such a great meeting. So we will change a little bit the environment that we will talk about. And first of all, maybe just before I start, few nice pictures of Slovenia, the neighboring country of Italy where I come from. But what I want to talk about today is soil bacteria. And we work with soil bacterium bacillus satellites for quite a while. And the approach we use in our work is actually that we isolate bacteria from a small space. Like from soil grains. And from each grain we isolate different strains of bacillus satellites. And the aim was to really understand social interactions between these strains. At that time, when we started to do this work, we were studying quorum sensing and diversity of quorum sensing specificity types that we find in bacillus, namely ferotypes. And from this picture, you can see from the color codes that even within the species you have in a small area, many so-called ferotypes. And the same is true if you look at the rhizosphere of one plant. It is the diversity of these social types is present. Now, in addition, when we have now these populations of bacteria, we try to understand how they behave in multicellular groups. Namely, we study swarming or also biofilms, being colonies or biofilms on plant roots or actually as pellicles. And the two social behaviors we addressed most often is one which I already mentioned to you. This is so-called quorum sensing. I don't need to repeat what it is, but it's a cell density-dependent behavior, which depends on accumulation of signals. And in-gram positives, the signaling molecules are peptides. And I just want to mention my student, Miha, Shpatsa Panku is in the audience. And he's here. He was presenting a poster on the role of these peptides in inducing proteases and bioform regulation. Actually, protease is mainly on this poster. And how these proteases regulate potentially the stability of these peptides. But today, I will talk about the other topic we do in our lab and try to explore. And this is so-called kin discrimination. And the work was presented, part of it, by Maya Bolyashic, who is also in the lab. And if you have questions about it, you can also ask her. But a lot of work on this topic that I will talk about today has been done by my more senior, two senior researchers in the lab, Apalonsa Stevonich and Barbara Kreiger. So kin discrimination is a mechanism where you have recognition between cells and then differential behavior upon this recognition between the interacting species or strains, actually. But so it is all based on cooperative behaviors which have been discussed during this conference as such that are susceptible to exploitation by cheaters. And a long time ago, Hamilton proposed that kin discrimination might be a mechanism, a sortment mechanism which can protect these populations from invasion by cheaters which are not kin. And so the process then results in the fact that two populations can, if they are non-kin, differentially behave towards the other. Or if they are kin, they can assist each other and thus better coordinate the group behavior. Now, what I would like to talk to you about today are three topics. First, I will explain how we discovered this phenomenon in the population of Bacellus satellites, actually community of strains that we isolated. And I will talk about a mechanism that we find occurs during strain interactions that are non-kin. And at the end, I would like to touch upon on the topic what are the social consequences when these strains interact in multicellular settings. Now, if we have a population of strains which are coming from the same environment, we ask ourselves, could we detect that between these strains a differential treatment which would correlate also with their phylogenetic relatedness? And so the first thing, of course, that was necessary to do was the determination of phylogenetic relatedness between these strains.  And therefore, they are highly similar at the level of phylogenetic genes. But still, the 39 strains that we use to address this question have been previously determined by us and in collaboration with Fred Cohen from United States that even at this small area of one cubic centimeter, you can find three ecological types of Bacellus satellites. So we took these strains. And then we asked ourselves whether we can, during their cooperative swarming, evaluate interactions. And at that time, it was already known that, for example, during protus mirabilis swarming, you can detect these boundaries that come between strains when they touch upon each other, as well as in a mix of bacterial compatibility tests. So we used the strains. We inoculated them at different spots or areas of the agar plate that induces, an agar medium that induces swarming. And so we indeed found this phenotype that we predicted. It will be there. So there is this boundary, dramatic boundary between strains that are different. And we don't see such a boundary. We see merging between the swarms of the same strain. So Barbara went on and tested in all possible combinations the 39 strains that we had. And of course, she did it many times. And at the end, she came out with the following metrics. Before that, I would also like to say that when we did these interactions, actually, Barbara, I did them. There were three different phenotypes, very strong boundary, a