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Bacteriophages: Genes and Genomes

Transcript of Part 1: Bacteriophages: What are they?

00:00:01.17		Hello. My name is Graham Hatfull. I am a professor at the University of Pittsburgh
00:00:05.27		and a Howard Hughes Medical Institute professor,
00:00:08.24		and we're going to talk today about bacteriophages, their genes and their genomes.
00:00:13.25		First of all in Part One, I'd like to just discuss what bacteriophages are,
00:00:20.10		some of their biological properties, and how they were discovered.
00:00:24.17		So bacteriophages are viruses that infect bacterial hosts.
00:00:30.21		So just as we and many of our animal cousins are infected by viruses that are specific for us,
00:00:39.29		bacteria also have viruses that infect them, and they are called bacteriophages or phages for short.
00:00:46.18		They were discovered or co-discovered by Felix D'Herelle and Frederick Twort between 1915 and 1918.
00:00:58.05		And they were discovered as agents that when added or present in a culture
00:01:03.25		of growing bacteria were capable of killing the bacteria,
00:01:09.01		and essentially having a bactericidal effect.
00:01:13.09		It was really Felix D'Herelle who developed an assay called the plaque test where he was able to take samples
00:01:24.01		of bacteriophages that were able to kill bacteria and he made dilutions,
00:01:31.03		increasingly greater series of dilutions of the bacteriophage sample
00:01:37.10		and then plated that out in the presence of the bacterial host on solid media using Petri dishes, as shown here.
00:01:46.24		And what he saw was that when he diluted out the sample sufficiently
00:01:53.01		he could see individual areas of killing as you can see here
00:01:58.05		with smaller and larger areas where the cells are dead and where the viruses have grown.
00:02:05.21		These are called plaques and each one of these individual plaques
00:02:09.07		has arisen by a single particle which was capable of infecting a cell,
00:02:17.20		and as the bacteria grew across the surface of the agar dish,
00:02:22.25		the virus propagated itself, multiplied, until... and the plaque here then
00:02:30.06		may be perhaps a million or ten million or more individual phage particles.
00:02:36.07		This plaque test was really important because they could tell
00:02:42.19		from looking at cultures of, or looking at lysates of bacteriophages,
00:02:48.10		that there was nothing to see in there.
00:02:51.00		They could filter the samples, they knew they still had the infectious property,
00:02:56.16		but the tube looked completely clear.
00:02:59.25		There was nothing to see and when they placed these particles
00:03:02.26		with this capability in the light microscope, there was nothing to be seen.
00:03:09.09		So these were mysterious entities, but Felix D'Herelle showed conclusively that they really are particulate in nature.
00:03:18.10		It was somewhat later, in the late 1930s and 1940s, that the electron microscope was developed,
00:03:27.17		which has a level of resolution way beyond the light microscope,
00:03:31.17		and was able to show for the very first time what viruses including bacteriophages actually look like,
00:03:37.25		and I'll show you some pictures in a minute.
00:03:41.17		But this just shows a stylized example of what one of those phages look like.
00:03:48.06		At the very top here you can see is a structure that is referred to as the head. Sometimes it is called the capsid.
00:03:56.00		That is attached to this longer structure here which is the tail.
00:04:02.26		And the DNA, or the genetic information that the virus carries in order to multiply itself,
00:04:09.08		the instructions for its own replication, are carried here in the virus head.
00:04:17.07		And so we can see and we now know of our understanding of what this particular virus and these types of viruses look like,
00:04:28.24		is that this structure at the very bottom which is the tip of the tail,
00:04:32.06		that is the region that recognizes and binds to the outside of the bacterial host.
00:04:39.29		During the process of infection, the structures that we see principally constructed
00:04:47.07		or made up of protein components that stuff stays on the outside of the cell,
00:04:52.22		and the DNA, or the genetic information is what passes from the head through the tail, into the cell.
00:05:02.28		and then reprograms that cell in order to make more copies of the virus.
00:05:08.24		By electron microscopy we can see and we now know that a very large proportion of these naturally found viruses
00:05:19.23		are in an order which we refer to as the Caudovirales, of which these are primarily phages that contain tails,
00:05:27.16		as I just described and shown in these examples here.
00:05:30.29		And they contain double stranded DNA.
00:05:33.14		So there are lots of different types of viruses with different shapes,
00:05:38.26		but the vast majority that you find in nature fall into this particular order.
