This Week in Microbiology
With Vincent Racaniello, Michele Swanson, Elio Schaechter, Michael Schmidt
Episode 177: Microbial sibling conflict
Aired June 1, 2018
Vincent: This Week in Microbiology is brought to you by the American Society for Microbiology at Asm.org/twim.
(music)
This is TWIM, This Week in Microbiology episode 177, recorded on May 24th 2018. I am Vincent Racaniello and you are listening to the podcast that explores unseen life on earth. Joining me today from Ann Arbor, Michigan, Michele Swanson.
Michele: Hello.
Vincent: How are you? We missed you for a week, right?
Michele: Between now and the last time we talked my son was married in Kalamazoo so that was a lovely event.
Vincent: Congratulations!
Elio: Mazel tov!
Michael: Nice!
Michele: And it’s golf season here in Michigan so I’m happy.
Vincent: That’s what you like. Also joining us from Small Things Considered, Elio Schaechter.
Elio: Hello there.
Vincent: Welcome back. You were here last time, right?
Michael: He was.
Vincent: And from Charleston, South Carolina, Michael Schmidt.
Michael: Hello everyone!
Vincent: I wasn’t too far from you yesterday, I was in Birmingham, Alabama.
Michael: That’s still pretty far.
Vincent: You know in the US scheme of things it’s closer than California, right?
Michael: That is indeed true.
Vincent: Do you remember the New Yorker map of the world?
Michele: (laughs) I was just going to say that you New Yorkers think it’s like really a long way.
Michael: Ah yes.
Elio: There’s nothing between the Hudson River and the West Coast, right?
Vincent: That’s right, you have New York, the Hudson River, and then California.
Michael: That’s why we refer to them as the fly over states.
Michele: Central Park is larger than the Midwest. (laughs)
Vincent: You know at one time many years ago there were not too many people out west.
Michele: It’s true.
Vincent: There weren’t any sports teams, they were all here and then they left (laughs)
Michael: To go to the West Coast.
Vincent: Yeah, people started leaving the cities in the northeast and they moved west and everything else went with them. That poster comes from those old days. But yeah, when I travel I always say I’m close to South Carolina, not really but that’s all right. So I was in Birmingham, Alabama.
Michele: What was happening there?
Vincent: I gave a talk at the University of Alabama.
Michele: UAB?
Vincent: UAB.
Michele: Yeah, great.
Vincent: And if you’re ever there, go to Lucy’s for coffee in the morning. That’s the place to go.
Michele: Pour-overs?
Vincent: Yeah, and Lucy will make your coffee for you. She’s been running the place. She used to have a cart outside on the street and she made enough money to rent a space. It’s really good. I got my picture taken with her and she knows all the virologists that have come through UAB because they all get coffee there.
Michele: That’s great.
Vincent: All right. I think this is our last show before ASM Microbe.
Michael: It is.
Michele: Yeah.
Vincent: Let’s see, today is wow almost the end of May, and then next week, that’s right, and then next week we would normally record on the Thursday the 7th but instead we will be recording on Sunday June 10th, and let me tell everyone the time is 10:30 AM in Atlanta. So we will be in there live, all of us will be there with a guest, and you should come listen to us.
Michele: Say hello!
Elio: Don’t just listen, you can even look at us!
Vincent: They could look at us and I will bring some TWIM t-shirts to throw out to the audience.
Michele: Wow, I hope I catch one.
Vincent: But of course if no one is in the audience then I will just give them to Michele and Elio and Michael.
Michael: I think that we have to put to our esteemed executive director a T-shirt cannon into his budget for next year, Michele, as you’re going to be the president you can put in a T-shirt cannon.
Vincent: You know I looked into one of those.
Michael: You did?
Vincent: They’re not cheap and I don’t think you can bring them on an airplane.
Michael: You probably can’t. Bill Murray is one of the co-owners of our minor league baseball team and the minor league baseball team park is always selling out. And they do all these promotions and they have the T-shirt cannon going, Bill Murray goes out there, Bill Murray from Caddyshack fame and Saturday Night Live. He goes out there and fires the gun and people clamor for t-shirts. Every Friday night when they are in town there’s fireworks.
Vincent: I always wanted to fire t-shirts but I’ll just throw them out.
Michele: We could just bring a few lacrosse sticks. I’ve seen our lacrosse team here at Michigan use those to lob t shirts up into the basketball arena.
Michael: That would work.
Vincent: That’s not a bad idea. I have sticks left over from my kids, they’re sitting in the garage. All right, let’s talk about some microbiology because this after all is This Week in Microbiology and today we have a snippet and a paper, no emails, so we can spend all our time on these papers, and Michael tell us what you have.
