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.

CIHR proposal - mutant phenotypes

We've been going back and forth and around and around on the part of our CIHR grant proposal where we propose to ...  well, part of the problem is that we've not decided whether this section should just propose to do one unified set of analyses or add in various disparate analyses that don't fit elsewhere.

There is a unified set of analyses to be done.  We have a complete set of knockout mutants that have been only partially characterized (transformation assays, preliminary DNA uptake assays).  So we're proposing to do a more thorough analysis of all the mutants whose DNA uptake is defective (because DNA uptake is the overall focus of the proposal), using two new methods.

1. One problem of working with the standard lab strain of H. influenzae is that it doesn't make long pili that can be seen by electron microscopy (EM), which means we can't tell whether our mutants block the assembly of pilin subunits into the pseudopili that pull DNA into the cell.  (Well, we might be able to devise an assay for pseudopili, using crosslinking, but this will be a fallback.)  So we're going to use transformation to put each of our knockout mutations into the NP strain, which does make pili, and then use EM to see if the mutation prevents pilus assembly. We'll be especially interested in mutants with abnormal pili.
2. Our DNA uptake assays use centrifugation (pellet cells, resuspend pellet in fresh liquid, repeat twice) to wash unbound DNA away from cells, with or without first adding DNase I to digest DNA that's not been taken into the cell.  But a B. subtilis paper I read on Saturday described instead washing cells by filtration, using special 96-well plates that have a filter in each well.  This allows more thorough washing and is gentler on the cells.  We need to repeat and replicate the uptake assays on our uptake-defective mutants anyway, because we've decided these are critical to detecting whether the mutants can still bind DNA at the cell surface.  So we're now proposing to use the filtration assay to get very solid data for all these mutants.

These assays will let us distinguish proteins that are needed for pilus assembly from proteins that matter only after the pilus has been assembled.  We can describe what we expect to find, based on postulated protein homologies and our work so far, and explain how the results will be interpreted.  So far so good.

The hard part is deciding what other investigations to include in this section.  One issue raised by a reviewer of the previous submission is that knockout mutants are a very crude tool for investigating function, especially for processes that depend on concerted work by many components.  In competence, a knockout that eliminates the pre-pilin peptidase has the same uptake and transformation phenotype as one that eliminates the pilin subunits or the assembly ATPase or the outer membrane pore used by the pseudopilus.  We've added another phenotype to our screen (production of pili by strain NP), but we would like to have at least one experiment showing that we can use less-drastic mutations to investigate the specific function of a gene.

I think we should describe several analyses we know we want to do (each a short paragraph), and explain that similar techniques can be applied to other genes, depending on the phenotypes revealed by assays 1 and 2 above.  I was planning to organize these by the questions they address, but it might be more effective to organize them by the techniques they illustrate.

1. Different DNA substrates for the uptake assays:  DNA concentration, ±USS, short vs long DNA fragments to detect retraction and retention defects (e.g. for the comE1 mutant), end blocked with a bead to detect polarity... 
2. Double mutants:  To see if the DNA uptake by the comE1 knockout depends on DNA translocation across the inner membrane we'll test a comE1-rec2 double mutant.
3. Reisolation of DNA from the periplasm:  Test whether comF is blocked at the same step as rec2.
4. Truncation mutations:
5. Point mutations that change specific amino acids:  We can make specific mutations to change proposed DNA-binding residues in the secretin subunits that form the outer membrane pore (comE).  We can test whether pilF2 encodes a pilotin homolog by mutating the specific residue that should be its lipidation site.
6. Random mutagenesis of a gene or part of a gene, followed by screening for a desired phenotype:  We'd mutagenize the gene in an expression plasmid, and then put the plasmid into the corresponding knockout mutant to look for the desired phenotype.  We're considering doing this to pilB to screen for an effect on retraction, but this is a long shot.
7. Cross-species complementation and optical tweezer experiments will be described in separate sections, but they might be mentioned here.

OK, I think writing this post has given me a workable plan.

HI0569, gene of mystery

The RA's heroic project to create knockout mutants of every gene in the H. influenzae competence regulon has turned up one big surprise - the HI0659 gene.  This small cytoplasmic protein, whose mRNA was induced about 25-fold in competent cells in our old microarray experiments, turns out to be essential for competence.  The knockout mutant doesn't detectably take up DNA and produces no transformants.