little less pronounced one, and no boundary, which we call merging. And the relatedness, actually I will say this first. So when we compared these strains, and now the color codes are labeled according to the ecotype. And between ecotypes, strains are more phylogenetically related. What we saw is that these groups that you see here, so in here, the strains always merged. But when you have two circles, strains formed a boundary. And you can see that boundaries would always occur between ecotypes. We don't see any merging between ecotypes ever. But within one ecotype, for example, the yellow one, you can see that there are more kin groups, as we call them. So the diversity of social interactions at the level of recognition or this differential treatment is much higher. It's 12 kin groups among 39 strains. And this is very similar, which was found already by Voss and Belliser in 2009, where they looked at a mix of bacterial isolates from a soil patch of a little bit bigger patch of 16 centimeters by 16 centimeters. Now if we looked and compared the phylogenetic genes of these strains very carefully in pairwise combinations and ask ourselves what kind of identity will correlate with the merging. So this is here indicated yellow merging with the intermediate phenotype or the boundary, which is indicated blue. And from this picture, you can already see that majority of interactions were resulting in the boundary formation. And merging would only occur in higher frequencies. And here you can see the frequencies. When there was 100% identity between phylogenetic genes, and then it dropped significantly. And below 99.4% identity between two genes in the pairwise combinations, you would never, ever see anymore the merging. So phylogenetic genes are not involved in these interactions. They are just reflection of the evolution of this population, suggesting that as the population evolves, there is a higher and higher frequency possibility that they will be antagonistic. But even if they are 100% identical at the phylogenetic genes, you can still see, for example, here one boundary or several intermediate types. So the next question, when we saw this nice distribution of boundaries and merging, and which suggested to us that actually the mechanism behind correlates with the evolution of these strains, we asked ourselves, could we detect also mechanisms that are responsible for these phenotypes for so-called kin discrimination? And the approach we used here in collaboration with Roberto Coulter's lab in the United States, I would especially like to acknowledge Nikol Lyons. He's a very talented postdoc. And here is Barbara from my lab who has also done the same kind of experiments. And we both did transposon metagenesis in different strains and as well as reverse genetics and then compared our results and talked about what we see. And the approach we used with transposon metagenesis was such that a strain, a parental strain would be used. It would be mutated. And then we would look for boundary formation between ancestor and the mutant. And doing these kind of experiments, it was found that the law side that were responsible for this boundary formation could be grouped into three groups functionally. And this is toxin immunity genes, which is not that surprising. For example, in Bacillus Vap A is the most interesting, maybe for this in this respect, which is a contact dependent toxin immunity function and is also polymorphic between strains. And then these are like killing also toxin immunity genes. And then there is a group of surface components, genes that are involved, for example, in synthesis of tychoic acid, or genes involved in synthesis of extracellular polymers, the polysaccharides that are present on the surface. And finally, here are regulatory genes, many of which actually regulate these functions. So at the end, we could conclude that there are three groups of genes, antimicrobial cell surface, and the response regulation genes that are controlling this function. The question was then, OK, we see these antimicrobial genes that might be important. Are the antagonistic interactions the main drivers of kin discrimination between Bacillus strains? Is there any cell damage in the cell boundary that we see? And this is another picture of the cell boundary. So here, Nick has done an experiment where he looked at cells in the boundary or between two kin strains. And he found that between kin strains, there is less, for example, propidium iodide stain cells, indicating less dead cells than if you look at the number of propidium iodide stain cells in the boundary. And we have done an experiment where we looked with electron microscopy what's happening actually at the boundary. And you can see these cells here are much different than the cells in the swarm. So in the boundary, the cells look like kind of empty bags, suggesting that there is some antagonism going on. This correlates also with the transcriptional activities that were tested within the boundary. And I will not go into the details. I just want to show you one example, which is, for example, expression of sigma w gene between two strains when they interact and they are non-kin. Sigma w is a sigma factor responsible for the patient to the stress which occurs at the envelope level. And you can see that very high induction