Monday, March 5, 2018

BacterioFiles 330 - Polar Plasmid Produces Particles

Plasmid DNA
By de:Benutzer:Sec11, CC BY-SA 3.0
This episode: A plasmid discovered in Antarctic archaea can create virus-like particles, membrane vesicles, and transfer itself to new hosts!

Thanks to Rick Cavicchioli for his contribution.
Download Episode (17.7 MB, 19.4 minutes)

Show notes:
Microbe of the episode: Hypomicrogaster canadensis bracovirus

News item

Journal Paper:
Erdmann S, Tschitschko B, Zhong L, Raftery MJ, Cavicchioli R. 2017. A plasmid from an Antarctic haloarchaeon uses specialized membrane vesicles to disseminate and infect plasmid-free cells. Nat Microbiol 2:1446.

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    Episode outline:

    • Background: Origin of viruses is debatable
      • Start out as cells that degenerated into little dependent bits?
      • Or grow from lesser dependent bits of nucleic acid, like transposon?
    • Learn more by studying many different kinds of viruses
      • Eukaryotic kinds more familiar, esp mammalian
      • Also bacteriophages
      • But archaeal viruses less well-known, have some unique characteristics
    • Can also learn by studying plasmids
      • Relatively small bits of DNA, usu circular
      • Transmitted from cell to cell
        • Vs. viruses, encapsulated extracellularly
      • Otherwise sometimes hard to distinguish genetically
        • Not universally harmful or helpful
    • But seems straightforward to go from plasmid to virus or back sometimes; just add capsid genes
    • What’s new: Now, Susanne Erdmann, Bernhard Tschitschko, Ling Zhong, Mark Raftery, and Ricardo Cavicchioli, publishing in Nature Microbiology, have discovered a system of plasmid transfer in archaea that blurs the line between plasmid and virus even more!
    • Organism is Halorubrum lacusprofundi, salt-loving archaeon originally isolated from Deep Lake (thus the name)
    • Statement 2
    • Methods: That was Dr. Rick Cavicchioli, final author on the study.
    • Looked at culture under electron microscope
      • Saw tiny virus-like particles around cells
    • Statement 3
    • Separated VLPs from cells with filtration, isolated DNA from them
      • Circular, ~50000 bases
      • Same DNA was found inside archaea
    • Koch’s postulates: isolate, reinfect, observe symptoms, reisolate
      • Here, VLPs could infect other strain of H. lacusprofundi that didn’t have before
      • After infection, this strain produced them
    • Sequenced DNA and analyzed
      • 3 separate regions with different characteristics
      • 1: allows replication and maintenance of plasmid
      • 2 and 3: not sure
      • Almost no similarities to viral genomes, much more like plasmid
    • Analyzed proteins produced from plasmid genes
      • Many from region 2 seemed involved in particle formation
      • found in particles or cell membrane
    • Discovered that plasmid isn’t just transferring itself
      • DNA content suddenly went up after growing in lab 3 months
      • Seems to integrate into host genome, and then come out and take genes with it
      • Looked for it in other strains of host, and found some integrated
      • Region 3 of original was missing, replaced with other genes
    • Applications and implications: Use system in useful ways in other microbes?
    • VLPs of interest for vaccines and such
    • Cold-tolerant microbes can be useful for some things, have cold-tolerant enzymes
    • Useful for understanding ecology: Statement 1
    • What do I think: Implications about virus evolution: File 3
    • Seems like just happened to pick up the right genes for transferring
    • Not quite virus, no protein capsid
      • Genes look more like plasmid than virus
      • But it could acquire viral proteins at some point, no reason why not
      • Right now, somewhere in between sorta
      • Could be like steps in emergence of virus
    • CRISPR/Cas systems can defend microbes against both viruses and plasmids
      • Sometimes interfere with transforming strains with plasmid
      • All just DNA that replicates itself with help from host, can be more helpful or more harmful
    • Finally, here’s Dr. Cavicchioli explaining more about his research: File 4, 5

    Statement 1:
    One of the things we've learnt about microbiology in Antarctica is that viruses play a really important role in controlling the microbial food web. And one of the reasons for that is there are fewer higher trophic organisms, so metazoans, larger eukaryotes that can eat the microorganisms, so the higher organisms don't exist in such a quantity in some of the Antarctic environments that we study, like lakes in the Vestfold Hills region, which is near Davis Station in Antarctica.

