Competence development in defined media

OK, my repeat time course worked nicely, and I can compare how wildtype cells develop competence in rich medium and in defined media with and without a purine source.  The defined medium is the 50:50 mix of the RPMI-based and cMMB media.

 

The first graph shows growth of cells in the three media.  They all started from equal aliquots of cells that had been pregrown in the '- inosine' medium and frozen.  We see that the cells in the defined medium with inosine grew almost as fast as the cells in rich medium, but they stopped growing earlier and began to die.  The cells in medium without inosine grew slower.  You can't tell from this graph, but they didn't get much more dense than the final point shown here.

The second graph shows the transformation frequences of all three cultures together for easy comparison, and the third graph shows the same data for each culture separately, with the lighter lines showing their MIV-induced transformation frequencies.   The inoculum with which the cultures were started gave no transformants, so its transformation frequency is graphed as the limit of detection (2.5x10^-8)

The blue line/top panel is control cells in the rich medium sBHI.  They behaved normally, giving a maximum transformation frequency of about 10^-5 when the culture got moderately dense, and an MIV-induced transformation frequency of about 2x10^-3. Note that the cells were barely competent at all when at low density (TF 5x10^-8).

The red line/middle panel is cells in the defined medium plus inosine.  At t = 0 min these cells were 5.5-fold more competent than the control cells (TF 3x10^-7), although the growth curves show that both cultures were in log phase growth.  They were also about 3.5-fold more competent at the next time point, but then their transformation frequency began to decline while they still appeared to be in log phase, and at the last time point there were no transformants at all.  Transfer to MIV made them about twice as competent as the control culture, consistent with Ranhand and Herriott's finding that pregrowth in medium with lots of inosine enhances subsequent competence development in MIV.

The green line/lower panel is cells in the defined medium with no inosine.  These cells became even more competent in log phase than the cells in medium with inosine did, with TF 25-fold higher than the control cells.  But they didn't get more competent as density increased, and their competence started falling while their density was still much lower than that which gave maximum competence in medium with inosine.  The second-last time point gave only a single transformant, and the last time point gave none.  Transfer to MIV did increase competence, but to a level about 200-fold lower than control cells and 400-fold lower than cells with inosine, about 10-fold lower than I saw last time.  The increase was only about 40-fold relative to the cells at time of transfer, whereas the others had MIV-induced increases of about 1000-fold.

I should test the effect of adding cAMP to the cultures in defined media.  The media contain some glucose, but no fructose, so our simple model of competence regulation predicts that the PTS will have activated adenlyate cyclase, producing high cAMP levels.  However a former PhD student found that adding physiological concentrations of fructose to rich medium and to MIV had no effect on competence development.

Inosine is a nucleoside, the base hypoxanthine attached to a ribose sugar.  Amy's chart shows that inosine cannot be directly converted to IMP and then to AMP and GMP.  Instead the sugar must first be cleaved off, and the resulting free hypoxanthine is then converted directly to IMP in a single step.  So use of inosine as a purine source creates hypoxanthine in the cytoplasm, which could accumulate to high enough levels that it represses the purine biosynthesis pathway by binding to PurR.

 I don't think we've ever tested other purine sources.  Ranhand and Herriott found that deoxyinosine had the same competence-stimulating effect as inosine, and that hypoxanthine and ribose were ineffective.