 Let me. Victoria and Pete already talk about some basics and definitions of quorum sensing so that I don't have to repeat those. So I'd like to talk about what we are doing in our laboratory in South Korea. We work on plant bacterium, which is called a gloomy. And today, I will talk about the control of primary bacterial metabolism by quorum sensing in the bulk cold area. So bulk cold area cause disease in the rice because rice particle disease and also cause bacteria wilt on many field crops, like hot pepper. And this bacterium produces the toxin, is the notorious toxin, so-called toxopulavine. It's very toxic to plant because this toxopulavine is photosensitized. So under light, they produce lots of radicals. It can be very toxic to rice. So if you go out to the rice field, you will see lots of panicle bright like this. And if you look at the individual panicles, you will see a white thing and sometimes this coloration and darkness. In paddy field, in hot pepper field, you can see a devastating wilting on the infection. So this bacteria infect roots and stems and flowers. So especially this bacterium infects rice flowers. So under favorable weather conditions, they produce lots of toxins and kills all rice panicles. Produce toxins, toxopulavine, and has only one quorum sensing system. And the optimum gross temperature is 37 degrees. And this one quorum sensing system produces two kinds of signals, one is C6 and the other one is C8. And this toxopulavine is flowering. So it's yellow color. And you can see actually the crystals of this toxopulavine on the microscope, very shiny, small crystals. So they produce lots of toxins. OK, so this is some topics we study in our laboratory using percolary glumia as a model system. We study control of primary metabolism, especially metabolic slowing and regulation of glyoxylate cycle and regulation of activated metal cycle and control of bacteria to GST. Second project is quorum sensing dependence cell death. They have very distinct cell degradation mechanism that we have not published yet. And also we study oxalazone formation and deformation, lastly, quorum sensing and genome evolution. Today I will talk about mainly the primary metabolism. For the last 15 years, we published quite a bit of papers to describe the quorum sensing system in percolary glumia. So this bacterium has one luxi and luxal quorum sensing system. It produces two signals. We don't know anything about C6-HSL, but C8-HSL is major quorum sensing signal. And top R is luxal homolog is CHHR receptor. And this tox R and C8-HSL activate another transcription activate tox J. And tox J and tox R activate tox problem biosensitizes. So that tox problem biosensitizes under control of quorum sensing. This tox top R and C8-HSL also activate QSMR. QSMR is transcription activator which belong to ICLR homolog. This QSMR activates many different kinds of genes. For example, type 2 secretion and FLHDC. FLHDC is transcriptional regulator for flagella biosensitizes and catholase genes and oxalide biosensitizes and USP genes. So this is another introduction about percolary glumia quorum sensing. Percolary glumia utilize amino acid as carbon source. So in amino acid rich medium like AB growth. So they produce a lot of ammonia because they utilize amino acid as carbon source. So in wild type, the quorum sensing system is on. So this QSMR activates OVCA and OVCB. This OVCA and OVC complex actually use acetyl-CoA and oxalate to produce oxalic acid. This oxalic acid is being secreted to the medium. So this oxalic acid react with ammonium to neutralize the environmental pH. So in wild type, this oxalic acid acts like a public good. It can be produced from QS positive strains. But in QS minus strain, there's no quorum sensing systems. So there's no oxalic acid. But they still utilize amino acid as carbon source. So they still produce lots of ammonium. So these percolary species, not only glumia, but other percolary species are very, very sensitive to high pH. So they die. So quorum sensing mutant, if you grow them in AB growth, they die after one day because of the alkaline toxicity. So this oxalic acid is key element in percolary glumia quorum sensing systems. We have many questions, but I suggest two questions that I want to deliver today. One is individual cell to restrict neutralization on the cloudy condition as a function of cooperative activity. So on the cloudy conditions, do they really restrict nutritional acquisition? That was one question. And the other one is quorum sensing controls the primary metabolism of individual cells to maintain methoic homeostasis. So we used basically two key tools. One is transcriptome analysis. Everybody does it nowadays. And the other one is metabolomic analysis by C. top mass. C. top mass is capillary electrophoresis top mass. It's very, very different from other type of mass vector analysis. So C. top mass is very ideal mass-backed tools to analyze hydrophilic compounds. So based on our RNA-seq analysis, we found that the PTSD, which encodes phosphate protein for transfer agent, is down-regulated by quorum sensing. So we confirmed our RNA-seq data by QRT-PCR. So if you look at the wild type, this is normalized for the expression. Wild type is warm. This quorum sensing mutant is