Fantastic like a seminar speaker Dr Denis Brogan He's at the Biological Sciences University of Cincinnati been there since ninety four and it's been a lot of interesting work in different systems but primarily I'm Marcie all systems. It's really have an issue that he's working on it's really interesting that I think you might heard before. In some other you're saying your class is a major problem yet you'll be OK so I will give you that people today will be right. Thanks Patty. So I really appreciate the chance get out and come to Georgia Tech. I had never been here before and I've enjoyed working around campus and I look forward to talking to several people this afternoon. So I don't microbiologist and. All Read is relatively diverse The problem is that diversity tends to be cryptic. Microorganisms don't look very different from each other under the microscope. But when examined for molecular characters in other words some kind of a sequence is used to compare relatedness of all organisms which can be done for example with a small subunit ribozyme or in a you're able to compare all cellular organisms with each other find out there were laid in this and the picture that emerges is this picture here which I know you've seen many times in which also real life can be related. And so we do think that there is some kind of universal common ancestor by those by this measure brought that it's discontinuous a clump. Into the three large clade. So here are all the bacteria that are forming a nice coherent clade here all you carry out forming a nice coherent clade and that all makes sense in fact you can even say that. OK The fact that all animals are reduced to this little group here is maybe even Also logical because animals or you know that that's a relatively recent group and same for all plants and funds are a little bit deeper and so we can sort of understand that the most diverse and ancient lineages are the unicellular organisms in this clade here for example or over here. But what maybe a little bit hard to rationalize this why there should be a third clade that is also all single celled microorganisms in that group. We now call the R K. And so they are K. I represent a third domain of life attacks on that's higher the kingdom. And. This raises a lot of interesting biological questions. The sort of maybe just the what you might call the purely followed genetic or picture based on this sequence is also reinforced by physiological ecological observations and other words this group of organisms seems to be very diverse and you would expect that there should be they should have it diverse habitats and have diverse properties and we do see evidence of that some of these organisms here are found in extreme environments like. The vent fluids and associated chimney structures from hydrothermal vents at the floor of the ocean where they are surviving it and actually require temperatures of one hundred degrees C. or higher others of the art. I grow only in hyper salient environments and so you know this this is also consistent with the apparent divergence that you see here and they are in a DATA. We also have to keep in mind that not all are K.-A require extreme conditions there are plenty of them that are found in the soil or and ruminants and so on. It just happens that historically the R.K. of the grow in these more but non or conventional environments don't grow very well under way. Average or conditions most of them are uncultured and only finding out about them through non culturing techniques. Now I'm a moral experimentalist and so of the R.K. I do like the ones that grow in the lab and so I've been specializing on thermo sort of files this. So this was my one of my favorite study sites in Yellowstone National Park. This is what it looks like if you go along the boardwalk you go through this. Thermal field around the edges of it. What I find to be the best source of the organism however off the boardwalk in places where. The. The hydrothermal system the plumbing is sort of unstable and changing moves into areas of forest and you get this corresponding death and destruction but it's a good habitat for these organisms. So I will take samples of water and sediment from one of these hot springs the PH is usually three point five to two point five temperature can be anywhere from eighty five down to maybe seventy degrees and from those conditions we dilute put on special medium that doesn't melt at high temperature and find sometimes dozens or sometimes thousands of colonies. The the organisms in these colonies of these irregular shape cells with the funny kind of cell. On a follow up and so this is these are all in the genus so FLOTUS they grow optimally at about eighty degrees and Ph three and. You know that raises the obvious question those conditions are lethal to organisms that we understand very well they're actually harmful to the cell components of D.N.A. They are now the proteins of most cells they tend to denature very quickly. So we like to know how a cell functions under those conditions since it's obviously this this genus is obviously adapted to those and we find a lot of unusual cellular features in these organisms. So if you notice here in this thin section the margin of the cell has kind of a funny appearance. It seems to be sort of two layers with a white zone and between that outer layer turns out to be a kind of Sakya was formed by glycoprotein subunits. When you look at the no in the normal direction. It actually has a very regular periodic pattern sort of hexagonal symmetry and. So this is obviously a very unusual property you certainly don't find this kind of situation of bacteria but nice that membrane is a lip and by sort of not a by layer but a lip and layer that is composed not a fossil the with fatty acids S. terrified to the glycerol but instead forty carbon I suppose