Natural transformation is a mechanism for genetic exchange in many bacterial genera. It proceeds through the uptake of exogenous DNA and subsequent homology-dependent integration into the genome. In Streptococcus pneumoniae, this integration requires the ubiquitous recombinase, RecA, and DprA, a protein of unknown function widely conserved in bacteria. To unravel the role of DprA, we have studied the properties of the purified S. pneumoniae protein and its Bacillus subtilis ortholog (Smf). We report that DprA and Smf bind cooperatively to single-stranded DNA (ssDNA) and that these proteins both self-interact and interact with RecA. We demonstrate that DprA-RecA-ssDNA filaments are produced and that these filaments catalyze the homology-dependent formation of joint molecules. Finally, we show that while the Escherichia coli ssDNA-binding protein SSB limits access of RecA to ssDNA, DprA lowers this barrier. We propose that DprA is a new member of the recombination-mediator protein family, dedicated to natural bacterial transformation.The topic is important because DprA is a ubiquitous and highly conserved bacteerial protein. In naturally competent bacteria it is induced when cells become competent to take up DNA, and without it the incoming DNA gets rapidly degraded. But most bacteria with DprA aren't known to ever take up DNA, so we suspect that protecting incoming DNA isn't its usual function (maybe just a side effect of its usual function). Because almost all bacteria have this protein, its function must be important.
Mortier-Barriere et al. mainly investigated the activities of purified DprA (from S. pneumoniae and sometimes also from B. subtilis) in various combinations with DNA and with two other proteins known to interact with DNA, SSB and RecA. SSB is also often competence-induced, but it's known to have a primary function in DNA replication and repair. RecA is competence induced in some bacteria but not others (not in H. influenzae) and has a primary function in DNA replication and repair.
What the authors' experiments showed:
DprA binds single stranded DNA: They use band-shift assays to show that purified DprA binds single stranded DNA. The lanes with 5-10-fold ratios of DprA to the 90 nt test DNA fragment have about half of the fragment shifted (actually stuck in the wells). The authors never discuss how many bp of DNA a DprA fragment might be expected to interact with. It's a medium-sized protein (about 30 kD) so probably about 10bp? So 10 DprAs might be enough to coat a 9o nt fragment along its length. Once complex had formed it was difficult to disrupt; even a 1000-fold excess of cold fragment added to a previously formed DprA-DNA complex didn't displace all the original fragment from the DprA.
They also examined DprA binding to the circular ssDNA plasmid phiX174. These assays used 4 micromolar DprA and a DNA concentration described as 8 micromolar (nucleotides), so there was one molecules of DprA for every two nucleotide of the plasmid. As phiX174 is 5386 nt long, its molecular concentration was 0.0015 micromolar, giving about 2700 DprAs per plasmid. But considering the ration of protein to nucleotides probably makes more sense.
DprA (and Smf, its B. subtilis homolog) would not bind linear or relaxed-circle double stranded DNAs, but they did bind supercoiled circular DNAs. The authors conclude that this binding is to regions that are locally single stranded (because of the supercoiling?), but the evidence for this isn't very strong (what are "sleeve-like complexes"?). However the evidence is good that, given a DNA molecule that is partly ssDNA and partly dsDNA, DprA binds only to the ssDNA part.
DprA molecules stick to each other: DprA molecules interact with each other in a yeast 2-hybrid assay. They also appear to bind DNA cooperatively - at a ratio of one DprA to 20 nucleotides of phiX174, some DNA molecules formed complexes with DprA while others remained bare. In electron micrographs, distinct DNA molecules were often seen to be attached to each other by globs of DprA. The DprA-coated DNA is lumpy when viewed by TEM, unlike the thick smooth filaments formed by RecA. The authors describe the lumps as "secondary structures/intramolecular bridges".
Abundant DprA can protect DNA from nucleases: DNA that was pre-incubated with a 1000-fold excess of DprA could no longer be digested by the nucleases ExoT and RecJ (opposite polarities) or by the mung bean endonuclease. But this is a LOT of DprA. Probably because DprA molecules stick to each other as well as to DNA, they form what the authors call ' tightly packed discrete complexes' (I'd describe them as 'globs'); these spread out a bit when the salt concentration is increased.
