Monday, October 23, 2006

Musing on Sxy

In my last blog posting I said that both Sxy and cAMP-CRP are limiting to competence development during growth in rich medium. However, I would like to make a more precise statement about how these limitations may change as culture conditions change. Here I expand my previous graph showing the correlation between increasing Sxy levels and increased transformation in both KW20 (blue) and hypercompetent mutants (red) during log (OD 0.2; filled circles) and late log (OD 1.0; empty circles) growth in rich medium (see "A strong positive correlation"). The black line plots the correlation, and the dashed black line is a hypothetical leveling-off of transformation frequency when Sxy levels become saturating. The dashed green line is a prediction of how the plot would look if cAMP were added to the culture medium.
Redfield (1991) showed that adding cAMP to hypercompetent sxy-1 cells increases transformation frequency in early log but has no effect in late log growth. Therefore, cAMP is not limiting to competence development at ~OD 1.0. The green line is not predicted to have the same slope as the black line because cAMP is only limiting during log growth; presumably, as PTS sugars are depleted in late log growth, cells are making their own cAMP. Thus, the two lines converge and level-off when Sxy becomes saturating. We know that Sxy isn’t saturating during late log because some hypercompetence mutants have more Sxy than others, and this correlates directly with increased transformation frequency.

This predicts that titration of cAMP during log growth will have no effect in KW20, but will increase transformation frequency in hypercompetent mutants such as sxy-1.
This hypothetical graph shows that due to insufficient Sxy levels in KW20 during log growth, no competence develops even at high cAMP levels. On the other hand, there is sufficient Sxy in sxy-1 to activate competence genes, thus adding cAMP increases transformation frequency. Hypercompetent strains are transformable in log growth, so there must be some cAMP produced by cells in rich medium. In the graph, the cyaA- mutants cannot make cAMP thus even the sxy-1 cyaA- double mutant cannot become competent until cAMP is added.


At 7:45 AM, Blogger Rosie Redfield said...

In the second paragraph you wrote: "Therefore, cAMP is not limiting to competence development at ~OD 1.0. " But if that were true shouldn't your empty-circle points lie on the dashed green line?

I'll send a more complicated response by email.

At 9:33 AM, Blogger Some call me Tim said...

Hi Rosie,
What my hypothetical graph is trying to depict is that during late-log growth, in all but the hypercompetent mutant with the highest amount of Sxy (empty red circle where the green dashed and black line converge - which I now notice is obscured by the thick lines), Sxy is limiting but cAMP is not. How else would we explain that adding cAMP to hypercompetent mutants in late log does not increase transformation frequency?


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