noid. Hydrocarbons. Linked via ether linkages to glycerol of the glycerol is are also in the opposite in answer marriage configuration from fossil upwards. So those are very unusual membranes the enzymes of these organisms are all intrinsically storm a stable and they don't really have they're not really catalytically active at you know room temperature or body temperature they don't really start working till you get to sixty or seventy degrees. The organisms have a circular chromosome which. Reminds us of bacteria but unlike bacteria in this genus there are three origins of replication. We think that the replication is carried out by C.D.C. six homologues In other words there's no D.N.A. a protein encoded by the genomes. There's no F.P.S. Z. protein so cell division. Also must operate in a very different way from bacterial cell division. So this whole list of unusual. So there are features and as I look at these organisms I raise the question like this. Namely with all these other sort of features are very different have these organisms also evolved from one usual genetics and so that's the kind of theme. To the research in my lab that's one way to sort of describe what we're interested in and just to give you a feeling for the sort of things we have to do in order to get to the point of asking questions is there was really no genetics available for these organisms and it all started on my postdoc in eighty nine where I actually started playing them for the first time. And so it's been a pretty slow laborious process to be able to ask genetic questions but the pace is been pretty steady in every couple of years we have either in my lab or someone else's lab we make a little bit of progress and so it's getting more interesting as time goes on. Just to give you an idea of what's been going on in my lab. What kind of things we have done with this we have first of in the early years we look at the accuracy of D.N.A. replication. And so flow was a sort of cold areas and it's a remarkably high. So despite the fact that you would predict that the D.N.A. should be decomposing at rates of at least a thousand times faster than D.N.A. decompose in a call. Y. Nevertheless the mutation rate is actually even below. Probably the spontaneous. Right in the cause. So they are very good at replicating their D.N.A. and keeping it repaired. That's led us to look at the some work situation other extreme of files extreme hail of files for example also some bacteria that grow at high temperature. So that's the work done by Mach one and collaborators at the I.H.S.. We examined transposable genetic elements in another species so full of us this land because this one. We have samples from all over the northern hemisphere Salissa loused to look at the at the genetic diversity and and what's out there. We've examined the genetic structure of specific populations this was collaboration with a couple of people and and the picture there is that these sorts of Lobos populations. Seem to really have true biogeography there always do seem to have. Genetic differences that correspond to different locations in the world is consistent with the idea that it's hard for these organisms to disperse and so you have a certain amount of and Demick genetic character to two specific populations. And in general what we're doing now focuses a lot around this theme of D.N.A. repair. I can I don't want to get into it because of time but D.N.A. repair is quite mysterious in these organisms and very intriguing for us. I don't know if anybody in the audience recognizes that name but that's a that's a Georgia Tech graduate who came to my lab did his master's degree. He worked here with Patty as an undergrad came to my lab did a master's degree in Zachary then has and I want to give you a progress report on Zachary so here from our lab he went further north. Still up to. Michigan State use Lansing. And he may not be publishing tons of papers of the ones he's putting out are pretty good. This is his advisor was inducted into the National Academy last year. And the inaugural publication that he published in P.S. was was from Zachary's work and just make sure you drink a cup of coffee before you read this paper it's. Makes you tired. But you should be proud as acarya next of you if you come back to campus make sure you give them you know some respect this time ragging on him because he's not here you know I don't want to go to his head but I feel safe and bring him when he's not here. All right so I'm talking about today is homologous recombination in the species so followed was a sort of called Arius and hope August recombination really is an important phenomenon so I feel like it is worth some attention. It is universal all cells and even some viruses encode recombination systems on the other hand it's not essential to their viability and so it's something that can be studied in the laboratory using genetics for example I mean you can inactivate home August recombination in the cell still survives but apparently it would not survive in nature. It affects genome stability on one level it allows the organism to replicate its genome efficiently with few failures. All right. But on the other hand the ability to recombine D.N.A. can also lead to genome rearrangement So it has kind of the historic two faces two aspects in with respect to stability and evolution in practical terms it's how we engineer microbial genomes and the R.K. I have not. We haven't really focused on this in the our care so we don't really know in what respects is recombination maybe unusual in these Finally practical thing is that in this species we can quantify it with genetic selections because a phenomenon of a phenomenon which I call a marker exchange. This is a generic term just to signify that there is a gene exchange specific seems to be a form of conjugation and. Here is an