elevated. If you add the quorum sensing signal to the eye mutant, it's covered to the wild type. QSMI mutant is still a high expression of PTSD gene. This is complementation. So PTSD gene is down-regulated by quorum sensing in percolate glumine. So PTSD is very important for glucose uptake. So we did actual glucose uptake experiment using C13-labeled glucose. So we measured total C13-labeled compound by using C14-MRI. So this is the wild type, 250 micromoles. This is the quorum sensing mutant. This is complementation. And this QSMI mutant, this is complementation. So at six hours, that is just before the quorum sensing onset. So after 10 hours, concentration reaches above 10 to the 8 cells per male. So if you look at the amount of C13-labeled compound, you see more C13-labeled compound in quorum sensing mutant. This is complementation. This is QSMI mutant. So quorum sensing mutants, top five mutant and QSMI mutant, they do uptake more glucose. OK, so if they uptake more glucose, we expect they should grow much faster than the wild type. So when we monitor the growth of wild type and the quorum sensing mutant, we did see the faster growth of the quorum sensing mutant, the green and the pink. They grow much faster than the wild type. So then we were curious what happens when we culture the quorum sensing mutant and the wild type. Do they outcompete the wild type? Yes. This is the quorum sensing mutant, top R mutant now. This is the wild type. So top R mutants grow much faster and better than the wild type. So quorum sensing mutant outcompeted the wild type in the early births. So quorum sensing downregulate glucose uptake. So we wanted to conform other genes that are involved in primary metabolism by QRT-PCR. So there are a lot of genes, but this is some examples like PGK and PYK, NUOV, and this is my ATP synthase and other kinase, NDK, which is nucleoside-diposible kinase. In all cases, the origins are elevated in quorum sensing mutants. So there is a higher level of teaching expression in quorum sensing mutant. And it did complement it by adding the small quantities of signals to the mutants. So there is downregulation of whole variety of primary metabolism genes in percolate gloomy. So if there is such a wide range of downregulation of the genes that are involved in primary carbon metabolism, there should be some big changes in major metabolites in percolate gloomy. So we actually quantified some key elements by CE top mass analysis. This is some part of analysis data. If you look at the glucose 6-phosphate, for example, you see a lot more glucose 6-phosphate in quorum sensing mutant, BGS2 is top i mutant, BGS9 is QSMR mutant. So the unit is picomal potential of the nine cells. So another thing is at the ATP, if you look at the ATP levels, you see almost 3 to 5-fold increase in quorum sensing mutants. So this is a really serious problem in quorum sensing mutants. There is a huge bias in primary metabolism in quorum sensing mutants like the GTP and the PP also. So there is a huge metabolic imbalance in quorum sensing mutants. So this is negative control of primary metabolism by quorum sensing in percolate gloomy. So this is TCA cycle, and this is oxalate cycle. We call it oxalate cycle because the OVCA and the OVCB use oxalic acetate and acetyl-CoA as a substrate. It produces oxalic acid in here. Citrus synthase use same kind of substrate, but they do the conjugation reaction, the enzyme. So the blue color indicates down regulation by quorum sensing. Glucose transport and ATP synthesis oxidative phosphorylation and the PPP pentasposphate pathway and also adductor pathway. And also diatose metabolism are down regulated by quorum sensing. This is another example of down regulation by quorum sensing in gloomy. It's glutamic to uptake. So if you look at data here, this normalized C-13 labeled glutamic contents in micromole. So wild type, they uptake like 1,000 micromole after quorum sensing onset. For quorum sensing mutants, they uptake much more. So if you look at the glutamic uptake gene expression, there's a huge difference here, quorum sensing mutants. These two are complementation. So this is just another example of down regulation of primary metabolism. So if they uptake a lot of glutamate by quorum sensing mutants, there should be some problems in cellular osmolarity. So we measure the cellular osmolarity at different time intervals. After 14 hours, we see huge differences in osmolarity. These two are quorum sensing mutants. So if there is huge cellular osmolarity, something should happen to the cell. So we harvest the cells and cross section and look at the inside of cell under electron microscope. You see lots of hyperhydration here. So this is a wild type. You see intact cell envelopes. These two are quorum sensing mutants. You see lots of hyperhydration. So they do have osmolarity problem and hyperhydration phenomenon. So if we rotate GLTI, which is glutamine transporter, can we avoid the hyperhydration? Answer was yes. So this is the GLTI mutant. There's no hyperhydration. This QS minus mutant along with glutamate transporter. There's no hyperhydration. So quorum sensing mutant have several osmolarity problems. But if you knock out the glutamate transporter, you can avoid it. So glutamate uptake is really