 Start, so the first speaker of the last day is Vittorio Venturi from mycgb who is gonna talk about Quorum Sensi. Quorum Sensi. Il titolo è un po' specifico. Io sono Vittorio, sono un bacteriologo completamente esperimentale. L'anno con i nostri studi, bisogna pensare più. Alla la linea di modulazione, la linea di cosa che sta succedendo, spero che ti mostri. Qua che faccio oggi, faccio un po' di un storico di mio lavoro. Come puoi immaginare il scienziamento, sono quasi antico. E il modo in cui stavo facendo research, quando ho iniziato nel 1989, è completamente meraviglioso il modo in cui stiamo facendoci oggi. Se qualcuno veniva a me nel 1990 e ha detto, vedi che nel 2020 potete fare qualcosa. A, B, C, io credo che sia Fanta C'ènsa. Il scenario ha cambiato completamente. E anche, sentendo alcuni conti, e parlando di alcuni di voi, sono molti pensanti. E cosa è un po' intimidata? Perché nel mio mondo, pensiamo più o meno. Per un esempio, ho un studente di PHD che è inizio l'octobre. E vogliamo capire a qualsiasi proteine interaggono con le proteine che abbiamo. Non so come funziona. Abbiamo fatto alcune interaccioni. E ha lavorato per un anno, incontrando l'esperienza per tentare di capire a qualsiasi proteine interaggono. Il mio studente, per un anno, non ha pensato molto, perché lo ha sempre fatto, ha preparato l'esperienza per portare le proteine che interaggono. Questo è molto il nostro mondo. Il tempo di pensamento non è molto, in my view, compared con il tuo tempo. Quindi, a volte, abbiamo bisogno di ricordare di pensare, e anche i miei studenti e tutti noi, abbiamo bisogno di pensare a piccole foto, perché abbiamo avuto l'esperienza con problemi tecniche di ogni giorno. È tornato meglio, perché la tecnologia è molto cambiata. Questo è il primo messaggio che voglio te dire. Il mio mondo è molto diverso da te, ma ora che ho diversi studenti, posso spendere più tempo pensando. E' una cosa che abbiamo fatto, e anche un'idea che posso collaborare, o che potete illuminare a noi, in alcune delle questioni che stiamo cercando di rispondere, specialmente in quei recenti anni. Ho iniziato a lavorare nel 90' nel senso di quorum, e ho iniziato con questa piccola qui. E potete vedere qui i squids, che in realtà illuminano, che faccio il bioluminescence. Questi ragazzi vivono in l'E.C., in cui c'è la luce o non c'è luce, ok? In i US, alcuni scientisti ha scoperto e isolato la bacteria, che fanno una relazione simbiotica con alcuni di questi squids. La bacteria è chiamata bubio fischerei, che non ha cambiato il nome. I taxonomisti stanno parlando di meravigli. Le nuove specie sono scoperte ogni giorno. I nomi sono cambiati. Se guardate a Google, c'è il fischerei di vibrio e si chiama alio fischerei. Questo è un bacterio di gramma negativo e potete vedere qui. È una famiglia di vibrio. C'è un colore di vibrio, che si chiama colora. È molto vicino, ma ovviamente non è un papagen. Se si chiama nel lab, nel piatto o nel liquido, sono davvero interessati a questo. Perché si chiama e come? Per fare una storia molto lunga, qui è 15 anni di lavoro per provare a scoprire questo. È che lo che si scopri è che questo vibrio producia un signo, un signo chemical, che è diffusabile e può essere in e out of cells senza transporti. Questo signo è basicamente prodotto all'anno. E se come la popolazione, come il numero di celli aumenta la concentrazione del signo e a un certo punto, a un certo concentrazione si chiama il bioluminescence. Si chiama la produzione di litro. È un comportamento sociale. È una cosa che noi bacteriologi devono completamente cambiare il nostro mindset. Quando sono andato alla università ho pensato che i bacteriogliani sono unicellili, che sono unicelli per il loro survivor, ma in realtà abbiamo potuto imparare i bacteriogliani social. I bacteriogliani producono un po' di fatti che sono secrete. Se guardate il steptomyces, può produire up to 200 proteini che si tronca in medio, può produrre 600 metaboliti che si tronca. Poi, se si tronca tanto l'energia di fare il suo corpo e il tuo colore nel 2013, il termine di microbiologia sociale si tronca, perché abbiamo realizzato come social questi organismi sono. Poi, nel 1994, Clay Fuqua, Greenberg e Steve Winans in the U.S. tronca il termine corum-sensing, quindi si tronca il corum-sels. E questa è una rivoluzione in il nostro mondo e io l'avevo, perché era una nuova cosa completamente nuova. E nel momento in cui questo era come un'eccezione come una nuova cosa per questa bacteria, ma in realtà, il scenario è che, a mio punto, tutti i bactieri che vivono i organismi comunicano. Perchè non vivono i organismi come un bactere non comunicano? La comunicazione è importante per la vita. E quindi, qui abbiamo un sistema di comunicazione che regla i comportamenti gruppi. E' davvero una rivoluzione tra il mondo bacteriologico e vi mostreremo alcun slide di alcuni fenotipi. In particolare, il Vibrio Fischera, l'ultimo modello di oggi, è che questi bactieri hanno un organo lite nel loro pelle o sotto. E quello che questo organo lite fa, non mi chiede perché ho trovato che è fascinato che il c'è fuori dei micropi, ma questo tipo è capito di questo tipo, specificamente. L'ultimo ha una rivoluzione, questo tipo lo cresce e la densità di cellulazione è upto 10-9 cellulari per millilitre perché è una bella fiamma che il squid già e in exchange il bactere lo farà la densità di cellulazione e la stessa storia è che la fiamma nel tuo pelle lo avverà un shadow che il mondo lo farà. E si chiama e se qualcosa è shadow lo farà. In questo modo, la teoria lite è che questo permette di survivere e di combattere i predativi. Questi sono alcuni dei signori che questo freddo si è tirato completamente in my view, è ancora il tipo dell'iceberg. Abbiamo trovato molti signori, c'è molti, molti più per essere trovati. Micropi, fangia, bactieri, che è il loro linguaggio. Questa è una piccola, si può vedere. Questa è uno dei signori e si può vedere che sono semplici. Non sono un chemist, ma posso dirvi che sono semplici e diffusibili. Non necessitano molte geni per essere sintesi e si muovono abbastanza rapidamente nel media. E si può vedere qui in un slide. Sì, il scienziato è stato meraviglioso. Questa è una nuova cosa. E si può vedere qui i fenotypes, i comportamenti che sono regolati per questa comunicazione. Ci sono le sfogolazioni in questa meeting. Ci sono le pigmenti di luminescence. Abbiamo belle materiale in un labio. Non sono pigmenti. Gli sviluppano e sono colorati. Il signore regola la pigmenta, che la pigmenta aiuta a presentare i produttori resistenti. L'antimacrobial attività aiuta a resistire i materiali, il UV-light, etc. Le informazioni biofino. I fenotypes sono relativi per la comunità, non per la singola cellulata. Ok? E, importantemente, ovviamente, bisogna lavorare con i pathogeni. Se vuoi fare research in Italia, e io faccio research in cittadini environmentally