Field of Science

The 'fraction competent' problem

The new graduate student is considering potential research projects, and he's keen to work on the long-standing puzzle of why all the cells in a 'competent' culture aren't equally competent.  I don't think this is something we should be focusing our efforts on now, so below I'll try to explain why.

First I need to describe the initial observation and what we now know about it.  I originally blogged about it 5 years ago; that post explains the assay and the usual result.  Briefly, we can asses the variation in transformability of cells in a culture by transforming them with two selectable markers on separate DNA fragments.  If all the cells are equally competent the frequency of double transformants should be simply the product of the frequencies of single transformants; for simplicity I'll call the double-transformant frequency the 'expected' frequency.  Usually we find that the frequency of double transformants is much higher than the expected frequency, and we conclude that some cells in the culture are much more transformable than others.  (If we had found that the frequency of double transformants was instead much  lower than the expected frequency, we would conclude that many cells could only take up one fragment of DNA.)

Refs from my old notebook:  Goodgal and Herriott 1961, J. Gen. Physiol. 44:1201;  Porter and Guild 1969, J. Bacteriol.  97:1033; Bremer et al 1984, J. Bacteriol. 157:868.

The relationship between the observed and expected frequencies of double transformants can be used to calculate the 'fraction competent' of the cells in a culture.  But this calculation requires the simplifying assuming that competence is an all-or-nothing state.  If instead there's a gradient of competence, or if one subset of the cells takes up ten times more DNA than the rest, then the calculation doesn't apply.

That said, what we and others have found is that, whenever a culture has a high enough transformation frequency that the frequency of double transformants can be measured, the double-transformation frequency indicates that most of the transformation is being done by a subset of cells that are just as transformable as the transformable cells in a fully-induced culture.  This is true for all cultures with intermediate transformation frequencies (late-log growth, induction in log-phase with cAMP, partial competence of the sxy-1 mutant in log phase).

Here's what we've learned from these experiments:  Wildtype cells in log-phase growth give no transformants.  Cells at the end of log phase ('late-log) have a transformation frequency about 100-fold less than that of MIV-induced 'competent' cultures (~10^-4 vs ~10^-2), and the 'fraction competent' is about 0.5-1%  In MIV-induced cultures the 'fraction competent' is about 50% (well, 10%-50%; it partly depends on which markers are tested).  Adding cAMP to log-phase cells raises competence to the late-log level, with a 'fraction competent' of about 1%.

We've also examined double-transformant frequencies in the hypercompetent sxy1 mutant.  These cells are as competent in log-phase growth as Rd is in late log, and their 'fraction competent' is similar (0.5%) They are as competent in late-log as Rd is in MIV, and again the 'fraction competent' is similar (10-90%).  Competence in MIV is like that of RD, and the 'fraction competent' is similar too.

I mustn't forget that the 'fraction competent' measure is probably an oversimplification - the double-transformant analysis can't detect more subtle variations.  Here's a figure to make that point.

So, why don't I think this is a problem we should be actively working on?

I.  The most likely explanation is the least interesting:  The proximate causes are probably the combined effects of (1) the details of interactions between regulatory sequences and regulatory proteins, (2) random fluctuations in the cellular levels of regulatory proteins and metabolites and (3) differences in the cell-cycle stages of different cells (replicating DNA, dividing etc.).

II.  Investigating these factors would be very difficult.

III.  The phenomenon may be irrelevant to understanding why cells take up DNA:  The distribution of competence we see in lab cultures may well be an artefact of the culture conditions we use.  Understanding it is unlikely to help us understand how competence is controlled in the human host.

IV.  There may not be an ultimate cause at all - the proximate factors may not have been under direct selection.  On the other hand, if they have been shaped by selection to optimize the distribution of competence in the cell population (the subject of some just-so-story speculation), the nature of this selection will be very hard to characterize.

1 comment:

  1. You could get at the "subtle variation" question by looking at triple transformation frequencies (of course the numbers may be too low).


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