key factor to maintain cellular osmolarity. So this is a small conclusion. In part one, quorum sensing act as a metabolic break on individual cells when cells begin to mess. And quorum sensing might have evolved to ensure homeostasis of the primary metabolism of individual cells under crowded conditions. And finally, quorum sensing dependent glutamate uptake is very important to maintain cellular osmolarity in a cooperative population. Now I want to change my subject to positive control. So I was talking about negative control. Now I want to talk about positive control of quorum sensing in Berkeley gloomy. So this is TCA cycle again. When I was talking about TCA cycle and people was laughing at me, it's very old classical stuff. And TCA cycle is dynamic anyway. Why do you study TCA cycle? That's true. But nobody really studied what happened to the TCA cycle in terms of population biology concept. So there is glyoxalate cycle here. And this is the oxalate cycle. The blue is negative control by quorum sensing. But glyoxalate cycle and oxalate cycle are upregulated by quorum sensing. So I'm going to talk about this. So HA is isocitrate synthase gene. And GLCP is malate synthase gene. These two enzymes are in the TCA glyoxalate cycle. So if you look at the gene level expression in the wild type, this is quorum sensing mutants, much lower than the wild type. Also GLCP, same thing here. If you add the signal to the quorum sensing mutants, it recovers or even the higher level of expression of SA and GLCP. And this is some DNA binding experiment to confirm that QSMR protein actually binds to the promoter of SA and GLCP. And this is metabolic rewiring by QS at the branch point of glyoxalate TCA cycle. So we actually measure the enzyme activity of isocitriase. And the other one is isocitrihydrogenase. So if we go back to here, this is the enzyme that are involved in isocitrate to glyoxalate. This isocitrihydrogenase convert isocitrate to alpha ketoglutarate. So here's negative control and positive control. Quorum sensing, negative control, this isocitrate dehydrate activity, I mean the transcription activity. And isocitrate liase is upregulated by quorum sensing. And also this oxalate cycle is upregulated. I didn't talk about this because it's all the stuff. So what happens when we mutate glyoxalate cycle? Why this bacter has glyoxalate cycle? Even in E. coli, we really don't know much about what the glyoxalate cycle does. What we know about glyoxalate cycle in E. coli, it is important for two carbon metabolism, like acetate metabolism. That's the only thing we know. If you look at the text book, that's the only thing you can find. And why there is glyoxalate cycle and why this glyoxalate cycle is activated by quorum sensing? That was the very, very curious question for us. So we mutated glyoxalate cycle and see what happens in terms of cooperative activity. So this is population density. This is the wild type, the blue. They grow very well. They reach over 10 cells per meal after one day. And they maintain good population density even after one week. This pink line is the OVCA mutants. This is oxalic acid minus mutant. They die. They collapse. This huge population collapse because they don't make oxalic acid that's probably good. The pH increase. This is the pH. If you look at the pH, which is up to 10 to the pH line. So on the high pH condition, they cannot survive. But these glyoxalate cycle mutants, they don't die. But there's this amount of decrease in population, this green line. And if you look at the pH changes, pH also increases. But not like OVCA mutant. The wild type, the pH goes up and goes down again and come back to neutralized pH. If you look at the oxalic acid production, the wild type produces lots of oxalic acid. Of course, the oxalic acid mutant do not produce anything here. Glyoxalate cycle mutant produces about 50% compared to the wild type. So what is the connection between glyoxalic cycle and oxalic biosensuses, which is the important for public good biosensuses? So first thing we did was we compared the transcriptional and translational levels of OVCA in wild type and glyoxalic cycle mutant. We didn't see much of difference of OVCA expression at transcriptional level and at translational levels. So in wild type and glyoxalic cycle mutant, OVCA was expressed at same level. Then why we don't have much of oxalic acid biosensuses in the glyoxalic cycle mutant. So this is the oxalic acid biosynthetic activity. This is actually the specific activity we measured. So after column sensing onset, the wild type shows about 40 nanomores per minute per microgram specific activity. But in the glyoxalic cycle mutant, about 20. So about 50% decrease in the glyoxalic cycle mutants. So why is that? What is the mechanism behind this data? So with these RNA signal analysis and glyoxalic cycle mutant and compared with wild type, we saw lots of differences in stress-responsive chaperone and chaperone engines like GroE-S, GroE-L, and DNAJK. OK, this is a wild type. And this is a mutant, glyoxalic cycle mutant. There is a huge difference between these two. So there was good indication that the chaperone might be involved in this business. So we wanted to confirm the RNA data by QRTPCR. This is wild type. This is a