, while here, this strand is replicated by the polymerase complex, DNA polymerase complex. So the plasmid in the donor is reinstalled. And in the recipient, the relaxase, when it's there, is able to find again the three prime end on the entering strand, DNA strand, and it cleaves and is a DNA strand transfer reaction, which makes this DNA to circle again. This is known in very detail, but I only have this cartoon here to show it. So we have this strand of the plasmid here, and then the replication machinery of the recipient cell is able to replicate the lagging strand here, right? So okay, that's the way it happens. So we have here at the end of conjugation, you have two copies of the DNA, one in the donor, one in the recipient. So the plasmid is again intact in the donor and the other goes to the recipient. So the elements of bacterial conjugation are then the most important ones are the components of the channel, which are like 12 to 15 or to 20 proteins, depending on the specific plasmid, the coupling protein, the relaxase and the relaxase, okay? But all plasmids, which are transmissible by conjugation, must have a relaxase because the relaxase is the only protein that can cleave the ority in the donor DNA to bring about the conjugation. But several many plasmids instead of having to code for everything in the transfer channel, they code only for the relaxase and maybe the coupling protein. And these are called mobilizable plasmids because they need a helper conjugative plasmid to conjugate, right? So we have the key proteins are the relaxase, which is present in all transmissible plasmids and the typhus efficient system, which is present only in the conjugative plasmids, right? So when if we use the relaxase as a proxy to a transmissible plasmid and very before, let's say it's one of the largest ATPAs in the typhus efficient system as the proxy for the transfer region, we know that we can we can decide that transmissible plasmids in general will be conjugative if they contain the relaxase and the vir before and they will be only mobilizable if they contain a relaxase but not a vir before, right? So when we check the database like here, it was REFSEC, I don't know, 81 that contain at the time a 1700 plasmids and we draw the size the sizes of the plasmids. What we find is not let's say a normal distribution but by modal shape like this. And that is and when we check then what plasmids have are conjugative, mobilizable or not transmissible no more. We find that conjugative plasmids are in general like 100 kb with a broad distribution here. Mobilizable plasmids are in general very much smaller like a five to 10 kb and then there are also very, very large plasmids which are maybe also already a secondary chromosomes or something like this and then there are these non transmissible plasmids that means 50% of the plasmids in the databases are do not contain relaxases. In some cases a few is because they lost them, in others is because they rely on other methods for propagation but that was with 1700 plasmids but now we have 20,000 plasmids here in REFSEC 200 and the by modal distribution still holds like this. So that's interesting. So why are there two basic plasmid lioids? Why some plasmids are mobilizable and some plasmids are conjugative, these large ones. And the main reason for this is that being conjugative is very costly as I explained before. So this you have this DNA and this DNA when you express it it costs a lot. So some plasmids decide it's not important to have already all these if we have the relaxase and can jump on a conjugative plasmid to allow us for transfer. So many mobilizable plasmids which is 50% of the transmissible plasmids do not have this burden. So when you have this small layout a plasmid these plasmids which are usually less than 10 kb you have only four kb or so dedicated to the mob region and then the rest can be cargo. You don't need anything else because the replication is again well this is a bit complicated replication of rsf1010 but in general is one protein or none but it's a fragment of 0.5 kb or one kb. So when you're small you can have high copy number and small plasmids like coli1, rsf1010 and so on are in the order of 20 copies per set roughly, very roughly. While if you're large like r388 your copy number has to get down a lot and the copy number of large plasmids is usually about one per chromosome. It could be a little bit more but in general is one. Now that we have the sequences of many, many, many genomes of bacteria you can compare the copy number of plasmids and chromosomes and it's more or less the same for most of the large plasmids all the time and so if you're low copy number you can have if you're low small size you can have high copy number and then you don't require systems of partition but if you're high copy number you require a large size you have low copy number and then you require a system to partition between the two daughter cells because if not the plasmid will be unstable and will be lost and now some detail about what's the difference between plasmid and ICs. ICs stands for integrative and conjugative elements and integrative and conjugative elements which were thought at the beginning at least by me to be a rarity. In fact there are we have shown that there are twice as many ICs than plasmids in sequence genomes. There are a lot and in the branches of the phylogeny they are undistinguishable so that means that in in some branch there can be some plasmids and some ICs. That doesn't mean that they exchange very very frequently but under evolutionary terms it can happen. So when you have a plasmid or a plasmid in a permissive host it can always integrate in the chromosome always not but any portable region of homology makes this plasmid able to integrate in the chromosome and therefore you have a nice here and the mechanisms of integration is something that we can discuss later but when it's integrated in the chromosome it can go to hosts which are non permissive for a plasmid because for instance it doesn't contain the replication region which is active here. So