of the sigma w in one of the strains. And that's what happens usually. When two strains meet, one responds widely, the other actually doesn't change its genes that much. So it seems like one is attacking. And the other is less, is then responding to this attack. Now, if strains are compared, which are phylogenetically, they are still very closely related within the species or subspecies. But if we compare strains and say, OK, let's compare in such a way that they would decrease in phylogenetic relatedness, we see that the boundaries here, they become more prominent with the distance. So the very closely related strains, they still merge. And then you can see some boundary and the increase in boundary prominence. And Nick did one very interesting comparison using bioinformatics, taking the genome of a lab strain that has been used to study biofilms a lot in the bacillus community of scientists. And here are listed genes that are actually known genes in coding antibiotic or antagonistic functions. Doesn't really matter their names. What's important here is that here are the strains listed also in the order. So that phylogeny relatedness drops in this direction. And the gray areas represent the similarity, and white areas represent like missing, that the strain is missing, the gene. And you can see that as the phylogenetic relatedness drops, so does the common number of antagonistic genes in these genomes. Compared, of course, here towards to the reference strain. And so with the boundary prominence, as I told you before, the boundary prominence increases. Now, we wanted to know if we, for example, have two non-kin and then inactivate genes that we predict are involved in one, could we actually merge them back? Could we make non-kin, kin again? And that has been a very, very difficult task. And the only time we were successful to do this was when we used very closely related strain, PC216, which is only different between this lab strain in this area of sp-beta-prophage, and mainly the sublensin locus, which encodes also in antibiotic. So here is the interaction between wild type strain and PC26 strain. You see some boundary not very strong. If we now inactivate the sublensin gene, which is present in this focal strain, then we found that, indeed, you can reverse the phenotype and the strains become like mergers or kin, as we call them also. So if we found now these three types of groups of genes, how does this knowledge correlates with the knowledge that is already available in the literature on kin discrimination in other organisms? And there are two mechanisms which have been studied so far. Mostly, for example, polymorphic receptors have been indicated as means that strains, which are kin, can find each other and therefore increase their probability of interaction. Indictiostelium, in myxocococcus santus, for example, the trareceptor responsible for the outer membrane exchange or flow 1 gene in saccharomyces cerevisiae. Or there are also indications of immunity to the effectors strategies in proteos mirabilis. For example, this group which has done the contact-dependent killing as proposed, the contact-dependent killing as the mechanism to line formation, but also from the laboratory of Karina Gibbs, who proposed that actually these IDS genes that she discovered in collaboration with Pete Greenberg not the killing machine, but rather proteins that are able to help two strains to identify each other as kin if they are the same. But in bacillus, we have a little bit different system as at least at this point when we look at it. And we call it combinatorial system. Many loci are involved in kin discrimination. That's what we find. So far, we have not found any such strong receptors that would bring the cells together. But they might exist, that it's very possible. And so what we believe is that strains forming boundary encode a different set of kin discrimination loci. And this is not one or two or three. We think it's many more loci that are involved. And that these loci are highly dynamic in their existence. It's not that one strain will have them three loci all the time, they will change due to horizontal gene transfer. That's our hypothesis. And why we think there are many loci? Because it's very difficult to switch from non-kin to kin because mutations in kin discrimination loci, and these are the same mutations that we introduce into different strains of bacillus satellites, can give very different phenotypes. So that suggests that they have other partners with them. And that there is also a symmetry in the expression of KD loci when transcriptomic is performed. But I have not talked about this much. So to conclude with this, I would say that number of shared antibicrobials and immunities is the key determinant of the bacillus satellites kin discrimination system. OK. So why? What would that do? Maybe this kind of system would prevent mixing, random mixing, which would be detrimental to the community of different strain and would stabilize the cooperation within the group of kin cells. So if we propose now that when kin meet, they actually are happy they are doing well. And when non-kin meet, then the outcome is a bit different. They don't want that non-kin get close to them. What would actually happen in more real settings if we now really mix them together and not