The Phantastic Phage Phind: Rise of Mycobacterium smegmatis

Let us begin!

Just before we get into the nitty-gritty of this microbiology stuff, I’ll go through a couple of frequently asked questions for any of you new to this phantastic phage blog. Well of course the main one is; what the heck is a bacteriophage (or phage for short)?!?! My sentiments exactly. To put your troubled mind at ease, I think of them simply as a virus that infects a specific bacteria. It is also interesting to know that bacteriophage undergo a lytic or lysogenic cycle depending on the bacteriophage and it’s environment. These little organisms are the most diverse and abundant living organism in the world and to put it in perspective, my super lab leader once quoted; ‘if you were to gather up all of the phage in the entire world and weigh them, they would exceed the weight of every single elephant combined by 1,000 fold’. Wow, that really puts them into perspective, especially considering the average size of a phage is around 1-5% the size of its host bacteria (which is extremely tiny in itself!). Did I hear you ask just how abundant these little creatures are? Well studies suggest that there are around 1031 phage present in our environment and viral ecologists suggest that they infect at a rate of 1023 viral infections per second.

So what have I got in common with this super virus? Well I am one of New Zealand’s newest phage hunters! This year Massey University teamed up with the Howard Hughes Medical Institute and offered this delightful experience as a paper in the Bachelor of Natural Sciences, working with Mycobacterium smegmatis – the sweet sister of bad-boy Mycobacterium tuberculosis. So let’s get down to the business of what I have discovered so far!

After initially isolating 23 plaques (holy moly, am I right!?) from 8 soil samples collected around Auckland and completing a series of direct plating an enrichment, my initial positivity was shorted lives with the knowledge that these plaques had not further developed. It is important to note that a plaque is a clear (or sometimes cloudy) zone representing an area within the media in which the bacteria that has experienced viral lysis – in other words plaques indicate the presence of a bacteriophage that is infecting the bacterial cell that was once present. Never fear, 5 new soils samples later and I had found something! 3 little plaques on the direct plating of sample #11, from the silage plot on our farm.

Location of soil sample containing phage - 36.671139 S, 174.662436 E.

Location of soil sample containing phage – 36.671139 S, 174.662436 E.

Fast forward several weeks later where I examined my plates after the initial titre (a titre is the counting of the number of plaques present in order to work out the concentration of bacteriophage). There looked to be some relationship between size of the plaques and the dilution. As the dilution increased from 10-4 to 10-3, the plaques observed on the plate also increased in size. This is interesting as my phage plaques are actually decreasing in the number of plaques as the titre plates become more diluted but should in fact be remaining the same size. So why is this happening?

As the titre dilution for 11.4 increased, so too did the size of the individual plaques. Dilutions 10-2 and 10-1 were too webbed for photographic comparison.

As the titre dilution for 11.4 increased, so too did the size of the individual plaques. Dilutions 10-2 and 10-1 were too webbed for photographic comparison.

After discussion, a few plausible hypotheses were concocted.

  • Can the bacteria detect the phage?
  • Do the phage detect density and not grow to a larger size to conserve nutrients? Phage signalling
  • Is the bacteria signalling to the other bacteria with the increased concentration of phage?
  • Do the bacteria detect different phage and restrict growth based on that particular phage?

Any of these seemed possible. Yet elaborating on phage detection, we came to the conclusion that phage are relatively stupid and thus is it extremely unlikely that it is the phage detecting density. Yet anything is possible in science? Hmmmm back to the drawing board!

Just to conclude today’s blog, I would like to draw your attention to a second observation that I have seen. It looks as though there is a ‘halo’ morphology around these plaques. Quite interesting considering this morphology actually became present after 10 days, rather than the 24-48 hours as the guide suggested! The picture below illustrates my plate 11.5.3 on the left, 48 hours after the first streak was completed, and another 10 days looking at the same streaked plate.

Unique 'halo' morphology observed on the same plate after 10 days incubation at 37 degrees Celsius.

Unique ‘halo’ morphology observed on the same plate after 10 days incubation at 37 degrees Celsius.

Click here to read a short blog of Debbie Jacobs-Sera (University of Pittsburgh) who observed a similar phenomenon on her plate of M. smegmatis phage and the discussions of possible hypotheses below her article. In brief, some of her hypotheses include;

  • After 5 days M. smegmatis runs out of nutrients and stop dividing
  • Phage initially in lysogenic stage (clear plaques), then it becomes efficient to enter lytic cycle (turbid) as nutrients are depleted
  • Clear plaque outline represents access to more nutrients further out

I’m hoping to keep looking into some of the possible hypotheses as to why these plaques are doing what they’re doing. There should be some interesting finds (I hope!)

In the meantime, keep phinding those phage!

Courtney 🙂

UPDATE:

We have had an interesting discussion in regards to further possible hypotheses below. 🙂

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About Courtney Davies

Current hunter of phage!
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2 Responses to The Phantastic Phage Phind: Rise of Mycobacterium smegmatis

  1. drhhnz says:

    Hey Courtney! Interesting stuff! I was thinking as I read your post: Maybe I had misunderstood part of what Debbie’s post had meant.
    The discussion there was talking about a time dependence to the phenomenon in terms of phages that had first been lysogenic (integrated into the cells)and then becoming lytic after the cells run out of nutrients. I had supposed that this referred to the time from the initial phage and bacterial pairing (centre of plaque) vs. the later stages of infection seen at the perimeter of the plaque.
    (((0)))–late
    |early

    What if instead, she was referring to the cells in the centre of her plaques looking turbid initially and then looking like the cells had lysed later on? There are really two different ways then of thinking about time as represented by a plaque morphology. That observation might be true of Debbie’s plaques then but not yours. So, did you plaques always look “clear/ ltyic” in the center and just the perimeter changed?

    (((0)))<–later and days later: (((0)))<–later + days later
    ^early ^days later

    Let's talk more!

    • Hi Heather,

      What a great comment! Hmm I never though of it like that!

      In hindsight that makes a lot more sense. Mine would seem to be the opposite of Debbie’s. My plaques were initially lytic (around the 24-48 hour observation mark) and noticeably changed to the halo/turbid ring morphology discussed above after about 10 days. Would it be possible that my phage has started in the lytic cycle before transitioning to the lysogenic cycle? It seems more common to begin lysogenic and then head into the lytic cycle (as Debbie’s phage exhibited) if a change was to occur.

      This got me thinking some more, because my plaques were relatively small, the equipment I had on hand (ruler) was not sufficient enough to provide an accurate measurement of the plaque to compare to any changes I may have observed later on. So quite possibly, the turbid ring I am observing may not necessarily be a change in the cycle (from lytic –> lysogenic) in the initial phage plaque, but an extension of the plaque with new phage starting out lysogenic – if that makes sense? (will discuss this more in the lab tomorrow!). It does look as though the plaques on the halo morphology plates are slightly larger than they initially were, supporting the hypothesis that in the beginning, the now turbid area was once just uninfected bacteria.

      So to briefly conclude, the turbid outer ring was initially just bacteria, before the lytic phage ‘burst’ out to infect and thus those phage that were previously lytic now infected the M. smegmatis in the lysogenic cycle resulting in the centre of the plaque remaining clear/lytic (no change) and the outer ring appearing more turbid than the surrounding bacteria.

      Will keep these plates around and see what happens in another few days.

      Thanks for the comment!

      Courtney.

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