early experiment that that I photographed on a white box just to show you what happens you have to have oxytocin mutants. All right so you have a in this case we have two mutants one of them is called one of the one of it's called one of the nine Genesis are very creative about how they name their strains. OK. And both one eighty eight and one eighty nine require yourself for growth. They have mutations in Boston set of genes and so these plates that I'm sure on your do not contain any yourself. So about ten of the seven cells of one of the just added to itself was played on this half of the plate when eighty nine by itself was played here I was saving plates combining things together and here is what happens when you mix those two strains together before your plate so hundreds of colonies form on the plate that do not require still so they lack both of the mutations that were present in the two original strains. The frequency of these things is about ten of them on a six to ten a minus fourth Purcel plated. This process is not blocked by D.N.A. So protease it is not mediated by the super Natan of the culture so it seems to require contact between the cells. However unlike some of the conjugation systems that we're familiar with from bacteria. There is no obvious differentiation that one strain is always going to be the donor and the other strain is always going to be the recipient and that since it's a bit unusual. So some of the properties that all summarize students have studied over the years. Eric and far not were to undergraduates and they saw an effect of U.V. radiation the experiment is very simple you take two of those mutants and you're going to do one of these mating experiments but before you do it you irradiate the cells with U.V. before mixing and then determine how many were comments you have versus survivors and then number goes up show I probably. DOS I didn't draw any lines here prices on that but you can see how the trend goes so nice exponential function. Of U.V. dose. Another property that we observe later on. Katie Schmidt did this work primarily that that stimulatory effect by U.V. isn't persistent it decays pretty rapidly and this is very easy to demonstrate simply by holding the two strains apart for a while before you allow them to recombine and exchange D.N.A. So here we have the two parental suspensions have been irradiated and then they've been incubated separately before being involved before being combined. So those of the dark symbols here are the the these are two different strains. So you have two different symbols and these are the irradiated control so this is kind of the background level recombination and after about five or six hours the hours are shown here on this axis but you know the whole process stimulation the case pretty quickly under physiological conditions. So this this change from being stimulated to being non stimulated requires time is shown here and also requires recently high temperature so we think is going on is that we are seeing evidence of some processing of D.N.A. damage in vivo and the way we've explained this to the results so far is that the U.V. converts an intact chromosome forms per million dinars per me dimer would by themselves have no effect on recombination or any other process you have to operate on them somehow per million dimmers we think are being converted to some kind of a lesion the does stimulate recombination but that's an intermediate. And eventually they'll be converted to an intact D.N.A. So we have a kind of a competition here. There is an intimate processors repairing these come recommissioning collegians or mating can convert them into a recombinant. But the stuff I want to focus on for the rest of the talk then has to do with an observation we made several years ago. This is Michelle Riley. She's a she was a master student in the lab. And that is this sounds like a very simple innocuous sort of statement that most Piri F. mutants former communists. OK the background that I need to give is that there are these two genes. There are very small they're next to each other they overlap so that from one end to the other this whole region is only about twelve hundred fifty base pairs. But these two genes of this very nice property of this compound called Five floral erotic acid or F. away. Plus you're still will select mutants in which one or both of those genes is in activated they in code to Boston that enzymes and if you block that box and set it pathway the F. away is not converted into a toxic compound. All right. So given how close together these things are then this observation. This is thing has more significance. All right so let me just talk through what we did. Michel isolate a large number of independent mutants and that's easy to do with with microorganisms you basically for example you can pick a colony pick a series of colonies each one comes from a separate cell. You can grow culture expand the population to be several million cells and then from each population you keep you know you put them on the selective medium and keep one mutant. So you have a collection of independent mutants they're not siblings of each other. And so Michelle tried testing just randomly drawing pairs of these mutants from this collection and asking whether they would form recombinant with each other and she did quite a few of them so we did this. Procedure where one of the pair get smeared on the surface of the plate and the other pairs just get spotted onto it. And it's looks kind of ugly but I don't know if you can see that everywhere a spot. You know here a spot has been placed on top of the lawn and a whole series of of recombinant colonies are now forming the. You know the plate does not contain your still so the original two mutants cannot grow the recombinant can grow in form colonies and so you see that most of these