Developmental Biology of a Simple Organism: Bacillus subtilis
Transcript of Part 2: New Research on Multicellularity
00:00:04.20 Hello, my name is Richard Losick and I'm a professor at Harvard University 00:00:10.11 and this is the second part of a three part presentation on 00:00:14.25 the developmental biology of a simple organism. 00:00:18.18 In this part, I'll be telling you about multicellularity in a bacterium, 00:00:23.09 the spore-forming bacterium Bacillus subtilis. 00:00:27.16 Bacteria have traditionally been thought of as solitary creatures 00:00:31.16 that go about their business on an individual basis 00:00:35.14 but increasingly we've come to recognize that bacteria can form 00:00:38.16 complex, multicellular communities known as biofilms. 00:00:42.21 I'm going to tell you about biofilm formation in Bacillus subtilis 00:00:48.28 where you'll see that the ability to produce a complex community 00:00:54.06 is linked to the process of sporulation. 00:01:00.05 The first topic that I'll cover is the formation of these communities 00:01:04.29 and a visualization of spore formation taking place in aerial structures. 00:01:11.11 Then I'll tell you about how these communities are held together, 00:01:16.04 what's the basis for their architecture? 00:01:18.05 And you'll see that there's an extracellular matrix 00:01:20.17 that glues the cells together, kind of the mortar for building these structures 00:01:25.18 and that the synthesis of this matrix is governed by 00:01:29.19 an intricate regulatory circuit that I'll describe. 00:01:32.24 And then finally, we'll look into a biofilm and we'll see that it resembles a tissue 00:01:40.11 with different kinds of cells in different places and with dynamic changes 00:01:44.22 in which different kinds of cells either grow or diminish 00:01:48.27 in their relative abundance in this tissue-like structure. 00:01:52.17 Well, that B. subtilis was capable of forming architecturally complex communities 00:01:59.29 was missed for many years for the following reason. 00:02:03.10 In the many years since this bacterium was discovered, 00:02:07.17 itâ€™s been worked on in the laboratory and, as a consequence, 00:02:11.20 over time, itâ€™s inadvertently become domesticated. 00:02:14.15 And so the standard laboratory form of bacteria don't form robust, multicellular communities. 00:02:22.18 Itâ€™s as if we've bred this out of the bacterium over time. 00:02:26.03 Here, for example, is a colony of B. subtilis on a plate 00:02:30.07 and you can see it forms a relatively unstructured colony. 00:02:33.23 And here is a culture...a standing culture of cells in which the bacteria 00:02:39.14 have collected as a very thin film at the surface of the culture. 00:02:44.08 But once again, not much architecture there. 00:02:47.03 But now if we go back to a wild strain of the bacterium 00:02:51.06 we see something that's dramatically different. 00:02:53.19 In the colony you can see a rich architecture with thick veins 00:02:58.13 of cells and other kinds of structures 00:03:01.06 and likewise, at the air liquid interface, a thick mat of cells known as a pellicle forms 00:03:09.03 that also has a distinctive architecture with all kinds of detailed features. 00:03:14.20 Let's look more closely at the biofilm structure and in particular 00:03:22.20 I'd like us to focus on the outer edge of the colony where you can see 00:03:27.17 that there are aerial structures that rise up from the surface. 00:03:30.24 I'm going to show you next one of these aerial structures in a cross-section view 00:03:36.22 with cells that harbor a fusion of the LacZ gene for beta-galactosidase 00:03:42.18 to a promoter that's under the control of a sporulation transcription factor. 00:03:47.26 So we'll be able to see where spore formation is taking place in this aerial structure 00:03:53.25 by staining the structure with a chromogenic dye 00:03:56.25 that turns blue when the beta-galactosidase product 00:04:00.19 of the reporter is produced. 00:04:02.21 And as you can see there's a striking, intense blue staining near the tip, the aerial tip 00:04:10.00 of this surface of this structure that rises from the surface. 