Michael: So I picked a paper from the Journal of Applied and Environmental Microbiology, one of the ASM’s flagship journals, and it is titled “A decade of Streptococcus thermophilus phage evolution in an Irish dairy plant”. The paper was authored by Lavelle, Murphy, Fitzgerald, Lugli, Zomer, Neve, Ventura, Franz, Cambillau, Sinderen, and Mahony.
Vincent: Let’s Elio and I tell you how to pronounce Lugli.
Michael: Loogie?
Elio: Loo-lhiey
Michael: I don’t think my tongue is connected like that.
Elio: It’s from Parma.
Vincent: Parma? Yeah.
Michael: he’s from Parma and the paper is from the University of Cork, Cork Island, the school of microbiology. And Parma, Italy, they are pro genomics in the department of chemistry and life sciences, and the department of microbiology and biotechnology from the Max-Rubner Institute in Kiel, Germany, and I picked this paper principally because I think most of our audience will appreciate that phages are viruses of bacteria and can result in one or two behaviors to the bacterium, either death as a consequence of lysis as the phage produces their progeny, or integration of the phage genome into the microbe.
And often lysogeny can confer behaviors within the host, and in the world of fermented products either lysis where the cells are effectively wiping everything out, where the phage are wiping everything out, or lysogeny might result in a bad outcome to the people fermenting the product. And in this case, since we are talking about cheese, they’re interacting with milk and so here the authors were concerned with the phages of Streptococcus thermophilus, as it is one of the most extensively employed commercial starter microbes used widely for the manufacture of these fermented milk products, such as yogurt and various cheese.
In this particular strain, or as they abbreviated throughout the paper as ST, it is used to produce a particular Irish hard cheese similar in style and flavor profile to cheddar, and it is at this particular plant that they study it. It is only produced three months of the year and the rest of the time the plant is producing whey protein, and many of our weightlifting friends out there use whey protein to help put on weight and to bulk up and to do all sorts of other things.
Vincent: Is that what you use to put on muscle, Michael?
Michael: No, I just put on weight, it seems. The reason they are concerned is a phage infection of an SD starter culture may result in an incomplete or failed fermentation and so the cheese may not develop the flavor profile. It may develop a different profile, the texture might not be right, and thus it can have considerable economic consequences to the dairy industry.
Vincent: Michael, when this happens, where does the phage come from?
Michael: The phage is literally in the air and it just falls in because they are ubiquitous. And part of this is, and this is one of the reasons I want people to not only look at this paper for the results that they observe, but to use it as a guide to think about all of the stuff that is coming out about the microbiome and some of the important controlling studies that we must do as we begin to take the microbiome apart when designing experiments, because a lot of the papers in the microbiome world are not considering the viruses of the microbiome. And it’s talked about as a virome but we have not yet seen it, and so I picked this paper principally because it shows you how an industry where the starter culture is important, the behavior of the starter culture is important in terms of creating a product, and in the case of looking at the microbiome and all the other things we want to think about how we need to design this experiment.
So this is a nice, straightforward paper that is rigorously analyzed. And that’s the hallmark of this paper, they have 10 years worth of data packed into this paper, and the other reason is once upon a time I was humbled by a fugitive phage infestation. It was during a phase of my research career where we were using a culture of methylotrophs, methylobacterium at a cell density of around an OD of 10 and the methylotroph I was using had this beautiful color. It looks like a strawberry milkshake and at an OD of 10 it looks like I was mixing up strawberry milkshakes in the lab. And while I was staring at the fermenter, and we were feeding this methylotroph methylene chloride from a vapor phase, what happened is a phage got into it and I literally watched an OD of 10 go to an OD of 0 right before my eyes.
Michele: Wow. So it was a lytic phage?
Michael: A lytic phage came in and lysed it, and of course–
Elio: Instead of having a strawberry milkshake, you had a strawberry soda.
Michael: It was devastating. And so you can understand these cheese factories use an incredible amount of milk and these authors elected to undertake an analysis of the phage-host interactions in order to understand how problematic phages recognize and infect their host bacterium. And as we were talking before we started this podcast, Vincent brought up, this effectively was how CRISPR was discovered. It was because of a phage infestation and two microbes that were resistant to the page actually had CRISPRs. And so what we can learn from looking at phages and other things, it is really pretty clever.
So the paper is straightforward but it required a significant amount of work on the behalf of the authors. It is organized into four parts. Part one they describe how they isolate the phages, and this is something that you might want to do if you are looking at the microbiome or something else. So they give you a method and the method is relatively straightforward, we will get to that in a second. Then they look at the genome organization and again you renew your understanding of the elegance of viruses and how they do things in a logical format unlike the bacteria, where everything is just sort of thrown in with some rhyme and some reason but it’s harder to figure out.
