Persistence and competence

Here’s my plan for testing why transformed colonies pop up later.

4 plain plates w/ 10^-6 NovR cells

4 antibiotic plates w/ 10^-6 NovR cells

8 antibiotic plates w/ 10^-3 of mixed cells(10ul of NovR into 10ml equivalent CFU NovS – 1 in a thousand dilution)

1 plain plate with NovS cells

1 antibiotic plate with NovS cells(these last two are controls)

I’m not sure yet if I’m going to plate directly from sBHI or do 100 minutes in MIV. From what I’ve read persister percentage in cultures varies depending on growth stage. Ideally I’d do both. It would be good to do it side by side of a transformation experiment so I can directly compare to transformation. Still, the most likely explanation for the phenomena I’ve observed, colonies appearing later on antibiotic/transformed plates than on plain plates is an effect of the antibiotic or cell density, but this test should be easy to figure out what’s going on.

I’m reading a Persister cells annual review of microbiology. I’ll write this post as I’m reading it(and heading to the bench every once in a while to do some science). The review includes a nice section about the history of persistor cell research.

– They propose a model for the recalcitrance of infectious diseases. Antibiotics wipe out reguler cells, then the immune system cleans up the remainder along with persister cells. However in biofilms antibiotics can penetrate but elements of the immune system can not, so persisters in biofilms allow regrowth of infection after antibiotics have dissipated.

– 1 in 3 people carry a latent form of tuberculosis! 10% of those will develop an acute infection at some point in their lives. It’s one of the main reservoirs for the disease, and very little is known about these latent pathogens. Ask thompson lab about this. Not super surprising due to what I learned at the humna microbiome conference – diseases can almost never be found to be caused by the presence or absence of bacterial species, but more complicated relationships of these microbes and their host. Still I assumed tuberculosis was exceptional and could not be considered commensal in any form. I really should bug the Thompson lab about it. There are three of us in the office usually, I wonder which one of us has it.

– Screening transposon insertion knockout libraries of Ecoli has not been able to find a non-persister phenotype. Therefor the mechanism controlling persistance is unlikely to be a single gene

– Interesting hypothesis: there is no mechanism for persisters, they just arise due to the accumulation of misfolded proteins. Doesn’t seem likely

– There are few persisters in early log, increasing sharply as the culture enters mid exponential. Keeping a culture in early log conditions by serial reinnoculation prevents persisters from arising entirely(presumably diluting out the ones that were initially in the culture when it aliqouted from a late log stock)! So competent cells and persistent cells have something in common – They are formed under similar growing conditions

– A larger Ecoli knockout screen found some global regulator genes that lowered persister formation, as well as two very interesting genes. YgfA can inhibit nucleotide syntesis. Digging into the Hansen 2008 paper:

“The mammalian homolog of YgfA, methenyltetrahydrofolate synthetase, catalyzes the conversion of the stable folate storage form 5-formyl-THF into the rapidly degraded 5,10-methenylTHF, thereby depleting cellular folate pools (2). If the E. coli enzyme has the same function, then in a simple scenario, the stochastic overexpression of YgfA will lead to folate deficiency,which will impair the biosynthesis of purines, thymidilate, and methionine (7) and will contribute to persister cell formation.The artificial overexpression of YgfA indeed produced a phenotype of increased tolerance to ofloxacin. The knocking out of ygfA would be expected to decrease the level of persister cells,which we have observed.”

ygfA is involved in purine nucleoside biosynthesis process. The annotations in the gene bank are confusing – it’s not very well studied. It’s an ATP and nucleotide binding ligase, it degrades folate pools which are needed for purine and thymine synthesis. Stochastic overexpression of ygfA will lower purine pools, causing occasional persistance. So when it’s knocked out folate always remains high and therefor purine pools always remain high-> purines repress persistance? purines repress competence.

It might not mean anything. It makes sense, there’s logic to why the same conditions could induce both competence and persistance

I did a blastp search, there’s a partial match to an unknown KW20 gene called Hi 0858, 54% identities 66% positives, evalue= 9e-48.

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