00:04:14.11 And as I go on, I'll argue later that it might be appropriate to think of these structures 00:04:19.09 as fruiting body-like structures that perhaps have been selected in evolution as... 00:04:25.22 for the purposes of dispersal of spores and I'll further suggest 00:04:30.18 that sporulation in the context of this community 00:04:35.00 depends on the very formation of the biofilm. 00:04:40.11 Let's look even more closely at this aerial structure 00:04:43.00 by scanning EM...by scanning electron microscopy. 00:04:48.02 And what you can see is that the biofilm consists of long chains of cells 00:04:52.17 that are cemented in parallel fashion to each other. 00:04:56.22 Long chains of cells, each one is glued to each other. 00:05:00.22 The glue that holds all these chains of cells together is known as the extracellular matrix. 00:05:08.13 The bacteria export a matrix material that cements the chains of cells together 00:05:15.09 so that the architecture can be built. This matrix consists of a polysaccharide 00:05:21.16 or a so-called exo-polysaccharide and a specific protein. 00:05:26.12 The polysaccharide is produced by enzymes encoded 00:05:30.28 within a large operon known as the eps operon, 00:05:34.06 for exo-polysaccharide operon. And the protein is encoded 00:05:38.15 with an operon that's referred to as the tasA operon. 00:05:42.06 Let's consider how these two operons are turned on under the right circumstances 00:05:50.28 that lead to the formation of the multicellular community. 00:05:53.12 This is mediated by an elaborate regulatory network that involves fully six regulatory proteins. 00:06:01.06 So on the right are the two operons, the one for polysaccharide synthesis 00:06:06.12 and the other for the protein and circled are the six regulatory proteins 00:06:13.07 that govern the expression of these two target operons. 00:06:18.01 This looks bewilderingly complex but, as I hope to show you 00:06:23.00 at its heart it has a simple logic. 00:06:25.24 So to simplify things, first of all let me get rid of the regulatory protein on the right 00:06:31.10 and let's just focus on the remaining five proteins; 00:06:35.00 SinI, SinR, AbbA and AbrB and Spo0A. 00:06:42.06 SinR and AbrB are repressor proteins, they each cross-repress both target operons. 00:06:50.08 So SinR represses the polysaccharide operon and the protein operon. 00:06:55.02 Likewise, AbrB contributes to the repression of the polysaccharide operon and the protein operon. 00:07:01.06 So two different repressors help to hold both of these matrix operons off. 00:07:07.01 In order for the operons to be de-repressed, 00:07:10.18 we need to antagonize the action of these two repressors. 00:07:14.27 So let me first convert the names of those two proteins into repressors, 00:07:20.00 the red repressor and the blue repressor, to make things look simple. 00:07:23.09 And now let's consider the SinI and AbbA protein. What are they doing? 00:07:27.25 Well, SinI is an anti-repressor that binds to its respective repressor to inactivate it. 00:07:36.08 Likewise, AbbA is an anti-repressor that binds to its repressor to inactivate it. 00:07:42.12 So let's convert SinI and AbbA into anti-repressor and anti-repressor. 00:07:50.21 So now you can see that there's a simple logic here 00:07:53.20 in which two parallel pathways of repression and anti-repression govern the expression 00:08:01.23 of the two operons that are responsible for matrix production. 00:08:06.28 This redundancy probably helps to ensure that the matrix genes are held totally silenced 00:08:13.22 until the right time and the right place for a community to form. 00:08:18.07 How does this whole system get going? 00:08:20.28 Well that's the role of the master regulator Spo0A. 00:08:24.12 Spo0A is a master regulator both for sporulation 00:08:29.16 as we saw in my previous presentation on sporulation 00:08:34.17 and itâ€™s also the principle regulatory protein 00:08:37.26 that's responsible for triggering biofilm formation. 00:08:41.14 Spo0A turns on the gene for both anti-repressors: 00:08:46.03 the SinI anti-repressor and the AbbA anti-repressor. 00:08:50.13 So, when Spo0A becomes activated, that leads to the production of two anti-repressors. 00:08:57.03 The two anti-repressors then bind to and inactivate their respective repressors, 00:09:01.22 and that finally leads to de-repression of the two operons that are responsible 00:09:08.02 for matrix production. 