DprA helps complementary single strands anneal: (Fig. 4C, S3CDE) The authors mixed DprA with labeled ssDNA and then added unlabeled complementary DNA strand. The DprA-bound strand left the DprA and instead base-paired with its complement, faster than it would have base paired in the absence of DprA. The ratio of DprA to the 80 nt DNA fragment DNA was 250, which seems very high. The SSB control used a ratio of 100. This experiment tells us that ssDNA would rather associate with SSB than its complement, and rather associate with its complement than DprA, but it also tells us that associating with DprA helps ssDNA find its complement. This may be just because DprA molecules bound to DNA like to stick to each other, or it might be due to something more sophisticated.
DprA interacts with RecA: When cells expressing His-tagged S. pneumoniae RecA were passed over a metal-chelate column, both RecA and DprA were bound. A yeast 2-hybrid screen for proteins interacting with DprA also identified RecA. I don't know whether this interaction depends on both proteins binding to DNA; the cell lysate was not treated with DNase before being passed over the column.
E. coli RecA has an ATPase activity that reflects its ability to unwind dsDNA when annealing a complementary strand. Addition of a low concentration of DprA to RecA bound to a ssDNA filament slightly increased RecA's ATPase activity (by about 20%), but higher concentrations reduced it (to about 50%. when there was twice as much DprA as RecA). E. coli SSB consistently reduced it.
DprA also made it easier for RecA to form filaments on ssDNA. At least some of the DprA remained on the DNA with the RecA. If the DNA was initially coated with SSB, the presence of DprA helped E. coli RecA to form a filament on the DNA. In this case the filaments didn't contain any DprA.
RecA that has formed filaments on ssDNA can help the DNA invade a double-stranded form of the same sequence. But if the ssDNA is first coated with SSB, RecA can't form a filament on it or promote strand invasion. Presence of DprA helps RecA get around the SSB barrier, and allows RecA to promote strand invasion even if the DNA has been previously coated with SSB.
Quibbles and complaints:
The authors cite their 2003 paper as evidence for the competence decrease in a ssb mutant, but this paper only briefly mentions a result and says the data will be in a manuscript in preparation. This means I can't check on how sick the ssb mutant cells are. The introduction also doesn't mention that both S. pneumoniae and B. subtilis have two ssb genes, and that only the one not induced in competence is orthologous to the well-studied E. coli ssb. H. influenzae only has one ssb homolog.
The authors never tell us where the SSB they used came from. I think it is likely to have been E. coli SSB, which can be purchased, rather than either SSB-A (its S. pneumoniae homolog) or SSB-B (a competence-induced S. pneumoniae paralog).
What the authors concluded:
The authors firmly believe that recombination is the reason for DNA uptake (that transformation is the function of competence). They also omit any mention of the consensus that DNA replication and repair are the true function (the evolutionary cause) of RecA and other proteins that contribute to recombination, leaving the naive reader with the impression that these proteins too must exist to create new genetic combinations. Thus I'm not surprised by their conclusion that "DprA is the prototype for a new recombination-mediator protein dedicated to bacterial transformation".
They go on to consider why DprA is so ubiquitous, present and highly conserved in almost all bacteria except those that live as intracellular parasites. Most of the bacteria with DprA homologs are not known to ever be naturally competent. I and others have interpreted this distribution as meaning that DprA has a primary function that is independent of DNA uptake (see previous post). But the authors go the other way. In their last paragraph they use the "it is tempting to speculate that..." qualifier to put forward the idea that all bacteria with DprA homologs are naturally competent.
What I conclude:
In competent cells DprA is likely to slow degradation of incoming DNA by binding to it, rather than by directly inhibiting a nuclease. This is consistent with our unpublished evidence that neither DprA or ComM acts by inhibiting the RecBCD nuclease.
Does anything about these experiments support or contradict the idea that DprA has an important function in non-competent cells? I don't think so. Induction of DprA in competent cells is unlikely to interfere with RecA's recombinational repair activity, but these new experiments don't provide strong evidence that DprA will enhance it.
One critical future goal should be to find out what goes wrong in dprA-knockout cells when they are not competent. As I described in an earlier post, an attempt to do this in E. coli found that the knockout had no detectable effect under a wide range of conditions. The authors of this paper summarize evidence that this is also the case for S. pneumoniae and B. subtilis. Perhaps our 'alternative' perspective on competence will enable us to think of tests the others have overlooked.
As the dprA gene is strongly induced in competent H. influenzae, maybe we should test whether cells with a dprA knockout survive competence (with or without DNA uptake) as well as do dprA+ cells. I may even have some old data that addresses this.