I've been rereading an important 2002 paper by Finn Erik Aas et al. (Mike Toomey's group; Molecular Microbiology 46:749-760). They dissect the roles of the Neisseria type IV pilus components PilE (the major pilin), ComP (a minor pilin important for DNA uptake), and PilT (the retraction ATPase). The results are important and need to be considered in the CIHR grant proposal we're rewriting.
I was about to try to summarize them here, when I wondered whether I might have already posted anything about this paper. A quick search for 'Aas' found a whole post from last summer, which I had completely forgotten about! So here I'm instead going to edit/annotate/rewrite that post.
An important difference between this paper and previous papers is their careful attempts to separate of DNA binding from DNA uptake. DNA binding is usually measured indirectly by giving cells radioactively labeled DNA and comparing cell-associated cpm with and without pretreatment with DNase I, which removes DNA that has bound to cells but not been taken inside them. And that's how it's done in this paper - the 'binding' values are how much of the total cpm is removed by DNase I treatment (i.e. they're determined by subtraction).
The Aas et al. paper showed that, when 5x10^8 wildtype Neisseria cells are incubated for 30 min with 500 ng of 32P-labeled plasmid DNA containing the Neisseria uptake sequence (the DUS), only about 1-2% of the DNA sticks to the cells (i.e. is still there after 10 min on ice and three washes with cold medium). They found that more than half of this DNA is taken up during the 30 min incubation, because it wasn't removed when the cells are incubated with DNase for 10 min at room temperature before being washed instead of being stored on ice. If the DNA doesn't contain a DUS the cells still bind a lot of it (25-100% of the +DUS binding amount), but almost none of the bound DNA is taken up (> 0.01% of the 500 ng). This says that cells will bind any DNA but can only take it up if it has a DUS.
The minor pilin-like protein encoded by comP is normally expressed at a very low level (so low that it's barely detectable by protein assays), but the level is high enough to show that the ComP protein is incorporated into pili along with the major pilus protein PilE. Cells lacking PilE don't have visible pili and they didn't bind DNA or take up DNA, but cells lacking ComP have normal-looking visible pili and they bound just about as much DNA as wildtype cells. But they hardly took up any of this DNA even if it had a DUS (they interacted with DUS+ DNA the way wildtype cells interact with DUS- DNA).
This strongly suggests that the ComP protein recognizes the DUS at the cell surface, but that ComP is not involved in the non-specific DNA binding step. Consistent with this, overexpressing the normally-scarce ComP protein increased by 20-fold the amount of DNA bound and taken up, and proportionately increased the transformation frequency. This increased uptake was specific for DNA containing a DUS, although a modest increase was also seen for DNA that lacked DUS. Overexpression also greatly increased the amount of ComP protein in the pili. I think this result says that ComP can cause DUS-specific DNA binding, on top of the normal non-specific binding. ComP, when assembled into pili, would be able to bind DUS-containing DNA but not other DNA.
However, the authors found that, when they purified ComP, it did not bind specifically to any DNA. They tested both 'recombinant' ComP (+ His-tagged) and 'overexpressed' ComP, but they they don't show this data and they don't say how the binding assays were done. Does this result mean that ComP really doesn't interact with the DUS? Maybe. The authors suggest that it acts by modifying some other protein so that it binds the DUS, but another explanation might be that ComP loses its DNA-recognizing function when it's purified away from the T4P complex.
My tentative model: ComP is processed by the prepilin peptidase as is the main pilin (PilE), and is assembled along with PilE into the pilus filament, where it forms a very minor component. Once DNA binds non-specifically to the PilE part of the pilus, it can interact with ComP and, if the DNA has a DUS, be taken up when the pilus is retracted. If the amount of ComP in the pilus exceeds the non-specific binding capacity of the PilE, DNA will bind directly to ComP (even if it can't be taken up, see below).
What about PilT? In most bacteria with T4P, PilT provides the power to retract type 4 pili filaments, by disassembling the subunits from the pilus base. (The exceptions are H. influenzae and its relatives in the family Pasteurellaceae, who don't have a pilT homolog.) As pili are thought to bind DNA at the cell surface, PilT would then be responsible for pulling the pilus and its DNA into the periplasm. PilT mutants had already been shown to have the expected phenotype: abnormally large amounts of pili, and unable to take up DNA or be transformed. We might then expect that having more pili would make pilT mutants bind more DNA than wildtype cells, but Aas et al. found that they bound <1% of the wildtype amount. They took up about 20% of what they bound, but I think this may be at the detection threshold.
Overexpressing ComP in a pilT knockout increased the DNA binding (but not uptake) by about 5-fold. This added binding was DUS-dependent. I could add this to my tentative model: The nonspecific binding by PilE requires PilT, but we have no idea why (I'll call this the magic PilT effect). Specific binding by ComP does not.
They then made transcriptional fusions of pilE and pilT to the E. coli lac promoter (in different strains) so they could keep the genes turned off or turn them on by adding IPTG, as desired, and measured transformation. When each gene was on throughout the transformation experiment, transformation was close to normal, as expected. When it was off, there was no transformation, again as expected. They then tried turning the genes on halfway through the transformation experiment, to find out whether PilT was only needed to pull the pili in
When pilE was off during the DNA incubation step and then turned on after the cells had been washed free of unbound DNA, transformation was down >500-fold. This is consistent with both nonspecific and specific binding happening only in assembled pili - it's no use making the pili after the DNA has all been washed away. When pilT was off during the DNA incubation step and then turned on after the cells had been washed free of unbound DNA, transformation was down >32-fold; this says that some DNA can bind to cells in the absence of PilT, and then be later taken up once PilT becomes available.
Does this mean that the binding that happens before turn-on of PilT is specific for the DUS?
The authors then did the same experiment in cells overexpressing ComP. When there are no pili (pilE off) my model predicts that no DNA will bind even though there's tons of ComP, so turning pilE back on after the cells are washed shouldn't give any transformants. In fact it gives some, but not a lot. When there are pili but no PilT, my model predicts that having tons of ComP will increase DNA binding, and that this will increase transformation frequencies once PilT becomes available. My model predicts that this binding will be DUS-dependent, but that isn't shown.
Summary of the PilT bit: Having pilT off while DNA is binding to the pili reduces transformation frequencies 32-fold when most binding is initially nonspecific, because most of this binding is caused by the magic PilT effect, but only 7-fold when more of the binding is by ComP to the DUS, because the magic PilT effect doesn't act on ComP binding to the DUS.
But uptake requires pilus retraction. So maybe I should conclude that, in wildtype cells, most DNA initially binds non-specifically to the many pili that have no ComP but have been magicked by PilT. DNA bound to these pili is not released to the solution, and these pili are not retracted. But if a DNA fragment has a DUS, the DUS can bind to one of the fewer pili (or pseudopili?) that have ComP (binding to directly to ComP or to another protein). Once a ComP-containing pilus has bound DNA it can be retracted, pulling the DNA in and initiating uptake. This pilus might then elongate and bind another DUS, or other ComP-containing pili might be continually assembled and accept DNAs from the PilE pili.
I'm going to stop here, because the more I read the confuseder I get. I'll talk this all over with the RA tomorrow morning and see if she can sort me out.
Why are unfalsifiable beliefs so attractive?
2 days ago in Epiphenom