Field of Science

What RecA has to do with it

I spent parts of yesterday happily responding to requests for information from a colleague supportive of ideas, who was deep in an email discussion with another who thinks we're dead wrong. This had me digging through the Bacillus subtilis literature looking for information about the competence phenotypes of recA mutants.

The other colleague had expressed amazement that anyone could ever think that cells take up DNA for anything other than recombination, whereas we think they take it up for food. The idea that bacteria take up DNA for the (assumed*) evolutionary benefits of changing their genes is widely believed by microbiologists. (I say 'believed' because, like religious believers they are very reluctant to consider alternative explanations.) I've posted about why this is an important issue, and why we think the way we do, here.

As is typical for believers in "competence is for sex", he supports his conclusion by one piece of information. In his case it was that the B. subtilis recE gene is one of the genes induced in competent cells. RecE is a homolog of E. coli's recA, and functions primarily as a DNA repair protein by both detecting DNA damage and contributing to its repair by a strand-exchange process. RecA (and RecE) can also perform strand exchange on DNA strands brought into the cell by direct DNA uptake or by plasmids or phages; in its absence competent cells cannot be transformed by the DNA they take up.

In H. influenzae we know that the RecA homolog makes no contribution to DNA uptake - in recA mutants all the DNA that cells take up gets into the cytoplasm, where it is degraded by resident nucleases. We know this because if we use radioactively labelled DNA we see the label randomly incorporated into the cell's chromosome.

I was trying to find direct evidence that the same is true for B. subtilis. It's unlikely that anyone would bother to do this experiment now that we know a lot about RecA. The experiment would either have been done many years ago, when researchers were still uncertain about what RecA does, or done as a control for some other experiment. I couldn't find any experiment where researchers had looked for chromosomal incorporation of label in a B. subtilis recE mutant, but I've emailed the person most likely to have done this to ask if he can help**.

But I did find other evidence that B. subtilis recE mutants have normal DNA uptake, from a genetic analysis that's independent of recombination. Wildtype B. subtilis can be transformed with circular plasmids, which can replicate in the cytoplasm without having recombined with the chromosome. But like H. influenzae, B. subtilis only brings linear single stranded DNA into the cytoplasm, and linear strands are degraded if they can't recombine. But B. subtilis cells can take up plasmids that start out circular because they cut the DNA before taking it up. If the plasmid circles are monomers the resulting linear strands are degraded in the cytoplasm. But if the circles are not monomers but contain two or more copies of the plasmid sequence, the strands can from different molecules efficiently reassociate in the cytoplasm to produce viable circles (sorry if this explanation isn't clear enough; I don't have time to do it deeply). In B. subtilis recE mutants, transformation with plasmid preps containing such multimers is normal, confirming that DNA uptake is normal.
*“Allow people to make assumptions and they will come away absolutely convinced that assumption was correct and that it represents fact."
Randi the Magician, quoted in a recent New York Times article
**The B. subtilis expert emailed the other participants (but didn't reply to me!) to confirm that yes, in recA mutants the DNA is degraded inside the cell. He also expressed strong disagreement with our hypothesis that cells take up DNA for the nucleotides, claiming that these couldn't possibly make a significant contribution to the cell's nutrient supply.

1 comment:

  1. I'd be interested to know, is RecA upregulated in competent cells per se, or only when competent cells are given DNA to take up?

    Also, what is the mean level of recA production in non-competent cells compared to competent cells? Any relevant references gratefully received!


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