try to compete them one against each other on the agar surface as we did here? So we did experiments with plants which are more realistic systems. Like we used Arabidopsis thaliana. And we inoculated these plants. We actually put the plant into the medium that was then inoculated with strains. And after a certain period of time, then the spatial distribution of these strains has been looked at. And you can see that strains were labeled with fluorescent markers constitutively expressed. And here the names of the strains indicate if they are the same. So here are kin or self. So you can see that in this kind of combination, you see always both types of, I mean, both cells present. They are kin. Or if you have even different strains, but they are kin defined by the swarming assay, they also coexist and form this cluster on the root. This is not the case when non-kin are mixed in this setting. Because then we see only one strain prevailing on the root. And here again, another one has one in this competition. If we do these assays on the swarming plate, this time mixing strains and inoculating them in the middle of the plate, again, the strains are labeled with different markers. So the prediction would be that, again, there would be competition between non-kin and maybe coexistence of kin. And after 24 hours, if we look under the dissecting microscope at these mixtures, yes, indeed, the kin always stay together. They swarm together. And that's why the color on the plate of the colony is yellow. But we don't see this phenotype if we mix non-kin. In non-kin combinations, always one or the other wins. Sometimes we see sectoring. But this sectoring is always present only when we mix non-kin, not when we mix kin. So these strains, these cells are moving. They are not fixed. They are mixing. But not very well if they are non-kin. They then move also sometimes in these separate sectors over the plate. The territoriality is achieved, obviously, through competition. Because who wins in the assay between non-kin is very much dependent on the frequency of one of the strains. So if one would be here, it's only 4 to 1 ratio, and this one would always win. You see, if you mix kin, this does not happen. You just see the ratio preserved between kin. And finally, maybe I will say a few words also about mixing of kin and non-kin in a static culture. Here, a static culture, we grow cells statically, and they form pellicles on the surface of the liquid medium. Again, they are labeled with different fluorescent markers. And Maya, Barbara, and Istok have done this work. What we see here is a little bit different than in swarming assays. We see coexistence of kin, of course, that's predicted. They mix well. But also, we see that non-kin coexist in this pellicle. Only the size of the patches is very different. It's larger when non-kin are mixed. So here, mixing is very, very close. And if we go at 12 micrometers distance, we would, in kin combinations, always see two colors. But this would not be in non-kin combinations, where it would be one or the other. And such mixing also has consequences for fitness. Not as dramatic as in swarming assay, where one is just wiped out almost most often. But here, what we see is that if we mix kin, so the fitness does not change much. Both strains have the coexist well. And in non-kin combinations that form like this very strong boundary, one would be a loser. The other wouldn't gain so much, but one would be the loser. In this kind of boundary pairs, there is still good coexistence. So with this, I can slowly come to my conclusions. And this is that kin discrimination that we discovered shapes multicellular behaviors. It shapes how they coexist, how they swarm. They swarm separately. So it also affects territoriality. And finally, we believe at the moment that antimicrobials and immunity pairs, which have to be the same in kin and are different in non-kin, are the main molecular determinants of kin discrimination in bacillus. So with this in mind, I would leave you with a take home message. And that's gains and losses of social life depend on who you are interacting with. So pick wisely.


What's up everyone take down here happy New Year's. I'm super excited because this is the first video. I'm uploading for 2019 Today I decided I'm basically just going to recap my 2019 everything from my personal life and my channel and I'm also going to give you guys an update of what to expect to see moving forward in 2019 new videos and stuff like that coming soon. So let's get right into it Okay, so I'm actually going to start off with my personal life which for 2018 for me at least It was a really roller coaster years So to start off I've already made videos on this before but it actually started nearing the end of 2017 And that was where I worked which I worked there for five years and it was my first job They started cutting my hours and mistreating me. This went on for about six months up until my 22nd birthday Which is in May and then just two days after my birthday. I became physically sick My doctor didn't know what I had they still don't know what I had and I was basically out for two weeks And I was unable to work. I couldn't make videos at this time either I kept going to the doctors and basically all my tests came back normal They didn't know what was wrong everything