Phage Biology Question
by Deborah Jacobs-Sera
Observe the picture below. You will note small, turbid plaques, of Cali, and large haloed plaques of unknown origin. Describe what you see as the two plaques collide. Hypothesize what could be happening there and develop an experimental plan to demonstrate your hypothesis/explanantion.
Here is the .jpg of the photo for a closer look!
My hypothesis is that this is competition among phages. The halos of the large plaques became larger over time and overlapped with the plaques that Cali had made. The areas surrounding the Cali plaques, something I'm affectionately calling "Cali-o's," contain an element that is somehow inhibiting infection by the halo forming phage. My favorite explanation is that this element is a protein coded by Cali (who has tons of ORFs of unknown function). This protein is binding something that is required for productive infection of the bacteria by the halo-former. This protein is released from the cell upon lysis and diffuses through the media more readily than Cali, thereby "claiming" nearby bacteria for Cali infection. Additionally, Cali lacks an integrase gene (as we know integrases) and I have been unable to show that it can form lysogens, so I don't think this is a very good explanation. I'm still trying to figure out what to support my hypothesis, but a couple things off the top of my head: 0) Photograph a plate over the course of 7 days to see the development of this phenotype. (in progress) 1) Purify each of the phages separately. (in progress) 2) Grow in pure culture bacteria from various parts of the plate, from the halo, the Cali-o, and the lawn, and infect these with both Cali and the halo former 3) Identify the halo-former as a previously-sequenced phage or one that hasn't been sequenced 4) Identify Cali mutants that don't make Cali-o's 5) Identify the protein in the Cali-o's that isn't in the wildtype smeg. These just came off the top of my head, I have to finish a paper, more later.
Drew--that's really cool! Are you going to keep us posted on this? ;)
I'll try to post any progress as it happens! Tim couldn't log into the site, so here's a response that he sent me: Hey Drew and Debbie, Drew sent me this earlier, so I've had a little time to think about it. The blog isn't letting me login so I am e-mailing you the response...perhaps one of you could put it up there? My thoughts about this: 1) Cali itself codes for a diffusible protein, OR genes that create a diffusible element. This can then prevent infection by other phages. Essentially sequestering cells for Cali to infect. Perhaps this is a repressor like protein to block replication or a protein which blocks a/multiple receptors preventing adsorbtion or injection. (I am not a fan of this model) 2) Cali induces the expression of a host protein or host genes that create a diffusible element. This could be like an alarmone that the host uses to warn other cells, and activate expression of hypothetical "phage defense genes" (CRISPR??) or turn off the expression of receptors for the phage. I am most curious to know if the halo-former is DEFINTATLY a Cali derivitive. If it is, model 1 makes little sense unless there are multiple mutations to create the phenotype. If model 1 is true, and the halo-former is a Cali derivative THEN the halo-former would have to have mutations that affect the diffusible element (since it doesn't have a "zone of protection" AND it would have to have mutations that do not allow it to attach to the Cali-protected cells (an unlikely scenario...shave with Occams Razor). So, in this case either model 1 does not hold OR the halo-former is not really Cali. Also, going against model 1 is that as far as I know, Cali does not continue infection across a plate. If it were sequestering cells for itself to use later, it would likely use those cells at some point in time. It makes little sense to protect the cells around you from other phage, and then never use them. I think that model 2 is more likely. That somehow Cali infection induces a host response. We know that environmental stress can induce a variety of cellular responses that can diffuse and cause action in nearby cells. Perhaps the cells that first recieve a Cali infection turn on this alarmone to signal to other cells that infection is emminent and they need to do something. I know Cali doesn't make lysogens, but there really is no need for a lysogen for either of these models to hold true. As long as the gene is expressed at a certain threshold level (probably small) we can see a response. -Tim
Hey all. I'm new to this phage stuff so whatever I add may sound uninformed, though my hope is that I'll get lucky and sound uninformed like a fox. One question: is it worth co-infecting some plates with Cali and other halo-formers besides this unknown one? My thought was to see whether this Cali-O effect is specific to these two phages or if in fact it's a more general protection of cells around Cali plaques.
