Today I’m giving you the gift of a post on Griffith! I had a lot of fun yesterday reading through Avery, MacCleod, & McCarty’s classic paper where, using experiments with strains of a bacterium called Pneumocci they were able to show that DNA holds genetic instructions. Avery’s whole experiment involved testing to figure out what was the “transforming principle” – what component of stuff they pulled out of dead, virulent (disease-causing) bacteria (S strain), when mixed with live, non-virulent bacteria (R strain), could “transform” the R bacteria into the S form.
And they were looking into this because of Griffith’s discovery – Griffith discovered the existence of a “transforming principle” and Avery figured out what that principle was – DNA! They built their work upon original findings by Frederick Griffith, an English bacteriologist, so I decided it would only be fair (and likely fun) to read his paper too – and today I want to explain it to you!
I have to admit – it actually wasn’t very fun of a read – no offense to Griffith, I didn’t enjoy this paper nearly as much because the whole “bacteriology” thing isn’t really my jam. Griffith was a public health officer and his interest in the bacteria stemmed from trying to better figure out, treat, and stop the spread of pneumonia. He was really interested in epidemiology and this whole transformation thing was a chance finding that was kinda out of the scope of his normal stuff. The paper’s really important and he did a lot of important work that I’m glad he did, but the paper reads like a lot of mice-killing… here’s a link if you want to read it http://bit.ly/2q1mmYy
The bacteria Griffith worked with came from actual human patients (as you might have guessed from the name, Pneumococcus bacteria can cause pneumonia). He would take bacteria from patients and plate them onto blood agar plates and/or inject them into mice. He would sometimes find that sputum from patients contained more than one strain of bacteria. And sometimes it would seem like there was only one strain, but after subculturing it or injecting it into mice, different strains would emerge. He didn’t know whether the bacteria were actually “transforming” or whether the other strain was already there, just hiding.
He was interested in finding weakened strains that might be useful for making vaccines – so he’d inject mice and see if they got protected. Some of the weakened strains he was looking into were these weird strains of the bacteria that, instead of forming nice Smooth colonies when plated on blood agar plates (S strains) grew as Rough colonies (R strains). And these weren’t just “aesthetic differences” – the S strain looked smooth because it had an outer capsule made of polysaccharides (big sugar chains). And the capsule was important for protecting it from the hosts’ immune system.
The reason the R strain looked rough was because it didn’t have that coat – and since it didn’t have the coat it also didn’t have the things it needed to protect itself & infect cells. It had everything else it needed to be a full-on diseases-causing bacteria – so if it could just get the genetic instructions for making that capsule it could be transformed into S-like cells.
And, indeed, Griffith found that heat-killed S cells (which couldn’t infect anything) could give these capsule-making instructions to live R cells and those R cells would transform into S cells. And those S cells could cause disease in mice just like normal live S cells do.
But that came later – he tried it out because of something he saw occasionally happen naturally – He saw that sometimes R strains would “revert” to their S form. Different strains “reverted” more easily than others and if you injected a ton of it, not just a little bit, into a mouse, it was more likely to revert – Griffith thought this might be because the R bacteria still had a little bit of “S-ness” that got released when the crowded bacteria broke open and when there are a lot of R cells close together, the little bit they each release adds up to enough for one of the unbroken ones to “build up their rudimentary S structure”
So he theorized that if he stuck some dead S cells in with live R cells he might be able to achieve the same effect. So he decided to try it out – he took some S bacteria, grew it up in glucose broth to get a nice dense culture, then steamed it at 100°C to kill it, & spun it in a centrifuge to concentrate it even more, then injected this and living serum broth culture of the R strain into the mice. Those mice died within 3-5 days and their blood was chock full with the live S.
The most obvious explanation would be that some S survived and then thrived and it wasn’t actually a transformation occurring – so he went to great lengths to confirm that no S bacteria could survive the heating – including plating and plating and plating lots of plates of it – none of which grew anything
And he knew it wasn’t just that there were some spare capsule parts the R cells could take and “reuse” because the transformed R cells were able to replicate (multiply themselves) and make enough of the capsule to coat all of those cells. And they could do this indefinitely – they were able to extract live S cells from the dead mice and infect healthy mice with them.
But the really cool part was that he was able to show that the bacteria were gaining a new “power” not just recovering a power they’d lost…
In addition to judging them based on looks, Griffith used agglutination reactions to test what strains of bacteria were present. There are different “types” of pneumococcus classified by how they react or don’t react to different antibodies in sera – these “serotypes” include Types I, II, & III & they’re based on what sugars stick out from the bacteria. And you can have R & S versions of the different types (e.g. IIIS & IIS are both virulent whereas IIIR & IIR aren’t but IIIS & IIIR will both react to the same antibody and IIR & IIS will both react to the a different antibody.
He could figure out what serotype the bacteria were by mixing them with serum from patients with known strains – the sera contained antibodies against those strains. Antibodies are little proteins that recognize specific features of other things (like specific sugars sticking out of bacterial membranes), so if they encountered bacteria of that type they’d react and clump up. The clumpy cells would settle to the bottom of the tube, leaving the liquid clear. But if the bacteria was of a different strain it wouldn’t have that specific thing the antibody was “looking for” so it wouldn’t react with it and would be free to grow throughout the liquid and the liquid would look cloudy.
He did this with multiple type combos – for example, when he injected live III-S it’d kill the mouse but if he heat-killed the III-S first the mouse would live. And the mouse would also live if he injected live II-R (it would also live if he injected III-R, but then, the bacteria would react to the “Type III” sera the same whether or not the cells had actually transformed. You see, from mouse-kill-ability he could tell if the strain gained virulence, and from the shape of the colonies, he would be able to tell if an R to S conversion occurred, but that wouldn’t tell him if it started making new stuff (type III stuff) or it just regained the ability to make a capsule.
When a bacterium acts differently from its parent, it’s usually because it’s acquired a mutation that messed up the instructions for making something the bacteria needs. Mutations aren’t always bad – they provide the foundation for evolution – and much of biochemical knowledge has been gained by studying mutants!
Sometimes, a mutation can “un-mutate” – acquire a “reversion mutation” that basically changes back the mutation. But, because he used R & S strains of different types, Griffith knew what he was seeing wasn’t just the R strand randomly fixing itself. Because when he added heat-killed S the R cells didn’t just turn smooth and virulent, they transformed into cells just like the cells the “transforming substance” came from – they changed serotype.
So even though he called it “reverting” it was actually a much more “wow” type thing happening. But remember, Griffith was this epidemiology-focused scientist, so he was more worried about changes that were happening in patients’ strains, etc. And he left it to other scientists to figure out what was happening at the molecular level. Avery and friends took up the challenge – by process of elimination (adding selective ingredient destroyers like proteases to chew up proteins and DNAses to chew up DNA), they was able to show that the transforming principle Griffith had discovered was DNA (only DNA chewers could destroy it).
more on Avery’s lab’s experiments: http://bit.ly/33mcB5I
more on antibodies: http://bit.ly/31PvrlO