13:05:13 what we're going to talk about in a way that will hopefully resonate with many of you. And then we will do the handoff and I will give you an example of the larger principles that he will be talking about that comes from my labs research, So with No further
13:05:33 ado, Avi farm Holtz is going to have the floor in just a second and I will just tell you something about him that's really quite I think amazing.
13:05:42 There are many things about Avi that are amazing but in terms of his scientific background there's something really special, which is that he started out as a computer scientist, he went to Princeton as an undergraduate where that was his major, although
13:05:55 he had a very heavy dose of Applied Mathematics and I believe even a certificate to prove it.
13:06:02 Following that he worked at Google, and you know honed his computer science chops but became interested in in biology and as a way of entering this he spent time at the vitamin Institute in Ron Milos lab and also working with many others is he'll tell
13:06:31 doing really phenomenal quantitative biology, mostly from a theoretical perspective but very, very impactful and beautiful stuff. But what is special about obvious that he's not just content to live in the world of theory and interact collaboratively with experimentalists he actually
13:06:40 experimentalists he actually wanted to have it all. And so he took the difficult decision of going to take a PhD in laboratory experimental science at UC Berkeley and Dave savages lab where he really became outstanding at doing molecular biology at the
13:06:58 bench, in addition to being able to analyze things you know from a framework that was much more physical and quantitative.
13:07:04 So I think this is something that is fairly rare to have actual genuine, you know knowledge that's deep on both sides.
13:07:13 So he's a perfect person to help me make a bridge to you guys, I am very much a laboratory scientist and love you will help us tell you about our research in a way that hopefully should be engaging for this audience.
13:07:26 So take it away.
13:07:29 Yeah.
13:07:34 I'll stop blushing in a second.
13:07:36 We'll get to the, to the, difficult to pronounce words in a minute.
13:07:40 So I just, I just wanted to say some things that I think we all know already, but maybe I think in a slightly different way, I guess, and with the purpose of then giving you like a background that I think speaks between the divides like between the chemistry
13:07:56 and biology and the physics people in the room will help set up Diane's work and maybe help you think a little differently about Redux chemistry and natural environments and stuff that nature so the first thing I want to say, I think we all know Terry
13:08:10 talked about this to some extent and lots of people talked about this is that, you know, we do lab experiments typically we do them in. In, ambient oxygen concentrations but the real world doesn't have ambient oxygen concentrations by ambient I mean,
13:08:24 water and equilibrium with with atmosphere with press the atmosphere 21% or two Henry's ball gives you about 300 micro molar oxygen and water to 70, something like that.
13:08:34 at 25 see so I don't reference temperature.
13:08:36 But if you go out in the world and you make measurements so this this is a classic paper where they made a measurement with a fistula that they made into the room and have a sheep, they're reaching into the woman or the sheep are collecting gas, and they're
13:08:47 making measurements of the gas composition, what you see is, so I'm just going to put up what the atmosphere at concentrations of all these gases are, and you see that in the sheep room in the concentrations are very different than the atmospheric concentrations
13:08:58 right there's much more co2 and much less oh two and much less nitrogen, then you would find it atmosphere, simple fact right and i love this paper. Certainly one this is human rectal gas.
13:09:12 What they did is they flushed, I think it was 17 male subjects. Their rectum, with argon, to get a gas composition of the rectum out and then they made him gas composition measurement is exactly the same phenomenon, right, that there's there's more co2
13:09:35 And this is a basic thing that arises when you have a diffusion barrier right if there's something preventing the oxygen from the atmosphere getting into our victims.
13:09:37 Right. Okay, I just I want to emphasize this point so I found this paper that's a lot of these older papers they don't actually report, the data in a form that I can use from scraping plots and stuff so here's a modern paper where they built this gas
13:09:40 and less.
13:09:49 sensor device.
13:09:51 Very impressive work, and they take it out into the field and then they bury the sensors in different depths and they make measurements in the field of oxygen and temperature and co2.
13:10:01 And this I think the data here just really emphasizes the point right that co2 and those who are correlated with each other and there's some relationship with depth in the soil.
13:10:10 Right. And that I think what this says is one that if it was just about oxygen, right, then you would expect to see less co2.
13:10:21 When there is less oxygen but that's not the case right when you see less oxygen you see more co2 that means that there's other terminal electronic sectors in the soil, other ways of making co2 from organic carbon.
13:10:32 That's the first thing I wanted to say about this.
13:10:35 And, and the second point is it is kind of it emphasizes this diffusion barrier issue right that at some at some level in the soil it's harder for the oxygen to get there but the co2 accumulates, and that's why they're there are varying sympathetically
13:10:46 with, I think, I mean, or at least that's my intuition when I look at this stuff I it's not they don't actually address it that way in the paper, but there's quite a lot of data in here and it's a very nice work.
13:10:57 Okay so many natural environments contain some ambient Oh, too.
13:11:07 And I think another thing that we've thought about in this course is that you can get oxygen gradients if you have bacterial metabolism and you have also some source of oxygen in there, those rates have to be balanced with each other, then you can draw
13:11:14 down the oxygen concentration.
13:11:16 But I wanted to just quickly highlight this work from a collaborative, Sergei Vinogradov. I just think his stuff is super impressive, where he shows that you don't need bacteria to get the oxygen gradient.
13:11:27 So, you know, so that the inanimate Environment Matters. Also, so he's developed he spent about 20 years developing these phosphorescent oxygen sensors you measure the posh restaurants lifetime, and the lifetime is related in through quenching to the
13:11:40 amount of oxygen the environment. They do these just incredible calibrations where he showing here, you get the same signal as a function of oxygen in many different media.
13:11:50 They encapsulated these sensors in, in, in some sort of bead and then they're doing calibrations in different media and they get exactly the same results.
13:11:57 These are very impressive physical, chemical tricks.
13:12:01 And here, So, this is really to me striking.
13:12:05 They measure the oxygen concentrations along the intestinal tract of living mice.
13:12:10 And what they see is that the conventional mice conventionally house mice have similar oxygen concentrations along their intestinal tract to germ free mice, meaning you don't need the bacteria to get the, you know, some form of anorexia right that the
13:12:25 the amount of oxygen is lower than ambient and all these cases. And it's lower in the germ free mice in some parts of the body.
13:12:33 Okay.
13:12:34 And they just, they make a very convincing argument as to why this is so here they're taking sequel contents parts of its you know taking matter out of the mouse's digestive tract, they put it in a sealed tube that has some oxygen in it, and then they
13:12:48 put their little beads in there and they measure the oxygen composition of the tube as a function of time and you see that there is in the conventional nice the oxygen decays very quickly because they're there bacteria in there, and the germ free mice,
13:13:00 it the case but more slowly. And they show with some very nice experiments that this is due to some spontaneous reactions that are happening in their oxygen reacts with some components of the stuff that is in the masses just track right so you don't even
13:13:12 need the bacteria to make that happen in other words, we should expect there to be oxygen gradients everywhere in nature and in many environments that we look at.
13:13:21 Not, not a huge surprise I think this is something that we all kind of knew already.
13:13:26 So then want to just talk a little bit about how we should think about or at least how I think about what should happen to metabolism in this circumstance.
13:13:34 This is going to take me a minute but I hope it's worth it. And I just want to say that this is work that I did with r&b, Evan who unfortunately passed away about a year ago.
13:13:43 Much too early.
13:13:44 So read newspapers, really excellent metabolism research.
13:13:51 So what I want to highlight here is what I'm showing you is data, these are measurements of equilibrium constants for carbon Redux reactions.
13:13:59 And if you have questions about what that means.
13:14:03 Ask me, and I'm grouping them by the functional group that is oxidized. Alright, so I'll just go through what kinds of things we're talking about here so you have oxidation of a carbon the old group.
13:14:12 This is like the oxidation of a sugar. Right. And this might be you might think of as the oxidation of a, of a methyl group and Olympic.
13:14:20 Right, so these and these have consistent patterns they have different characteristic Redux potentials from each other. Right. That's just an observation.
13:14:31 So aren't called this the rich get richer meaning that the more reduce carbons have more positive reduction potentials This is chemical phenomenon that I can't explain to you maybe if there's a chemist in the room they can tell us why this is the case.
13:14:44 But another way to think about it is that the more electrons that the carbon atom has this is like as as a complicated concept here but the more electrons that it has the easier it is to add another electron, just confounding to me, hopefully somebody
13:14:57 can explain it to me someday.
13:15:03 You, these are phenomenal logical that people made measurements of Redux potentials which are you know you have a you have an electrode and you try to transfer electrons from electro to the molecule.
13:15:13 And you have to pause the electrode in a particular potential in order to do it you kick you measure the midpoint potential and that's what we report to you on this graph.
13:15:19 So, yeah.
13:15:22 Yeah, exactly.
13:15:24 No no no no it's not a kinetic, this is not this is these are all equilibrium.
13:15:29 And, you know, we talked about auto told us about this this equation for converting the difference in.
13:15:38 in reduction potential into a free energy. Right. And so we just we multiply the number of electrons already constant in the gap. And we get some number, and it should be negative when it's favorable and we remember that when you're going downwards towards
13:15:40 Okay.
13:15:50 more positive potentials that it's a favorable reaction. Right.
13:15:56 So what I want to emphasize is that when oxygen involved all these gaps are very big right when oxygen is the terminal acceptor all the gaps are really big, but for example, in space work where you're using, nitrogen oxides as a terminal acceptor or in
13:16:13 lots of natural environments where iron and sulfur compounds or the or the acceptor.
