Fighting Tuberculosis – lions – Jan 12

biotin is vitamin B7 or vitamin H, which ever you prefer. it’s necessary for all living cells, but only in real low amounts (someone estimated it’s about 10 molecules/cell for e. coli, which is ridiculous). it’s made by most bacteria, some plants, and some fungi, the rest (including humans) either get it from the environment, from eating plants, or from commensal bacteria.

tuberculosis in particular has a really weird metabolism (actually most things about it are weird, the overriding “philosophy” or whatever in our lab is that people did all these experiments in easier-to-work-with bacteria like e. coli or whatever, and that we’ve always assumed that all bacteria are alike, but that a bunch of classical genetics concepts that were hashed out in coli aren’t applicable to tb or in fact quite a lot of bacteria. part of the reason tuberculosis has such a weird metabolism is that it lives inside of human macrophages, which are actually immune cells meant to kill it. it’s kind of like living in the barrel of a gun. but the macrophage does a bunch of things to try to kill TB, including oxidizing it, trying to cut it up, acidifying its compartment, and isolating it both from the rest of the macrophage (on a cellular level) and from the rest of the lung (on a organismal level). tuberculosis is able to recognize these external stresses through various mechanisms and drastically alter its metabolism and lifestyle in order to survive. we found a protein in the lab we believe is necessary for stress response and our lab has been studying it for quite a while, one of the things that’s messed up in mutants which lack this protein is biotin biosynthesis, but not the part of biotin biosynthesis (the last four steps) that we know about.

canonically there are four enzymes in biotin biosynthesis BioF, BioA, BioD, and bioB. those are the last four steps, which take pimeloyl-CoA and transform it into biotin. steps to make pim-CoA are generally pretty mysterious, in coli there’s bioC and bioH which shunt a weird substrate through fatty acid synthesis, in b. subtilis there’s bioI and bioW and whoa are those guys weird (they break down fatty acids into pim-CoA). in some fungi, there’s a couple of enzymes in the peroxisome which function sort of like bioI. in the rest of life, it’s all a black box. we want to understand what is disrupted in our mutant, which generally requires to know what you’re looking for. we found one thing which is likely to have a role, but it’s definitely not the whole story.

our “strategy” is to infect a tb family bacteria with a defective bacteriophage which can’t integrate into the tb genome. it can only integrate via a transposon excision and reintegration. basically we give the bacteria a little DNA element into its genome which likely will disrupt and nullify one of its genes, and also gives it kanamycin resistance so we can isolate it. we do that about 40,000 times (lol) and get what’s called a library of mutants, which is a classical genetics thing where you literally have a culture of cells where at least one cell in the culture has literally any mutant in the genome. then you test all of them one by one to see if they can make their own biotin. i’m running into trouble on the last bit because i can’t get good conditions to make that distinction.

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