The first question to consider (and maybe to answer) is whether this protein plays an essential mechanistic role in DNA uptake or has a regulatory function that's needed for effective expression of the other genes.  It's only 98 amino acids long, and most of that is a single helix-turn-helix (HTH) domain (see figure).  HTH domains typically regulate gene expression by binding to specific DNA sequences, but they usually are only part of larger proteins whose activities are in turn regulated by other effectors such as sugars and amino acids.  But HI0659 doesn't have much room for other interactions, decreasing the likelihood of a regulatory function.  On the other hand, the protein doesn't have much room for DNA uptake functions either.  And it doesn't have any targeting signals that would send it into the cell envelope.

The postdoc speculates that it might bind RNA rather than DNA, perhaps interacting with sxy mRNA.  Apparently some HTH motifs do bind RNA.  But ssRNA has a very different structure than dsDNA, so I wonder if what these motifs bind is actually dsRNA.  

One thing the microarray summary doesn't tell us is the basal level of HI0659 mRNA expression.  This is of interest because, if it's a regulator of competence that's needed for expression of the other genes in the CRP-S regulon, it should be active before they come on.  Maybe it's active constitutively at a moderate level, and induced even higher in competent cells.

It's just downstream of HI0660, another tiny protein with no known function.  HI0660 is even less conserved than I0659, and knockouts of it have normal competence.  Surprisingly, the 'marked' knockout of HI0659 (the same deletion with an inserted SpcR cassette) retains some competence.

Here's a graphic of HI0660 and HI0659 aligned with homologs.  I asked the database to find homologs in other Pasturellacean species (H. ducreyi, Mannheimia succinoproducens, Pasteurella multocida) but none were found even though I lowered the % similarity cutoff to 30% from the default 40%.  This is a bit surprising, because A. pleuropneumoniae is a more distant relative than M. succinoproducens and P. multocida.  The operon is also in M. haemolytica, a close relative of A. pleuropneumoniae not shown in the figure, and I think the grad student who did the analysis also found that it has a CRP-S promoter in these species.

For the CIHR proposal we're going to propose to do 'RNA-seq' of the HI0659 knockout and controls, to look for changes in the mRNA population.  RNA-seq is the shorthand term for measuring the abundances of all a cell's transcripts by doing deep sequencing of a cDNA prep.  It's pretty straightforward; the only big problem is avoiding wasteful sequencing of ribosomal RNAs, which are by far the most abundant RNAs in bacterial cells, but the postdoc says there's a good kit for that.

Before doing this we should confirm that the HI0659 mutation we've made is responsible for the competence defect we see by 'backcrossing' the unmarked mutation into a clean genetic background,.  This would be much easier if the mutation is closely linked to a marker we can select for.  We could instead transform it into the marked HI0660 knockout and screen for loss of SpcR; in principle this wouldn't be as efficient as selection but it might be easier.

The RNA-seq experiment will be useful in other ways.  Because the genome (and transcriptome) are small, we can afford to include lots of controls.  At a minimum we'll do wildtype and mutant cells in log phase and after competence induction, but we might also include crp and sxy mutants, and maybe even one or more of the mysterious hypercompetence mutants of HI1133 (murE).  This analysis will complement our previous microarray analysis, putting our identification of competence-regulated genes on a very solid foundation.

Back to the CIHR grant proposal

We've submitted four (!) papers in the past two weeks: the arseniclife paper, now under review at Science; the RA's E. coli competence paper, submitted to PLoS One after being bounced back to us by Journal of Bacteriology, the postdoc's DNA uptake paper, submitted to PNAS Plus; and the visiting grad student's paper about Gallibacterium anatis competence, submitted this morning to Applied and Environmental Microbiology.

Our big CIHR grant proposal is due at the end of the month.  This is yet another (improved) variant of the DNA uptake proposal we've submitted several times over the past few years.  On those occasions it would have been a second grant, but our current grant will end in September so this time the proposal will be for the renewal of the current grant.  That means its success is more important than in the past.  Again we're fortunate to have a colleague critiquing our draft for us, arranged through a in-house peer-review program that used to be called HeRRO but now might be called something else.

The figure is one we'll be including in the Background section of the proposal.  It shows the predicted cellular localizations of all of the proteins of the H. influenzae competence regulon, colour-coded to indicate the effect of each knockout mutation on DNA uptake.