spots are showing positive the only three here in the middle that are negative overall Michelle found that greater than ninety percent of the random pairs drawn from this collection would in fact yield recombinant suits with each other and the point is that all of them have to have their mutations in this one short region of the chromosome. So the implications for this are focused on what modest recombination so we think is happening on these plates is that you know you have two different mutants mutations in different places on the chromosome. And this process has to go on. They have to get together and stick together at least for a short period time some kind of a channel has to open up D.N.A. has to transfer from one cell to another we don't know how much D.N.A. transfers we don't know how many you know what percentage of the time it transfers all these are all unknown but once the once the two D.N.A. has come into contact then some kind of recombination of an occurs so that you know this region here which is intact gets combined with that region there this intact and that intact chromosome forms a colony on the plate. All right. If we look at other genetic systems we see that these kind of events are very sparse or very small widely spaced in the human genome you get a typical spacing of about sixty million base pairs between crossover events. And yeast. It's about one. And every quarter million base pairs and call it's once about every one hundred thousand base pairs what we're seeing is you know that the spacing seems to be on the order of it seems like on the order of a few base pairs. All right so there's this several orders of magnitude difference between the behavior of these model systems and what seems to be happening in so flow with the so-called there is so this marker exchange process is unusually frequent the problem is of course that we have relatively few ways to control what I talked about the fact that these early steps you know pairing of a cell creation of some channel transfer the D.N.A. we can't control them experimentally right now. Yes. Yes that's right. I'm not saying what kind of recombination OK So recombination means everything not I'm not talking about the mechanism yet. I'm just talking about the functional properties of the system and you're exactly right. That's exactly what we think is happening which will come up in another couple sides. All right so the next question I want to ask and can we be more quantitative about this. How does the distance between the markers affect the efficiency of recombination. And at this point by this time we have a number of mutations which are known which have no motivation. So there are sequences been determined so we know where they occur in these two genes and so we can do a kind of experiment to test the effect of distance between the markers on the frequency of recombination and the motivation for doing this you know is one of the few things we can control in the system. One of the few things we can manipulate and it does have the potential to allow us to compare this recombination system to these other model systems. And this is how they behave. They they show the sort of characters. Behavior you're familiar with the idea of genetic linkage where the farther apart two markers are the more frequently recombination the more frequently they will recombine which reflects the fact that some kind of cross or event occurs between them. And so the more distance there is between them the more often recombinant will be formed as you get farther and farther apart that reaches a limiting value because a second recombination event becomes possible and that will subtract a recombinant will undo the effect of the first one and so the current then begins to sort of break over and reach a kind of Asymptote here a very long distances in the other direction as the distances become shorter and shorter in most systems you reach zero. And this threshold this finite distance would still is not really yield recombination. Has been interpreted as a minimum efficient processing segment or any P.S. and it's just not you know mechanistic it's a bit difficult to interpret but it has been observed for most systems for the all as far as I know that have you know reciprocal recombination so so we have this model of this is the textbook example of homologous recombination those reciprocal events the crossover events and we can compare our data. With that in the data don't really fit very well. Our distribution is not really uniform. We focused on very short distances on very long distances we don't have a lot of data points in between but certainly the Statistically there is no positive intercept from these data it's a very flat sort of function and you get reasonably high recombination rates even at these very short distances although there's a lot of scatter here. If we expand at the very close. Distances and compare them to literature values in the dark symbols we have bacteria phage T. for recombination and with the triangles we have an equal why plasma recombination system and they show this classical behavior of extrapolate a zero at about forty forty base pairs or so and also flow this data extrapolate over here in the negative direction. So it's not really a very good fit for these classical systems another way to look at the data is with the log logarithmic plots. And there are the behaviors is a little different. The M.E. P.S. shows up as a breakpoint between two regions of a low slope out here and higher slope at short distances and there is a literature values plot on the scale and you see they're mostly pretty steep dependences on distance and so follow this as a very flat thing and of the possibility of any P.S.. If there is one is about three or four base pairs so extremely short. We've also tested for genetic linkage. Again we're very