è basicamente una funghi, 98% della funghi è in cittadini biomedici. Ma comunque, la produzione di produttori di virali, se fai un pathogeni e fai un posto, un planto, un uomo, un animo, l'ultimo che vuoi fare è rivelare yourselfi al sistema immunale. E, normalmente, rivelare yourself is when you start firing. It's a bit like humans go to war, if they start firing, the enemy knows you're there. So they have to fire at the right time, and the right time is really when there is a... If you're able to enter a host, for example, I'm interested in plants, and bacteria enter in plants, and then they can hide and grow and reproduce, and at the right time throw out their enzymes or their toxins, and that's the time when the plant knows oh wow, and then the plant can defend. So there's this Troyer horse strategy of defense that bacteria can use through communication, so initially they grow and establish, then they recognize each other and then they attack. And attacking when there's a lot of you, you're more likely to survive, just like humans go to war, it's basically a similar scenario. When you're a large number, you kill the other cells and you release food for the community. Okay, but the attacking, so the making of the enzyme, or the making of the toxin, then that's the moment that the plant perceives responsible signals and then produces factors which will then counteract the bacteria. I don't know, antimicrobial peptides or reinforce the plant cell, those plant cell wall. So the recognition, the fighting between the pathogen and the plant, for example, is, because I think the major reason for that is growth. They need it for growth. Yeah, they kill the cells, the cells release, they kill vegetable cells or whatever cells and the cells release food and the bacteria can grow more and more. I'll show you some more examples later about this food thing. How is it, how do, how is it possible, sorry, to show that it's current sensing and does not like, for example, in this case, they are in higher density so they exhaust nutrients around them and reason signal is not like a molecule they emit, but rather sort of like a change of the environment that comes with the cell density but that is not properly spoken current sensing. How do we differentiate this? Well, there's a lot of data on this. You know, we have mutants that don't make the signal, we have mutants that don't proceed the signal, there's a lot of data on that. There are ways to stop the signal, to degrade the signal, so there's a lot of strategy. There's over 100 patents now blocking the signaling and they show that it's less virulent, so there's a lot of data there, okay. And a second question, sorry. Other documented cases where another species of bacteria could use this current sensing to sort of lead the... I'll come to that. Yeah, it's a very good question, I'll come to that. This is one of the things I'm getting into. Okay. So you can imagine the excitement that we lived. Our world as a microbiologist is a bit of a sin that I love science and if you go to university departments, a bacteriologist, but then PCR, enzymes, PCR, now genome editing is old thanks to discovering bacteria. But we have this kind of life, a bacteriologist. We go down and then we discover, we make a revolutionary discovery, we become popular again. So this you can imagine, and also program cell death was discovered first in microbes as well. So this was an incredible excitement and communication group here and you can imagine the applications. Because here you have a strategy to control an infection but not killing the bacteria. Because killing the bacteria like antibiotics does, the pressure for resistance is enormous. But stop the bacteria but stopping virulence, gene expression has much less implication of resistance. So there's a lot of, as I said, a lot of patents, a lot of scientists studying ways to block this communication because then in a way you render the pathogen much less pathogenic and you can control the disease. Okay. So I just want to get in now what I'm trying to get, what my work here in Trieste is I'm working with this signal it's called homocerylactone. I just don't want to talk it all about the chemistry. All I want to say is that it's very simple to synthesize and there's lots of different dialects to this language. The R2, the chain can be up to 20 carbons and the R1 you can substitute. So within this family of signal you have a lot of specificities so a lot of species can make its own type of homocerylactone. And it is so simple and so powerful. You need one gene to make this signal because this enzyme opportunistically takes precursors that are present actually in every living cell. If you take this gene and put it for example in a human cell or in a plant cell it will make the signal. Right? So what you have is that you have one gene coding for one enzyme and basically making the signal all the time and of course what happens, as I already showed you before high cell density will be more signal and it's diffusible and it will interact in exactly one to one stoichiometry in a very strong interaction with a regulator, a sensor regulator that will go and then when there is this interaction the activator becomes functional or I'll show you later or maybe not functional and affect target gene expression. It will switch on bioluminescence, it will switch on genes related that will control phenotypes community phenotypes. But one very important thing is the positive feedback loop. So the first gene that regulates is the gene for the synthesis of the signal. So there's a very fast strong amplification to make more signal. And this mechanism is so simple you have this activator actually if you're interested in biochemistry binding to the signal will create a homodimer so two polypeptides come together each polypeptide is bound to one signal and in most cases 90% of the scenario then this homodimer activator will bind to a DNA promoter and activate transcription of the target gene. There are also scenarios where the homodimer will bind DNA which is when it's unbound to the signal so it represses and binding to the signal will make it unable to bind DNA and release release the repression and then the gene will be transcribed. So you have both and there are implications of the two which is better to use depending on the gene bla bla bla, but I don't want to just to show you that bacteria can use both but by far activation is the most common. And what I've done, I started to work in the late 90's I just want to show this picture I worked with rice as the model system of my plant. Sorry short question. Oh sorry, do they pump the molecule out or is it just diffusing? Very good question, very good question. As I was saying before there are different dialects it is it is diffusable but there are reports that the larger molecules the one with