13:16:19 It makes a huge difference, right because certain reactions are more challenging, quote unquote, and some of them are inaccessible right because there's overlap between the ranges.
13:16:30 Over here, for example.
13:16:32 Yeah.
13:16:36 So, thinking very nicely about this more electrons carbon atom has more.
13:16:42 Could it have anything to do with this naive idea for your you're trying to fill your electron shell configuration and the closer you get to I don't know, eight or at know yeah yeah, it could have something to do with that I'm not gonna make any bets
13:17:02 Okay. I suspect you're right, I don't know.
13:17:06 Okay, so now the other thing I want to just remind all of us and I think it's another thing that we already know kind of is that real organisms they don't just like cover the gap in one step and the way that I drew it like it's going from liquid oxidation
13:17:18 to oxygen that's not how it happens they have multiple steps right, do a bunch of reactions on the lip it and then you enter the TC a cycle then you extract electrons the electrons go through electron transport chain and there's lots of individual steps,
13:17:30 and you know each of these steps needs to be thermodynamic with favorable.
13:17:34 Second Law.
13:17:36 And there, if you have a big gap right there's lots of ways of making it favorable meaning that there's lots of slop in the concentrations of the intermediates and the pH and so on, and so you could imagine moving up and down, either by changing concentrations
13:17:48 are changing some, some extensive variable about the system and everything is still sort of working right.
13:17:54 But then as I move along. Right, so say I'm oxidizing a sugar with iron rather than with oxygen I have somewhat less slop in the system right I can still do it it's still the net reaction is still favorable.
13:18:06 But there's maybe less range of motion, or all the variables of the system.
13:18:11 Then if I do this last thing right.
13:18:14 If I have a limpid and I'm oxidizing it with iron is a terminal acceptor right it looks if I plug the net reaction in to a calculator and in fact we we made this calculator together aren't in a lot night, you can go online and you can make calculations
13:18:27 of what the net potentials are various reactions are.
13:18:31 I can tell you how that works and so on.
13:18:34 It looks like this net reaction is very favorable but the problem is that the first step of this. You have to go to some, you have to perform this reaction here where you take electrons out of the liquid and you give them directly to iron right.
13:18:47 That's like, you know, if iron is my only terminal acceptor I have to do each individual piece of this cascade with iron as the acceptor, and that first step just doesn't look all that favorable.
13:18:57 And so I have to do something like this where I move the potential of this down by altering some variables the system right like have to change the concentrations or something of that nature.
13:19:05 Right. So, I, you know, we can talk about formalizing this intuition, but basically this, this kind of system is challenging to make favorable. and I want to highlight this paper that Diane and I read together by Scott fenders group, where they where
13:19:32 Okay, so how do organisms cope with the ubiquitous low oxygen environments that they find in nature, I'm just going to highlight strategies that we know about empirically from microbiology.
13:19:34 formalize this into a sort of theory for arguing about which organic carbon molecules are going to get oxidized again to get eaten in in nature.
13:19:45 The first one is that there are many organisms that just use very low concentrations about two.
13:19:53 You do all those curses monotonic Lee decreasing. Yeah. You know it, you know spends an ATP to push it up, maybe I could put it into an easier pathway and then it could get four back or something.
13:20:05 So you're you're thinking about the ATP as being outside of the reaction scheme, and just imagine it internalizing the ATP into the reaction scheme and then it's downhill again.
13:20:14 See what time is it has to the right, it's just a question of how you draw the box.
13:20:18 Right. But, but if I draw the box that way then it has to be done.
13:20:23 That makes sense.
13:20:25 Okay, so the first strategy which is very common is that there are many organisms that just, you know, they get by on very low amounts of go to. It can use super, super low mass.
13:20:33 It can use super, super low amount. So here's an example from a guy know Daniel Stolper. He's measuring the growth of Nikolai anaerobic Lee not anaerobic Lee but they're starting with low oxygen concentrations and they're watching it draw the oxygen down
13:20:45 and you see that there is still growth at sub detection limit levels of oxygen.
13:20:50 Right, so they have less than three an animal or two which is approximately the detection limit in the system and they still see growth in that period in this bioreactor.
13:20:57 So, the point I want to emphasize here is just that what anorexia means depends on our limited detection typically we call something anoxic when we can't detect any oxygen, but there are still levels of oxygen that are significant that are beneath our
13:21:13 a nother strategy. Yeah.
13:21:14 NASA growth human growth rate growth you gurus, like that curve on the right hand side, how am I, oh, that was cells over time so that over time. Yeah, the number of cells is increasing.
13:21:22 detection,
13:21:30 Okay.
13:21:31 Another strategy, right, again, like, like the stuff that's fa is doing is to, for example, use nitrate or use Mangan eight or something like that as as a terminal electron acceptor.
13:21:45 So I gave you some examples right there so there are abundant discussions with this and the microbiology literature.
13:21:53 Another way that we all know about right all these can be sort of contextualize with this with this tower is to ferment right and so what fermentation is is using an electron acceptor that's a metabolic product of the carbon source and we can think about
13:22:05 this in familiar terms with this graph that I stole off of Khan Academy diagram right you take electrons off of glucose put them on meds you donate them back to see the ball to hide the form ethanol that's a fermentation classic fermentation for making
13:22:19 beer.
13:22:20 Right, that's what fermentation is. And that's a way of making energy through substrate level phosphorylation making ATP over here without having any extra cellular Terminal Terminal electronic sector.
13:22:32 And the for strategy which was discovered in the last 25 or so years, I think, is what's called extracellular electron transfer, which is reaching out for a better acceptor So one example of this phenomenon there's lots of examples of them is literally
13:22:47 making something that is conductive some sort of appendage of the sale that reaches out and finds an acceptor at a distance, physical distance and it might even ultimately be at the electrons go to oxygen, the oxygen is far away.
13:23:00 Okay.
13:23:02 So, Diane is going to tell you about Scott's paper. That's going to be the meat of what she's going to talk about Scott's kind of like my science brother he did his PhD with Diana now he's doing his postdoc at from Philip so I can only aspire to be as
13:23:14 good as him.
13:23:17 And she's going to tell you about a strategy that's kind of a mix of all four of these things.
13:23:22 She's working there they're working with an organism Pseudomonas Aeruginosa that for whatever reason doesn't really ferment in the classical sense it doesn't like make ethanol doesn't make lactate or anything like that in any substantial way, but it makes
13:23:35 a molecule that Diane will tell you about that it uses to the molecule is a product of. It's a its carbon source that it uses as an alternative terminal electron acceptor so it makes its own acceptor that except are ultimately donate electrons go to.
13:23:52 And it's kind of a fermentation because the acceptor is a metabolic product of the, of the donor. And it's clearly an extra solid electron transfer because electrons are being transferred to the molecule that's outside the cell.
13:24:02 And I just this paper kind of blew my mind because it really emphasized the range of things that might the lengths that microbes will go to to survive in realistic natural environment.
13:24:13 So, I think you'll all enjoy hearing about it.
13:24:16 And with that I'll, I'll give the whole thing to Diane.
13:24:20 All right, thanks so if he can everybody hear me okay.
13:24:24 Can everybody hear me okay. I think we need to wait for you to switch the plugin right now.
13:24:31 Okay, now you're on and you can go to whatever slide.
13:24:35 Okay, excellent. Awesome. All right. Well, that was an excellent intro Avi thanks so much. And so to everybody else Hello, I wish I were there with you in person, unfortunately that's not possible, but I really am excited to share with you guys.
13:25:08 to share with you is, I think, imparting an appreciation and respect for the extraordinary diversity of mechanisms that microbes have to cope with oxygen and Avi just ran through, you know for really of the major categories but I think even there it just
13:25:28 really scratches the surface of what microbial metabolism is capable of and part of the joy for me and being an experimental microbiologist is being able to constantly learn and get new perspectives on very old things.
13:25:42 And so, I am going to now tell you why we titled our talk that we did here.
13:25:51 And in particular, actually I want to talk to you about this word, a gap okay ecological, which has become one of my favorite words, and if nothing else maybe you'll walk away from this talk with a good vocabulary word.
13:26:01 I learned about this word from a friend of mine who over the pandemic amused himself and many of his friends, by sending a word from the Oxford English Dictionary to us every day in an email, and he asked us to send him a page number, just at random.
13:26:19 And on the page that we sent him he picked a word.
13:26:22 And so happily for me on my page turned up the word again okay logical, which means composed of both good and evil.
13:26:31 And I really liked this because.
13:26:35 Okay.
13:26:36 Now I need to be able to advance my slide here.
13:26:40 I think I've changed it a little bit. That's all right.
13:26:44 In the next year, you'll get to see the full.
13:26:49 Hopefully, let's see this. There we go. Now you can see it. So the reason I love this word graphic ecological so much is that it immediately reminded me of our research on extracellular electron transfer.
13:26:59 So let me make that connection for you.
13:27:33 And so, with the.
13:27:36 The caca logical idea in mind. I want to point out that though, you know, typically this is thought of as a rendition of the scream something of horror something is bad for me looking at this, it very easily could be seen as sort of an expression of excitement
13:27:49 enthusiasm of joy.
13:27:51 Recognizing that these pigments actually are probably doing something for the microbes that make them. And it's fun to start to ask yourself, you know, why is something colored and go one more step at to ask, why did they colors here actually change.
13:28:05 And so the story I'm going to tell you today hinges on color change that you can see in this movie that's repeating on the right. This is a movie of a typical culture of the organism Pseudomonas Aeruginosa that we grow in my lab.