00:05:44.14		And these have names according to the types of tails that they have.
00:05:49.00		These are the Myoviridae, and they have contractile tails so the tail actually contracts like a syringe
00:05:56.28		when the DNA is injected into the bacterial host.
00:06:01.16		These are called the Podoviridae which have little short stubby tails.
00:06:06.22		And these ones on the right here are the Siphoviridae, and these have long, non-contractile and flexible tails.
00:06:17.17		So these are the main forms of these viruses.
00:06:21.27		There are however a variety of number of different types of viruses that have different shapes
00:06:26.21		and have, indeed, different types of DNA or RNA genomes within them.
00:06:33.18		So we can think about the various steps that are involved in the propagation
00:06:39.29		of a bacteriophage during this process known as lytic growth.
00:06:44.23		The phage starts by adsorbing to the outside of the cell.
00:06:52.10		The DNA is injected inside the cell in this process of penetration.
00:06:57.29		There is a set of early proteins which are encoded by the phage, but which uses the host machinery to express them.
00:07:07.19		Viral DNA is replicated to make lots more copies of the virus DNA.
00:07:13.10		And then these late proteins are expressed that make the structures that I just showed you in the electron microscope.
00:07:22.27		The capsid structure gets assembled. The tails get assembled.
00:07:28.08		And then in the process of DNA packaging the DNA is stuffed into those heads until the heads are full.
00:07:35.15		The tails are attached. And in the very final step of this process the bacterial cell
00:07:42.01		is going to lyse with enzymes encoded by the phage genome to break open the outside of the cell
00:07:49.10		and the progeny viruses, typically 50 to 100 new progeny viruses, will be released
00:07:55.16		and will go on to repeat this cycle whenever they find another bacterial host.
00:08:01.03		So while many phages go through a lytic growth cycle,
00:08:08.00		and that is essentially simply how they reproduce themselves,
00:08:11.03		that is not the only type or form of growth cycle that phages can enjoy.
00:08:17.06		And there is a large class, perhaps even a majority of bacteriophages
00:08:20.08		that enjoy what is referred to as a temperate life state.
00:08:26.21		What that means is that when a temperate phage infects its bacterial host,
00:08:32.22		there are two possible alternative outcomes.
00:08:36.24		One of those outcomes is lytic growth in which they propagate themselves
00:08:43.02		in exactly the same way as I described to you in the previous slide.
00:08:49.07		And normally that occurs perhaps 80 or 90% of the times that the host cell is infected by the bacteriophage.
00:08:57.10		Ten to twenty percent of the time there is an alternative outcome.
00:09:02.11		And that alternative outcome is referred to as lysogeny.
00:09:06.09		In lysogeny the lytic genes, the genes that are required to propagate itself and lyse and kill the cell,
00:09:14.28		they are all switched off.
00:09:16.18		They are repressed, and the phage DNA establishes itself so that it can be propagated
00:09:23.28		in the long term within the subsequent growth cycles of the bacterial host.
00:09:29.25		And that is usually, although not always, accomplished by integration of the phage DNA into the bacterial chromosome.
00:09:38.29		So the phage genome itself becomes incorporated and becomes a part of the bacterial chromosome.
00:09:45.26		It gets replicated just like all the other of the bacterial genes do.
00:09:49.27		So we can think of lysogeny as a form of parasitism
00:09:54.02		where the phage is simply going along for the ride in what is otherwise a very healthy cell.
00:10:01.10		It is basically a free ride through many, many generations.
00:10:05.27		So lysogens tend to be stable, but they are not going to, they don't have to remain in that state forever.
00:10:20.14		They can go through many, many, many rounds of growth,
00:10:23.27		however, as a process that happens either spontaneously or can be induced by DNA damage,
00:10:31.11		such as UV light, lysogens can leave this... the comfort of that life cycle, and they can be induced into full lytic growth.
00:10:44.25		There's a relatively easy way to distinguish between phages
00:10:49.22		that are temperate and those that are lytic and can only undergo the lytic growth cycle
00:10:54.26		by the types of plaques that you see on agar plates.
00:10:58.27		Lytic phages form simply just clear plaques, as shown in the bottom right hand corner here,
00:11:05.03		where all of the cells within that infected area have been killed.
00:11:11.12		Killed by phage infection.
00:11:14.00		Over on the left on the bottom here, you can see what temperate phages look like.
00:11:18.20		They form turbid plaques.