The phage are really good about that. They then talked about tail and tip functions and how they harbor carbohydrate binding domains and receptor interaction between the viruses and host. And then they talk about morphological analysis, and since we payed tribute to Allan Campbell two podcasts ago, these phage that actually infect this particular Streptococcus is actually in the family of COS phages, which COS stands for cohesive ends. And so you have a linear molecule of double-stranded DNA in the phage head and of course the sticky ends upon the DNA going into the cell become circularized and of course it can integrate or it can go lytic.
And so what this work entailed is they took whey samples, and whey is a byproduct of the cheese manufacturing product process. It is the liquid remaining after the milk has been initially curdled and strained, and that is where the phages effectively are. And what this manufacturing plant does in addition to making cheese is they make whey protein. And the way you make whey protein is you effectively spray dry it which means you take the liquid and you put it in the equivalent of a pressure washer and you blow it out and then the decompression as the liquid comes out at the high-pressure nozzle, it just falls out.
So the phage are effectively given a tremendous amount of kinetic energy to remain aloft in the atmosphere of the cheese manufacturing plant, and that’s how they get in to the process of making cheese. The cheese is the classic Irish hard cheese, or the Dubliner cheese from the county Cork, and that’s where the authors are from. It is made from cow’s milk and it is aged over a year and has the hard texture similar to cheddar. So starting in 2006, they isolated a single phage, and they did this via the classic double agar plaque assay. And I will put into the show notes the reference to the method that they used, commenting on how they did it along with the illustration about how the double agar plaque essay is used. And they have a couple of tricks that you need to know about when isolating phage from this particular group, you have to help things along by putting calcium chloride into the plating medium and the soft agar, and the plates have to be dry, and they have to be the right nutrients.
And so they were able to effectively get a number of phages out of the system and in all cases they use the same master stock of host or ST. And it was the parent strain and they continuously obtained this from the manufacturer of this inoculum, so that it was always the same source of the host, so they knew what they got was indeed appropriate. They also then went out and they got whey samples from other plants. And the second approach that they used to isolate phage involved 2,043 whey samples over this 10 year span. And they tested them against a panel of 52 variants of ST, and they asked the question: did any of the whey samples plaque on these particular strains? And they effectively report out that they did 106,000 characterizations.
And so they got a bunch of phage out of the system and one of their most interesting findings is that throughout the testing period of the decade, despite the fact that the strain is only used for at most 1/3 of a year, namely the parent host strain ST1, that the phage are present throughout the year. So where are they coming from? And the answer is the whey protein production, which is present in the plant, is likely the source of the phage. And the phage isolates with minor modifications were characterized by restriction analysis, and then if they differed a little, they further tested them by Multiplex PCR, and if they were really different they used genome sequencing–they show a beautiful series of figures in which they were able to illustrate 17 genetically distinct phages, 8 of which were isolated from the factory, from cheese and whey samples in 2006 and 2008 and 2015. And between 2008 and 2015 they got over a hundred phage isolates that were nearly identical to the first phage they isolated back in 2006.
Elio: Can I interrupt you for a second, what’s the take from this, what have we learned?
Michael: What we’ve learned is that the inoculum starter culture is pretty stable in terms of its phage predilection, and that the phage effectively are adapted to this particular dairy plant, that not these peculiar pages are able to get a foothold into the factory, they continued to isolate ST1.
Elio: Isn’t this kind of predictable, isn’t this what you predict?
Michael: You would predict this, yes indeed, but what they then did, and this is the last aspect of their paper, in order to assess the extent of phage diversity that were acquired from these other factories, because they are trying to understand if an aberrant one gets in and should it be a better infector or it will change the flavor profile, they want it to really understand it. And so they then determine the phage lineages a la what we see with a lot of different species going on in the microbiome. And they only found four different phage lineages, two were very similar to what they found in the factory. The other two lineages were distinct and as you say, what is news here? And to greatly distill the results from the genome organization of their manuscript is that they learned from a focused analysis of the genomic region that encodes the host recognition functions, which is the tail tip. This highlights that the associated proteins harbor a variety of carbohydrate binding domains. Which then corroborates your notion that the phages of this Streptococcus thermophilus recognize carbohydrate receptors at the initial stages of the phage cycle and these carbohydrate receptors don’t seem to change all that much hence the reason I think why we don’t see much of evolution going on.
Michele: So there’s a strong selective pressure for maintaining that carbohydrate binding and any variance would just be lost from the population, is that how we should think about it?
Michael: Correct, that is how you should think about it, and that is basically the paper in a nutshell.
Vincent: Michael, they’re not trying to make resistant bacteria at all are they, right?
Michael: No, they’re just asking who is there.