limited we have really limited markers and so on available this is a kind of variation on the three point cross or three factor cross and just to summarize quickly what we observe is that if we force. The recombination event to be in a certain orientation by having these of the two selected markers so we're going to force a recombination event that includes that region from that strain and this region from this terrain and that way you would expect that that would bias. The region of D.N.A. to one side and it should only come from one of the two parental strains in fact we don't really see a bias we see essentially equal numbers only when we of of that illegal and either the mutant form of the wild type form in the progeny. And we did it cause I. Well way and got a very similar value and so there's really not any evidence of genetic linkage in these these markers this region here is only about five hundred base pairs away from this region where the quote crossover would expect you would expect it to occur. And so it seems as though the these were comments that were recovering are not you know forming from this sort of classical reciprocal kind of recombination the fact that the marker spacing has very little effect and the fact that we don't see any genetic linkage over this short distances just suggest a kind of non or super bowl form of recombination. That is for example like gene conversion and fungi were just sort patches of D.N.A. are being transferred. So another way in a small diagram here. If you can see this you know here's our starting situation A minus B. plus and one's parents a plus B. minus on the other and instead of getting both sort of given these reciprocal events we have one double mutant and one wild type. We think that probably what's happening is that simply genetic information is being transferred in a unit direction away to one of the. Key here this other strain in forming an A plus B. plus without a corresponding A minus B. minus. Again the most common situation for that is gene conversion which is observed primarily in fungi. Here's another phenomenon that kind of reinforces that idea that little pieces of D.N.A. are quite active in recombination this organism this is all more artificial this is genetic transformation by synthetic all going to create tides. This is a phenomenon that has been observed in other systems. This is not unique to sulfa Lobos it was first described in yeast. It's been reproduced in a male in cells and he call I under certain conditions. Name what you call i asked me expressing recombination genes of bacteria phages lambda. What all these situations have in common is that the the sort of primary recombination protein is not required for this kind of recombination. So in you. CARIO it's read fifty one or an bacteria wreckage A is not needed for this kind of a combination. D.N.A. mismatch repair tends to oppose it. And so it's much more efficient in a mismatch repair. Mutant. And there's usually some strand bias. So one only going to clean tide usually performs much better than it's complimentary all going to be tight even though it shouldn't matter in terms of you know making the change and putting it into the into the strain. So I'm calling this process. So it's hard to say illegal nucleotide mediated transformation. I'll just call him T. so nice generic term and cover that encompasses all these other phenomena. And this is interesting because this is a nice essay for a combination and involves a very precisely defined substrate we can really control. One of the two D.N.A. is that are interacting. To generate these are comments. And so as an experimentalist I really like control. All right. And just so this is how it works. This is what one of our first plates looks like this is more for historical interest these colonies here are are we common it's produced by electro praying a synthetic oil going to be tied into the cell. I mean you literally just you know type in a sequence or IT course has to be a sequence. Well it's a sequence that will correct a mutation in the chromosome right. It's proportional to me out of D.N.A. up to a point. So there is a region of linearity that you can use if you want to study it. Sufficiency. The only go must overlap the chromosome mutation we established that early on. So it's not just mutagenesis they only go. Not just simply mutating at a certain rate and there is a strand bias that we observe at least in the case of point mutants in the pure. Alright so. Another undergraduate in my lab Christy Stangl spent a lot of time electro pro-rating D.N.A. into so FLOTUS and she worked on it so she she was studying. So you so followed us and we had a couple of mutations that allowed us to vary slightly the position of the Taishan relative to the all going to create a tired without having to buy quite so many all of those and so that's why I'm showing you these diagrams two mutations they're both insertions of a G. and they happen to occur in the sense of the same region. So while Christie was doing was was asked saying these kind of all are going to tides. Right. So there's what she's putting in it's shown in the opposite direction because it corresponds to the opposite strand here right. This is the scent Strand. And what we're putting in are the or the complements that sequence. And in the meantime I designed a similar system many call eyes so we can compare eco lied to so followed us very closely. I mean they have essentially the same kind of genetic assaye the same kind of D.N.A. is replacing a piece of the chromosome in both these organisms in the core as well as self lobes. So I'll just show you here on the sofa lobes data we you know you can control the substrate you can control how long it is so there's a mutation that you're replacing and there's the D.N.A. that you're putting in and we can control its overall