13:28:18 This is a ubiquitous organism it's also a notorious opportunistic pathogen that's very important in a variety of chronic infections.
13:28:27 But the name originals comes from the fact that it makes this type of molecule called a sentencing. I'm going to show you it structure in just a moment.
13:28:36 Actually, I'll do that right now.
13:28:38 And the fantasy that renders the liquid this lovely Aquamarine shade, which is what Original Sin means it means copper rust, which has the color of this sort of Aquamarine.
13:28:52 It is due to the Redux chemistry the electron transfer chemistry of a specific variant of the general class of molecules called fantasies, and this variant is known as pi assign and and that's the structure I've drawn for you here.
13:29:07 And what's happening is that it high school density, these bacteria produce this pigment. They excrete it, and it's reduced state where it's colorless, then if you area to as the hand is about to do.
13:29:20 Introduce oxygen. This colorless pigment reacts with the oxygen, reducing oxygen to Super oxide and becoming oxidized in the process. And in this state.
13:29:31 It appears blue.
13:29:33 Now, for many many decades, this reaction was thought to be the reason for the major biological role of these molecules they were viewed as antibiotics because, as you can see here they generate super oxide.
13:29:48 And this is you may know is a broadly reactive chemical that can damage lipids protein DNA, you name it, you name it, it can be havoc on itself.
13:29:58 However, what is also the case and something that had been observed back in 1931 by Ernst free time, who's one of my scientific heroes.
13:30:10 Sadly, this work was promptly forgotten, after it was published, and I'll tell you why in just a moment but in a paper with Leonor McCandless as in Nicholas Menton kinetics Ernst figured out much of what my lab went on to really study for the following
13:30:26 or for my labs history, which has now been over 21 years. And what Earth hypothesized is that in the absence of oxygen these pigments might serve an alternative function he called them accessory respiratory pigments.
13:30:40 And while they don't exactly do respiration per se, what they do do is help cells conserve energy in the absence of oxygen, and I'm going to tell you how that works, as we go.
13:30:52 But one of the reasons that these pigments for sort of forgotten about is because shortly after 1930s You know what, then comes World War Two. Then comes to the discovery of antibiotics from the soil, led by Sir Alexander Fleming, and continued ably here
13:31:09 in United States by Simon Waxman where it became appreciated that excluded products by microbes that you could isolate from the soil and grow on a plate could have enormous potential benefits for medicine and human health.
13:31:23 And here's the classic example of a fungus on the bottom of this plate that's this big white mass, excluding something that is preventing this bacterium above it from growing, all the way up to its edge, as you can see this halo of inhibition, and that
13:31:39 is resulting from the production of what we now know to be penicillin.
13:31:44 And so this was ushered in the golden age of antibiotic discovery, it was done by taking organisms out of their native context, looking at what they made and realizing that under conditions on the bench top.
13:31:56 Many of these compounds could have antibiotic activity and of course you know this has been a major incredibly impactful discovery and was critical to the advance of of medicine in the 20th century and such a profound way.
13:32:11 But what I think is important that we not lose sight of is the fact that these organisms actually hail from the soil, and in the soil conditions are dynamic and in fact, these organisms are microbes and what they're doing at the scale that's relevant
13:32:25 to them the microbial scale, may be very different from what we infer them to be doing when we just grew them up in the laboratory.
13:32:33 And so it's complex because it's, it's also true of course that in nature these molecules are indeed acting as antibiotics in fact, the person who's another one of my scientific heroes, Dr.
13:32:45 Linda Toma show at the US Department of Agriculture in eastern Washington State, she demonstrated with her colleague David Weller as early as 1988, that a particular type of finishing, produced by Pseudomonas species in the soils have.
13:33:04 In fact, the wheat rises sphere, that means the zone of the soil and the vicinity of wheat roots was critical for the biological control of a particular fungus that causes.
13:33:16 Take all disease. And this was really the first demonstration of in-situ antibiotic activity, to my knowledge by anyone. So it was a very important observation, and here you can see dramatically in the middle, this image of wheat labeled the control this
13:33:32 is where it's being taken over by the fungus.
13:33:37 However, if this week is incubated in the presence of a bacterium that can produce the fencing carb oxalic acid, his structure I show you on the right.
13:33:47 It is able to resist being taken over by the fungus. And yet, if you remove the ability to make this molecule.
13:33:56 Then, the fungus comes back in stride. And so you can see here in this very elegant genetic experiment convincing demonstration that it is the production of the specific metabolite that is really responsible for bio control.
13:34:10 So this was a very seminal contribution, made in the late 80s.
13:34:17 And recently, because my lab has been interested in exercise electron transfer and trying to think about how these molecules is free time postulated, you know, might be important to when oxygen conditions go low, we decided to ask a whole variety of questions
13:34:36 but one began with just ecologically how widespread are these type of compounds Is this something that we really should think about that. This is globally significant in terms of their chemical ecology.
13:34:50 And so I'm not going to take you through how this was done, but former brilliant postdoc from my lab Daniel dar did some work that showed us by looking at the meta genomes available at the time, and there were over 800 of them, that when he looked very
13:35:06 specifically for the sequences that would tell us whether or not.
13:35:11 Within this microbial community from which the meta genome was sequenced was there the capacity for an organism to make fantasies and if so, which organism was making it what he found was that actually present in every environment were indeed organisms
13:35:25 that could make these fantasies, and they were enriched relative, you know, to the population, specifically within soils in the rises sphere and so you see those are the green boxes here that are rising above this estimated percentage of the population
13:35:42 of point two 5% with the dashed line showing us that we have an enrichment over like a nine fold enrichment greater than point 5% that are capable of making fantasies within the rise of spirit compared to book soil.
13:35:58 So this is an important molecule, many different places.
13:36:04 And yet, what we want to know is, why are the organisms making it in these environments, is it strictly to serve as a competitor or could it be more nuanced and so for that we've been working hard and we're very much in the thick of this it's really early
13:36:19 days I'll see to develop methodologies that allow us to look at the micro scale within diverse context, both within the rises fairies you see here on the left, where I'm showing you the roots of in this particular case this is amazed plant.
13:36:39 And we know that in the soil around roots such as this, the concentrations are quite abundant of this molecule, up to 1.6 micrograms per root in fact what we're trying to do is localize where these molecules are made where the organisms are within this
13:36:55 root structure.
13:36:58 Again, as I said, this is work we're just starting so I'm not going to belabor this where we've done more work until now has in fact been in the context of human infection.
13:37:07 I'm going to give you a quick tour here.
13:37:09 And I'm going to shoot an example of the way these organisms that make fantasies reside within this environment, and also just to ground truth is it's on a different scale now it's within the micro molar scale.
13:37:24 But here too. We have appreciable concentrations of fantasies.
13:37:28 In this case we have a dominant financing being the blue one I introduced to you earlier pipeline.
13:37:33 So let's take a look, this is going to show you a glimpse a tour through the mucus filled lungs of an individual living with cystic fibrosis. And historically it's been these individuals who've gotten a lot of attention for the role that Pseudomonas Aeruginosa
13:37:49 sadly plays in there.
13:37:51 And then morbidity and mortality.
13:37:54 And so we care to know how is Pseudomonas surviving in vivo okay so here you see, I'm going to stop it now this pink cluster is aggregates of Pseudomonas Aeruginosa embedded within this orange leptons stain mucus and in blue here is deputy staining polymorphous
13:38:11 nuclear leukocytes which aka go by the word neutrophils This is a type of human immune cell that's coming in to try to get rid of these infecting organisms and unfortunately it's not very successful.
13:38:26 Alright so we just got rid of the mucus so you can see this better. And what you'll see in just a moment that comes into view is that this is an environment that has both Pseudomonas originals and pink and also another organism in green, not surprising,
13:38:40 you know, many environments have microbial communities, and depending upon the environment they're organized around each other in different ways.
13:38:49 So, the question of interest to us to start was asking in this environment how quickly as Pseudomonas Aeruginosa or other pathogens how how quickly are these pathogens growing.
13:39:02 And so I'm just going to show you a little glimpse here.
13:39:05 This is work done by a former students Sebastian cop when he was in my lab in collaboration with Alex sessions, talented organic geochemist at Caltech where they came up with an ingenious way of measuring the growth rate of an organism in any natural
13:39:22 environment, by looking at the incorporation of deuterium into specifically particular lipids, that were made to novo by the organism. In this case we're actually using a slightly different technique we're not looking at particular limits per se but just
13:39:53 So in this column here on the left side, you see in red, the organism stained just by a bacterial probe here in the middle. This is a positive control this is looking at the iron counts for 14 and 12.
13:40:14 See, and in right here you're looking at actually the fractional abundance of deuterium.
13:40:22 And what you can see interestingly is despite the fact that these clusters are so close together.
13:40:28 You know, separated by no more than 20 microns or so from one another.
13:40:32 Using this methodology, we can see that one is an active, and the other is active.
13:40:37 And if we were to go further and look at the individual cells and actually quantify the degree of activity as a function, you know, of how much they're incorporating of this isotope over time which we can infer to be a measure of growth rate.
13:40:58 The point simply is that there is a profound range, it varies and oftentimes it's slow, and this is true regardless of which patients mucus sample we looked at.
13:41:09 Here you can see the tick marks for individual cells what their growth rate is and note this is a log scale so it's really really different ranging from three hours on the right to nearly a year, all the way over on the left, and you can see the density
13:41:24 distribution function here smooth.