    Statement 2:
    So my group set about isolating viruses so that we could characterize the interaction between viruses and their hosts in the laboratory. And Susanne Erdmann, who is a EMBO fellow, had joined my group, and she had expertise in isolating viruses, so she set about isolating what we call virus-like particles.

    Statement 3:
    The virus-like particles, or one of them that we isolated, turned out to be, in fact, not a virus, but a plasmid, and this plasmid is special in that it encodes proteins that go into the membrane of the host cell, and those proteins then help to form membrane vesicles, and the plasmid is then carried into the membrane vesicles, those specialized membrane vesicles that we call plasmid vesicles can then leave the host cell and they can float off and then infect other cells. So that process whereby the DNA goes into some kind of encapsulation, and in this case the membrane vesicle is very much like a virus, so when a virus produces copies of its own DNA and the DNA is then captured inside the head of the virus, and then the virus that is released from the host cells and can go off and infect other cells. So these specialized membrane vesicles perform a very similar function.

    File 3:
    Viruses and plasmids share many similar properties. So they're both small DNA particles that are separate from the main chromosome, and they can transfer from organism to organism, so bacteria to bacteria or archaea to archaea, and one of the remaining distinctions between plasmids and viruses is the ability of viruses to encapsulate their own DNA and go on to infect other cells. So the discovery we made about the plasmid really blurs the boundaries between plasmids and viruses, because now we've found a plasmid that also has the same capacity. And so this is of course very interesting, because it makes us reflect on the distinctions between plasmids and viruses, and think about just how these things evolved over time.

    File 4:
    One of the things that people often don't appreciate or just don't understand is that a large proportion of the Earth's biosphere is cold. In fact about 80% or more of the Earth's biosphere exists at temperatures of 5 degrees or lower. And a lot of this is based on the deep ocean, so a very large part of the biosphere, the deep ocean, is cold. And Antarctica is a very special environment that's also very cold, so the research that we do in Antarctica informs us about Antarctica itself, and also about cold environments on the planet. One of the things that has really helped to gain an understanding of life forms in cold environments, and particularly Antarctica, is using in terms of the microbial studies, metagenomics. So these are studies where we harvest biomass onto filters, so we literally filter water onto filters, we then extract the biomass from the filters, get the DNA, and get that sequenced. And the sequence data then tells us about the communities that are present. We can infer the functions that they perform. And so we start to learn about the microbial processes that are occurring in Antarctica, and also of course then can also occur in other cold environments. So it's a very empowering technology.

    File 5:
    One of the really valuable things we've learnt about the metagenomics is letting it describe to us about the systems that are present. So instead of having a preconceived idea of what sorts of microbes will be present and what functions they'll perform, it's letting the data tell us. It's like David Attenborough turning over the rock and saying “hm, here under this rock we find”, and so the metagenomics is a great tool for allowing us to discover. Now in the context of viruses, just for example, we've identified a virus in one lake that's a predator of another virus, so it's called a virophage, and by examining the interactions of that virophage with the virus, and the virus's interaction with its host, actually predict that the virophage has a positive impact on the ecosystem leading to a higher level of carbon turnover in the lake, and so hence the actual health of the lake system. Now in another lake we identified a microorganism called a green sulfur bacterium, that grows under very low light levels. And this green sulfur bacterium has a very important role in the ecosystem. And what we discovered is that the levels of the bacterium were very high, but the levels of the viruses that were present in the same fraction were very, very low. Now this is very unusual, because usually viruses are present at about 10 times the numbers of microorganisms, so archaea and bacteria, that are present. So what this indicates is that the green sulfur bacterium has in fact evolved mechanisms that enable it to be resistant to viruses. And so this also means that the system is very specialized, is very fit for its purpose, but it also means that it's quite likely to be susceptible to the introduction of foreign viruses. And so this is one of the things that we're very concerned about for Antarctica, is the introduction of foreign species. So when we discovered the virus that wasn't in fact a virus but was in fact a plasmid, it just was an illustration of another novelty and what can be gleaned by using basically metagenomic analyses and then following them up with laboratory studies like the isolations that Susanne Erdmann performed. And so we're beginning to learn a great deal about Antarctica and the life that's evolved there, things we would never have known unless we had performed the expeditions and performed this kind of research and let IT tell us about what is exactly going on there.