a plasmid can always go to a non permissive host by integration into the chromosome so I think the interplay between plasmids and ICs is because of that because they allow the colonization of non permissive hosts and here they can evolve to be able again to acquire a replication region which is as I said a 1kb region to be able to replicate here or mutate their own replication system. Okay so very quickly there are several ways in which plasmids replicate. You have rolling circle here I will not explain rolling circle you have the rep dependent replication which is a ceta replication and in which a rep protein interacts with the origin and opens the origin so that the DNA polymerases can land there. Then there is a strand replacement which is what RSF1010 does and then there's this RNA primer replication which is used by Koli1. Koli1 is a member of the prototype of a family of plasmids which is the family the mob p5 family for us and it doesn't require a protein for replication it just uses a primer RNA which is called RNA2 which binds to the origin and it helps opening the origin so that the DNA polymerase 1 can start replication here. So Koli1 doesn't require a protein for replication that's important but it's narrow host range Koli1. So because of replication plasmids can be incompatible two plasmids are incompatible they is said when they cannot reside in the same cell why not because if they have the same organization of the replication machinery they the cell cannot let's say cannot distinguish between one another and then they cannot assort independently and therefore plasmids when they cannot be in fact for the cell or for the replication protein are the same plasmid there is random selection of the DNA molecules for replication and for and in partition and in cell division and therefore the plasmid becomes unstable and it disappears one or the other right so then these two plasmids are said to be incompatible. About a partition I will only say that it's always and there's always an ATPase and another protein which binds to the partition side in most cases it's like that and there are different families used by different plasmids but in general you can say that the mechanism is similar and there are mechanisms by which the two the copies of the plasmids are moved to the let's say the balls of the cells or separated between them so that they can then be stable inherited even if you have only two copies because one will go to one side one will go to the other and the system is about two three kb which is required to do that. Okay and the last section I'm going to talk about is about plasmid costs as I said having a plasmid is costly for the cell so there has to be reasons for the plasmid to stay there and there will be all kinds of interactions between plasmid chromosome plasmid and plasmid and so on and the different plasmids that's the important thing about this slide is that the everything which is involved here in the excision or insertion of plasmids in chromosome interactions between plasmids entrance or persistence of the plasmid in one strain will be more or less different and specific of each plasmid or ptu as I called right and it's important to to know this because there will be differences very important differences between different plasmids you can find something which happens for one plasmid and doesn't happen for another for an example that comes to mind that came to mind when I was preparing this was the example of liquid versus surface maters some conjugation systems allow the plasmid to be transferred when the cells are in liquid and these are called liquid maters the example is plasmid f or r1 r100 and so on which is very good because you can transfer in the middle of growth while most plasmids are only surface maters that means that they need to be together on a surface of a biofilm or a plate or whatever an agar agar plate so in close contact so that they can transfer the DNA probably because the links the breaches between donors and recipients are brittle so liquid mating is an important invention and it was invented at least twice one is in the mob f class and another one in particularly the mob the mob f12 family which is the family of f many ptu's in the enterobacteriaceae and in the eye plasmids like eye one eye eye one and so on and other ptu's around that and these two can transfer in liquid the others have to transfer on surface and and the mob age as well have it has invented this liquid conjugation and so that is a radically different ecology of these plasmids with respect to others so the success i think of the f like plasmids in enterobacteriaceae is because of liquid mating and that's something that maybe in the talks after that will be stressed and as will also be spoken later on there are many mechanisms affecting plasmid fitness that i have no time to talk about here but you all know about crispers about restriction modification systems and so on that impede some plasmids to be transferred to recipients for reasons when we took in one experiment i think it was like 200 clinical isolates of enterobacteria and we transfer plasmid f388 compared to the lab recipient dh5 alpha 80 percent of them transfer to frequent at frequencies 10 times lower or much lower than that in 80 percent of the cases that means there are many barriers in recipients to the conjugation of plasmids and we can discuss that later so these these are some of the barriers like restriction crisper cast bacteriocins exclusion systems and fertility inhibition and i will only put an example on this fertility inhibition is when a plasmid interferes in the transfer of a co-resident plasmid for instance a phib A is a protein produced by incand plasmids that makes in p-plasmids non-transferable and so on and you can see here how many each plasmid has many systems to avoid competition with other plasmids and that's only fertility inhibition i told you there are many barriers one of them is fertility inhibition and you have an idea of the complexity of the interactions and the plasmid wars because of this okay so i think i i wanted