13:41:28 For each of these patients.
13:41:28 But the exact numbers are not the point. The point is that the majority of these organisms across many of these patients, and really, in general in nature to not just an infection, but in nature.
13:41:41 Most organisms are not doubling very rapidly. Okay, doubling time have less than two days is much more common.
13:41:49 And so what we need to ask ourselves is what constraints growth in situ and there can be many things that can constrain growth of course but one of the most, I think important as obby, introduced to you, is whether oxygen is present.
13:42:04 And ironically, this is even true in an environment like the lung, which is where we take in air to breathe and so it seems ironic that that environment would be oxygen limited, and yet again This reinforces the point of thinking at the scale of the microbe
13:42:21 because the microbes are embedded within sputum, and as the sputum is sitting there, and not mixing.
13:42:31 Very with drawing, you know they're taking down the oxygen faster than it can diffuse and so one can actually do very simple calculations to ask for instance, if you were to model a bronc he lists.
13:42:46 That was filling in with mucus and I'm going to show you in the next image of slice through this tube with different levels of mucus. So here you see, imagine this is the bronc your lesson, as you go down there's more mucus and as you go from left to
13:42:59 right, theoretically were postulating they're more cells.
13:43:04 And we're assuming just very generic respiration rates for oxygen, what falls out of these type of steady state oxygen models, immediately is the fact that you would expect the full spectrum of oxygen depending upon the condition.
13:43:19 So it would be very reasonable if you had low cell density and not very much mucus, or if your density was really low and doesn't matter so much how French mucus is there.
13:43:29 This environment would still have oxygen present. If on the other hand you had sticker mucus and a higher density, you would expect the organisms to consume it much more rapidly than it could diffuse.
13:43:41 And this is borne out when we actually make measurements in this environment here what you're looking at is a tube of sputum on the left, that we collected from a patient at Children's Hospital Los Angeles, and my former technician at least Kelly took
13:43:55 these different types of electrodes and drilled into it.
13:43:59 As immediately as she could after expect duration.
13:44:02 And here are just representative plots of what she would see. And so I'm going to focus here on the oxygen profile. The point is that after just a few millimeters at the top of the tube sputum oxygen is depleted and the remainder is anoxic, or at least
13:44:20 the levels of oxygen are not detectable with our electrode.
13:44:24 Okay.
13:44:26 So I hope this makes the point that at least at the. millimeter scale, we have a lot of heterogeneity and oxygen. But I think what is even more striking is to actually realize this is true at the mic Kron scale as well.
13:44:40 And I love this is my favorite slide in the whole talk because it's funny. This is a gift that. Come on.
13:44:49 Yeah, go ahead.
13:44:51 So if there's not enough oxygen for the microbes. How come the patients are still alive if their blood vessels are on the other side of the mucus, great question because the mucus itself gets coughed up and jostled around, and oxygen can make its way
13:45:07 into the epithelial cells you know between the mucous sloshing, and one of the very most important treatment regimens for these patients is every day, they have to wear a jacket that literally just holds them to help them cough or expect rate as the clinical
13:45:25 term is this mucus up. And so again it's just a question of scale.
13:45:30 But if you're a microbe, even within mucus that is being coughed if it's sufficiently viscous and this very viscous this mucus.
13:45:38 The local micro environment may still be utterly devoid of oxygen.
13:45:43 One other quick question from the deuterium measurements. It looks like there's no alternative electronics adapter for the Pseudomonas in this environment to oxygen but not necessarily the determine and measurement is simply showing you how quickly or
13:45:58 slowly they're growing it's just a measure of an apple ism it's agnostic to what other electron acceptor might be in the environment, and we'll get there and just a second.
13:46:09 But, though, as I'm going to suggest one option.
13:46:13 Maybe fantasies. Another very good option. Maybe night nitric oxide or its oxidation products nitrate and nitrate and and the reason for that is that the neutrophils part of the way they attempt to kill these pathogens is that they have, they make nitric
13:46:32 oxide and nitric oxide can then in this environment, wind up getting oxidized back to nitrate and my trade.
13:46:41 All right.
13:46:42 Thanks for the questions, any others or should I keep going. Yes, so hey Dinah have a question, given. So can you because you measured how stupid that oxygen gradient is course, and can that observation that explain the early observation you had which
13:46:59 a tool, you know clusters of microbes there. Maybe 20 microns or less apart for the ones. Yeah, I think, probably for being honest, that probably not that would have to be very lucky if that if that image was like just on a knife edge of where they'll
13:47:17 be talking to each ingredient dropped. Although it's possible, I wouldn't say it's probable.
13:47:22 I think that what is making those two at that scale which I remind you is here I can show you.
13:47:28 You know this scale was between these guys this is here's the scale bar 10 microns.
13:47:35 I don't know, is the short answer. It's a mystery, it would be impossible for me really to find out if all I have is this image, but what I what I might argue, what I could plausibly argue, based on other data from my lab is this guy is really proximal
13:47:51 to this massive neutrophils on the left which might be admitting a concentration of super oxide and nitric oxide that is sufficient to take this guy out, whereas this cluster is far enough away that possibly it's not as harmed.
13:48:07 And it could be that there is an entirely different explanation for this. So, I think, you know, we're entering into the realm of other speculation but it's it's a good exercise to try to come up with all the possible explanations that might be relevant
13:48:22 here.
13:48:24 And the answer is I, I don't know, for this particular image but my goal in this talk is to help you all appreciate that. One of the many things that can limit microbes in a whole diversity of habitats and skills that might surprise you, is oxygen.
13:48:41 Okay.
13:48:43 All right. It's okay for me to keep going.
13:48:47 All right, I'm gonna go for it. Okay, so it's it's good that I actually just talked about all the neutrophils surrounding these Pseudomonas biofilms you can see that here on the left in a slightly different image taken from the same environment.
13:49:02 But what I want to emphasize about this picture at the moment to you is just how densely packed these pink Pseudomonas cells are. And so now, in the middle of this and black and white.
13:49:14 I have a different version of the Pseudomonas file phone that was grown. This is a picture for using scanning electron microscopy, and and the gift is just to make a humorous point this is I actually don't watch Game of Thrones but if I think if you do,
13:49:30 maybe you'll recognize this is my students Scott was really into the game of thrones for a while. And so he gave me this. And the reason that I like it so much is that I find it illustrating this existential crisis that really must be faced by any organism
13:49:45 in the middle of these massive biofilms where they're surrounded by you know other organisms exactly like them, that are struggling for the same resources.
13:49:56 And that's not an easy situation to be in.
13:49:59 And so, the great thing about microbes, is that they've had a long time to figure out how to solve it.
13:50:05 And before I get into my favorite solution I just want to take one step further and really impressing upon you, not only how many souls are present in these biofilms but how heterogeneous their micro environment must be as we can infer from taking a snapshot
13:50:21 of what they're doing. Okay so this goes back to my excellent former bisect Daniel, who is now about to start at the Weitzman Institute of Science in Israel his own lab.
13:50:36 So happen in the fall and you can ask. avi to tell you about this poster behind him later.
13:50:42 But what Daniel did while he was in my lab was developed a new method that he calls Parsi fish which stands for parallel sequential fish. I'm not going to go into the details about how it works if you'd like.
13:50:55 The papers out on the bio archive.
13:50:58 But the upshot is what he figured out a way to do was leverage a methodology that had been honed in the laboratory of my colleague lon Chi, in order to be able to actually measure.
13:51:13 Concurrently, the abundance of over 100 transcripts at the single cell level in the Pseudomonas biofilms okay and so what you're seeing here is just in the middle black and white, that's just a picture of the cells here on the right he's using a segmentation
13:51:28 algorithm, which was going to allow him then to quantify the transcription of profile in in the cells, And then analyze it. Ok so again I'm not going to go through the technical details of how this works because it's way too complicated and really not
13:51:45 the point for this talk, but what I want to impress upon you is that if you look at this, you know, I might have looked at this and thought, oh, they're probably all doing basically the same thing it's not very thick they're just attached to the surface
13:51:56 how different Can it really be.
13:51:59 And I would have been really wrong.
13:52:02 Because what Daniel was able to do was, apply statistical method called the you map, which stands for uniform manifold approximation and projection, which is a way of taking the expression space that he can measure quantitatively through images and map
13:52:19 it into a phenotypic space, and he did this with over 150,000 cells, and what he was able to do was defined different physiological you map clusters that he could then map spatially alright so that's what you're looking here you see all of them and their
13:52:36 glory it's kind of like modern art here to make a little easier on the I'm showing you just select the maps of some of my favorite physiological state if you will.
13:52:47 So in blue cells that are primarily engaged in transcription of processes that tell us that they're doing fermentation in yellow de nitric vacation, which is the pathway where we start with nitrate as an electron acceptor, and it gets rapidly converted
13:53:04 to different in oxides. And then in red my favorites cells that are producing fantasies.
13:53:12 And just as an example of the D nitric vacation here what I'm showing you is what this label comes from is the fact that we have all these raw reads for over 100 different genes that we can cluster and do statistical analysis and ask Okay, what is preferentially
13:53:27 and rich in the cells. Here you can see an example of one of those jeans near s, which is in the DNA clarification pathway.
13:53:34 Okay. And here's an example of two of the finishing biosynthesis genes. And I like to say is that this demonstrates the fantasies really are everywhere, and that's why we should focus on them.