Vincent: Is that something they might want to do, because as you’ve mentioned, the CRISPR originated from a yogurt plant trying to identify bacteria resistant to phages, because the phages are causing problems in the production, and that’s where they discovered CRISPR. So could they do a similar thing or maybe it’s not an issue in their production.
Michele: So a host mutant? You’re thinking about a host mutant?
Vincent: Yeah. Well, the simplest thing you could do is simply remove the carbohydrate.
Michael: Well here the tail and tip function of the phage protein which harbors the carbohydrate binding domain is the important business end.
Vincent: Yes, so you could remove the carbohydrate from the host or you could just put a CRISPR array in that is known to inhibit this phage if they wanted to do that.
Elio: You can certainly try to make bugs resistant to the phage but the phage is going to mutate again and soon you’re going to have a warfare that has no end.
Michael: Yeah, and so they just wanted to know what was actually going on, and there were principally three remarkable events that they talked about. First is that COS phages predominate in dairy samples where the Streptococcus is the predominant starter culture. Second, phages were abundant in whey protein powder. Third, these ST phages are synonymous with modular exchange in their evolutionary developmental pathways. And I didn’t get into the nitty-gritty of looking at all these open reading frames.
Elio: Thank God!
Vincent: (laughs)
Michael: I did that for you!
Elio: But I don’t know what the take-home lesson is other than this is a practical study about something that is very important to their industry, but I don’t know that I learned anything that I didn’t know before, to be honest.
Michael: But again, this is a major commercial industry, and in the end, longitudinal studies such as this one are important to understand the natural evolutionary processes that are at play in this industrial context.
Elio: Sure.
Vincent: Elio, what I learned is that bodybuilders use whey and they are probably eating a lot of phages.
Michael: They are indeed eating a lot of phages!
Elio: That’s right.
Michael: So I’m sorry it wasn’t as exciting as it should be but it’s a good object lesson of looking at some of the methods and some of the other aspects.
Vincent: I like the title that had Irish dairy plant in it, I think that’s pretty cool.
Michele: (laughs)
Michael: The Irish dairy plant. And you can get this cheese, I spotted this paper a few weeks ago and I was in Costco this past weekend and there the cheese was, sitting in the Costco Dairy case. So I was ready to pick up a chunk.
Michele: I was wondering, and I didn’t pull it out of the methods, whether every time they sampled, did they just pick one plaque of phage or did they choose 10 and make sure that at any one time point there was only one type?
Michael: It wasn’t delineated in the methods itself and I didn’t go to see if there were supplemental methods.
Michele: Because in Legionella surveillance methods it is becoming clear that there is typically heterogeneity in any given population, so if you’re satisfied with just one bacterium and then next time you sample you pick another you may not have realized that there is variety. And you’ve got to pick like 10 so that you sample the whole spectrum each time, and then you can begin to understand what’s evolution and what’s just sampling error, essentially.
Michael: So that’s, in a nutshell, phages are everywhere and whey protein is loaded.
Vincent: The way to make whey.
Michael: The way the whey to make way.
Vincent: Okay, thank you Michael. (laughs) I want to tell you about the upcoming meeting called ASM Microbe, the annual meeting of the American Society for Microbiology. This year it is in Atlanta, Georgia and ASM has a special opportunity for our podcast listeners. Get $50 off registration for Microbe 2018, which is June 7th thru 11th in Atlanta using the promo code ASMPOD. Delve into your scientific niche in eight different tracks. Visit ASM.org and use the promo code ASMPOD, all one word, for $50 off registration. When you go to register you’ll see “do you have a promo code” and you type in ASMPOD. All right, now from Elio we have a paper. What do we have, Elio?
Elio: Alright, I’ll read you the title which is a paper appeared in MBIO, a journal that is thriving by the way. The title is “Physiological heterogeneity triggers sibling conflict mediated by the type 6 secretion system it in aggregative multicellular bacterium.” That is quite a mouthful. And the authors are Vera Troselj, Anke Treuner-Lange, Lotte Sogaard-Andersen, and Daniel Wall. I know Daniel, he is at the University of Wisconsin and does some amazing work with the subject of the paper which is on myxococcus–
Vincent: Wyoming.
Michele: It is actually Wyoming, the other W state.
Elio: –and aggregated multicellular bacterium. So the paper is remarkable in that there is some very nice experimental work and it shows something novel but is also geared towards an understanding of the whole idea of multicellularity in microbiology. So the paper has a beautiful combination of experimental work and some philosophy and I must say I liked it very much. I enjoy this paper. So since we have a few minutes I have to go back and talk a little bit about differentiation and multicellularity in bacteria a subject that does not occupy the minds of this traditional E. coli oriented people that I used to represent, I used to belong to. E. coli is not supposed to be a multicellular organism since E. coli is the paradigm for everything, or at least it used to be. Multicellularity became a subject sort of on the side on the side, however, in one sense multicellularity in the microbial world is all over the place and increasingly we have the sense that most bacteria in the universe are sessile as they sit on the surface. They either make biofilms or something else, which represents a different idea than bacteria going in L growth in a flask and being all by themselves.