to finish now because my time is out just to acknowledge the people in my lab and that's all i wanted to say for the moment. Are you still there or have you all gone? We're here that was fantastic. I quite like to watch it all again. I don't know if i maybe i wanted to cover too much of the field but i wanted to give you an idea that a plasmid biology has some complexities so a plasmid is not just a circle that you can put here okay anyway that's it well that's great thank you very much are there any questions i don't have the chat here yes gikwin one has the hand rise right if you want to say something okay good yes i have a question thank you very much first my question is about during the conjugation once the single strand DNA from the donor cell entered the recipient cell so there's some genes which are required for establishing the new incoming DNA in the recipient cell like single strand DNA binding protein and the protein psia psab for the sos reaction in the recipient cell so my question is are there other unknown function protein maybe or the mechanism is already very clear is there any protein we need to explore more in the maintenance region of r388 which is one of the smallest conjugative plasmids there are 12 proteins one is an ssb and there are others which have apparently unknown functions and they are not required for for transfer between nicolei and nicolei in the lab but maybe they are required in some other for instance some of the anti sos proteins that you talk about that are for instance in the and that's very important in the i ptoi one and several and similar plasmids they are involved in transmission to recipients which are of different gene general that was already known by Brian published by Brian Wilkins many years ago so you know these proteins won't be essential for transfer between nicoleis but they will be essential for transfer and there are some anti restriction systems in nicolei or in in several plasmids and and i'm sure there are many to discover many functions but they won't appear essential in every case you know it will be maybe for transfer here or there for instance we know rp4 and other plasmids like r388 have very few restriction sites for their size so you would expect a number of restriction sites and there are no so that means that they are able to conjugate to strains having type 2 restriction systems because they won't be affected and in fact is known that they transfer better to strains that contain those restriction systems and and in general there there you can bet there will be many functions like this but the thing is that we don't know in what cases these proteins will act so it's worth pursuing this system problem is you will find one with a lot of effort while bioinformaticians are all all the time having results without doing anything so functional genomics with functional plasmid genomics is now a a field of very much sacrifice so i abandoned it sometimes well i do something but not me people working with me but uh i like bioinformatics now more easy thank you very much okay i don't see okay there i mean cony i have okay yes so i enjoyed your talk very much fernando and i really liked that in the last part of your talk you addressed this aspect that some plasmids transfer much easier on surfaces others more in liquid and actually in 1996 we have published a paper from a study where we introduced e coli carrying either in w in n in p1 in f or ii into a soil microcosm together with a recipient and interestingly and the recipient was in e coli and we followed the transfer and it was very interesting because the conditions were actually the same the donor background was the same the recipient was the same and we did not see any transfer for ink f and ink i really uh supporting what you said and we saw very high frequencies for instance for ink p1 or in w or ink n and at that time and this is what i wanted to add we speculated a little bit that the pillow shape might be responsible for this ecologically interesting aspect because the long flexible p like ink f they are more efficient in liquid than these short rigid ones and i'm not sure and this is my question now to you is there any new data on this because at that time we felt this is quite intriguing because it helps us to predict in which environments which type of plasmids are more important but the plasmids you used were a wild type plasmids no okay for instance your plasmids in n so we we used 3d plasmids pkm 101 or a 3d pa are let's say natural plasmids but for instance a r1 r101 which is what people use for f is at the repressed plasmid of r100 so if you use r100 r100 is heavily repressed for transfer why and r64 is the same so in the lab we always use r100-1 which is at the repressed version of r100 or r64 drd 11 for instance those are the ones which transfer to high frequencies in in the because in and to if you used i don't remember that paper if you use wild type plasmids or no did you use r100 the repressed version of and no we received this f plasmid and in guy plasmid from varniga rhoda from helmut schepa at the time and so i i would have had to look it up as well but because one possibility is that is that those plasmids were repressed and therefore you need a high concentration of donors and recipients yeah to bring about this this transient the repression but we had a high density because we introduced e coli and yes but we did it exactly the same way for the different plasmid types and at least at that time i thought this is quite cool that eventually this plasmid pilus type or shape is influencing the efficiency to transfer so but uh you're right maybe also repression might be play a role but i i will send you the paper because or or yeah yeah sure but uh well i i probably read it a long time ago and and it was fine but what you say and that's very interesting maybe you know the pilus or the pilot of the f and i they stick to something else and then they are not good for this transferring soil you see because in soil you have many interactions absorption to things to the material of the of the soil