I'm not sure what I think about a "diffusable element". Is there precedent for bacteriophage in general making secreted proteins, and if so, what are their functions? I'm currently thinking of a CRISPR explanation, CRISPR being an adaptive immune response by bacteria where they incorporate random portions of a phage genome into a region of their chromosome, and then through a yet uncharacterized method can repel infection from that phage. Bacteria at the edge of Cali plaques may develop immunity by picking up a portion of Cali's genome when they are infected but don't lyse (abortive infection, quick action of CRISPR, slow Cali infection, etc). These immune bacteria then multiply around the plaque to form a halo of cells that are difficult to infect. If the second phage has sequences resembling Cali's, then these bacteria may also be immune to infection by it. One aspect supporting this just by looking at the plate is how the halos of cells form around Cali plaques and then intrude upon the plaques of the other phage, but the other phage does not have a ring of cells that intrudes on Cali's plaques. There are several issues with this theory though. (1) Why don't bacteria develop immunity to the other phage through CRISPR, or do so readily to Cali? Perhaps the other phage tears through cells so quickly that smeg replication can't keep up and there's no time to acquire immunity, while Cali replicates and spreads more slowly, allowing cells time to replicate, perhaps even while they are infected. But I don't know about Cali's or other phages' replication times, so I defer to your judgments on this one. (2) How is it that all Cali plaques generate these immune bacteria that are then also immune to the other phage? The bacteria would have to always pick up very similar genes that both phage share! The plate does seem to have every Cali plaque surrounded by a halo of cells, but then if the cells around a plaque were not immune, would we see the Cali plaque after the other phage got to it? Towards the top of the plate photo there are two big plaques that have merged; in the bottom left of their plaque, a small hole the size of other Cali plaques is visible without a halo of cells--maybe a Cali plaque? Perhaps we don't always see the Cali plaques lacking a halo? One way to kind of examine this would be to look at the density of Cali plaques on the plate, and compare that to the density of halo'd Cali plaques in the other phage's plaques; just count the number of Cali plaques for a given area, then count the Cali halos in the other phage's plaques for a similar area and compare the numbers. If there are less halo-plaques than there are plaques on the rest of the plate, then they halo of cells may not be the default result of their plaque overlaps. (3) Can you guys come up with any other flaws with the theory? There's gotta be more :) My one suggestion as to how to investigate CRISPR's potential role would be to research the CRISPR sequences for smegmatis and make primers to amplify the regions from the smeg genome. It may be possible to sequence these regions and see if there are any Cali phage genes present. If there are, you can look for homologs of those genes in other phage genomes to narrow down the candidates for the other phage. This might be difficult though, so any other ideas how we might research this theory?
Also, I put a lot of spaces in there that didn't show up apparently...so sorry for the giant paragraph :(
Well, my initial thought was that if Cali is lysogenic in M. smegmatis, the presence of a Cali lysogen in a cell might provide immunity to infection by certain other phage, namely including the unknown other present on this plate. However, since Cali does not have an integrase this hypothesis seems very unlikely... it is certainly possible that Cali has some completely novel type of integrase, but that is improbable in light of the fact that we only know of two major classes of DNA recombinases (serine and tyrosine recombinases). One thing I was wondering, on the side, was how I might be able to check on the predicted function of phage genes-- that is, is such information available on NCBI or something like that? In this case, Drew, your prompt response made checking about the presence of a Cali integrase unnecessary for me, but this is something I was curious about for future reference. I think it would be helpful to see if the second phage is a previously characterized one or not. I imagine it would be rather time-consuming but one could take halo-generating-phage plaque samples and then test them with PCR primers specific to various plaque-forming phages we are familiar with. Perhaps an EM picture could be taken of a concentrated sample of the unknown phage to narrow down the known-phage candidates. Nonetheless, this approach would probably be rather expensive in terms of ordered primers and the plaque-former might very well be an unknown phage. That's all for now; I'll try to come up with some more ideas soon.
Kim, Steve has incorporated something to take out the spaces. Spaces crash the website, so you won't see them often.
Dan, To your reply, there are 60 phages to choose from - which ones would you like to try?