So multicellularity in bacteria can mean something as simple as making a fairly nondescript biofilm, although biofilms are complicated and heterogeneous, it is still a fairly simple sort of thing. Contrast this with what happens in the world of eukaryotes where I’m looking out my window, I see palm trees, I see a dog walking around, and bacteria never develop into something as complicated as that, right? They stayed relatively simple. But within the bacteria, there are some which are capable of significant differentiation and these are the myxobacteria. Myxo means slime so myxobacteria is something akin to myxomyces which is slime mold and they’re not of course–
Michael: And they are motile, like gliding motility.
Elio: That’s right, they are motile. And let me say something about that, perhaps the best study or one of the best studies of the myxococci is a bug called Myxococcus xanthus , xanthus which means yellow, and this has been studied enough in quite a few years in some wonderful laboratories and added tremendous amount of knowledge. Myxococcus xanthus is capable of two kinds of differentiation, not just one. One of them is in its motility. It has two kinds of motility, one is fairly straightforward, the single cells follow a trail of slime. This is called adventurous motility.
In contrast to this, there is one called social motility, which is when cells get together in what is called a raft or a swarm and move along the surface doing that. So why are they doing it? So there are two kinds of motility, one is simple the other is differentiated. So they do it because they’re looking for prey. The prey are the bacteria. They eat other bacteria so when a single celled or a swarm of cells finds a certain bacteria, they gobble them up. The swarms by the way are also known as a wolf pack because they act like one.
So this is kind of fascinating in its own way. Okay, by the way, the way they eat the other bacteria is by inducing them to lyse by secreting hydrolysates that destroy the bacteria, and they use the food to develop. This is a very cooperative sort of thing because of the secretion of the metabolites by the dead bacteria is used by the whole swarm. So it is really quite interesting. But when food is scarce and they can’t get any food by swarming around what they do is they aggregate into what amounts to fruiting bodies. Fruiting bodies is a term that is used in mushrooms, a mushroom is a fruiting body of a fungus. These are not fungi but they form fruiting bodies partly because they stick out from the surface and these are seen on dung and soil particles all over the place. They are not uncommon at all. The fruiting bodies of the myxococci can be very simple, like in Myxococcus xanthus it’s really just a hill. It looks like a little hill of cells. Or it can be quite complicated in which case it looks like beautiful forms, shapes which branch with branches and so forth. I will put this in the show notes to show people.
Michele: Kind of like mushrooms
Michael: Yes.
Michele: They have beautiful structures of mushrooms.
Elio: That’s right, mushroom like structures of things or fungus-like. Anyhow so this is social motility and it is a form of multicellularity. Remember the title said the study is on an aggregative multicellular bacterium so aggregative means here that it makes an aggregate of alike cells. So here is a bit of a story. When we think about multicellularity that is in higher forms of life, complicated forms of life, we start out usually with an egg that was fertilized and that is a single cell that becomes two cells, four cells, etc., with all of them identical until something unusual and unlikely happens and that is somatic mutation. So by and large when we start, when you look and contemplated animals and plants, they are all clones of a single cell and that is required for their aggregation ability. It is if you want to aggregate into a structure like the stem of a plant or the organ of an animal, you are going to have cells which are alike. So that is solved in multicellular organisms by starting with an egg. This is not solved in bacteria because the aggregation requires the coming together of potentially different cells, unlike cells, and if you did that you would not have a very successful aggregate because the cells are distinct. They are each going to pull their own way. They are not going to say let’s all pull in the same direction. Some are going to pull this way, some are going to go the other way.
So this is a problem and that is the problem that is addressed in this paper. So I would say that generally speaking one can learn about differentiation by studying bacteria even though higher forms of differentiation are not common, they do exist. By the way, as an aside, these guys bugs that are capable of this kind of differentiation are notable for having the largest genomes in the microbial world. There is one guy called Minicystis rosea which has a genome of 16 million bases.
Michele: Wow.
Elio: That’s better than half of what we have in terms of genes. So why do they all have large genomes?
Vincent: What was that number again?
Elio: 16 million bases.
Michael: 16 million. That’s four times the size of E. coli.
Vincent: E. coli, right, okay.
Elio: And this is the world record in terms of genome size among microbes and bacteria. I hope you’re impressed.
Michele: I am, it’s just a very versatile eater and then this multicellularity must take a lot of their genome.