13:53:46 That's just a joke. The The point is that there is diversity here at a level that to me is really quite striking. And I think the gets raises the challenge to all of us to figure out, You know how is it that the micro environment is changing so much at
13:54:04 the small spatial skills, and to be able to develop methodologies that will allow us to actually quantitatively measure the environmental parameters at these skills, and that's something that I'll be seeking out to do in his postdoc in my lab which is
13:54:19 a very challenging task but I think he's the guy for the job, and it's going to open up a lot better ability for us as a community to contextualize microbial activities going forward.
13:54:32 If we can simply say here's how much oxygen is present, you know, here's what the pH is at the micro scale. That alone is going to be quite transformative shopping with a quick question.
13:54:42 Yes, please. Um, so in the middle panel here I guess I'm trying to understand what we should find unusual about this this heterogeneity. Should I be thinking that all of these cells without seeing these pictures would have been doing all three of those
13:54:57 fermentation identification fusing processes at different times or do you think these are sort of spatially localized populations that are doing picking one and staying that way for for a long time.
13:55:07 Yeah, so I'll go back to this image to give you my answer this question be curious what you and others listening to this talk would say my gut feeling when I look at something like this is I asked myself okay.
13:55:21 Is this an environment that is probably chemically and physically conserved maybe because of just the wrong word uniform is probably the better word, and very naively I would have said yeah you know they're all attached to a glass slide.
13:55:36 They're not that well developed this is young biofilm there's surely plenty of oxygen that all of them are seeing. And since my personal worldview of microbes is very much shaped through the lens of microbial metabolism instinctively I would have thought
13:55:49 Oh, probably all of these guys are engaged in some type of aerobic respiration, and they're pretty uniform.
13:55:58 And that would have been my naive view. And so to answer your question, when I see this and I recognize that oh my goodness, you know, within 30 microns, here this circle here, the length of it is about 30 microns by my scale.
13:56:13 This little cluster is primarily doing the nitric vacation and yet if you just extend the ruler, you know, to the left or right or below, then you see, you know, different programs going on below it you see this cluster fermentation, you know, to the
13:56:31 right you see a mixture of, you know, got fantasies going on there.
13:56:36 For me that was shocking because I would not have expected those differences for this type of relatively what I had thought would have been a relatively flat biofilm to be engaged in metabolisms that are so distinct.
13:56:52 Does that answer your question. Yes, thank you. I think there's one more question after me.
13:56:58 So I just want to understand how experiments were done by so these will not patient the samples, these were growing from a population that may be exponential cells and grow on top of about Yeah, we're right in the face.
13:57:08 Right, exactly. So this was done in two different media in lb and then in the medium be called synthetic CF speed of medium that mimics the composition of the CF speed up, but this is a very reductionist experiment here Our goal was just to honestly to
13:57:27 figure out the method, more than anything we were trying, Daniel was trying to come up with a new way to do what's called spatial transcriptome x, which had never been done before on bacterial populations.
13:57:41 So that was our only goal but what our hope is now going forward is that with this amazingly powerful technique will be able to actually apply it to much more complex environments, to actually apply it to speed on or to apply it to microbial communities
13:57:58 in the rises fear for instance, this yeah this was very very reductionist, and that's why I urge you with the paper is going to come out next week it's going to be published, not sorry not next week, next month sometime in mid August, but right now it's
13:58:13 on the bio archive and look it up because it Daniel did it in a very very careful job to try to develop this method, I see. So then the question is, in early growth phase, do you expect that you won't see the heterogeneity.
13:58:29 Is that correct yeah yeah okay so here's the thing well this is what I don't have time to show you but it's in the paper, you would expect right okay so for instance and not even early growth phase yes early growth phase, whether it's in planktonic or
13:58:44 attached to a surface style, you know, maybe I would think so sale potentially you have edge effects you might have ways of intuitively I would have thought that would have if there was going to be heterogeneity I might have expected more when it was
13:58:56 attached than what I thought was a seemingly well mixed planktonic culture.
13:59:01 Probably the most stunning thing in some ways of Daniels work is if you go to the paper and you look at what happens over the course of the growth curve.
13:59:10 The degree of phenotypic states that you see at different stages going along a batch growth curve, are really amazing.
13:59:19 And in a way, it's surprising and in a way it's not surprising, because it's not a key misstep, it's a batch growth, and in batch you have the nutrients evolving and time.
13:59:29 Right. So I think if you step back and you ask yourself how is the environment the local environment, whether it's in a liquid culture, whether it's in a station and attached community.
13:59:45 How's that local micro environment changing in time. That's the way you're going to be able to make sense of how heterogeneous the transcription on response, turns out to be.
13:59:53 Does that make sense.
13:59:56 Can I ask another question since long guys on this paper.
14:00:00 So there's a lot of inherent stochastic city already in gene expression, if you look at stem cells and even the most conditioned material, media you'd see something like this with local founder backs, little patches, I think, by.
14:00:15 So how do you, you keep emphasizing the micro environments, how do we know that it's not just diversity.
14:00:23 Great question. Yeah, great question. And, indeed, I'm sure some of that is going on.
14:00:28 So if you go to the paper I don't want to get bogged down in this right now because I got that I wanted to talk to you guys about that as much more in my domain of expertise.
14:00:38 But that is addressed very explicitly in in the paper and I think it's probably better for me to just leave it at that for now if you don't mind.
14:00:46 All right, but it's a point we'll take it.
14:00:50 Okay. Is it all right with all if I, if I now go into the fantasy story.
14:00:56 Okay, I'm gonna go for it. Alright, So, we were just talking about batch culture.
14:01:01 Here's an example of batch culture, this is looking at the growth of originals over time here in these black diamonds. And the point simply is that if you're measuring in a batch culture what you'll find is that at this late stage of growth as they're
14:01:19 entering into what we call stationary phase.
14:01:23 This is when the fantasies get made. And the timing of this is well understood it is because the biosynthesis of these metabolites is in large part under quorum sensing control.
14:01:37 It's also under other things, but quorum sensing dominates that at this point when oxygen is disappearing from the culture. That's when we get these metabolites appearing.
14:01:48 So, this idea is I said the idea that these metabolites could maybe replace oxygen and serve as accessory respiratory pigments was first articulated in 1931 by our tree time, he was thinking about this in the context of rat tumors actually not microbial
14:02:06 biofilms. And so, sort of the small but important conceptual extension that my first graduate student Mario Hernandez and I made. When we were starting my lab was that actually the physiological context where this really matters for the producer may be
14:02:19 when they're forming communities that are have a high cell density, and they're, you know, within the core of these communities, they're, they're unable to access oxygen that that would be a potential limiting nutrients.
14:02:34 Okay, so, so without idea in mind I want to now spend the rest of my time talking about what we've come to understand about how this works in the context of a biofilm and, and this is what I mean by fantasies are a gap okay ecological.
14:02:52 It means that they are both good and evil at different times if you want to be simplistic about it.
14:03:01 And yet it is the duality of the toxicity at an early stage, and their ability to serve as a conduit for electrons at a later stage that make them a metabolic Lifeline that allow these organisms to be able to remain viable in biofilms, as they develop.
14:03:22 Okay. And so, again, the notion here is that fantasies are molecules that get made it a particular stage as bacteria are developing into a critical density at early stages, they can actually be toxic when there's some oxygen present in this environment.
14:03:41 Indeed, that's a true fact about their reactivity they do indeed generate reactive oxygen species. But as these biofilms develop over time, and oxygen is consumed at the periphery faster than it can diffuse into the interior.
14:03:57 Then there becomes a more nuanced role for these metabolites that allows the interior cells to conserve energy. Okay, so that's the overall idea and and this is just stating this explicitly that it is the ecological nature of these molecules that underpins
14:04:16 the fitness of Pseudomonas Aeruginosa populations in vitro, and in situ and I'm going to illustrate this by talking about biofilms.
14:04:25 But, you know, we can talk more about how this might work in the larger world.
14:04:31 We wrote a, an essay where we were speculating.
14:04:35 A couple other postdocs and my lab crew das Drummond Darcy macros. That would be fun to discuss with you later if there's time. Okay, so let's focus on the very reductionist case, the simplest case we could do, which is to grow biofilms in the lab.
14:04:50 As you know, a population.
14:04:53 No mixed species here is just Pseudomonas Aeruginosa and try to figure out what role fantasies play in this process of biofilm development.
14:05:03 So from my laboratory many years ago we had shown that if you make a mutants that can't make sentences that's what this delta fuzz means it's genetically altered strain that is unable to produce these molecules.
14:05:16 And we compare the shape of the biofilms that it makes to what we call the wild type, that means the natural version of the strain, as it was from the wild.
14:05:29 What we see here is under these particular conditions which are ones where bacteria are grown in what we call a flow cell where they're allowed to attach to glass and then develop into these kind of dome like looking structures here.
14:05:47 Over time, as nutrients are flown by them. What we see is that if they can't make fun of scenes they are unable to make these thicker biofilms, whereas if we add back to the solution.
14:05:59 The missing by a sign and then that in part restores their ability to develop into a thicker film.
14:06:07 And in addition, what we know and what many, many other labs had observed is that if you if you stay into these biofilms with different stains one that could in blue here below, show you where the cell biomass was.
14:06:22 And in the middle here in yellow dye that is specific for extra cellular DNA, and then we overlaid them here on the right.
14:06:30 What's very clear is that there's a lot of extra cellular DNA and these biofilms.
14:06:35 So this is just setting the stage as a, as a fact. There's a lot of exercise their DNA in these biofilms, and without fantasies biofilms don't develop okay so that's what we knew and we wanted to.
14:06:52 I tried to see if we could understand that better.