a long side chain they do, there are some transporters which help pumping it out it can still diffuse but there is some help to pump it out for the bigger ones. So I work with rice for obvious reasons it's the most important zero crop in the world it's two and a half billion people eat it three times a day so I thought why not work with rice? Which are the bacteria which are most important that interact with rice, both good and bad because most bacteria that interact with plants are sure in a second are good absolutely, we live with three kilograms of bacteria especially in our gut so do plants in a second. So I worked a lot with pathogens and beneficial but I don't want to get into historical data and publication what I want to show you is that in our world, experimental world very often you start with looking for something and you look and you find and you have to move away, you find something that you didn't expect and then you get excited and then you investigate and this will happen so when I was looking for these I isolated these bacteria and we're talking about the pre-genomic era here so some of you young guys have to click a little bit in your head that we had no genomes there was no automatic sequencing I did all my sequencing by hand no oligonucle ties so anyway so I was taking these bacteria from friends all over the world send me these bacteria that you isolated and I got these bacteria and I was looking for these signals using sensors or chemistry and we found a lot that most of them have except these two a very important pathogen I don't want to get into the very important pathogen and very important beneficial bacteria that live next to the roots 1 millimetre, 2 millimetre of soil is called the rhizosphere and it's basically the gut of plants it's a perfect parallelism it does a lot, so many important things that microbial community I'll show you in a second but these guys, I couldn't find the signals they didn't make them so it was the late 90s I thought these guys don't communicate now we know that so many signals can be made I was just looking for one I was looking for one language in other languages anyway what we found, what I want to go fast what we found because then genome started coming out is that these bacteria they really puzzled us immediately we found that they have the gene basically the protein for detecting the signal but they didn't have the gene for making the signal and these two genes are in fact always next to each other in chromosomes so we had scenarios that bacteria that lost the gene that makes the signal but retain the gene to respond to the signal and this is when I we coined the term in 2009 oh but we coined the term this is not the new slide that I made looks are solo, we coined that term looks are a regulator which is a solo that doesn't have a cognate signal because you have the signal gene generating gene which makes that signal and you have the specific regulator to respond best to that particular structural signal so then we started hypothesizing with these guys do these bacteria respond to signals made by other bacteria? so like eaves dropping or do they respond to a plant because already in the early 2000 there were groups in the US saying oh plants could make these same signals as bacteria makers so they could interfere or they could mix up with the signaling and to make it long story short what we found is that oops, is that actually these bacteria that we were studying have lost the ability we've shown that they don't respond to almost any lactose but they respond to a plant signal which is not almost any lactose so basically what I'm telling you here is that then we did a lot of work and we showed that this is basically an inter-kindome signaling system bacteria is plant communicating with the bacteria e in the case of the path one is a pathogen in the case of the pathogen the pathogen perceives the signal ah, I'm inside the plant and it starts behaving like inside the plant and the good guy living in the in the rhizosphere next to the root oh, I'm next to the plant I start doing something good for you for the plant in exchange for food so this is what I'm saying but then you know genomics exploded so we went back to the this particular regulator which has specific primary structure amino acid sequence features is very well conserved in bacteria sorry, you can't read them but all these bacteria are plant associated so it's only homologous to plant associated bacteria so basically what probably happened is that this system was a classical signal producer signal sensor lost the signal generator there's a few changes of amino acids in the domain here, I didn't show you here this is a modular protein these looks are about, sorry again the figure is not complete, 250 amino acids and there is at the end terminus a domain that responds to the signal and the C terminus the domain that binds the DNA so there's been changes in that domain that then is no longer able to bind to almost an ectome but I can bind to a plant single and then this guy then distributed among plant associated bacteria and now we have a new signaling system between plant and bacteria and then to make I don't have the slides but then one of my students went to Greenberg in Seattle who is basically the father figure of quorum sensing and she identified the signal and she published really PNAS papers and so on, so forth about which is the signal and what are the mechanistic aspects of this system but what I want to tell you here is that the genomics here really changed us this is why we beautiful to do this work as technologies come in you really your view of science you know the things you can do are basically amazing so basically we discovered this setup that we have a regulator that no longer produces a signal and we coin the storm looks our solos and then we started going to the genome for example we went to our favorite so the monas is a general bacteria that are friendly very friendly and live in the gut of plants and in fact they are so abundant in the gut of plants they do so many good things like help plants get food keep pathogens away produce plant hormones so the root grows better blah blah so on and so forth we looked at 612 genomes of these scientists including us we sequenced 50 or so but in the sequence of these beneficial guys and we found that 82% have looks our solos compared to 17% having a complete looks IR system so a system that makes a signal basically what's happening is that it's far more common to have a looks our solos than being a bacteria that makes the signal and responds to the signal don't think that it doesn't communicate maybe it makes other signals then we do even more genomics here close collaboration with Azaf Levine in Israel who is a microbial genomics fantastic microbial genomic scientist and we could we could identify 9 different