Vincent: Elio I’m not impressed because our genome is 3 billion bases. (laughs)
Elio: That’s the implication, if you want to make something complicated you better use a lot of genes. But the meaning of that we will let go for the time being so let’s–
Vincent: Let me just say one thing, you know the genome of some flower, I can’t remember it now, is huge, bigger than ours, bigger than the human genome. So size doesn’t always mean complexity, there are some other things involved that we don’t understand.
Elio: That’s right. You’re right but we’re talking about bacteria here. So you know we’re impressed. Some plants have an extravagant number of genes. So, okay, anyhow, so what’s the problem here? The problem here is how do these cells recognize their kin and how do they avoid making aggregates with non-kin? So the experimental setup they use is very simple. They take the bacterial strain of M. xanthus and mutagenizes it to make auxotrophs, that is mutant which are not capable of making some amino acids and they can not grow unless you supply them with amino acids. So they have two strains, the wild type which is capable of growing on minimal media that doesn’t require amino acids, and a mutant which requires certain given amino acids.
Michele: That was a beautifully elegant strategy, wasn’t it?
Michael: Auxotrophs always are.
Michele: Yeah I just thought that was a lovely strategy to control the growth.
Elio: I want to do another little bit of philosophizing because the lab of Wall’s has found another thing which is quite unique among the myxobacteria, and that is when the way they recognize each other is by something called outer membrane exchange which consists of cells binding to each other to effect a give-and-take of outer membrane proteins. And remember these are gram-negative so they have an outer membrane. So this is a way of communicating between bacteria by exchanging outer membrane proteins. I hope you agree that this is interesting and it serves either good things, like it can help damaged cells recover, or not-so-good things like delivering toxins to neighbors but this is an aside, I’m sorry, just to show you that these are interesting bugs I’m plugging myxococcus here.
Michael: (laughs)
Elio: But what we discussed here is something different. The recognition of non self is carried out by a different method than the exchange of outer membranes. It is called type 6 secretion. So what is Type 6 secretion? It is one of many types of secretion which we have discussed numerous times here and it was discovered in Mekalanos’s lab in Vibrio cholerae where he found that strains of Vibrio cholerae will kill other bacterium, and they do it by having an injection apparatus which is characteristic of several secretion systems, not just this one. But this is separate and different and here the apparatus pierces the target cell to deliver toxins or other cargo.
So here this is where the conversation takes place between bacteria, by the type 6 secretion systems. So the authors examined the interaction between genetic siblings that differ physiologically, namely the wild-type and the auxotrophs. So when you put this in minimal media, the wild-type will grow, the parent cell will grow, and the auxotroph will not. So let me quote what they say, they say “we asked whether the kin cells antagonize, cooperate with, or act indifferently towards one another. We found that antagonism occurred between 10 M. xanthus strains only under conditions in which they were physiologically distinct.” In other words, kin likes kin.
Nothing happens if you have just one kind. If you mix the two kinds, as they say, we show that this antagonism is depending on the type 6 secretion system, and they say that we identify the effector immunity pair that mediates this interaction. Let me stay with that for a second. If you are going to produce a toxin you have to do something else. You have to become immune to the toxin otherwise you keel over and die. So when the effector is injected it will probably inject both a toxin and an antitoxin.
This reminds you of the subject of programmed cell death in bacteria, well it should. Okay. Because the system there of toxin antitoxin, just to remind you, it requires that the toxin be neutralized by an antitoxin. But the toxin turns out to be stable and the antitoxin unstable which has enormous consequences. So how did they arrive at the conclusion kin likes kin but it destroys the non-kin, that is the wild-type cells which are capable of growing on minimal media will kill the non growing–let me remind you that non-growing does not mean you are going to die right away, so an auxotroph put in a minimal media will not die to a significant extent for a while, even though it can’t grow. But if it is put together with the wild-type, the wild-type will inject its bad stuff into it and it will die.
So this is, they show very nice experiments that this is a type 6 secretion system, and as expected xanthus carries a gene cluster for the type 6 secretion consisting of 13 core genes and this is required for both killing and immunity. So what is it that it transfers? The putative toxic material is called TSXE. Turns out it consists of proteins involved in peptidoglycan degradation and other hydrolytic activities; in other words, the toxic material will destroy a recipient cell unless it becomes immune. So wait a minute, why isn’t the auxotroph protected by the immune component that it is supposed to be carrying? The reason the authors suppose is that all these experiments are eventually done on the condition of starvation, which is where if you remember what I said earlier, the fruiting body formation requires starvation. So under these conditions there is a reduction in the synthesis of the toxic and anti toxic material, and since the material involved in immunity is likely to be unstable, the level decreases when protein synthesis is inhibited. So when you starve and the wild-type cells inject their toxin antitoxin, the recipient cells or the mutant will die because eventually the toxin remains stable but no new antitoxin is made, and this is the way they see it. So are you with me?
