The Research Associate's experiments, part I

While I've been paying attention to other things (teaching, writing, proposing), the Research Associate has been doing a lot of work on the projects we're funded to do.  Below I'll highlight in purple the experiments that I might take on.

Recombineering:  The goal was/is to create marked (selectable for antibiotic resistance cassette) and unmarked deletions of all the H. influenzae competence genes.  This has required an enormous amount of work, much more than was originally expected.  The last step, converting the marked mutants to unmarked, turned out to be particularly troublesome because the counter-selection doesn't work in the standard lab strain.

But I think we now have marked knockouts of every gene in the competence regulon, but unmarked mutants for only some.  These are on the back burner for now, though she's still creating knockouts of comI and comJ (implicated in competence though not regulated by it) and mutS (for study of recombination).  She also knocked out the sxy gene of a related species, Actinobacillus suis, for a collaborator.  One project for me is to follow up on the characterization of these competence mutants (begun by an undergrad), rechecking their starvation-induced transformation frequencies.

Competence differences in H. influenzae strain 86-028NP ('NP'):  This is the low-competence strain the postdoc is using for his sequencing of recombination tracts and the planned mapping of genes responsible for competence differences.  The RA noticed that, although this strain has homologs of all the genes known to be needed by H. influenzae strain Rd for transformation, it lacks several competence-induced genes with unknown functions.  So she's putting the Rd genes into the NP chromosome to see if this changes their competence.  This should be easy as she already has the Rd genes cloned in the recombineering vector, and the counter-selection that fails in Rd works fine in NP.


Regulation of competence by purines and purine nucleotides:

The big project that's most important is understanding how purines affect competence in H. influenzae.  The new work on this project has two fronts, repression of CRP-S genes by PurR and post-transcriptional control of Sxy expression by purine nucleotides.

Front 1, regulation by PurR:  The lesser front is finding out whether the purine repressor PurR affects transcription of any competence genes.  When one of the purines guanine and hypoxanthine is available, they bind to PurR and enable it to bind its recognition sequence.  This sequence is present in the promoters of most genes involved in de novo synthesis of purines.  I initially thought it was also present in the promoters of several CRP-S genes but the RA and others have analyzed this more rigorously and find that the only strong candidate is in the rec2 promoter.

Our previous work on the role of PurR in competence has been compromised by problems with what we thought was a good PurR knockout strain.  First we found that strains had been mixed up, and ten that the knockout didn't remove the part of the gene that codes for the DNA-binding domain of PurR.  Now the RA has made a clean mutation that removes the whole gene.  She's also knocked out the purH gene; this gene codes for the last step in purine synthesis, and the knockout allows us to disassociate competence effects due to PurR regulation from effects due to synthesis of purines.  She's put the two knockouts together and also combined them with some sxy-hypercompetence mutations (see below).

One simple way to test for PurR regulation of rec2 is using some fusions to the E. coli lacZ gene, made years ago by Michele Gwinn.  We have her fusions to two CRP-S genes, comA and rec2 - comA makes a good control as it doesn't have a candidate PurR site.  The RA has made the purR- derivatives of these fusion strains and tested them in rich medium, using cells in log phase, at the onset of slowed growth, and after overnight culture.  None of the conditions showed much difference in expression of the fusions with or without PurR.

...pause while I diagram what we should expect...  First, we expect PurR to be active (repressing) throughout growth in sBHI, because our microarrays showed no induction of PurR-repressed purine genes.   We also expect little or no expression of comA, rec2, and the other competence genes (CRP-S genes) in log phase, because sxy is not transcribed much or translated efficiently.  When growth slows we expect low-level expression of the CRP-S genes.  If PurR represses rec2, we expect to see higher beta-galactosidase expression in the purR- rec2 fusion strain than in its purR+ control.  But she didn't see that. 

This might mean that PurR doesn't regulate transcription of rec2.  But the expression levels of the fusions were very low (mostly less than 40 Miller units, whereas starvation-induced cells produce several thousand units).  It would be good to repeat these experiments after adding cAMP to all the cultures to give higher expression of the CRP-S genes; if we don't see any effect of PurR, probably there isn't much effect.  She's now also done careful measurements of the baseline expression of these fusions, data we need to interpret the results of whatever manipulations we now do.  I don't know if we've ever monitored the effects of cAMP on expression of these fusions in rich medium - it might be easier to just do it now than to find out whether we've already done it...