subgroups based on the primary structure and the adjacent genes of these solos in pseudomonas so we think we have 9 different types of looks our solos we think 3 are binding AHLs so they listen to the eaves drop to signal AHLs almost inactive produced by the bacteria but the other 6 one of them is the green one that binds to the plant signal but then the others we don't know what they do maybe they bind to other plant signals maybe they respond to fungi maybe we don't know but the message I want to give you is that you can see here how I'll come to you later how bacteria which live in a very complex community basically are specializing their cell to cell signaling systems and then of course we just wrote then we get excited with Azaf and we just published a big study big review where we looked at 23,000 genomes we mapped all the looks our solos in proto bacteria and we can see and we try to combine looks our solos with the ecology so maybe we find a subgroup of looks our solos we found one among plant associated bacteria we find one with interic bacteria we find one with soil born bacteria or water born bacteria in order to try to see if specifically a solos will have a specific function in an environment I hope I'm not boring you with too much but anyway but the the message I want to give here is now through our studies and also the other scientists are also studying looks our solos is that we have at the moment 5 scenarios one scenario is that it's been shown also by Pete Greenberg that you have a looks our solos in a bacteria that actually has a looks IR complete system and the response to the signal that it produces so you have two sensors one is a solo and one is a cognate the response to the signal it just amplifies the ability your ability to respond to the signal that you make we have a looks our solos the response to HLs almost in lacto in a bacteria that doesn't make almost in lacto the classic example is the coli salmonella, salmonella in coli you have looks our solos the looks are solos that respond to signals which are made in dodgy but are not HLs they have a different structure slightly different structure we have signal looks our solos we discover the response to plant signals and we have looks our solos that work in a ligand independent manner they just activate all the time so these are the 5 scenarios we have at the moment so the message here I want to give is that we think that this quorum sensing system cell signaling systems in particularly classes of bacteria like these bacteria that live in very complex community in soils I'll talk about now which you are very unlikely to have isodensities because the microbiome of the soil microbiome today is the most bio diverse microbiome there is tens of thousands of species also in there so we have a scenario which is very different from the Vibrio fissure I showed you and maybe there's a specialization going on in terms of cell to cell signaling there's a different requirement that these bacteria need to use in order to survive in this very complex mixed communities and so the rhizosphere and the soil is something that interests us a lot and as I was saying to you we're doing microbiome studies in these environments and the complexity is enormous so yeah so we have to think of this so we have to really think we are now finding as I show you finding that quorum sensing is actually the decision making the on-off system is much more complex and there's a lot of specialization taking place and also what we are seeing and I'll show you now what we are seeing and we want to advocate is that pathogens pathogens have a different requirement compared to maybe a soil or a soil or rhizosphere bacteria per esempio we looked at diseases of rice different diseases answering your question some diseases cause rotting we did what's called the pathobiome so we determined the whole bacterial and fungal community of these diseases and what I could tell you for example in one of these diseases here one of the diseases that causes grain rot which is sorry, sheet rot which is very common in Africa when you grow rice at high altitudes you get this, you know the sheet is what covers the panicle just before the panicle forms and that rots and it rots because the bacteria make toxins and enzymes cells die and get food so we went there and what I can tell you is that if we look at a healthy sheet a healthy sheet is basically very little pathogen but a six sheets where you have the symptoms of the disease 30% of the bacterial load of that community is the pathogen so here we have a dramatic change right and then we do all the analysis and we see that that when the plant is healthy there is a very biodiverse community when the plant is sick 30% of the bacterial load is the pathogen and the other 70% is affected dramatically and then we looked at this more carefully and what we found in studying another disease is studying a disease of olive called olive knot disease and this is here, you see this is an olive, one year old olive plant and here we infect with this pathogen and after 60 days you get this knot, it's also called a tumor where you can call it a gall and this is a bacteria and this bacteria we've shown that it communicates through the signaling system and if we knock out like the question that somebody asked me if I knock out the ability to communicate produce a signal or respond to the signal you can see and you infect here this is a bacteria that doesn't make a signal this is a bacteria that doesn't respond to the signal you can see if you infect the basically the knot is basically non existent but it's very small so if you cannot communicate then you don't produce the virulence factors that make this knot you send the plant in apoplasia the cell division goes crazy and they start giving a lot of food to the bacterial community but what I want to talk about here is that you know when I was contacted by a scientist from Morocco working in Perugia in Italy he said can I come to your lab to study whether this pathogen, pseudomonas savastanui uses this cell to cell signaling system for pathogenesis and I said I wasn't too excited because there already a plethora of articles of other pathogen other plant pathogens that use the system for pathogenesis so if we find it we just join the family but we don't find anything new but what really excited me is that when I go to the field take the knot isolate the pathogen from the knot I always isolate basically I always isolate another bacteria called the virulence factor which was actually published the species was actually coined in 2004 in spain in fact Toletana means Toledo and the Spanish scientists said that Rina Toletana is an other species associated with the savastanui induced three knots so I thought wait a minute here we have pathogen that comes in takes over 34 in fact here we did the microbiome 