00:11:20.15		You see plaques because there are cells that are being killed through the lytic growth, propagation of the virus.
00:11:27.11		But there is that important and key subset of cells that are...
00:11:32.07		in which lysogeny is established, and those lysogens are able to survive
00:11:39.19		and to grow. And they can grow perfectly happy even though they are bathed
00:11:44.03		within this density of bacteriophage particles.
00:11:48.28		So the fact that lysogens can actually grow quite happily even though
00:11:54.01		there's lots of viruses around to infect them is referred to as super infection immunity.
00:12:01.02		Lysogens are immune to super infection by a phage of the same or closely related type.
00:12:09.00		This slide just shows an example of how that can be studied and what that looks like in the lab.
00:12:17.09		At the top left here are plaques of a turbid phage, a temperate phage,
00:12:23.22		growing on a lawn of bacteria. Using either a toothpick or a wire you can go and pick cells from the very center
00:12:33.06		of one of those plaques. Streak it out on an agar plate, as shown on the bottom left here,
00:12:38.23		in order to generate single colonies, each grown up from a single cell,
00:12:45.04		that will have come from the turbid plaque.
00:12:48.05		And then these individual colonies can in turn be tested for their immune phenotypes as shown in the top right.
00:12:57.26		In this example we are looking at, we are comparing, a non-lysogenic strain with a lysogen
00:13:05.00		and in the top we have just spotted on dilutions of a bacteriophage sample.
00:13:11.14		And you can see that these serial dilutions from the most going down to the least number of particles
00:13:20.24		gives you the titer, i.e. the number of particles per milliliter of sample on the non-lysogen.
00:13:32.05		But when you compare it with exactly the same dilutions of that particular phage onto a lysogen of itself.
00:13:38.00		So this particular phage is called Giles.
00:13:39.29		On the right is the Giles lysogen, and you can see that there is essentially very little infection
00:13:45.25		except perhaps a little bit of clearing at the very highest concentration
00:13:49.14		of phage at the top left-hand corner of that panel.
00:13:53.18		A control phage in this case below it, showing L5, which is also a temperate phage,
00:14:00.08		but it does not share immunity with Giles,
00:14:04.03		and therefore you see essentially the same number of plaques on the lysogen of Giles and the non-lysogen.
00:14:12.03		And therefore bacteriophages can be grouped together according to their immune specificities.
00:14:19.11		And it can be done just by comparing the infectivity of a whole set of phages on lysogens and non-lysogens.
00:14:28.14		So this is an important parameter that enables us to group together phages
00:14:34.22		that may be similar by sharing these types of parameters.
00:14:38.27		I mentioned that phage DNA can integrate itself into the host chromosome.
00:14:46.10		And this is a common feature of temperate bacteriophages.
00:14:50.07		They do it by a process which is referred to as site specific recombination.
00:14:55.14		This process is catalyzed by an enzyme which is called integrase or Int for short.
00:15:03.19		The integrase is encoded by the phage genome,
00:15:07.20		and integrase catalyzes recombination between two specific sites or segments of DNA.
00:15:14.29		One is the so called phage attachment site, or attP for short,
00:15:20.06		and the other is a specific site within the bacterial chromosome
00:15:23.28		which is called the attachment site for the bacterium or attB.
00:15:29.11		Integration results in the formation of what is called a pro-phage,
00:15:34.26		an integrated phage DNA that has become part of the chromosome,
00:15:40.11		and it is now the pro-phage DNA is flanked by the left and right attachment sites
00:15:46.02		referred to as attL and attR respectively.
00:15:50.10		This reaction does use host functions quite commonly,
00:15:56.26		Integrase works together with a host protein, a bacterial protein called integration host factor or IHF for short.
00:16:05.15		And you'll recall that I told you that lysogens can undergo spontaneous or induced induction into lytic growth,
00:16:15.10		which means that there has to be a process for this to come back out again.
00:16:21.13		A biological reversal of this overall reaction.
00:16:24.17		That is called excision, and excision is again catalyzed by integrase,
00:16:30.04		and there is a second phage encoded protein called excise or Xis for short.
00:16:38.15		And Xis is a protein that essentially dictates and determines the directionality of how these reactions will occur.
00:16:48.28		In the absence of Xis you do integration.
00:16:53.09		In the presence of Xis, then the integrase is only capable of doing the excision reaction.
00:16:59.11		A few years ago people started thinking about how many bacteriophages there really were out there in the biosphere.
