The arena is empty except for one bact, not driving or striving as fast as it can

A very important implication of stochastism I didn’t mention explicitly in my bringing the noise post are persister cells. Not my fault. Since 2005 there are 1,760 papers that mention “stochastic gene expression”, 847 on “persister cells”, but only 41 that include both. Nevertheless persister cells are ruled by stochastic forces, and are very relevant medically so this post will be a brief introduction to them.

Persister cells are genetically identical to normal cells, but have a non-growing or very slowly growing phenotype. There’s usually a small percentage of them in most bacterial colonies. We don’t like them because this ubiquitous strategy is super effective against antibiotics, which usually target some aspect of growth such as mRNA translation. Antibiotics kill all the growing cells, but the persisters are unaffected, and then when some trigger comes along(or no trigger at all) the persisters spring to life and the infection starts anew

In terms of our research I can’t imagine them being an issue for any of our experiments. They aren’t competent, so they won’t be taking up DNA, they are antibiotic resistant so they could show up on antibiotic plates but by their very definition they won’t grow. The antibiotics on the plates aren’t going anywhere so if they did switch to a growing phenotype they would be killed, and by the time the time the antibiotics are no longer effective the vitamins have denatured and HI wouldn’t grow anyways(HI is super wimpy, it needs very fresh vitamins to be cultured). Maybe they could affect plate counts? They would have no effect on antibiotic plates, but maybe they could affect colony counts on plain plates based on when the plate is counted, which could be as early the next morning up to a few days later and can vary a lot based on how busy/lazy I am. But since I usually see colonies growing at ~ consistent rate on plain plates, and again the vitamins are done within a day or so of being out of the fridge, I don’t think persisters matter. Still next time I have a plain plate I’ll circle the colonies and leave it on my bench for a few days to see if the count changes instead of just garbaging it.

There’s this interesting 2010 paper entitled Resonant activation: a strategy against bacterial persistence. The idea the authors present, although in a computer simulated very hypothetical often over-my-head physicy way, is that the current way we treat infections, with a constant dose for a long time, is dumb. Instead a smarter way to deal with the reality of these persister cells is using intermittent high doses of antibiotics. The idea is that a constant dose of antibiotics keeps cells in a persister phenotype, so by matching up the frequency of antibiotic administration to a bacterias natural average frequency of revision to a growing phenotype the entire population can be wiped out more quickly and with less total antibiotics involved. The authors also mention that this method could be used for cancer treatment. Spencer et al have reported that bacterial-like persitence occurs in cancer cells.

I think my next big post will be on biofilms(where persisters are found at higher percentage). There’s a 2008 paper ‘Haemophilus influenzae biofilms: hypothesis or fact?” I want to read. We seriously still aren’t sure if HI makes biofilms? Seems like a problem that could be solved by looking, so I assume there’s more interesting is going on. In lab news I’m on campus anyways so I’ll drop by the lab to prepare some over nights so I have fresh cultures rollin’ for tomorrows experiments.

edit:  How about this for an experiment? Allowing a bacteria to live for a long time, and continually adding differently marked DNA to see how long the cycle between competency and and non-competency is. The average amount of time a cell spends in competency should be roughly equal to the percentage of competent cells in a culture under the same conditions right? To achieve this I’ll have to stop the bacteria from replicating, which could be difficult without killing the bacteria. Hmm. I could stick them in the fridge but that won’t work b/c I don’t want to slow the metabolic rate. Maybe I could use a ‘bacteriostasic’ agent(thanks worldbook encyclopedia). Antibiotics can be bacteriostasic, and I recognize one as something we use in our lab often, tetracycline. It works by blocking aminoacyl-tRNAs from binding to the mRNA-ribosome, inhibiting translation. But inhibiting translation would definitely inhibit competence so I can’t use that. Embedding in a semi solid? It won’t stop replication completely, atleast not in any material I’m aware of. We were discussing doing a 1 day experiment involving embedding bact into agar a while back, what was the point of that again? Alternatively maybe a KO of a gene that only affects replication, but how would that work, it wouldn’t be culturable

Maybe I’m thinking about the approach wrong, why stop replication when 1) it interferes with what the bacteria normally likes doing and 2) I can learn the same information by tracking TF’s of isolated colonies. I could restreak/culture single colonies  and test the transformation frequency of their descendants over time. Would descendants from a single non-competent colony, a colony that grew on a plain plate but can’t grow on a marked plate, ? If it has roughly the same competency rate as the culture in general it means competency fluctuates relatively quickly under those conditions. If the competency rate is much lower, it means that the epigenetic inheritance of competence is very strong. Lots of problems with this approach, #1 being that we can’t even really tell if a cell is non competent by using a marker, it could be competent but just not have picked up that piece of DNA. And #2 because transformation is already so rare, at the very best 10-3 under the most favorable lab conditions, it would be difficult to detect anything unless we let the cells divide a lot prior to transformation, which means we wouldn’t be able to resolve a frequency of competence on/off switching quicker than several generations.


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