50% of the community inside the knot is the pathogen but there's this guy he's known to be harmless it's a bacteria that lives together with harmless or beneficial there's not a lot of studies but definitely not a pathogen so then we started asking question what's going on between this guy is the pathogen, is the niche maker he uses all his energy and he's evolved to make this beautiful this really nice niche this nice living quarter and this guy, is he a cheat is he a bacteria that takes advantage and cheets and takes the resources from the solivno or is he a collaborator so we started working with this and again you know this is the idea but then you have to work a year too so the first thing we found out which was immediately interesting is that the pathogen and the Ervinia make the same signal so they speak exactly the same language so the Ervinia also has a signal generator and a signal responder exactly the same just like the pathogen and we thought that was interesting and then we inactivated we made all the possible mutants and what I show you here is infections or co-infections so as I already showed you this is the knot volume of one year old olive plants that we infected and we infected with the pathogen just the pathogen alone we have up to 60 days the knot as I shown you before in pictures when we when we infected the pathogen with the Ervinia toletana the harmless we have bigger knots so already that was an indication significantly bigger knots so it means that when they are together they probably both benefit then we did an experiment which really blew me away and I already showed you when we infected with the pathogen they cannot communicate cannot produce generated signal but when we co-infect with the mutant of the pathogen that doesn't make the signal with the wild type Ervinia toletana we can rescue the knot so basically that was telling me these guys are living together and now the pathogen that doesn't make the signal can use the signal made by the Ervinia by the harmless bacteria when we co-infect with the pathogen that doesn't make the signal we don't have the knot and what is important if we do the same experiment but we use the mutant of the pathogen the regulatory mutant the mutant that cannot sense the signal even if we put the wild type that makes the signal we don't rescue the knot so basically this was evidence that we were sharing a signal that here it was in vivo in planta there was these two bacteria living together and they share their signals they speak the same language and they talk to each other and in fact then we went and this is a very also important experiment we did we measured through the 60 days of the knot cell counts how many cells in the knot and here in open circles this is the pathogen you can see that as the knot increases you have more pathogen then we measure the pathogen you can see we have more pathogen so the pathogen grows better when there is the ervinia and this is even more dramatic if we measure the ervinia if we infect with the ervinia in fact we put the ervinia in the plant alone basically it disappears in 60 days it cannot grow very well but if we measure the ervinia when we also have the pathogen look at it the ervinia is so happy it can grow so we went to Cairo Ramos fantastic guy really nice guy in Malaga who is basically the world leader starting this disease and he has all the model he has the plant, he has the epifluorescence see a few per gram the knot is bigger but this value is see a few per gram of knot so then we went to Cairo one of the PhD students spent some time in Cairo who has a beautiful system he is a incredibly nice guy in Malaga professor in Malaga and he has the epifluorescence he has the confocomacroscopy and we did all these confections then we make the red and green again another revolution in bacteria thanks and look at this I mean I get so excited look at this this is a biofilm inside the olive knot look at the red and green they are making love they are so beautiful together you know they don't kill each other and basically our data shows that the pathogen goes there I take over I make the niche I am 50% of the population but with some of the guys there I am going to have a good time because actually at the end of the day we both benefit and I don't know if I put this slide no I didn't put this slide and then we got an embo fellowship from Cairo lab because we put the genomes together and we saw some metabolic complementarity so we saw that some metabolic pathways were more complete when the two genomes are together so we were looking at ways we were looking whether the two bacteria together could be metabolically stronger and we did again so much work, mutants, confection and unfortunately we were not able to show because our belief, our hypothesis is that there are some metabolism which is much more complete when the two bacteria together compared to them being separate there are compounds in plants especially phenolics that need to be degraded for food and we think that together they can do it better but unfortunately we didn't show that after a lot of work that happens a lot in our job so I just have 3-4 slides to finish so here just some takeaway messages and to finish I will give the takeaway messages in a couple of slides I just have a few minutes left and what we are now finding also because we isolate so many bacteria from the soil, from the rhizosphere pathogens we find bacteria more and more that have quorum sensing systems like this beautiful bacteria we work with actually Misha is working here which has two quorum sensing systems which we cannot switch on in the lab so basically they work take the genes and remove them from their regulatory components and we just fire them and they work, they make the signal, they respond to the signal they bind the signal I mean they are functional, they are not pseudogenes but we cannot switch them on in the lab well Misha was able to do that so it goes against the initial concept of quorum sensing basal production and then a certain cell density switch on it's actually not like that the switching on mechanism is something that is really baffling us and the rest of the quorum sensing community because we are understanding in many cases there is much more to it than that and one experiment that Misha did here and these just come out of the microscope these are, if we take these pseudomonas and we make a green with constitutive green this is the wild type right we are looking at one system in particular and then we give it the signal that the system that the gene we know produces we give it exogenously you can buy these signals we give a lot of it we saturate the quantity of the signal you can then see sorry I am a bit electronic the difference between these two panels this is a if it's on it's yellow it's just a different color but you can see here so if