To make sure that all culture tubes started with the same medium and the same density of cells, I mixed up a big batch of no-phosphate medium, added cells (2 x 10^5 cfu/ml), and divided the culture into thirds. I then added NaPO4 to two of these parts, to give 3 µM and 1500 µM (in addition to whatever phosphate might be contaminating the medium), and added 5 ml of each part to each of 15 screw-cap galss tubes to which I had already added the appropriate NaAsO4/NaCl mixture. So I had 45 tubes in all, 15 with no added phosphate, 15 with 3 µM, and 15 with 1500 µM.
I incubated the tubes at 28 °C with gentle rocking, and checked the optical density after 24 and 48 hr.
Conclusion: Arsenate stimulated growth, but didn't affect the final densities of the cultures. The stimulation is not because the arsenate is contaminated with phosphate, because the effect was strong only in the cultures with 1500 µM added phosphate, and because it didn't affect final density in the phosphate-limited cultures.
There's still much more variation in final culture density than I'd like to see. This might be due to minor differences in trace contaminants in the tubes, although they were all last used for similar cultures and all thoroughly washed the same way. One solution would be to use only new tubes, but these tubes are not cheap and I don't want to take money from our transformation work.
I'm not going to do any more work on this - not going to do experiments to find out why arsenate stimulates growth, unless the mass spec shows that there really is arsenic in the DNA of arsenate-grown cells. The growth stimulation I'm seeing isn't a replication of Wolfe-Simon et al's report that their cultures grew with arsenate but not without it, but it might reflect the same biological process.
I would think that the potassium in the potassium arsenate could be stimulating the growth. If you changed the NaCl to KCl, you could try to rule this out.
ReplyDeleteI'm using sodium arsenate, not potassium arsenate, and my AML60 medium includes 10 mM KCl. I made these changes to ensure that differences in potassium don't confound the effects of phosphorus and arsenic. See Two mistakes discovered.
ReplyDeleteWell what do you know, maybe there is something to this "flim-flam" "bad science" after all.
ReplyDeleteRosie,
ReplyDeleteI saw one of your comments on one of the recently published articles, notably the deposition of the GFAJ-1 sequence. It is remarkable how FWS and Oremland seem to be doubling down on their statement that we will find As incorporated into biomolecules.
I don't have a critical understanding of the sequence, but I wanted to ask:
1)Did I understand the sequencing author correctly in that the sequence looks a lot like e. coli and possesses no obvious or known genes to deal with As toxicity. Does "looks a lot like" meant to be understood in a taxonomic sense?
2)You can certainly grown the bugs in high [As], if indeed this is an e. coli variant, is the high [As] notable to begin with if e. coli can grow in it?
No doubt you will talking about these things in an upcoming post, but just thought I would ask.
Hi
ReplyDeleteCongratulations for your great job (I just posted a brief summary in my blog). Concerning the stimulation of growth by arsenate, I am not a chemist but I have a wild speculation. It could be that GFAJ-1 uses As in ATP?, I mean, in high As levels it could exist ADP-As? Maybe the chemical bond P-As gives as much energy as P-P, and the unstability is not a problem there.
Just a thought. Regards
Do you think high As levels might be turning on a high affinity PO4 transporter. I'm sort of thinking of how cationic peptides can displace divalent cations from the Mg2+/Ca2+ sensor PhoQ tricking the bacterium into thinking there's less phosphate than there is. Alternately, it could promote other PO4 scavenging pathways like production of non-phospholipids (like ornithine lipids). This happens under limiting PO4 growth with Pseudomonas. I've been following this with interest since last December, but I don't really have time to devote to it on the bench.
ReplyDelete@Joe: Yes, for sure something like that could be going on. I don't have the time to devote to it either, but maybe Felisa Wolfe-Simon will figure it out in her new lab.
ReplyDelete