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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		it’s been worked on in the laboratory and, as a consequence,
00:02:11.20		over time, it’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		It’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 it’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 it’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, it’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.

This material is based upon work supported by the National Science Foundation and the National Institute of General Medical Sciences under Grant No. 2122350 and 1 R25 GM139147. Any opinion, finding, conclusion, or recommendation expressed in these videos are solely those of the speakers and do not necessarily represent the views of the Science Communication Lab/iBiology, the National Science Foundation, the National Institutes of Health, or other Science Communication Lab funders.

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