you give it a lot of signal 5% of the cells go on not all of them and then Misha was playing with different molecules to understand how we are responsible for the different molecules and here we have a scenario where we identified the genetic element which was repressing the system which is a repressor in case we activate that that guy we have 100% of the cells on so here we don't add any HL we just remove the repressor then the bacteria make a lot of signals but in this case all of them are on but if we give exogenous signal the same that is making here 5% are on so help me guys maybe you guys have you can model this you can give us some idea ok so here does this system repress repressor because a response to an environmental stimulus or we have like a stochastic switch on some of the cells repressor just doesn't work switch on and then throws out a lot of signal in that particular area and then it switches on also the neighbors so we're trying to understand and these scenarios are getting more and more common we're finding them so we're trying to understand this switch on off mechanism which is not as as straight forward as first postulated by this quorum sensing because again don't forget the Vibro Fischera it is really a unique scenario where a homogeneous population is desired so we want all the cells we want to maximize bioluminescence production community scenarios where you want to do some bed edging you want to do some division of labor you want to switch on off the system in a different way in order to optimize your chance of survival so we must not take the Vibro Fischera as the in my view as the paradigm example it's really an exceptional example what we're finding now is that if we look at the signaling system I've shown you specialization through looks are solos much more complicated so we're now thinking ways we're now trying to understand how this cell to cell signaling system really works in much more complex communities associated with plants or in soils and so we just published actually last week so we think that actually quorum sensing can regulate adapted traits is much more than a cell density dependent switch we really we begin to argue that in the wild we think quorum sensing promotes phenotypic terogeneity so it doesn't go towards a homogeneous response but it goes towards a heterogeneous response which has obvious advantages in a complex community or in an environment with a lot of different types of food a lot of different types of competition scenarios so basically quorum sensing could enable some societal organization in bacteria basically we published this giving an idea that we should really rethink and re-envision the way we see quorum sensing in bacteria so basically I finished this last slide since this is a really thinking environment the thinking the messages that maybe I can give here is the role of quorum sensing in complex plant and soil microbiome versus the disease environment I think it's very different and I've shown also that in the disease environment there's also sharing of signals which has not been shown yet very much in complex communities looks are solos extremely widespread much more than complete signaling systems and that more diverse much more specialized sorry I just did this morning my spelling went environmental effects on the signals these signals can be degraded by the environment can be made unstable we know that for example arcali ph will degrade the signal so this signal can be affected a lot by the environment and that will change things a lot as well many different phenotypes are regulated by these systems ok not all cells produce and respond to the signal if the system is saturated that is really baffling to us we give saturated level of signals but only 5% of the cells are on I just finished and then phenotypic heterogeneity and what I want to say for all these things that you guys are big thinkers we have working models so actually we think we have we can do quite simple experiments to validate maybe some of your simulation, some of your modeling so we are very interested to to get together with you thinkers and modelers that maybe can help us answer some of these questions that are now because in my view this field needs a revival it's kind of quieting down because we find a lot of signals it needs a revival and we really need to understand the real function of this system how it's on and off and the real how it works in nature out there rather than in a very simple conical flask pure culture that has been studied so far up to now it's basically been studied like that ok so I finish so thank you very much thanks a lot Vittorio, we have time for questions I'll start here just because it's closed alright ok, maybe first just a short remark for the case where 5% goes on when you give a saturating signal maybe look at whether there are differences in growth rate in the single cells that distinguish which ones go on and which ones don't go on right so there is a stochastic variation but so the question I want to ask is it seems these cells put out hundreds of different kinds of chemical species as they're growing as you're saying septumizes 600 different so any one of these that couples to regulators inside other cells that can diffuse into other cells and couple to regulators there could act like a sensing signal for the presence of others so then I would sort of almost imagine that almost anything could be a quorum sensing in the sense that you sense who others are there because you see the molecules that others excrete but then in most of your examples there were like one or two families of regulators that were that were basically implementing this so this was somewhat so do you think that this is just because that's where you're looking that's sort of where the light is and that there is a vastly larger number of regulators that are also acting like quorum senses o do you think that there is something some reason that the sort of the same regulators are used for this over and over I mean one of the most studied probably the best studied organism for quorum sensing to the monosaryginosa we are now at six signals let's say we define a signal when you know it is true antibiotics have been shown to be signals ciderophores have been shown to be so molecules which have a biological function for the bacteria like taking up iron or fighting on they've also been shown to have some signaling properties but what I'm talking about here pure signaling the function is a pure signaling function so I agree I mean I agree with you it's much more complex, there's a lot of signals we try to simplify we try to use this model because we know it's a signal we have so we are trying to understand you know how this model works and how this model benefits a community of cells but again you know the Vasodipandora that you're opening is out there and