Vincent: Yes, got it.
Michele: Yep.
Elio: So the paper is, I think it is really an amazing paper. It has rich and interesting theoretical conjectures, they let themselves go with a lot of thought, and I like that. They point out that heterogeneity in a population can often be beneficial. For instance, bacterial cultures can carry dormant persistent cells that are resistant to antibiotics so they are heterogeneous with regard to sensitivity to antibiotics. So some are resistant which is good for business; however, in cases where the population fitness depends on cooperative and synchronized cell behaviors such as for social or aggregative multicellular organisms like the ones we are talking about here, here you want cooperation so the population heterogeneity is a challenge that hinders the ability to coordinate behaviors, in other words, if you have cells which are different you are not going to be able to make a fruiting body for it or you may not be able to make a raft.
Michele: So they wipe out the stragglers (laughs)
Vincent: (laughs)
Michael: They’re separating the wheat from the chaff.
Vincent: That’s it.
Michele: That’s right.
Elio: So the way to handle this in this case is simply to kill some of the guys, and a similar situation is found in Bacillus subtilis biofilms where cells make a matrix in order to make a biofilm, and cells that do not produce a matrix are cannibalized by the matrix producing siblings. So this is kind of the story. The mechanism of death is not clear and the mechanism of immunity is not clear. They need to really go in and study that. So right now we’re on hold as they go after this, but the conclusion is that multicellularity offers many advantages. Obviously if you are bigger, if you are multicellular you’re going to be bigger, you can avoid predation by relatively small predators. You can also exploit your environmental niche and develop specialized functions that allow complex and powerful behaviors. In bacteria this does not happen all that much, but when it happens we want to know why. This is a general question: why is it the bacteria did not learn how to become trees dogs or cats?
Vincent: Maybe they did.
Elio: We don’t know, maybe they did, but no.
Vincent: Dogs and cats and trees have—well, dogs and cats have bacteria inside of them.
Elio: They do and they’re part of it but they’re not, you can’t say a bacterium became a tree.
Vincent: No, they don’t make enough energy to make those multicellular.
Elio: That’s a popular view of it. The reason developmental biology happened in eukaryotes is because they had the mitochondria.
Vincent: Exactly.
Elio: And they make a lot of energy, that’s the view that’s held.
Michael: Also, oxygen. Remember oxygen is relatively new to the planet and the bacteria have been around much longer than there’s been oxygen.
Vincent: Yeah but they couldn’t use it as efficiently as a mitochondria can, right, that’s the difference?
Elio: Well, anyhow.
Vincent: Elio, let me ask you a question. So this is done using auxotrophs, right? How do we know that in the real world, maybe there’s something weird about an auxotroph that stimulates this killing. How do we know that the sibling killing would happen in nature with these bacteria?
Elio: That’s a very good question and I think that it’s possible to study this, but the nature–in a way an agar plate is nature also.
Michael: I’ll volunteer that in pathogenesis a lot of the pathogens that attack humans are auxotrophes. They lack the ability to make a particular amino acid or vitamin or some other growth factor and so auxotrophs are quite common as pathogens simply because the host is supplying them. And in the case of these organisms–
Elio: Thanks for pointing it out.
Michael: –growing in the environment, auxotrophy is a normal behavior because the thing that the microbe is interacting with, namely they are eating other bacteria, the other bacteria are going to supply them with a necessary growth factor or the necessary amino acid that they may not be able to make and that will give them the selective advantage. They don’t need to invest the energy into making something complicated and big.
Michele: But they also want to take care of cheaters to eliminate cheaters that aren’t carrying their weight. So I always thought of myxococcus as this very cooperative population of cells but boy they’re ruthless. If you are growing slowly or if you’re not as fit as the rest of us, boom, we’re killing you with a toxin.
Michael: You can give this paper to the graduate students who are not working hard enough and ask them to comment on it.
Vincent: (laughs)
Elio: It’s full of food for thought, shall we say. Very good. So I want to finish by telling you a good title for the show would be what my colleague in the blog, Christoph Weigel, he termed the strategy of the progeny wild-type killing the mutants “kill the loser” as opposed to “kill the winner”.
Michele: Kill the laggards. Yes so this work was led a PhD student in Dan Wall’s lab, Vera Troselj, and she describes her path to science as somewhat unconventional. She earned a bachelor’s degree in veterinary medicine at the University of Belgrade in Serbia where she grew up and then worked as a veterinarian for several years, but then she decided to pursue her interest in research and she applied to the molecular and cellular life sciences program at the University of Wyoming. She is now a graduate student there. She is remembering that during her first year she was able to rotate or sample four different labs: cell biology, cancer biology, microfluidics, and molecular biology. And through that process she realized that microbiology was her primary interest and also her passion. She is fascinated by the complexity of microbes especially by the intricate mechanisms and tools they use to communicate, cooperate, and compete to survive in an ever-changing environment.