mutant. There's a huge difference here. Also, we did the Western blood analysis using anti-GroE-L. This is after 14 hours. This is wild type. This is a mutant. This is complementation. There is a huge difference even in GroE-L expression at the translational level. OK, so we observed that there is a big differences of GroE-L expression at transcription and translational levels. So what is the connection between the elevated expression of GroE-L and oxalic acid biosynthesis? So we tried many, many different possibilities. And we found out that there is a physical interruption of OVCA by GroE-L, all right? So to prove it, we did immunoprecipitation experiment. So we harvested the whole proteins and immunoprecipitated with anti-OVCA and then separated on the STSGR and the Western blood analysis using anti-GroE-L. This is the result. So this is loading control. There's an elevated amount of OVCA, GroE-L here. So if you convert this plot to the data, it's a ratio of IP readings per input readings. There is a big difference between wild type and the quorum sensing mutant. There's a more GroE-L immunoprecipitation. When we submitted this data to MBIO, the reviewers were not happy with this data. So we had to revise it and with this one more experiment. So we actually measured the OVCA activity with the BSA control. This is a non-specific protein. So we did OVCA activity experiment in vitro. And this is kind of control, a non-specific protein. So BSA added a different amount of BSA to the reaction. It did not inhibit much. But if you increase the GroE-L at a different amount, we see dramatic decrease of oxalic acid biosensitizes. So this did prove that this OVCA activity is interrupted by GroE-L. So GroE-L did inhibit the oxalic biosensitivity activity of OVCA. So if we mutate oxalic acid cycle, this bacterium experienced lots of metabolic stress. So what happened? We grew them for a longer time. Do they can survive, or do they die, or what happened to the culture? So we grew them up to seven days and take some adequate spread on the LB aga plate and observe the colony morphology. So after four days, this is the wild type, the small colony. We are seeing the big type of morphology colonies appear. So this is five days, and after six days, or seven days, almost all colonies will look like the flat and bigger compared to wild type. This is the wild type, and this is the mutant. So this is the percent of QSMR plus or QSMR minus. And if you look at the data here, this is the minus QSMR mutant. They went up to almost 90%. So we took some colonies of the big colonies and sequenced the whole genome in the beginning. And we found there is a mutation in QSMR. So this QSMR is under control of the Luxal homolog system in North Korea. Some of them had insertion in the upper reading frame. It's an IS element insertion in the beginning of the upper reading frame. And some of them had big deletion and 780 base deletion and 100 base deletions. So we found these bigger colonies have mutation in QSMR. So under metabolic stress, they have mutation in QSMR. Why? So this is QSMR spontaneous QSMR mutants. So there is an elevated activity in the QSMR mutants. Isocitrate dehydration activity increase. This is a wild type. This is the original client oxalate cycle mutants, very similar. These three are three different kinds of QSMR mutation to the client oxalate cycle. They want to mutate QSMR so that they can express more isocitrate dehydrogenase, because the client oxalate cycle is blocked. So they want to convert isocitrate to alpha ketoglutarate more under stress. So what happened to that kind of QSMR mutants? So we measure the population density at different times. So if we look at here, yellow and purple and green, they die. QSMR mutants, because they cannot activate oxalate biosynthesis. So if you look at the biosynthetic activity of oxalate here, the mutants do not make much of oxalic acid so that the pH increases. So those mutants are very similar to QSMR mutants, which cannot survive after one day. So they try to survive under metabolic stress by mutation in QSMR. But somehow they cannot survive because they cannot make oxalic acid, which is the public good. OK, so this is a conclusion based on that experiment. Quarum-sensing mediated network rewiring is very, very critical to sustain the bacterial cooperation in buccalic lumi. And we identified new roles of client oxalate cycle in bacterial population biology. And this is one good example of how molecular chaperones play important roles in bacterial cooperation. OK, so here again, to write things that we are relatively recently added to this quarum-sensing circuit in here. So this QSMR activated client oxalate cycle but repressed it because metabolism and nucleotide metabolism and also glutamate uptake. All right, so before I finish up, I'd like to thank the postdocs, Eun-hae Gu and Yong-sun Gang and former members of the students and collaborators. Sang-gi Lee is the x-ray guy who did the OVCA structural analysis. Also, Pete Grimberg who collaborated with us about buccalia malei and pseudo malei. And this is the funding agents that we got money from Korean government, the Creative Research Institute. And I will stop here, and I will be happy to answer any questions. Thank you. Thank you.