00:09:12.03 OK, now let's look in more detail at the biofilm. 00:09:17.08 We've just considered in some detail the regulatory pathway that's responsible 00:09:23.13 for turning on the synthesis of the matrix. 00:09:26.25 So some of the cells in the biofilm, at least, are responsible for matrix production. 00:09:32.01 But now let's consider what other kinds of cells might be present in the biofilm. 00:09:36.29 As we saw earlier, spore formation is also taking place in the biofilm. 00:09:42.03 And there are also vegetative cells that are capable 00:09:44.24 of motility that are present in the biofilm. 00:09:47.17 So, what we're going to do is take one of these biofilms, cut it in half, 00:09:52.17 and then look at it from the side by confocal microscopy using three different fluorescence reporters: 00:10:02.28 one to a gene under sporulation control; another under the control of the matrix producing pathway; 00:10:12.03 and a third under the control of genes involved in motility. 00:10:17.09 So each of these reporters represent three distinct kinds of cells: 00:10:22.28 sporulating cells; matrix producing cells; and motile cells, motile, vegetative cells. 00:10:29.10 OK, so if you look now at the bottom you can see that matrix producing cells in red 00:10:39.03 and sporulating cells in green have distinct locations. 00:10:43.09 They occupy distinct positions in the biofilm. The sporulating cells are near the top 00:10:48.26 as we saw in that fruiting body-like structure 00:10:51.25 in the light micrograph that I showed you at the beginning. 00:10:56.08 And the matrix-producing cells are underneath. 00:10:59.22 Now let's consider the position of motile cells. 00:11:03.01 The sporulating cells for comparison, once again, are near the extreme top and the matrix... 00:11:09.27 the motile cells are near the extreme bottom. 00:11:12.22 So motile cells, matrix cells and sporulating cells occupy three different regions of the biofilm; 00:11:20.23 with the motile cells near the bottom, the matrix-producing cells in the middle, and 00:11:25.11 the spore-forming cells near the top. So we can begin to think of the biofilm as 00:11:31.03 a kind of tissue that's composed of different kinds of cells that 00:11:35.29 occupy different positions in that tissue. 00:11:40.04 But this is a dynamic tissue because the relative proportion 00:11:43.27 of these different kinds of cells changes over time. 00:11:47.10 And I can illustrate that for you with a simple experiment 00:11:51.27 in which we take these biofilms harboring these fluorescent reporters 00:11:56.16 and separate all the cells from one another, disassemble the matrix 00:12:00.08 so that the cells are separated and then we use a fluorescence activated cell sorter 00:12:05.22 to measure the relative proportion of the three cell types over time. 00:12:10.20 Let's first consider sporulating cells. So the arrow marks increasing time up to 72 hours, 00:12:19.21 and the axis that runs from left to right represents increasing fluorescence. 00:12:25.23 As you can see, sporulating cells appear as a distinct population of high fluorescence 00:12:33.22 only at about 48 hours into the process. 00:12:37.00 If we look instead at matrix-producing cells, a peak of matrix producing cells 00:12:42.01 appears between 12 and 24 hours and then diminishes somewhat over time. 00:12:48.03 Then, finally, let's consider the motile cells. 00:12:52.16 The motile cells are most abundant at the beginning 00:12:56.02 and then they gradually diminish over time and become less and less 00:13:00.20 abundant by 72 hours into the process. 00:13:05.20 So gene expression is dynamic in the biofilm. 00:13:09.27 Cells occupy distinct positions. 00:13:12.11 Cells of different types and their relative abundance in the biofilm changes over time. 00:13:19.11 Now, finally, let me come to what I think might be the most interesting finding 00:13:25.00 concerning having more than one cell type. 00:13:27.15 As we've seen, spores are produced near the top of aerial structures. 00:13:31.29 And itâ€™s appealing to imagine that perhaps these are primitive fruiting body-like structures 00:13:38.27 that perhaps have a role in spore dispersal. 