we're aware of it about the 5% the growth rate it's a possibility I'm not a big believer of that because we grow them here in defined medium and you know we think that the growth is pretty well controlled but again it's something we need to look at hi thank you for this talk I was curious about the experiment where you saturated them with the signal like are they grown in plantonic cultures like illiquid cultures and then you add the signal and see in the microscope what happens or did you try also maybe like the first one, so far we grow them in plantonic so we grow them in liquid saturated signal which we believe is it's very well distributed ideal homogeneous scenario and have you tried instead in like inducing the cultures like in other structures like biofilms because I was looking at all these pictures like it looks like the special structure is very important like in these notes we haven't done we haven't done sesal structures we haven't done other media but it's something that we again we're just doing this stuff so we start with the most simple defined way and then try to understand that before we move on to anything else already this very simple setup is creating us a lot of questions 2 very stupid questions so first one how do you know that the signal is saturated actually should be the internal concentration yeah we know we know the kind of concentration needed for the response so here we put far excess and the second one so you said that there are 9 subgroups of solos, right? well in surumon in that particular subgroup and 3 you know that there are 2 HL and the other 6 you don't know well one of them is responding to a plant signal and the other 3 plus 1 the other 5 we don't know but we don't have any evidence but based on we do cartography models so a lot of these proteins have been crystallized together with signal with the HL signal so there's a lot of structure information so then here I collaborate I'm a scientist and we do the modeling so based on the modeling we're pretty confident that 3 subgroups are responding to the HL based on the model also on the sequence in the modeling one of them we know it's planned because we have the evidence the other 4 they don't fit any of the scenarios of the HL or the plant so it might be something else in our view something else when I say something else another signal another molecule one question that I have is so these molecules are really they diffuse through the membranes and can they be used as carbon sources not just as regulators they can be used as carbon sources ok so these molecules are made for example by gram-negative bacteria you've heard a lot of Bacillus subtilis here which is a gram-positive bacteria and one scientist published in Nature actually Yanui Zhang with friend of mine published in 2001 that Bacillus make l'actonase enzymes actually in the case of Bacillus so the lactonase opens the ring but then as far as I know it doesn't eat it so it's like it might be regardless more or less like a defense so to interfere with the signaling of another group of bacteria but there are bacteria especially gram-negative that that will use them in fact even the producers at one point when the quorum sensing let's say when the window finishes like the expression then it starts degrading it for food very interesting so what you discussed the degradation of the signal is a factor that controls quorum sensing you make it a degraded to control it if you have the honor of but then there couldn't be the looks or solos or like I don't know a sixth explanation of the looks or solos related to this that they actually sense it to just he'll eat it why not why not it could be but what I can say to you the concentration these molecules work nanomolar concentration so you're not eating a lot of food here so they work even in some cases even 5 nanomolar concentration they work so it's not that you're going to make a big deal it's not a big buffet so I have a question about the regulator that you deleted I guess can you give some more insight into what you know about it what it regulates etc which one when you flood the system with molecules you don't see anything and then when you delete you suddenly see a very strong thanks a lot for the question remember at the beginning of my talk I said that I have a student that spent a year setting up so this is a repressor that represses the system and it's not a DNA binding protein so we're trying to find out going to your question how does that repression works because I showed you a picture that a repressor binds DNA blocks transcription this repressor is so strong and some US scientists was so fascinated because it's unique also in bacteria in fact we're just going to publish a review coming out in a couple of weeks it's completely unique so usually regulatory there are families in bacteria consisting of a lot of members and this is only a unique scenario we have only this repressor and we don't know how it works a scientist in the US was so fascinated he crystallized and he concluded from the structure not DNA binding, it's not RNA binding so we don't know how it represses transcription so in my view it works with other proteins it makes an interactome so we need to find out that by setting up the whole experiment so we spend a whole year doing all the constructs and now hopefully we're very close to try to understand which protein interacts to do this repression ok and then the second question I don't know if this is relevant but if you don't just flood the system do you see a different response so if you kind of try out different concentrations of the molecule that you think is sensed because it could also be that you somewhere like in between have an optimum for sensing that's a good point we haven't I don't know Misha can you answer that so it's I'm maybe a little bit confused still about this I think along what Eric was asking I hope at least is there a growth speed a growth speed dependency it seems like when you grow to 10 to the 9 cells you're sort of going into stationary and he sort of said that well you know your signal keep the abilities much higher suddenly so you need a lot less signal as well to do something is that something that's happening here right if you just take your cells and dilute them what I believe it's important is that when cells are growing fast and happy they don't do signaling this signaling happens at stationary phase when cells are not growing fast because the reason I say that is because what is regulated by this system are phenotypes related to survival they make things that it makes sense to make them when you are not growing or when you are in trouble so if you're growing fast in my view and data is there that cell to cell signaling is really form sensing behavior social business not happening as much so when you have stationary phase and then again you can