length. We can control the three prime side and apparently the five. Prime side we can slide the thing back and forth holding on constant and so on. So that's what is happening here just to give you an idea of how what the requirements of the system are you know what kind of what does it. What does it need. Either already have official recombination and it has amazingly few needs it's really quite flexible. There is a minimum length that required And on this graph here what I've done is I've part of the five prime length here on the vertical axis the three prime length in the horizontal axis and the size of the face of the symbol tells you how efficient recombination was this was like a three dimensional plot probably better ways are still the data I just couldn't couldn't generate. Some other alternatives here. And so the point is that if you follow this diagonal what you're doing is you're making the all those longer longer but keeping them centered over the mutation and so you see what your threshold is you have to be out to about here. So about twenty five nucleotides or so in overall length if you're symmetrically position but if you get out to longer links of about for example here on this diagonals everything is about is thirty eight nucleotides long and just sliding across this mutation and you can go to some pretty pretty far extremes just a few nucleotides on either the five prime and or the three prime and and get some detectable recombination so the system seems to be quite flexible. If you get it all going to clean tide of any decent length at all. Because these are being sent to you by Fed Ex from a company to synthesize in them. Chemical you can build various unnatural features into these things as well. And so it was kind of fun to put phosphor thought Wait linkages which block X. a nucleus processing and so we did you know. So there's the modified D.N.A. We have we put the the phosphor a thought away it's into the five Prime Min or the three prior and or both and compared the effects in so FLOTUS and in the call line and we can just focus on these parts over here they show the relative affects OK no. Phosphor thought away and then fire primarily three primarily both. And this is the relative rate of recombination. So the big picture here is that most of these modifications tend to help in a sort of called Arius especially if it's at the three prime end of the three prime and is limiting you get a big boost. You know almost forty times more and that makes sense. You know because you're blocking X. a nuclear activity or preserving the substrate. What was interesting is that you call why is more complicated than that. Many of the situations and he caught the phosphor thought it was either not helping or severely inhibiting the reaction so it was actually preventing recombination. And so that's the conclusion here e-coli is strongly inhibited by a phosphor of thought wait a limiting five Prime Min. And I know my talk is not supposed to be about equal laws. But it is kind of interesting that the reason for that is you call a appears to require a phosphate on the end of the all ago and we had been using an phosphor related all going to create tides and so there wasn't a phosphate on there originally and the phosphor thought it was preventing. Probably preventing X. in a cool little processing that would that would expose the five prime phosphate So there is definitely a difference between the eco I system and the self Loba system. All right. A new finished up here. So me just mention finally a current undergraduate John in Iraq who would who is looking at the products of homologous recombination in a more defined way not just quantitatively but also qualitatively. So what Johnny has done is we have come up with a an artificial peer E.G. in which has a number of sequence differences from the native Piri gene but is otherwise completely functional. All right so. That is a linear D.N.A. that is a lector pointed into a peer in a mutant and so it contains a number of sequence markers which are labeled here by D. symbolizing donor right so the mark donor D.N.A. is electroplated in the sofa Lobos which has a deletion it's Piri gene we force or so something mysterious happens the recombination occurs and we select for gene function so we require some of this donor D.N.A. to go in. Specifically it has to replace the deletion and then Johnny picks a bunch of colonies extracts the D.N.A. sample far as a period gene and then ask where these donor markers. How many of them are present and she does that with restriction and a nucleus of these markers are usually putting in restriction sites. All right so I'm going to show you now our Her first one hundred or competence. Or she has assessed. I think about twenty five genetic markers and each of these one hundred comments. Johnny has a very strong work ethic. How exactly in that regard I guess. All right. And so as we would expect where the deletion occurs the donor marker is there in every single case we forced that region to come from the donor by the selection. The question is what else came along with that and the answer is the bits and pieces and so these have been ranked in terms of how many donor markers they have bits and pieces. OK what you would be expecting in the normal situation to be something like this right a block of D.N.A. has been transferred from the donor to the recipient. And we find a fair number of those but they're actually the minority. The typical recombination event includes the region that was selected plus various discontinuous. Regions were maybe one or. Two markers have come in from the donor and then gaps and then maybe another region like that in the same event. So we see multiple discontinuous tracks and they're distributed throughout the sequence that we've provided for recombination so an incredibly random