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.