14:06:55 So let's see if I can advance my slide here.
14:07:03 My computer is being slow on me. Sorry about that. There we go.
14:07:08 Alright, so, bringing the specific hypothesis that I articulated earlier that these both sort of evil and good consequences of fantasies is a function of oxygen in the environment, play into these observations that I just showed you that they're necessary
14:07:27 for bio from development and that there's a lot of E DNA and biofilms, we developed kind of a two part hypothesis, which was that it early stages where there was a little bit of oxygen.
14:07:39 And the fantasies could react with it.
14:07:41 Under these conditions there toxicity promoted the license of cells, and that the DNA that was released at this early stage became a scaffold upon which the bacteria could client.
14:07:54 And I want to be clear that that idea was, was certainly not one from my lab, this was one that was out there in the literature through work of Cynthia Whitchurch Mike man failed at all, and others.
14:08:08 But we were putting it together now in a way that connects it to the whole developmental process with the fantasies.
14:08:14 But that later again as oxygen goes away, these fantasies are interacting now with DNA in a different way. And in fact that the DNA that's in the matrix might be important for retaining these fantasies and facilitating extracellular electron transfer.
14:08:33 Okay, so that's the hypothesis so let's break it down in different steps. Let's start with the first step.
14:08:38 If we're able to remove fanzines during an early stage of bio from development where there's plenty of oxygen, does that impact how these micro colonies form and is that related in any way to the fact that there's a DNA in them.
14:08:56 So it's a good question how can you selectively perturb How can you knock out fantasy and specifically. And so this is possible if we think back to where I began my talk about their ecological context and the fact that fantasy and producers come from
14:09:15 the soil, and knowing that fantasy and producers come from the soil, a former postdoc in my lab Kyle Costa thought that if finishing producers are in the soil, odds are in the soil, living in their vicinity somewhere or organisms that can eat fantasies,
14:09:30 and that they might even do this as a defensive move.
14:09:33 They won't go into all the details there I'm just going to fast forward through a lot of hard work that Kyle did Kyle's now a professor at the University of Minnesota, leaving his own lab.
14:09:44 But what he showed was that there indeed was an organism, he isolated and Mycobacterium, they could eat these fantasies, and after developing a genetic system in this environmental isolate and doing a lot of detective work he identified a particular new
14:10:00 enzyme pod a that stands for the finishing oxidative de methylation because it's the methyl pious sign in here. And the way it does this is really neat biochemically it winds up, reducing the Pio sign and and creating reduced one hydroxide fantasy.
14:10:20 And then the oxidation of that methyl group leads to formaldehyde to be a product.
14:10:27 Okay. But, forgetting the biochemistry, for the moment the key is that we had an enzyme in hand, that could take out pi assignments specifically selectively and this is an enzyme we understood very well.
14:10:41 And we could make an inactive variant because we had solved the crystal structure and we knew the residues that were critical for cartel assists. And so in the data I'm about to show you next what you just need to keep in mind is that we've isolated an
14:10:54 enzyme.
14:10:56 We've purified an enzyme from this bug that eats fanzines. And we've also purified a different variants that is inactive but otherwise it's, you know, it looks exactly the same.
14:11:06 Okay, so what we were then able to do was this experiment of asking if we add this enzyme potty at an early stage of biofilm development, what's the effect.
14:11:18 And so here qualitatively at the top, you can see the effect.
14:11:23 Visually hopefully it jumps out that the one on the left this panel here on the left has a lot more cells there stand here in Dappy by blue.
14:11:32 That's what the wild type looks like this particular image happens to be where we added inactive today, it looks just like the wild type it doesn't alter anything.
14:11:42 And then on the right, what you see is a lot more blackness which means that at this stage the biofilm isn't developing nearly as well.
14:11:50 And we can quantify this by just measuring the surface coverage. Okay, so this is an early stage, and most of these biofilms are, you know, not very thick, they're attached, mostly to the surface forming these micro colonies.
14:12:04 So if we want to find the brightness and measure the surface coverage we can approximate how much biomass is there. And what I want to walk you through is just a few different conditions that really clued us on that there's this relationship between fantasies
14:12:21 and DNA that's potentially very important. So on the left, this is the amount of surface coverage by the wild type.
14:12:26 Next, in white is the amount of surface coverage of a fantasy mutant.
14:12:31 Next is the amount of surface coverage when we add inactive potty, and it is statistically indistinguishable from the wild type.
14:12:42 And then, you know, the parallel experiment where we add active potty. And that's just in black that's statistically indistinguishable from the fantasy music.
14:12:51 Okay, so all that fits our expectations. These two at the end, are really the important ones that taught us something new.
14:12:59 And what we did was we compared what would happen when we added the inactive variant of potty plus DNA to these developing biofilms to what happened when we added pod a there was active and DNS at the same time.
14:13:17 And prior work from the Mansfield lab had shown that when they added DNA in their hands, it led to, you know, significant diminishment of the ability for the cells to build a biofilm.
14:13:30 And so the fact that we didn't see any difference. There was no greater effect when we added today to DNA, whether it was in the active or the inactive version, very much suggested that the effect of these fantasies was in a pathway that was linked to
14:13:46 the production of the DNA. And this makes sense because we know that these fantasies can trigger so license. And so this you know reinforce the idea that early on.
14:13:57 It is through these toxicity, the toxicity and fantasies that we're getting some of this DNA here, and there's a lot more data I could show you but I am going to skip it for now, just take my word for that we and others have demonstrated that fantasies
14:14:13 promote license, when you've got oxygen around.
14:14:15 Okay, so here's where I think it gets even more interesting. And that is asking as they develop what happens when oxygen starts being depleted in the interior.
14:14:26 And that's where fantasies come to the rescue as street time predicted almost a century ago.
14:14:33 Okay, so what we know about how extracellular electron transfer with financing works, I'm going to now show you here and just walk you through this as a cartoon and then I'm going to talk about how this actually works in the biofilm.
14:14:48 So this cartoon is meant to help you understand how fantasies, promote energy conservation. And I'm going to start by just reminding you of how the aerobic electron transport chain works in most, you know, organisms including ourselves and our mitochondria.
14:15:08 Here you have to membranes because Pseudomonas is a gram negative bacteria and they have to membranes, all that really matters is to just remember that as in our mitochondria, there are a series of electron carriers embedded in the membrane that are able
14:15:22 to pass electrons through the electron transport chain, and some of these carriers are able competently to translate quite protons, out of the membrane and that leads to this electric chemical potential gradient around the membrane that can be harnessed
14:15:39 to do work. So for instance, driving the oxidative phosphorylation of ATP of may have the phosphorylation of ATP to make ATP, right this is energy dependent, it is driven by the influx of protons.
14:15:56 Okay, so that's the ATP synthesis.
14:15:59 What is much less appreciated, is that you can have this weird kind of hybrid form of fermentation, and after say electron transfer, and ultimately you even need oxygen as Avi was saying this work kind of combines everything in one that supports energy
14:16:15 conservation so let me now just walk you through that.
14:16:19 So what we have shown through research this was done by my former student at the glacier and currently my lab Johnson, Jackie is carrying this on easily, is that there is a fantastic pathway within Pseudomonas where pirate eight being converted to acetate
14:16:35 is coupled to substrate level phosphorylation okay so I'm keeping the fermentation.
14:16:49 Quite simple and just showing you the fact that there's this pathway that leads to ATP to be made using substrate level phosphorylation. In order for that to happen.
14:16:52 However, we absolutely, and this is true in any fermentation. We need to have an oxidized co factor around and in this case we need to have any D plus.
14:17:00 And so there has to be a process somewhere in the cell where we're generating enough led to keep this flux going.
14:17:07 And the way it happens under these conditions, is that any DHS oxidized via an electron transfer scheme that we're still sort of sorting out.
14:17:18 But we believe it's coupled to the membrane, in some way, that balances that in precisely the Redux pool, and that it's fencing dependent. So, this we know that in order for this from a native pathway to proceed it absolutely requires fantasies, and it
14:17:34 requires their recycling.
14:17:37 Okay, so how does it sell recycle fantasies what synthesizes them. We know that it expects them to the outside through machinery whose name is not important.
14:17:46 Once these fantasies are outside, they can react with a variety of things in the environment, including oxygen, of course, but also they can react with other electronic sectors like iron three or manganese for.
14:18:03 And in this way they get recycles. All right, you can have them react with an electrode and they can get recycled. All that matters is they get recycled and these electrons get dumped to them, and then they get excluded.
14:18:14 They give up the electrodes they come back, so on and so forth. So this is what we mean by extracellular electron shuttling. Can I ask a quick question.
14:18:24 Please come.
14:18:24 outside the cell is there is there some obvious metabolic reason for that to be retained inside the cell to do deal with the accumulation of reducing equivalents and NADH, they need to be re oxidized so the question is, you know, how are they going to
14:18:44 get oxidized, and by sticking around within the cell, you know, the only way they could get oxidized is if you had an accident that was also coming into the cell, but we know that these fantasies are made when other accidents are depleted.
14:19:00 And we also know through laboratory measurements that they're actually physically excluded from the cell.
14:19:09 And therefore, in terms of both their timing of the production at high cell density.
14:19:14 The fact that their regulation is when accidents are low, and unavailable to the cell, that, and the measurement that they just accumulate outside in large abundance.
14:19:25 That's why we're driving it this way.
14:19:27 Does that answer the question. Yeah. Thank you. Okay, great. Any other questions on this before we move on.