Debbie, I was only thinking of co-infecting with other halo-forming phages. How many of the 60 are halo-formers?
This comment chimes in with others, but has a more specific ring to it. I have pasted the abstract of Molecular Microbiology (1995) 15(3), 415-420 Phage-exclusion enzymes: a bonanza of biochemical and cell biology reagents? Many parasitic DNA elements including prophages and plasmids synthesize proteins that kill the cell after infection by other phages, thereby blocking the multiplication of the infecting phages and their spread to other nearby cells. The only known function of these proteins is to exclude the infecting phage, and therefore to protect their hosts, and thereby the DNA elements themselves, against phage contagion. Many of these exclusions have been studied extensively and some have long been used in molecular genetics, but their molecular basis was unknown. The most famous of the phage exclusions are those caused by the Rex proteins of λ prophage. The Rex exclusions are still not completely understood, but recent evidence has begun to lead to more specific models for their action. One of the Rex proteins, RexA, may be activated by a DNA-protein complex, perhaps a recombination or replication intermediate, produced after phage infection. In the activated state, RexA may activate RexB, which has been proposed to be a membrane ion channel that allows the passage of monovalent cations, destroying the cellular membrane potential, and killing the cell. We now understand two other phage exclusions at the molecular level which use strategies that are remarkably similar to each other. The parasitic DNA elements responsible for the exclusions both constitutively synthesize enzymes that are inactive as synthesized by the DNA element but are activated after phage infection by a short peptide determinant encoded by the infecting phage. In the activated state, the enzymes cleave evolutionarily conserved components of the translation apparatus, in one case EF-Tu, and in the other case tRNALys. Translation is blocked and development of the phage is arrested. A myriad of different phage-exclusion systems are known to exist and many of these may also be specific for highly conserved cellular components, furnishing generally useful enzymes for biochemical and biomedical research. My point is that has to be many phage-exclusion mechanisms to choose from!
I agree with what Tim and Drew both said about some type of diffusable protein that may be claiming cells for Cali. What ever it turns out to be this is super cool. Is it also possible that somehow the two phages co-infected and are somehow inhibiting each other from lysing. Just a thought.
Ogechi, What do you mean by 'the unknown is so much bigger'? What is responsible for the size of the plaques? What hypothesis is best supported by the 'less defined in shape'? What is less defined in shape?
I really don't know what to think. It seems that there has to be a competion issue like Drew said. But normally in nature when there is competition one specimen loses out. The fact that the unknown and Cali can coexist much more overlap is weird to me. So my other thought is that the unknown is a harmless competitor. And why is the unknown so much bigger and less defined in shape?
I agree with Ogechi that the unknown and cali have a mutaulistic relationship. I guess the question is what is each benefitting from the other? Also why is the symbiotic relationship only apparent on some of the plaques of cali and not all?
Enyinna, When are two organisms mutualistic or symbiotic? Which one is appropriate here? Also, I am not sure what you mean by your last comments
These ideas sound very interesting. Drew have you already tried photographing the development of the plaques to see what happens? When I first saw the plate, I thought that maybe after the halo-forming plaque and Cali were present, the halo-forming plaque's halo grew larger and since Cali had already infect bacteria in the area with the Cali plaque and so the halo formation just diffused through and formed the halo around Cali as though Cali wasn't there. Whether or not the ring of cells around Cali has a function such that Cali can claim some cells to infect is unclear to me but if you look at parts of the plate where there are just Cali plaques, you can see the formation of the ring of cells occurs wherever there are Cali plaques. If the ring of cells was for competition, why would Cali form a ring of cells in areas where there are no other phages to compete for the bacteria? Isn't that a waste of cells?
I agree with the competition idea, as stated by just about everyone. But just an observation: the cali plaques form in the halo region of the unknown, but not ever in the dark center of the unknown plaques. So is the competition affected by the concentration of the phages (which is probably highest in the center of the plaques)?
Sorry I'm only now remembering to put this in! As some of you may remember from last week's meeting, Tim Sampson stopped by. He informed me that my theory about CRISPR was entirely incorrect, as M. smegmatis does not have a CRISPR system. So I guess I'm back to square one!