according to the tests seem to be okay So he just basically kept writing me doctors notes to get rest So that's why I was off for two weeks the whole time I was off for that two weeks My work was basically harass me wondering when I'm gonna come back and I just didn't know I was physically ill and I couldn't work and they basically didn't understand that so finally I was back pretty much a hundred percent and I was able to go back to work now This was on a Monday And I did tell them ahead of time that I was going to be able to start working once again on the Monday But whenever I went in for my shift my manager came up to me and told me my shift was cancelled They got coverage and my next shift is going to be on the Friday So I had already been out of work for two weeks because I was sick and the week I was coming back to return They didn't just cancel that day's shift They cancelled basically my whole week worth of shifts next day The boss called me in and basically called me up to the office and was yelling and screaming at me claiming that I told him I was not going to be back this week, which was a complete lie because I already missed two weeks of work Why would I want to miss another week of work whenever I'm a hundred percent? So I know he was lying right off the bat, but he was basically just yelling and screaming at me in his office So after I left that day, I made the decision that my next shift, which was supposed to be the following Friday I was actually going into quit and I actually went in and basically quit. They were pissed, but I just didn't care anymore I had enough with everything that they were doing with me And this is when my 2018 actually started to get a lot better for me the day that I quit I actually as soon as I got home I checked my email and I got an email from Canada's commissioner saying that I can start my security guard course right away Other people that I talked to that took this course said that they finished it within a month The reason that it took me longer than most people taking this course is because I finished the online portion for the course But I needed to get my first aid certificate before I could go and write the final for this course So it took me a while to get my first aid because every time I would schedule it the day before I had to go and do it it would cancel and automatically refund me my money So I'd schedule it again for the next time available and it kept happening over and over again It happened at least three maybe four times and by the time I actually got it done It was two months since I started the course The only thing left I had to do for my course was to write a practice exam Which he actually used as my final exam and then I had to go and write a Provincial test, which is an hour away and once I had that all done. I found out that I passed the course I had to wait for my security guard license, which I later got I got my certificate I got my first aid certificate and I have everything right now The one thing that stopped me from working is getting a vehicle, which I'm really hoping to get one really soon So that was basically a recap of my personal life for 2018 I know like I said, it was basically a roller coaster, but that's how my year went now for my channel for 2018 So right off the bat in 2018 and early 2018 I said I wanted to start doing new kinds of videos for you guys One thing I started to do more of is taste test videos with my jerky tests my munch pack and different ones like that I started doing unboxing videos which I still continue to do throughout the year another thing I started doing in 2018 was my vlogs I started doing them weekly then I extended that to one vlog every two weeks And then I extended that to one vlog every three weeks and for me They just became harder and harder to film just because every three weeks for example Canada Day is July 1st my Canada Day vlog never came out until I want to say it was around September so I was two months behind so my vlogs really didn't make any sense So I do have two vlogs left that it did not upload yet after I upload them.  It's not worth it I did enjoy making them but it took a long time to do and I'd rather focus my attention on other videos for my Channel nearing the end of 2018 I started doing more react videos So I started reacting to my own videos music videos movie trailers different things like that I started doing try not to laughs which are one of my favorite things to do and for my react videos my try not to Laughs, I'm going to be continuing to do them in 2019 because for me They are so much fun another thing I'm gonna be doing in 2019 is going to be doing would you rather and higher or lower? I know there's a website. That's what I've been basically using and recording off of that. So that is a lot of fun I already have three episodes of each done and it's so much fun. I can't wait to start sharing that with you guys I'm gonna be making more videos where I'm talking more in-depth on Different topics and I think I'm gonna continue doing conspiracies, but I'm also gonna be adding I'm as of right now I think myths and different things like that for example big foot and UFOs I might make videos talking about them because it is something that's very interesting. I know there is kind of a Niche for it people are interested and