I've spent most of this week just testing media. Not a rewarding activity, especially when the results don't make much sense.
Before I can test our purH mutant's phenotype, I need to establish conditions where its growth should depend on the availability of a purine. This requires using a defined medium, where I can control whether a purine source is present. I have been trying two defined media, but both are giving weird results.
The first is based on a medium called MMB. When we first tried it, years ago, our wildtype cells wouldn't grow. We suspected that the problem was lack of a required amino acid, so we tried adding some casamino acids. The cells grew fine in this modified MMB so we renamed it cMMB. (Derived from the protein casein, casamino acids contains everything except tryptophan.) Growth in cMMB required a pyrimidine source (uracil) or precursor (citrulline), and was higher if the medium was also supplemented with the purine inosine. This is what we expected, as H. influenzae can make its own purines but not its own pyrimidines.
As I described in the previous post, we didn't have any more of the MMB ingredient glutathione (a tripeptide), and just adding more casamino acids didn't allow the wildtype cells to grow. I then tried supplementing cMMB with two of the three amino acids in glutathione (glutamate and cysteine). The wildtype cells grow quite well in this medium provided uracil is present, but when I also add inosine they don't grow (done twice). I would suspect that there's something wrong with the inosine stock I made, if it weren't for the results with the other defined medium I've tested.
The other defined medium is based on the tissue culture medium RPMI 1640. I've never used this before, and I was going to just scrounge a bit of RPMI 1640 from one of the many cell biology labs in the building, but found I could buy 500 ml from Stores for only $12.
RPMI 1640 medium contains salts, vitamins and amino acids; for tissue culture it's usually supplemented with 10% serum (and pyruvate and glutamine) but serum isn't needed for H. influenzae. The H. influenzae recipe has hemin, NAD, uracil and inosine, as well as pyruvate and glutamine. With these supplements our wild type cells grow to high density, but if I leave out the inosine there's no detectable growth at all.
So the RPMI results are the opposite of the MMB results. Inosine appears to be toxic in MMB but essential in RPMI. And uracil supports growth in MMB but not in RPMI.
I could just go ahead and test the purH mutant, predicting that it would not grow in cMMB (or RPMI) without inosine but would grow in RPMI with inosine. But this wouldn't be a well-controlled test, and I think we really need to get a defined medium working cleanly so we can use it to test our hypotheses about the role of nucleotide pools in competence.
Might the problem with the cMMB be the absence of glutathione? Maybe glutathione isn't just serving as a source of amino acids. The authors said that glutathione wasn't needed if cystine (or cysteine) was provided, but they included it because it "promoted the most luxuriant growth". Of course, they also said that growth required inosine, but it doesn't.
So I've just dug into the glutathione literature - luckily there's a group in Belgium that's been studying its role in H. influenzae. H. influenzae cells need glutathione to cope with oxidative stress, but they can't synthesize their own and must get it from the human host. This doesn't create a big problem in lab cultures because they're not under oxidative stress. I also recalculated the cost, and it's not that bad (maybe my earlier calculation dropped a decimal point...).
The Belgian group used as their defined medium the original MIc medium developed by Roger Herriott, which I've been avoiding because it didn't give reproducible growth. But maybe now I'd better make some up, as the newer media are being equally problematic.
In the next post I'll get to the other media problem I've been trying to sort out - the difference between the two brands of brain-heart infusion.
p.s. I tried Googling "cheap glutathione". Glutathione turns out to be a favourite dietary supplement of weight-lifters and other athletes, hyped as an antioxidant by 'wellness' gurus, and advertised to whiten your skin (because it inhibits tyrosinase).
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