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The Wolfe-Simon et al paper reported that these cells were resistant to 40 mM arsenate, but in my experiments so far the only time the cells really appeared to be resistant to 40 mM arsenate was Expt. 5, when I grew them in screw-capped glass tubes.
Even if I set aside all the other experiments (in foil-capped flasks or in screw-capped plastic tubes), this experiment was compromised by tube-to-tube inconsistencies in final cell density, probably due to the presence of some limiting nutrient (perhaps not phospate) contaminating some of the tubes. So I've now acid-washed all the glass tubes and caps, rinsed them lavishly in distilled water, and re-autoclaved them, so I can see if this experiment's result is reproducible. These conditions are closest to those used by Wolfe-Simon et al., so if I can consistently get growth in 40 mM arsenate I can prepare DNA from arsenate-grown and control cultures for mass-spectrometry analysis.
I'm going to streamline the number of conditions (just ± 40 mM arsenate, combined with no added phosphate, 3 µM phosphate or 1500 µM phosphate) and do two replicates in glass tubes and one in plastic tubes. I'll make up 50 ml of no-phosphate medium, add cells (from my frozen stock of phosphate-depleted GFAJ-1) to about 10^4 cfu/ml, and split this in two. I'll add arsenate to one half (and water to the other). Then I'll put 5 ml into each no-phosphate tube, add phosphate to 3 µM to the rest (both parts), and put 5 ml into each 3 µM PO4 tube (using 3 replicate glass tubes). Then I'll add more phosphate to the rest, to 1500 µM, and put 5 ml into each 1500 µM tube. Then I'll put all the cells gently rocking in the 28 °C incubator.
Are they PET tubes? Is it possible that those tiny traces of antimony could be reacting with the arsenate? Or chelating compounds from the bacteria leach out antimony, to which they are less robust?
ReplyDeleteThis is a real puzzle.
ReplyDeleteThe fact that you have seen growth in 40mm As suggests that the cells are probably As tolerant and something subtle is going on. It's easy to imagine how one might observe a false negative for growth (e.g. some contaminant in the tube lining poisoning cells)..it is very hard to imagine how one could observe a false positive.
You've been growing bugs for a while, so you are pretty experienced at this...but my only guess is that it feels like a pH issue (as one commentor has suggested previously).
Have you confirmed the pH of the media before and after growth?
@Pat: I doubt that tiny traces of anything could overwhelm 40 mM arsenate, but I suppose they could react with arsenate to produce tiny traces of something very toxic...
ReplyDelete@NAA: I haven't checked the pH of the arsenate cultures, but it's been stable in cultures without arsenate. Also, the growth stops right away, while there are so few cells that they couldn't be influencing the pH.
Long ago in a previous life I did a little bit of microbe-metal research. The lab that I was working in made it pretty clear to me that it is very tough to get all contaminating metal out of glassware, no matter how much it is acid washed.
ReplyDeleteAs a long shot: Is it possible that some of the aresenate is getting chelated by contaminating metal associated with the glass tubes? If that is what is happening (and that is admittedly a very big if) then you have less bioavailable arsenate than you think in the glass vessels... so the critters grow in the glass culture but not the plastic culture.
Regardless, my understanding is that acid washing of plasticware is much more effective than acid washing of glassware. Consequently, I'd suggest switching all culture vessels to plastic and forgo glassware altogether if at all possible.
Maybe you just don't know what you are doing, because you have never done this kind of research before. People spend their whole careers learning how to grow extremophiles. Your foray into fame ranting against "bad scientists" doesn't make you good at growing some of the most difficult cultures. If these bugs are not As resistant why are they living in Mono Lake which has 200 uM As? There are bugs that have been shown to tolerate much more than this, why do you doubt that these bugs can do it? Maybe it is your methodology because you are a novice at this and you are out of your field of expertise, something that maybe you should have mentioned to Slate from day one.
ReplyDelete