Growth of GFAJ-1 in 40 mM arsenate (not)

My initial attempts to grow GFAJ-1 in media containing 40 mM sodium arsenate have given unexpected results.  The first time I thought it might be a screw-up on my part, but the second time suggests a problem with the cells.

The experimental design is simple.  I set up the six flasks shown below, and inoculated each with about 2 x 10^4 GFAJ-1 cells/ml.  If we ignore the presence of glutamate in the medium, flask 1 corresponds to the +P/-As condition of Wolfe-Simon et al.'s Fig. 1B (shown below the flasks), and flasks 3 and 4 correspond to  the -P/+As and -P/-As conditions.

Because I've already found that the cells grow in limiting phosphate without any arsenate,  I expected dense growth (~ 10^9 cells/ml) in flasks 1 & 2 (lots of phosphate), thinner growth in flasks 3 & 4 (limiting phosphate) and even less growth in flasks 5 & 6 (very limiting phosphate).  The graph below shows the results (blue line is initial cell denbsity).  Cultures 1, 3 and 5 grew as expected, but I was surprised to see very little growth in flask 2 and quite thick growth in flask 4 (both with arsenate).  

The most likely explanation was that I'd made a mistake in setting up the flasks - perhaps putting the 1.5 mM PO4 into flask 4 instead of flask 2.

So I set up the cultures again, this time starting them from cells taken from flask 4, because these appeared to have grown well in the presence of arsenate.  (The other cells had been growing without arsenate for about 2 months, and I was worried that they might need time to adjust to this stressor.)  These cultures started with about 10^3 cells/ml.  The graph below shows interim results after only 14 hr incubation; again the blue line is the initial cell density.

All of the cultures without arsenate have grown well; none have run out of phosphate yet.  But there hasn't been any growth in the cultures with 40 mM arsenate; instead most of the cells have died.  These numbers are the density of the cultures yesterday morning.  This morning cultures 1 & 3 were turbid, as expected, but all the arsenate cultures looked clear.

Because these arsenate  cultures looked unexpectedly thin yesterday, I set up another replicate set of 6, using phosphate-depleted cells from the freezer.  I'll have results from these tomorrow, along with the final results from the above cultures.*

So maybe I'll need to gradually re-accustom GFAJ-1 to growing in high arsenate, by first growing them in a lower concentration.  This will take some time, and will have to wait until I get back from Science Foo Camp next Sunday.  I'll also leave the present cultures for another week, to see if cells eventually grow.

Quick check that I made up the sodium arsenate correctly:  The molecular weight of sodium arsenate is 312 (this is the dibasic, heptahydrate version = Na2HAsO4 7H2O), so I put 31.2 g into 100 ml final volume, to give a 1.0 M solution.  I then added 400 µl of this to each 10 ml culture to give 40 mM.

*Tantalizing note added Aug. 8:  Well, my third set of cultures are behaving like the first set - the cells in 3 µM PO4 + 40 mM AsO4 have grown up slightly more dense than the cells in 3 µM PO4 (no AsO4), but the cells with more and with less phosphate haven't grown much at all.  So I probably didn't make a mistake with the first set after all.  I don't know what this could mean, but it is sort-of consistent with the -P/+As and -P/-As results in the Wolfe-Simon et al. figure above...  Unfortunately we'll all have to wait until next week for me to do any more investigating.