    1. What are some of the exceptions of Koch's postulates?

      1. Good catch! I wondered if someone would ask about that.

        The postulates again (from Wikipedia), are:
        1. The microorganism must be found in abundance in all organisms suffering from the disease, but should not be found in healthy organisms.

        2. The microorganism must be isolated from a diseased organism and grown in pure culture.

        3. The cultured microorganism should cause disease when introduced into a healthy organism.

        4. The microorganism must be reisolated from the inoculated, diseased experimental host and identified as being identical to the original specific causative agent.

        Each of these can have exceptions, since things are a lot more complicated in nature. Some examples:

        1. Sometimes people/hosts can be carriers for a pathogen without being sick from it. A popular example is Typhoid Mary, who was constantly shedding the bacteria that cause typhoid fever but didn't have any problems with it herself (but caused a lot of disease serving as a cook to people who did get sick from it). Some microbes don't cause problems for one animal, but can cause severe disease in another. And sometimes a pathogen will cause problems only in a fraction of those it infects; poliovirus can cause paralysis, but only in about 0.5% of people it infects.
        Also, some diseases can be caused by bacteria but not by an infection of the cells themselves but by the toxins they've left behind, like with some food poisonings by Staphylococcus. In that case the bacteria would still be responsible but wouldn't actually be found in the victims.

        2. Often we haven't figured out how to grow a pathogen in pure culture, or in the case of viruses it isn't really possible to grow them at all without host cells present.

        3. Similar to exceptions of 1, sometimes a pathogen only causes disease in a fraction of hosts it infects. Or if a pathogen only infects humans, it's definitely not ethical to try to go infecting healthy people to see if they get sick. (Though sometimes this can happen accidentally and confirm this postulate without ethical problems, as in the case of a couple of HIV researchers that got accidentally infected.)

        And 4 is similar to 2.

        Also, sometimes a single pathogen can cause different diseases depending on how or whom it infects, like anthrax is different depending on whether it comes in through the skin, inhaled, ingested, or injected somehow. And sometimes a disease (or very similar, indistinguishable diseases) is caused by multiple different organisms--an obvious example is the common cold, caused by many different kinds of viruses.

        So the postulates do have a lot of exceptions, but they can be a good starting point for thinking about discovering the pathogen behind an emerging disease, at least.

    2. Hello Dr. Noar,

      I find it fascinating and a little disturbing in straight from a science-fiction move type of way that these archaea can infect host in the same way that a virus does. It reminded me of the 2009 movie, "The Thaw" where a prehistoric bacteria was released through the melting polar ice caps. Even though the movie was far fetched, it isn't that far from the reality that new bacteria and their functions that we are discovering can lead to a whole new world of information (and hopefully not a bacterial apocalypse).

      Thank you,
      Zoe Wafford

      1. Hi Zoe,

        It could be a concern though! Who knows what lurks in the permafrost in Siberia, for example, waiting to thaw? People have gone looking and found things already like really big viruses (though not a very big threat to humans, fortunately), like in this episode:
        But a more realistic concern might be something like smallpox that can cause serious disease in a significant portion of the population now that many of us aren't immune either from exposure or vaccination, since it was eliminated from the wild.

        Thanks for writing,