have stationary phase at very low cell density so stationary phase is not related to cell density if you have a very poor medium you can have very low number of cells which are physiologically a low state but I believe there you probably have signaling as well so you I mean it's something I need to think about but it's definitely growth phase and cell to cell signaling is very closely correlated if you make movies and you do this when cells are growing happily doubling every 20 minutes you don't see this has it all the time do you have an idea of how many different types of such signaling molecules are used by bacteria the spectrum of bacteria how many such signaling molecules exist what he said a lot but we're really we're not discovering them as fast as we should but there's a lot I'm a believer that every bacteria makes at least one signal at least speaks a language but then it can speak other languages it can listen to languages of others but at least it has its own mother tongue right and it will have and then it might have more languages it might respond to other so it's what we're beginning to see that this bacteria that we studied in detail which is a human pathogen they already found six and what makes it more complicated they're interconnected, they're hierarchical so it's complex so just to continue a bit that you showed an example of where these two bacteria were having a mutualistic interaction now in general when you have microbiomes which contain a large number of bacteria is there a way to estimate this kind of signaling that is going on between how many pairs of species or does one have any idea at all I mean there is cross feeding I think Martina asked that question there's a lot of cross feeding there's hardly any data hardly any data we've written about this we've also written about this because the marvelous world of bacteriology in the last ten years discovered incredible fascinating cell-to-cell communicating systems both contact dependent and contact dependent I don't know if you've heard about the secretion system so now bacteria can make these needles inject proteins into other bacteria they are vesicles they are nanotubes here we have cell-to-cell signals so there are many mechanisms they share metabolites so there are many mechanisms by which microbes can communicate in a microbiome don't go home thinking oh this is the only way diffusable signals contact independent signals is one way there's contact dependent there's a plethora mechanisms and many microbes have all these these bacteria they can inject proteins in other bacteria they make vesicles so there's a lot of different signalling is one and there's a lot of other mechanisms they can complex which one to use when when to switch on one with respect to the other it's something that is extremely interesting and what is the role also in the microbiome we have very little data because as I said as bacterologists we're working in pure culture we need to move away from this start going into the wild as you're saying what is the role in the wild of all these mechanisms hardly any data on that it's not easy to do my question is are there any known cases of coupled quorum sensing and chemotaxes like whether signal could be an attractant or repellent for chemotaxes I understand you right now again is the question that a colleague said not that I'm aware of but it wouldn't surprise me unfortunately this world is we've had a if you look at the we've had a big surge of scientists studying these this topic and and now through funding especially in the US and Europe is now it's not going down a lot unfortunately we need to revive it a little bit any other question also because one of the reasons one of the reasons that I've lost a bit of a lost a bit of momentum because you remember the Trojan horse I showed you it's actually what is actually evident now is that if you're infected it's too late it's too late if you go with a drug that blocks quorum sensing it's too late because quorum sensing is important at initial phases of the infection of the establishment so if you have a biofilm in your lungs for example as in the case of for example cystic fibrosis patient the anti quorum sensing drug will not work it's too late so basically you need to work you need to take the anti quorum sensing drug all the time to prevent the formation of the biofilm so this has lost a lot of interest in terms of utilizing the system as an application if you follow me, okay which is a pity sorry very very little philosophical questions you said great presentation Vittorio you said that the quorum sensing could be a major driver of phenotypic diversification in metagenomics usually we cannot argue when you do the bioinformatics stuff you can find a lot of bacteria a lot of fungi, a lot of viruses et cetera but especially for the bacteria world we did the metagenomics we cannot argue this diversification of the number of species that within a single sample we can find sometimes we can find 600 species 300, sometimes one within the gut within the tongue or whatever and on the soil because you multiply that by also in the soil community so from the philosophical point of view the quorum sensing the looks are so the system could be a major driver for the diversification of the species in the terms because if we should have within a bacterial community two species, three species, four species four strains we could have a diversification an obligation of a diversification to converge within a single function for example survive within an environment why as in your opinion why a single species could not be sufficient to thrive within an environment for example the nose, the olive nose and if the quorum system could be a system that evolutionary is demanding and demanded is evolutionary to to share something within different species to thrive within an environment so it's a sort of trade off to diversify as a bottom up approach or top down and also answer the question of a previous colleague we are now setting up synthetic communities we are trying to simplify a root microbiome we have the data the number of protobacteria, the phyla so we are simplifying a microbiome up to 30, 40 species and we're beginning to work with this it's not easy with the simplified microbiome to try and answer questions like this so this is I think us and other scientists are working with the called synthetic communities in the lab to try and answer some of these questions and trying to look at cell to cell signaling in a more pertinent scenario that reflects to those acting in the wild so this is the way us and others are going we are making small microbiomes and we can start doing some of these experiments but happy to to team up with you guys for ideas, thinking and models and simulations which can help us these other experiments Any other question? Ok, that's thank you