and so on yeah so but of course we worked in the rhizosphere so it's a particular soil which is metabolically active because of the root exudates yeah so but um but that would be interesting to see you know what what are the the effects of a soil on the transfer efficiency of different plasmid types yeah as far as i know nobody has done that but i don't read that much yeah okay you're like bioinformatics no more another point on if i what i really think uh fitness i know there are more talks to come but i i always think that fitness costs of plasmids for hosts that live for instance in the environment are not that high as typically in an albiboroth where you have rapid growth because generation times in soils or in the rhizosphere they are about one to two days or even more so i do not believe that fitness cost is that type of an issue i mean for bloodstream infection it is but not in the environment where you have less growth so that's thank you yeah the plasmids i mean like f and uh are 64 or i1 plasmids they are residents of enterobacteriaceae that grow in the gut grow quickly well relatively but we find them also on the leaves as i said yesterday okay yes yes there i i do not believe that you have rapid growth like in an albiboroth yeah sure you're right okay okay let us have this Marco consentino has a question as well uh hi thanks so i had the curiosity basically you showed us that there's a lot of interactions towards the end of your talk between plasmids that i could define as ecological but and but um i don't know if you agree so that's i guess the first question um but they are they seem to be mostly negative so is there uh like are there examples of more mutualistic interactions like um pairs of plasmids for example that are codependent and they are only found uh together because they um complete each other gene in terms of functions or other examples of mutualism between plasmids well for instance mobilization in itself is a kind of a positive interaction right uh an extreme case of positive interaction uh i think the way in which these experiments because this is a summary this was that cartoon was a summary of many many papers but in general people have looked at when a plasmid a co-resident plasmid is there is affecting transfer in a negative way because in a positive way it was difficult to see at the beginning now okay i don't know now of any instance of a positive interaction but there was a paper by gamma et al uh in which they consider positive interactions let me see if i find the paper here so but so you don't exclude that they could exist but it's just the field you know in biology i am sure everything exists all the possibilities will be uh used by some guy or another because you know you see a but the point is what why you would like to help others to be transferred what's the logic behind that um no but maybe you you basically maybe it's not about transfer but it's more about presence like you want to be both present in the same species because then you have you get a fitness increase if the plasmid is also present because that they you know you can carry out functions that would not be possible alone for both of them that that i'm sure it happens because i remember there is a paper i have to remember if it's alvaro or somebody else that they see that a calling one like plasmids another plasmids there is a positive correlation so strings containing a large plasmid are more are more likely to contain a calling one that strings that do not contain this large plasmid i don't know if alvaro is here and can answer this question because i think it was one paper of his it's in ismi journal 2014 yes from alvaro yeah okay good you see it's important to have this a background information so this for for cooperation i'm sure there are many interactions like this which are positive interactions okay thanks and i remember that one but i don't think in that paper or even later they really knew the reason why this happened they didn't right no francisco d'unizio also published right with gamma also on yes d'unizio yes yeah yeah there they they looked at many many plasmid plasmid interactions and it was a very nice paper it was published in plasmid i think in the journal but it contains very interesting data thank you okay any other question then we have a break now or no no break alice what do you think hi guys i don't know if you were asking for me all right oh yes i kind of heard okay so you know the question or not i didn't i didn't get the question okay so the thing is marco consentino asked if are there instances in which there are positive interactions between plasmids not only because i talked about fertility inhibition and so on and they were all negative interaction and i remember one paper of yours right yeah yeah we look at that yeah so there's also a paper from isabel gordo where they found what their paper and our paper we both look at fitness effects of plasmids and and looking into possible epistasis between the fitness effects of of different coexistent plasmids and what we found we found instances of positive epistasis between plasmids in cell the monoceros genosa meaning that carrying to plasmid cost less than you would expect by their independent cost in isolation in the same in the same strain and the isabel they kind of found similar things they found both positive and negative epistasis and what we did we look into the databases to like genomic into the genomes in databases because we kind of predicted that if positive epistasis is common then we would find plasmid coexistent being more common than you would expect yes due to chance and we found that obviously there are other reasons for for that to happen but but but we found that yep and it's an ismy paper i think i think the link is in the in the chat now somebody put the link there i guess and isabel's paper was a plus genetics paper but you don't know the mechanism no for the call no one positive thing in this case we had we had we didn't we didn't uh we don't know at all no thanks okay and more anything else so then we what do we do at least