always looking up that stuff and it kind of interests me as well So I think I might move into that direction as well for me in 2019 I don't think I'm going to continue taste tests as much I think I already have five taste tests that I recorded in 2018, but I'm not gonna be uploading them until 2019 Those are basically as of right now. I think that's gonna be all the taste tests I do for 2019 It's just not something I want to continue to do I'm evolving my channel and there's other videos that are gonna be taking those places now if I do come across something in the grocery store Something online that I want to try and do a taste test video for I will order it I might do a video like that it'd be interesting to do But I'm not gonna be focusing on taste tests anymore and for unboxing videos Wish for me is completely out of it because it was not worth it I tried it twice and didn't work out and loot crate and geek fuel I've done those before and I don't want to do them monthly basically because I can't afford it But if I come across one month where it everything in the box is supposed to be of Topics or themes that I'm interested in or every couple of months Loot crate and geek fuel will do it whereas massive savings I think loot crate does 50 and 60 percent off every couple months and geek fuel sometimes will do it where You get the box for free. You just pay shipping, which is 11 to 15 dollars So whenever they have those events, I might do them, but I'm not gonna be doing them often I'm not gonna be doing a lot of unboxing videos unless I have a product or purchase a product that I'm super excited for and I Really want to share with you guys then I might do an unboxing video one thing I said nearing the end of 2018 was I'm gonna be giving you guys some more new beats But honestly, I don't want to get your hopes up for that because I haven't started making new beats yet If I do get back into that mindset of making new beats then I'll start uploading them But I haven't even started yet just because I'm focusing so much on my channel and everything else that's going on in my life I haven't sat down to make any new beats yet. I also would like to do a little bit more gaming for 2019 I know I now have battlefield 5 Fortnite pub g all on the ps4 So expect to see some more montage videos, which is stuff I used to do before I'm gonna be bringing them back because that's something that on my channel people like to see I have also brought back my retro gaming series, which for me is a lot of fun And I got everything on my ps4 for all my retro games It's a lot of fun for me to play the old games that I used to play all the time as a kid growing up So far I have the first five episodes recorded. I'm gonna I think as I've already uploaded this video I've already started uploading those ones, but for me I think I'm gonna continue it if the views are there if the views aren't there I'm gonna replace them with other videos and start doing other kind of videos Hopefully some more gaming videos So if something funny or something interesting happens in a video game I might put them together for video game funny moments different stuff like that But for me that's stuff I really enjoy doing also any games I might start making once again trophy tutorials because that's something I used to do whenever I started my channel And I haven't done it in a long time But I noticed for my channel right now games that were about two years ago that I made trophy videos for People are looking up those videos now trying to get those trophies So I might start making more trophy videos for newer games and start doing that I think people will be interested in that but my main focus for 2019 is to only make content that I want to make and content that I think is fun to make and stuff that interests me So even if it's a video that's potentially will only get five views or no views If I enjoyed creating the video that for me is what matters the most. I do have high hopes for 2019 I'm super excited to share a lot of new stuff with you guys new videos I might start doing monthly update videos where I'm sharing new videos of what's to come for the next month on my channel I might start doing that comment down below if you'd like to see that and also comment down below Anything else that you guys would like to see me do or attempt on my channel if it's something that interests me I will do it But if you guys have any ideas, let me know down below 2018 was so much fun I can honestly see the evolution that I did for 2018 because I went back and I did a Channel review slash memory lane style video where I look back at all the videos I did and I basically talk about them They're gonna be up very soon But from doing those videos just seeing how when I started my channel to how I am now with my videos and making all the Content that I do I've come a long way and I'm super excited 2018 for me was a lot of fun I did enjoy everything that I did for 2018 So 2019 expect pretty much the same thing just videos that I really want to do videos that I'm enjoying doing and that's basically What you're gonna see, but I am gonna leave this video here. I hope you all did enjoy I'll see you guys very soon. Hope you guys had an amazing 2018 and I hope you all have a blast for 2019 I will see you guys in the next video. Please take care. Peace