14:19:35 You know where I'm drawing where they're getting reduced in the cell that's a little bit more of a conjecture it's also very possible indeed we have some evidence that they can be reduced in the cytoplasm too.
14:19:45 But what is very clear is that there's machinery that helps them traffic in and out of the cell, and that extra cellular Lee external to the cell is where they're oxidized.
14:19:57 And I can show you and I will show you just a second a lot more data about that.
14:20:02 Okay, if there's no other questions I'm going to now move on.
14:20:05 We doing okay on timing.
14:20:09 You can go ahead, we've got maybe another 10 or 15 minutes if that's okay that's fine yeah okay so let me try to finish up so now the final part of this is to try to help you understand how it is once these fantasies are outside of the cell How do they
14:20:23 actually move around or how do their electrons. More to the point, how to. Had they move around.
14:20:30 So, a key set of questions to our model that fantasies are important for sustaining the interior relied on a couple of assumptions that you know weren't terribly well found it in one was that the fantasies weren't being lost in space, and the other was
14:20:50 that fantasies could be recycled sufficiently quickly, that it could sustain metabolism and we'd never really tested this and so this was the goal of my former students Scott for his PhD.
14:21:04 So he first set out to figure out what the concentrations of fantasies were in the biofilm matrix and whether they were retained. And if so, how. And then even harder thing to try to measure is the rate at which electrons effectively were able to be cycled
14:21:22 through the system, a measurement of extra sale electron transfer, and whether or not the financing recycling rate in the biofilm is faster than its physical loss from the biofilm.
14:21:33 So, let me take the first one, and just show you the data.
14:21:37 The bottom line is that different fantasies are made by these cells, and they have different chemical properties, this one on the left is an anti on it's negatively charged, these two are neutral.
14:21:48 And when we take biofilms that we grow on top of auger. And that are separated from the auger by a membrane and we quantify how much fantasy is present in these two different volumes.
14:21:59 What we see is that the two neutral ones accumulate in the biofilm, whereas the negatively charged one partitions equally.
14:22:08 And that actually makes a lot of sense because we know there's a lot of DNA in these biofilms that's negatively charged and so it would be thought to propel a negatively charged compound.
14:22:20 And we decided we would actually make a measurement in vitro of the affinity for these fantasies binding to double stranded DNA and turns out that you know those affinities actually correlate with the ratio of these particular molecules partitioning into
14:22:37 the biofilm versus the auger so the ones that have higher affinity for interpolating into DNA, they're the ones that this is pi assign and this is the best one, its present most in the biofilm versus the auger and PC.
14:22:53 On the other hand, doesn't bind DNA and it doesn't have a preference.
14:22:56 and it doesn't have a preference. Okay, I'm gonna move forward this.
14:23:00 For the sake of time.
14:23:03 Another question we could ask is, does the DNA, other than binding these fantasies can facilitate electron transfer through the DNA, and while that might sound like a really wild idea.
14:23:16 This is the life's work of my colleague Jackie Barton at Caltech, who has developed methods to measure what we call DNA charge transfer. And so this was done in collaboration with her blab was a former postdoc admins, say, who helps got do these experiments.
14:23:33 And what they did was an algae chemical technique called cyclical symmetry, where what you do is you have an electrode, to which you are able to associate and tether a double stranded bit of DNA, to which at the end, we have co violently linked, a fantasy.
14:23:56 That is for you know this purpose of PC and this is, you know, the one in the middle, it's able to interpolate it's not as good as pile but for synthetic chemistry reasons it was a lot easier to actually couple it to the DNA that pile.
14:24:12 the point. And what they had demonstrated proof for through, you know again decades of research in her laboratory is that when you have a system like this that you backfill in such a way that the lecture active species cannot come into contact with the
14:24:25 electrode.
14:24:27 You can measure a difference. If there is DNA charge transfer in the amount of current that's able to flow.
14:24:36 And these experiments, as a function of whether the DNA is well matched here on the left shirt and blue or mismatched that a mismatch strand will not be able to conduct these electrons as well, because the pie stacks that allow the electrons to flow,
14:24:51 you know are not as well aligned.
14:24:54 And so what you see here in experiments that were done in this very very controlled in vitro system, in the absence of oxygen is a measurement where we're plotting the current as a function of the applied potential where we're sweeping from more oxidize
14:25:07 potential on the left to more reduced potential on the right.
14:25:10 And what you see in the blue line is the trace of how many electrons in the system by measure of current are flowing into the fantasy.
14:25:25 They saturate, and then as we go back now to oxidizing potentials. Here you see them coming off and back into the electrode.
14:25:29 And you can see that there is more of this flow that's happening with the well match DNA, then with the mismatch DNA under these conditions, which are really single turnover they're limited by the amount of PCs in the system and the density of DNA that's
14:25:44 tethered in these experiments.
14:25:47 Okay.
14:25:49 Now, what's really neat. Is it if we perform these experiments. Under toxic conditions where oxygen can readily we oxidize, the fantasy and at the end.
14:26:05 Here you see a dramatic change where there's a ton of current being generated as we're going and the reductive direction, because these electrons can continuously like keep being dumped off on oxygen and it keeps being accepting it.
14:26:14 And yet when we go back, the electrons aren't held on by the fencing and so we don't go down into, you know, the electrode itself isn't receiving the electrons anymore so that's why you just see this unidirectional amplification of a peak but not in the
14:26:28 opposite oxidative branch. And the difference you're striking between the well match DNA the mismatch DNA and the control here that's well matched but doesn't have the fantasy tethered at the end.
14:26:40 Alright, So how does this work in a messy sloppy system.
14:26:44 When you have DNA in the biofilm, that was a very hard thing to do.
14:26:50 And so this was God's crowning achievement of his PhD was to attempt to do this and specifically to measure extra say electron transfer through the biofilm matrix.
14:26:59 And to do so he used these devices.
14:27:03 Here you see him in an optimistic moment.
14:27:06 This is a hard problem. And it wasn't always as optimistic as he is here.
14:27:12 But he persevered and he really did something amazing.
14:27:17 With a system that was created through a great collaboration with Lenny tender who's an excellent electric chemist at the Naval Research Lab. And what Lenny and his colleagues had done was to develop something called an entire digital electronics array
14:27:30 or an idea for short, which is this thing here, it's hanging down to spend it into these cells where we cultivate the organisms, and just zoom in on it what it contains it are two different electrodes that can be independently controlled the generator
14:27:45 and the collector, and they are interwoven they are literally interdicted like combs. And on top of them over time grows a biofilm.
14:27:54 So now let me show you what it actually looks like. So here it is so here's on the left, this is the generator calm. On the right, the collector and here now are off of those two base electrodes, the branches that are interwoven.
14:28:07 And on top of this develops this diminish of biomass and red and he DNA in blue, and it's highly a heterogeneous.
14:28:19 And that makes interpretation of everything here really very challenging, but we're going to do our best. So what I want to emphasize is that there are 130 electrodes in the system, 65 pairs of generators and collectors.
14:28:31 And here's what the biofilms look like if we zoom in.
14:28:35 So this looks similar to what I showed you before and other slowing saw biofilms where you have, you know, a mass of a biofilm collecting here, you can see that it's riddled with a DNA.
14:28:52 Merge here you see on the left the DNA middle cells.
14:28:52 If you go, just to the surface of the IDA again you can see the same staining patterns in DNA and the cells are present everywhere.
14:29:09 And now, just zooming in to an individual generator and collector pair here. I hope you can see just what really what's going on at least at this particular spot.
14:29:13 It's highlighting individual cells and they're a meshed within this DNA matrix. Okay, so I'm almost done. And what I want to now do is just show you a couple pieces of data and then give you the punch line, and then we'll stop.
14:29:29 And so the question that we're building towards answering is, what is the rate, the apparent diffusion rate if you will of the electron in the system how can we get a handle on that.
14:29:39 And is this enough to sustain metabolic current.
14:29:43 And is it due to fantasy.
14:29:44 So let's take a look first at whether fantasies are playing a role in just simple metabolic current. So, for this we can ask a question, and just use only one set of these electrodes.
14:29:55 Here I'm going to call it the collector electrode, as the electron acceptor, and this isn't an environment without any oxygen, and we're going to feed them and electron donor in the form of organic carbon, and we're going to ask his current that we measure
14:30:10 that is coming to this collector.
14:30:14 That is ultimately driving from the oxidation of organic carbon and the passage of electrons to the collector is this fantasy independent. And so here what you see is the profile of current over time in yellow, we don't see any current in a mutant, that
14:30:29 doesn't have fantasy, they can't pass it to the electrode here in blue, what you see is this kind of bumpy shape of current going up over time for the wild type.
14:30:43 And I can tell you that the reason for these pumps, is because of temperature fluctuations in the room over the days in which this experiment was performed, which is a nice sort of independent sign of life.
14:30:56 And now here is where while it's not as dramatic as the wild type we nevertheless see a rise and current, and it's also exhibiting you know this coming and going as the temperature in the room is changing.
14:31:08 So, what was clear was that when electrons come from oxidation of the carbon by the cells metabolic current can be measured in two minutes financing dependent, a different way of asking decoupled from metabolic current but just using a much faster way
14:31:24 of doing this these experiments can be done, you know, on the order of seconds to make the measurement where you're basically treating the biofilm like a material.
14:31:33 It doesn't even need to be living is, we can pin it to fantasies in a different way where we can ask the current that is flowing in this case now from the generator to the collector is that fantasy independent.