So as we saw here, she is focused on how bacterial interactions are influenced by genetic and physiological heterogeneity, and she wants to continue this line of investigation. She is considering possibly looking at microbiome and human health and disease or host pathogen interactions. She is grateful for the friendly, supportive, and stimulating environment that she has had with Daniel Wall’s group. They are at the University of Wyoming and she does have some advice for junior colleagues. She says, never lose your scientific curiosity or sense of wonder and joy of discovery, because it is those emotions that will carry you through and provide resilience through the unavoidable setbacks and challenges that experimental research has.
She also says that being an international student has had its own challenges. She’s away from her family, her friends, her home, and she’s found the best way to cope with that is to continue to cherish meaningful relationships and contact the people you love and to invest effort to build new friendships in her new temporary home. Outside of the lab, Vera enjoys art, books, and fitness and although she is no longer working as a veterinarian, she still devotes some free time to helping animals by volunteering in the Laramie Animal Shelter and also in one of Colorado’s farm animal sanctuaries. So that is our first author, Vera Troselj, who has led this work and this is her third publication in this general area.
Vincent: You said Colorado? I thought she was in Wyoming.
Michele: She is in Wyoming but she said Colorado farm sanctuary.
Vincent: Oh, it’ the name of it, it’s not actually in Colorado, then. Or maybe they are really close because I don’t know my geography, I guess. Let me just check (laughs) Maybe it’s a half hour away.
Michele: Yeah, I’m thinking maybe four corners, those make the four corners.
Vincent: I’m checking it.
Elio: It’s west of the Hudson.
Michele and Michael: (laughs)
Vincent: It’s west of the Hudson, what’s west? (laughs)
Michael: New Jersey is west.
Vincent: That’s right, yes.
Michael: You see it out of your window every day.
Vincent: I do, I do. So here is Wyoming. Now four corners doesn’t include Wyoming, Four Corners are Utah, Colorado, Arizona, and New Mexico.
Michele: Ah, my bad.
Vincent: So Wyoming, however, is north of Colorado. So she’s in Laramie, it’s right near the border.
Michael: Yeah.
Vincent: So she could drive right over and find the Colorado thing that you just said.
Michele: Apparently she does.
Vincent: There you go.
Michael: And Laramie is also home to the USDA arthropod borne animal disease research laboratory, for the USDA.
Vincent: Oh, okay. I haven’t been there. My knowledge of the west is lacking, clearly. But I do know Michigan. (laughs) I know where that is, and it has something to do with the palm of a hand that I can’t remember.
Michele: A mitten.
Vincent: Alright. Thank you, Michele.
Michele: You’re welcome.
Vincent: Very nice to hear some personal stories from our authors. Are you done, Elio?
Elio: I am done.
Vincent: Thank you very much.
Elio: My pleasure.
Vincent: So that is it for TWIM 177. The next one, 178, will be in Atlanta so hopefully you can join us there. If not, you can listen afterwards. And how do you listen? You could go to asm.org/twim or you can listen on your mobile device, like a phone or a tablet, in which case you probably use some kind of an app to play the podcast. You can just search for TWIM, now, you might find other podcasts with the same name, so make sure you are getting the correct one, This Week in Microbiology. You can subscribe, it is free, and you will get every episode automatically. We post about twice a month, so it won’t fill up your phone. So please do that. And also consider donating and becoming a patron of TWIM and all the other science shows that we do. You can go to microbe.tv/contribute. We have a number of ways in which you can help us out including, say, you could give us a dollar a month and that would really help. If every listener did that, boy, we could do all kinds of things. And of course, questions and comments go to twim@microbe.tv. Michele Swanson is at the University of Michigan, thank you Michele.
Michele: My pleasure, thank you.
Vincent: Elio Schaecter is at Small Things Considered, thank you Elio.
Elio: Again, thank you.
Vincent: Michael Schmidt is at the Medical University of South Carolina, thank you Michael.
Michael: Thanks everyone.
Vincent: And I am Vincent Racaniello, you can find me at virology.ws. And we will all be in Atlanta for ASM Microbe as we mentioned and I would also like to thank ASM for their support of TWIM, Ray Ortega for his technical help, if you want to see Ray he will be at ASM Microbe, and I want to thank Ronald Jenkees for the music you hear on TWIM. Thanks for listening everyone, see you next time on This Week in Microbiology.
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Content on This Week in Microbiology (microbe.tv/twim) is licensed under a Creative Commons Attribution 3.0 License.
Transcribed by Sarah Morgan.