Field of Science

Finding the elusive retraction motor

Sorry for no recent posts.  I've been traveling and grant-writing, but now I need to think through one part of our pending CIHR grant proposal (due Wednesday).

The overall focus of the proposal is the mechanism of DNA uptake by Gram-negative bacteria. Specifically, we will investigate the role of uptake sequence bias and the forces responsible for DNA uptake in H. influenzae.  One part of the proposal aims to identify the protein responsible for the force that pulls DNA into the periplasm. This force might be dispensable for ongoing DNA transport (a periplasmic DNA-packaging protein might pull DNA in) but it must exist for the initiation of uptake, when a kinked loop of DNA is first pulled into the periplasm.
 In other bacteria all evidence points to the 'secreton ATPase' PilT as the source of this force, but PilT homologs are absent from H. influenzae and all other Pasteurellacean genomes.  PilT is thought to act by using energy from ATP hydrolysis to forcibly remove subunits from the base of the T4P pilus or pseudopilus, shortening it and thus pulling on whatever the pilus or pseudopilus is attached to.

This section of our proposal aims to (1) test competence-gene mutants for the phenotype expected of a pseudopilus-retraction defect, and (2) find out whether the ComE1 protein acts to restrain DNA in the periplasm, either by DNA packaging or by acting as a 'pawl' for the retraction ratchet (some of its homologs have been shown to bind DNA).  This isn't a very strong section because we don't have any very clever strategies.

Aim 1.  The first problem is the expected phenotype.  Cells defective in the retraction motor should make pseudopili and bind DNA, but not transport the DNA into the periplasm.  That's what's seen in a Neisseria mutant lacking PilT.  But the standard H. influenzae lab strain Rd never makes visible pili at all, so we can't check for these.  Another strain ('NP') does make pili and we propose to transfer our mutants to that strain and look for loss of pili, but this is more problematic than it seems.  NP pili are difficult to observe even with electron microscopy.

A new paper from colleagues working on the NP strain reports the phenotypes caused by mutations in the major T4P genes comABCDEF and pilABCD, but the authors did not directly assess changes in pilus formation.  Instead they used proxy phenotypes: formation of a thick biofilm on glass surfaces and adherence to cultured cells.  But the effects were not very dramatic.  Biofilm thickness and biomass was reduced to 10-20% of wildtype for the pilABCD and comC mutants, and 50% of wildype for the comABDEF mutants; adherence was reduced to 40-50% in all mutants.  Work in other species predicts that these mutants will entirely lack pili, and the NP mutations did completely eliminate transformation, as do our Rd mutations.  The simplest interpretation is that these mutations do eliminate pilus production, but that the tested phenotypes are not good proxies for piliation, at least in H. influenzae

The other phenotype we plan to test is DNA binding and uptake.  (Our NP colleagues didn't test this in their mutants.)  But inferring binding is a bit tricky, as it can't be directly measured; instead we calculate the difference between total cell-associated DNA (cpm after washing without DNase) and internalized DNA (cpm after washing with DNase).  We've already done preliminary assays of this in all our Rd mutants, but the data are noisy and we're going to redo them (and more replicates) with a better filtration-based washing procedure.  Most mutants had greatly reduced total cell-associated DNA and even lower internalized DNA, but we're not confident that the differences are significant. 

Another problem is the mutations we will test.  We should transfer the comNOPQ mutants, and two other mutants that affect uptake (pilF2 and comE1) to NP and test biofilm formation, since our colleagues didn't test  these.  Finding that biofilm production is normal or increased would suggest a retraction defect.  I don't see any point in transferring our comABCDEF and pilABCD mutations into NP unless we're going to test piliation directly.  Should we propose to do the EM studies our more-expert colleagues didn't do?  We can test for pilin processing by looking for a change in pilin size, but this isn't really informative about pilus assembly or stability.

The postdoc is keen on the idea that the secreton ATPase that normally powers pilus assembly, PilB, also powers retraction, perhaps by coupling to a separate disassembly module.  I think this is a long shot, because there is no precedent for an ATPase powering two different reactions (or the forward and reverse versions of the same reaction), and nothing about PilB in H. influenzae or other species suggests that it might have the capacity to do this.  The H. influenzae PilB is homologous for almost its full length to the PilBs of species that have PilT.  This hypothesis is also extremely difficult to test, as we would need to identify some mutation or combination of mutations that inactivated the hypothesized retraction function while maintaining the assembly function, and we have no clues about what these mutations might be.  Our phenotype-based screen for retraction defects (pili+, binding+, uptake-) would find the hypothetical disassembly protein anyway, if it's in the competence regulon.

Which brings up another problem.  What if there is a retraction protein but it's not in the competence regulon?  It couldn't be a secreton ATPase, but in principle could act in some other way to power retraction.  The mutant hunts that have been done haven't turned up any candidates, and I can't think of any efficient way to select or screen for them.

Yet another problem is whether the retraction motor is really needed for initiation.  Might the initial loop be pulled in some other way, and the suggested DNA-packaging protein take care of the subsequent DNA transport into the periplasm?  In principle yes, but this begs the question of what's the point of the whole T4P system.  Almost all the genes needed for DNA uptake are needed for T4P production in other species.  The review that proposed that ComE1's homologs are DNA-packaging proteins suggested that the pseudopilus's function is just to make a hole in the cell wall and outer membrane that DNA can passively move through, but they overlooked the problem of initiation, which we don't think can be solved without active pulling.

Aim 2:  Does ComE1 restrain DNA in the periplasm, either by packaging DNA or by acting as a pawl for a pseudopilus uptake ratchet?  We have an advantage over other systems here, since the H. influenzae comE1 mutation doesn't entirely prevent DNA uptake and transformation as it does in other systems.  (Well, except in Neisseria, which has four identical copies; deletion of all four reduces uptake only about 4-fold (the limit of detection?) and transformation 40,000-fold.)  We can do several tests here: 

One is the comE1 mutant phenotype.  A mutant that is defective at restraining the DNA in the periplasm might be able to take up very short fragments but not long fragments, bur we need to think through this assay more carefully than we've done so far.

Another test is the phenotype of a comE1 rec2 double mutant.  Chromosomal DNA that comE1 mutants take up is efficiently translocated and recombined, and we wonder how much of this uptake is due to translocation.  In the absence of Rec2's translocation function, is uptake reduced?  The postdoc has done a preliminary test of this but the results are inconclusive.

The final test is overproduction of ComE1.  Especially in a rec2 mutant background, increasing the amount of ComE1 might increase the amount of DNA taken up.  This would favour the DNA-packaging hypothesis, but probably isn't inconsistent with the pawl function either.  These alternatives can maybe be distinguished by the optical tweezers force/displacement measurements we propose in the next section, since the ratchet/pawl function should be associated with jerky 10-20 nm displacements and the packaging function with relatively smooth displacements.

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