14:31:45 And we know we can test this by sweeping the potential from the generator and asking at what potential. Do we see current starting to flow. Okay so here we're going to sweep to from more positive on the left to negative on the right, potentials.
14:32:00 I'm going to show you now.
14:32:03 What's happening here in blue, we're going along, we see electrons start coming off and now we get a big rise here.
14:32:10 And this is happening at a potential that we know to be the midpoint potential through calibration with just the pure molecule of pi assignment.
14:32:21 Here, with a fantasy and mutants. We don't see that happening, and now when we add back purified pi sign and again we see it. And the reason this is just a mirror image is because we're looking at current that is going out from one electrode and into
14:32:33 the other from the generator to the collector.
14:32:37 other from the generator to the collector. So, sorry. Just one quick question the mutant biofilm has slightly different spatial structure is that correct, yes it does, which is a very good point.
14:32:48 That's correct. And so, its ability to retain the fantasy and is not going to be as good as the wild type because it's not going to have as much DNA.
14:32:58 And this could affect this electron transport on the DNA presumably, totally. Absolutely. That's a great note and I think it's, you know, very much is consistent with the fact that you see when we add back pi sign into this fantasy mutates.
14:33:13 It does restore current flow but it's attenuated.
14:33:17 Great.
14:33:18 Okay, so the final thing is, and I can tell you in more detail if you want later how we actually measure these diffusion content constants.
14:33:30 But what we were able to do with these watch chemical methods were to separate the term for the, what we call D loss that is what is the rate at which pi scientists lost from the bio film system, you know, coming off through physical diffusion from the
14:33:58 three dimensional biofilm matrix into solution, versus what we call the apparent diffusion of can think of as the electron and the system, which, you know, from electrochemical theory has to be a function either of physical diffusion AKD loss.
14:34:16 Plus, potentially, another term of some type of mediated diffusion the diffusion coefficient for self exchange reactions, if you will.
14:34:29 And so what you see here is if you look at the biofilm, certainly the gap is significantly greater than the de los for quite a few biofilms.
14:34:38 And so if you look just at that you might think, okay, that is evidence that in these systems, there may indeed be a role of self exchange reactions mediated possibly by DNA.
14:34:51 It's possible, where it gets a little hairy, is that if we do a blank, where there's no biofilm, and we compare using or electrochemical methods these different techniques for making a measurement of the apparent diffusion consonant for the electrons
14:35:06 in the system the app versus the loss here now we see it's the same.
14:35:10 So that leaves us with the state of the unknown here, which is that what we now know is it financings indeed are retained in the biofilm and they retained by DNA.
14:35:22 We know that financing recycling within the biofilm is faster than its loss. What we have not yet conclusively demonstrated is whether or not the fact that it's faster than it's lost in the presence of the biofilm is due to the, the mediation of electron
14:35:38 transfer through DNA, and speeding it up.
14:35:43 Or, if it's simply that you have physical diffusion going on within the biofilm and what's happening is that pipeline is being retained and not lost that tracks it in the helps make everything work.
14:35:54 And what's very very possible, is that it doesn't have to be an either or. It can be that we have both of these things happening and in fact if I were to bet, I would say that's that's likely.
14:36:06 And because this is such a heterogeneous and mixed and sloppy system, you know I would never expect there to be 100 hundred percent one or the other. We certainly don't have a direct conduit of DNA between every you know electrode site within the system
14:36:21 and the next which is what one would need in order to really, you know, be able to have a true separation of the rates of DNA charge transfer which are orders of magnitude faster than pure diffusion.
14:36:34 And so I think the truth is that, As usual, life is much more complicated.
14:36:39 But regardless of the mechanistic gory details what I think is hopefully important for this audience to keep in your heads as you ask yourself questions about microbial ecology, is that life is really spectacular at coming up with creative solutions.
14:36:57 And that mystery I've told you here with one particular metabolite these fantasies that we've studied a lot with one organism may well be much more generalizable, there are literally thousands of bacteria that may create accepted metabolites of different
14:37:11 types of whose structures I show here. And what I think is kind of existentially poignant and cool is that it is the duality of the effects these molecules have on the cells that produce them both good and bad that in the end, I think is what allows for
14:37:28 the populations and the communities to be structured, the way they are. So I'm done.
14:37:36 Here is the acknowledgments.
14:37:38 And I'm highlighting in this little sun, members of Team fantasy. And I won't take more of your time to call out each of them every one of them, but they're an incredible group.
14:37:50 avi should deserve sort of a little star here but he and others are broader than just fantasies. And, and I'll just say, I am fortunate to have really an incredible group of students and postdocs working with me and I am really happy to answer any questions
14:38:07 if there's time remaining.
14:38:09 Thank you so much.
14:38:17 Here comes a question. Okay.
14:38:20 It was neat to see how useful DNA is in this is it clear where the extra cellular DNA is coming from the exercise of DNA is coming from the cells that likes.
14:38:31 Yeah. So in this case it's coming from, you know, different cells within this population that lies, and if you mean, which specific cells within the population.
14:38:43 That's a great question and a current postdoc in my lab, Georgia Squires is actually pursuing this for her post doctoral project she wants to really understand when and where individual cells do different things and biofilms, and why it is that they do
14:38:59 it where they do and how that impacts the development of the whole community.
14:39:05 Maybe a follow up to that question about the extra seller DNA in the specific case of the biofilms that lack the ability to produce bio Sinan and don't therefore have the DNA.
14:39:17 Is there a way where you can either add the enzyme that degrades bio Sinan, but add extra seller DNA yourself and see if you can supplement the DNA, without having the pyre sign and president does that rescue the buyer from a particular point of development,
14:39:32 at which point and this electrode.
14:39:40 Yeah, nice that's really cool experiment.
14:39:40 We'll have to keep that in mind, we haven't. We haven't done that. But that is a nice way of separating out the feature of early on, it being important in maturation but not electron transfer.
14:39:51 I should I should know that other investigators have shown that in other bacteria license is important for bacteria to colonize and form these micro colonies and in some of those organisms they don't make fantasies.
14:40:08 So you can you know look to other organisms as corroboration of the fact that the DNA is important at this stage, even for organisms that don't necessarily make fantasies.
14:40:20 I guess the sort of the related question was, like, if you do the outback experiment with DNA, you can restrict the size of the DNA that you provide and ask if a particular link scale is needed for this sort of antenna stage that you need.
14:40:42 Yeah, nice. That's an interesting idea. So, I mean, I think that that's a great idea. How long does it need to be to make a difference, and well I think the answer would be what is the spatial scale over which you're trying to pass electrons
14:40:51 And so being able to yeah added of different sizes that's interesting, I'd have to map the scale of the DNA helix into the scale of the microbial biofilm, but I really like where you're going with that.
14:41:08 Oh.
14:41:08 Yeah, go ahead. Yeah, I've done that was fantastic.
14:41:11 So my question is this sort of balance to the heartstrings set up but that this DNA released as well as a lot of extracellular poly saccharine. Could that be part of the story as well that's question a question be is.
14:41:27 So most of what you've shown is with you know Pseudomonas and Palestine in a fantasy. But the story could be a whole more complicated if you have, you know, complex community releasing a lot of these so you, you could you can speculate on you know with
14:41:44 the whole community be sort of controlling the setup.
14:41:49 Thanks so much for both of your questions let me really quickly just show you data for the first regarding the xo poly American substances that are present in these biofilms besides DNA that's a really important point because it's very plausible, you
14:42:04 know, a priori that these other poly sack right other other xo polymers, and in this case what we know it's kind of nice because our organism only makes one other really important, extra polymer and that is this polyunsaturated called pill.
14:42:20 So we just do a quick genetic try and ask, is the retention linked to Pell yes or no. And the reality is, know if anything when you get rid of pill pipeline is retained better.
14:42:32 And this makes complete sense because Pell is known to actually bind at night. And so when you get rid of pal you've got more binding sites for pile. And so, actually going forward we're collaborating with Matt carsick who knows much more about these
14:42:46 And so, actually going forward we're collaborating with Matt carsick who knows much more about these extra polymers and it was Pseudomonas to tease this apart.
14:42:51 Back to your other question though which I love, which is important. I certainly it's really exciting to try to think about multi species and how different microbial communities are going to respond to fantasies, as they're made and and we're taking baby
14:43:13 steps into this right now. But that's precisely direction we're ultimately driving at.
14:43:26 We hypothesize and this was in this paper that I alluded to, you might find it interesting so we'll see if I can find it again. Yeah, this Keystone metabolites This is a paper then we put out as an essay it's publishing Current Biology, where we really
14:43:35 talk about it and it's very predictable because fantasies are such nice metabolites because we actually while there's a lot more to learn we know enough to be able to conjecture regarding what it is that an organism would need in order to not be harmed
14:43:52 by them. And what would make an organism particularly vulnerable to them and how their vulnerability would be impacted dynamically as environmental conditions change.
14:44:02 And so it's actually setting up in our heads a goal for both experiment and modeling, because we had some very clear predictions as to what might happen, depending upon which organisms we're going to mix together, and it is, you know, for the future for
14:44:22 us to do those studies but that's, that's really what we want to now begin to do. So thank you for that question.
14:44:26 Constant.
14:44:26 Thank you so much. Let's thank the speaker one more time.
14:44:33 Okay, I'm going to send the local participants to their cookies and it looks like Keisha has one question maybe he could just ask that to Diane over zoom, if that's okay.
14:44:43 Sure. All right, thank you. Thank you.
14:44:49 Hi Keisha.
14:44:58 I can't hear you.