00:17:12.01		And what they did was that they developed a technique called epi-fluorescence
00:17:15.09		where they could take a sample, let's say of seawater, which is easy to get.
00:17:19.28		Get seawater, add a sample of a dye which binds to the nucleic acids,
00:17:26.05		place that sample under a fluorescence microscope
00:17:29.21		and simply look and count for the viruses and other components that you see in that kind of experiment.
00:17:37.11		This is an example of what they saw.
00:17:40.16		There is a very large number of what you can see as small green fluorescent dots here.
00:17:48.16		Those are all the viruses that are present.
00:17:50.02		And there is a smaller number, a fewer number, of these large brighter spots,
00:17:56.04		which are larger objects, and they are the bacteria.
00:18:00.25		And so what it was possible to do was simply to count how many virus like particles are present in these samples.
00:18:09.13		And when that was done, it was clear that the viral population is indeed absolutely vast.
00:18:15.20		When you measure there is about 10 to the power of 6 to 10 to the power of 7
00:18:19.12		viral particles per mL, and this number seems to be reasonably steady no matter where you have looked.
00:18:26.22		It's true in sea water if you look in coastal samples, if you look in oceanic samples,
00:18:33.11		or the surface or the deep.
00:18:35.27		And there's a similar abundance, or related degree of abundance in terrestrial samples as well, it is believed.
00:18:45.16		So because we can measure the number of particles present in small samples,
00:18:50.18		and we can multiply the amount of sea water
00:18:53.23		and the amount of terrestrial components and when we do that we can conclude that the biosphere
00:19:01.05		contains a total of 10 to the power of 31 virus particles, the vast majority of which are bacteriophages.
00:19:10.01		This is an incredible number. This number would suggest that there's more bacteriophage particles
00:19:18.11		in the biosphere than all other biological entities added together.
00:19:23.00		Phages are in fact the majority of all biological things in the biosphere.
00:19:30.17		They are not only abundant, but this appears to be a very dynamic population as well.
00:19:38.15		You can see from the fluorescence patterns in this slide,
00:19:45.03		but it is true in most samples that have been examined that the ratio of bacteriophage particles
00:19:52.05		to bacteria is about between 5 to 1 and 10 to 1.
00:19:58.04		And that is important because it means that the bacteria are likely to constantly be subjected
00:20:06.00		to infection by the bacteriophages in their natural environments.
00:20:12.18		And in fact there are ecological studies that estimate the number of viral infections per second
00:20:19.28		that occur on a global scale.
00:20:22.29		And that number is estimated to be between 10 to the power of 23 and 24,
00:20:27.24		which is just an incredible number of activity.
00:20:33.24		A dynamic population. Indeed these numbers would suggest that the entire phage population turns over every 4 or 5 days.
00:20:42.20		It is a large and a stunningly dynamic set of biological items.
00:20:50.01		Not surprisingly perhaps the viral population is extremely diverse when we look at the genetic level.
00:20:59.16		Currently a number far short of 10 to the power of 31 phage particles have been subjected to DNA sequencing,
00:21:09.06		perhaps about 650 or so. And from these genomes we can look
00:21:15.09		and we can see how similar or different they are to each other.
00:21:19.05		And what we have learned from this is that indeed there's many, many different types of sequences.
00:21:25.05		And these genomes appear to harbor and to contain large numbers of genes,
00:21:30.15		which are unlike any other genes that we have seen before.
00:21:34.11		So, we can conclude then that bacteriophages represent the majority of all biological entities in the biosphere.
00:21:42.15		They're a dynamic population. They are constantly infecting bacterial hosts and generating more copies of themselves.
00:21:51.12		The bacteria must be struggling to maintain their survival through resistance to these infections.
00:21:59.10		And I think a compelling argument can be made that this population has probably also been evolving for a very long time.
00:22:08.04		Perhaps 2-3, perhaps even 4, billion years, extending right back to the very early days of when life evolved.
00:22:17.10		And finally, phages appear to represent the largest unexplored reservoir of new genetic information in the biosphere.
00:22:29.19		If you want to discover new genes, perhaps with new functions, perhaps with new structures,
00:22:35.20		the bacteriophage population I think we would argue is exactly where you should start to look.
00:22:43.11		In Part Two, we'll look in some more detail at the genetic structures of bacteriophage genomes
00:22:51.26		and see how that's given us some insights into how these genomes have evolved.

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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