kind of process seems to be occurring in this organism. All right so me just wrap it up here by saying that out there. On Earth. There is a geothermal micro biota it's very diverse that in to it's talk about many of the species but they're dominated at the very high temperature range by Arcadia. Is a very hardy diversion inches and they raise a lot of fundamental questions about how cells evolved and how they can function under physical and chemical extremes. Genome sequences argue that the hyperthermia feel like Arcadia with SARS what A.J. stands for should have some unusual genetic properties I didn't stress this but they are sensibly missing some very important D.N.A. repair systems according to their genome sequences. All right. We are getting some methods that I was to probes off a lot of species genetically. And we're able to do some quantitative experimental analysis this species a single called various has a natural mechanism that transfers chromosomal D.N.A.. As a form of conjugation and once the D.N.A. is transferred and it's recombined by a home August recombination system it's usually very efficient. Finally however the mode of that recombination at least on a gene size scale. So we're right we are admittedly focused on a very small scale but it does seem to be operating on very short segments of the genome. You know much shorter then then our constraints require. And so it's probably not a reciprocal recombination and it generates multiple short patches from these longer substrates. I don't feel. Interested in some of the questions that we're interested in. So I won't go in to a lot of detail but we're interested in and what determines this short patch size for recombination. We're interested in knowing how this relates to the fact that classical M.R. in any Our systems are not present here in these organisms. We want to know you know does reciprocal recombination occur. Because we don't really pick it up in this system necessarily but that's a little bit difficult to to assess what about ectopic or illegitimately combination is that a threat to these cells. How can they control recombination if they will accept such short D.N.A. sequences in order to carry out a recombination event. And you know can we take advantage of this to engineer mutations in the chromosome and so on. And so I want to also acknowledge and thank the National Science Foundation for its funding two different grants and thank you for your attention. Thanks. It's about the same perhaps a bit. It's a bit less efficient than a long piece of D.N.A. and it's probably a bit less efficient than you would get from a conjugation reaction but not you know not by orders of magnitude. Maybe it's maybe on a typically maybe you get one tenth of what you would get if you'd simply just made the two strains together. The tens of minus fifty would probably be a typical value. Yes We we have tried that a few times and so it does seem like it's going to be possible to for example integrate and insertion if you just have. Of you know fifty or sixty base pairs on either side. So the way they've done in the East for example seems to be feasible in this organism also we have tried it once or twice and it has worked in some cases it hasn't worked in all cases. So maybe about half of the time it is successful and the other the failures of course could be due to the fact that it might be an essential gene in some of these cases we don't really know we're just in the process of doing that now. Yes. So a deployed strain. Not that we have observed. So when I said that it might be difficult to measure or to to to assess reciprocal recombination lot of the reasons for saying that is because when we would put in a circular D.N.A. that should persist. I mean there are some telco complications because this species has a restriction modification system right. But we have a way of protecting the protecting the D.N.A. against that system. So the circular form should persist. And you know it should you would think it would recombine into the chromosome by a single crossover and persist in the chromosome for a while and then perhaps you know excise at a certain rate by a second crossover. And we have never observed. That intermediate formed by a single cross over. In some cases we looked you know right away as soon as we could by P.C.R. within you know maybe. I don't know five or ten generations or something like that and never observed it so I think that's that's very suspicious and you know there may be some unusual property that either. Interferes with that process or you know somehow makes it very unstable. You know. And usually unstable so that will be something we'll try and resolve but it is going to be able bit challenging from a technical standpoint. You know we don't know it's tempting to think but I guess the way I think about it is that there are probably a number of differences that distinguish R.K.. In terms of their D.N.A. metabolism broadly you know conceived. All right. And maybe you know that they're they are just you know they have several genetic difference of this is one of them and it might be that you know that sort of system is you know works fine but features of that cannot simply be transplanted into bacteria or vice versa. But these that over evolutionary time these different sort of strategies for maintaining your chromosome you know how important is recombination versus you know something else how rapidly is the D.N.A. replicated you know they don't replicate their D.N.A. very quickly. It's a pretty slow rate of D.N.A. replication and who knows whether there is some sort of special combination of genetic or molecular features that are stable. You know and the R.K. or solve this at least has this one suite of you know strategies that work well and you call it has a very different suite and used as yet another. Thank you.