00:13:43.12 Well, is the process of spore formation not only associated with 00:13:49.26 multicellularity, is it actually dependent upon it? 00:13:53.06 And we can ask that question by looking at sporulation gene expression 00:13:58.16 or the process of sporulation both in a wild-type biofilm 00:14:03.12 and a biofilm that's mutant for matrix production. 00:14:07.09 Actually, a mutant that can't make a normal biofilm. 00:14:11.01 And then we can use the fluorescence activated cell sorter to measure the 00:14:15.13 proportion of sporulating cells in the wild-type and in the mutant. 00:14:20.04 And that's shown in this slide here. You can see that in the wild-type case in blue 00:14:25.09 there's a distinct peak of cells, a distinct sub-population of cells 00:14:31.09 that are undergoing sporulation, that are expressing sporulation genes. 00:14:35.28 But when we look at the expression of the same sporulation gene in a matrix mutant, 00:14:42.13 well, that peak almost completely disappears. 00:14:44.20 In other words, in the context of the biofilm, spore formation 00:14:49.15 is substantially dependent on the formation of an architecturally complex community, 00:14:55.26 on the production of the matrix and the formation of these structures. 00:14:59.22 So, that plus the earlier result that making a biofilm, like sporulation, 00:15:06.27 are both under the control of the same master regulator, Spo0A 00:15:11.04 leads us to speculate that this has an important biological significance. 00:15:16.09 And that in nature, spores are formed not in the form of individual cells on their own 00:15:22.04 at least not all the time but are sometimes produced in the context of 00:15:26.07 complex multicellular communities in which sporulation 00:15:29.15 is itself coupled to the process of assembling the community. 00:15:34.19 Lastly, let me point to the future on an important challenge that awaits us 00:15:41.03 in the years ahead. Even though we think we understand quite a bit 00:15:46.19 about the regulatory network that governs the production of the matrix 00:15:50.23 we're a long way from understanding how matrix-producing cells assemble 00:15:57.15 macroscopically into the elaborate architectures that I've been showing you. 00:16:01.19 Well, this issue comes into even sharper relief when you consider the following finding. 00:16:08.24 My collaborators and I have been collecting wild strains of Bacillus from around the world. 00:16:14.03 And the striking finding is that frequently these different wild strains 00:16:19.11 each exhibit their own distinctive architecture. 00:16:23.10 You can see that in this slide. 00:16:25.11 Here is a collection of different, very closely related strains of Bacillus subtilis 00:16:31.27 yet each one exhibits its own distinctive architecture. 00:16:36.02 Consider this one at the top or this one over here, or this one down near the lower left. 00:16:43.08 They have their own distinct morphotype. 00:16:46.01 Surely, this must be dictated by genetic differences between these two strains, 00:16:52.22 unknown genetic differences and in principle it should be possible to identify 00:16:57.25 the genetic differences between these strains that give rise to these different morphotypes 00:17:02.27 and thereby obtain a clue into the larger challenge of understanding 00:17:08.07 how morphogenesis is controlled, how a multicellular community with a distinctive 00:17:14.06 architecture is created by the expression of genes involved in the formation of the biofilm. 00:17:23.19 Finally, itâ€™s important for me to emphasize that all of the work that I've told you about 00:17:29.18 has been a wonderful collaboration between my laboratory 00:17:33.02 and that of my good friend Roberto Kolter at the Harvard Medical School 00:17:36.20 on the Cambridge side of the Charles River. 00:17:42.10 The individuals that have been involved in this story 00:17:46.09 that I've told you are Dan Kearns, Win Chai, Frances Chu and Anna McLoon. 00:17:51.09 And on the Harvard Medical School side of the Charles River in the Kolter lab 00:17:57.22 the individuals who have driven this project forward are Steve Branda, Dani Lopez, 00:18:01.29 Claudio Aguilar, Hera Vlamakis and Ashlee Earl. 00:18:06.23 Thank you very much.