The presence of a functional purine-biosynthesis pathway in these cells shouldn't have mattered, but the purR- cells did differ from the controls in this, so it would be good to repeat the experiments with the purH knockout added to all strains. 

Another way to test for PurR repression of rec2 would be to put the H. influenzae lacZ fusions into E. coli, where it will be easier to test the effect of PurR and purines (because we can grow cells with and without various purines.  Unfortunately we don't have plasmid clones of the fusions, just  H. influenzae chromosomal fusions.  The RA thinks it might be easiest to just remake the E. coli fusions rather than cloning them from the H. influenzae chromosome, but I think that at least one of the fusions is a simple integration of an E. coli plasmid.  These cells should contain a low frequency of excised plasmid - if so, simple transformation of E. coli with a cell lysate might give the desired plasmid clone.

Another test would be direct assay of bandshifting.  The RA wondered if we might be able to get purified PurR protein (E. coli) from someone.  But I just looked at the old E. coli PurR paper (1988); they did bandshift assays using crude cell extracts, so maybe we could do that too. 

We also want to know how widespread competence regulation by purines is in other Pasteurellaceae.  The RA has found that the putative PurR binding site is also present in the rec2 promoters of other Pasteurellaceae, and E. coli.

Front 2:  Regulation of sxy expression: 

The RA has confirmed and extended our previous work showing that purine nucleotides (AMP and GMP) prevent competence development in starvation medium.  Most of that work used quite high nucleotide concentrations (5 mM), and some of our experiments found that these were high enough to inhibit cell growth.  So she's meticulously repeated these using concentrations of 1.0 and 0.1 mM AMP (3 replicates).  1.0 mM was enough to reduce transformation by 500-fold, and 0.1 mM by 40-fold.  But the 1.0 mM AMP also inhibited cell growth - cells usually double once after transfer to the starvation medium, but their numbers didn't significantly increase in 1.0 mM AMP.  Cells transferred to starvation medium with 0.1 mM AMP doubled normally.  This is excellent, as it gives effects on competence at concentrations that are unlikely to cause other problems.  But I'd still like to have some better controls, to confirm that the competence effects we see are really directly due to specific effects on competence genes, not to more widespread effects.  One possibility is to use a CRP-N-regulated gene (not responsive to Sxy) as a control.

She's done similar experiments in two other competent Pasteurellaceae, Actinobacilus pleuropneumoniae and A. suis.  Our collaborators have found strains of these species that transform at sufficiently high frequencies that the effects of added purines could be tested, and the RA has shown that AMP reduces their competence in the same way as in H. influenzae.

She also tested the new purR knockout (in H. influenzae).  Its transformation frequency in starvation medium is down 5-fold.  This may be because the cells are constitutively synthesizing their own purines and so are less 'shocked' by the sudden absence of purines from their surroundings.  They were also less sensitive to addition of AMP;1.0 mM AMP reduced transformation only 10-fold, and 0.1 mM had no effect.  This reinforces the hypothesis that sudden purine depletion contributes to competence induction, and that enrichment of the starvation medium with AMP decreases competence directly and not by some general perturbation of metabolism or gene expression.

These effects were reduced when the cells carried sxy-hypercompetence mutations that increase the efficiency of translation of sxy mRNA.  1.0 mM AMP reduced transformation of a sxy1 mutant only 20-fold, and 0.1 mM no more than 2-fold.  She's done 3 replicates so this is a solid result.  She also tested a couple of our other hypercompetence mutations; in both of these she only tried 0.1 mM, and saw no effect on transformation.  This is consistent with our hypothesis that AMP acts by preventing translation of sxy mRNA.  She has saved cell pellets of all the competent-cell preps she used, so these can be assayed for expression of Sxy.  An excellent control would be to assay the pellets for presence of some other protein that's induced by the starvation regime but not regulated by Sxy.  Again, one of the cAMP-induced CRP-N-regulated proteins would be good, but we'd have to make our own antibody first (either purifying the protein or specifying a synthetic peptide).

She's also modified our antibody-based assay (Western blot) for Sxy.  The grad student who developed it used a detection method that was very sensitive, but his blots had very high background.  She's found that, with a less-sensitive assay, the Sxy bands are still very clear but the background is greatly reduced.  It's not entirely eliminated, which is a good thing because the few background bands serve as excellent controls for normalizing the band intensity (provided, of course, that their proteins aren't altered by the starvation regime).


E. coli competence:

This is the second big research problem the RA has been working on.  I'll leave this for a later post.