Final checks on a surprising competence gene (Whew!)

We now have almost all the data in place for our paper about the roles of all the genes in Haemophilus influenzae's competence regulon.  We (really the RA) created deletion mutations of all the 26 genes except ssb, which is essential; these deletions remove almost all of each gene's coding sequence.  One set of mutations contains spectinomycin cassettes inserted at the site of the deletion; these are very useful because they let us select for each mutation by the SpcR phenotype it causes.  The other set is 'unmarked', and these clean deletions are 'in-frame', preventing disruptions of translation that could interfere with expression of downstream genes in the same operon ('polarity).

For each unmarked mutant we've examined (1) its growth using the Bioscreen incubator/recorder, (2) its survival after transfer to the MIV starvation medium that induces competence, (3) its MIV-inducible ability to take up radiolabeled DNA and (4) its ability to be transformed by genetically marked chromosomal DNA.  For all but one of the genes these phenotypes are at least roughly consistent with what we expected from the phenotypes of known mutations in H. influenzae or other bacteria and from the predicted properties of the encoded proteins.

But one gene's phenotypes surprised us.  HI0659 is predicted to be a small cytoplasmic protein, and it has a predicted helix-turn-helix that would be expected to bind to DNA, probably at a specific sequence.  It's mRNA is induced about 20-fold on MIV treatment.  We expected it to either play no role in DNA uptake and transformation or to have normal uptake but reduced transformation.  But our unmarked mutant (∆HI0659) doesn't take up any detectable DNA and doesn't transform at all, which suggests that it is required either for assembly/function of the uptake machinery or for continued expression of the competence regulon after initial induction by Sxy and CRP.  That's of course very interesting, and we've thought of lots of cool experiments we could eventually do to find out how it acts.

But there's one wrinkle that needs to be cleared up before we publish this result.  The phenotype of the marked (SpcR) HI0659 mutant (∆HI0659::spc) is not the same as that of the unmarked mutant - its transformation frequency is much higher, though still substantially lower than that of wildtype cells. (I don't know if its DNA uptake has been tested.)  This is unexpected and suggests that there's a problem with the structure of either the marked or the unmarked mutation.

The structure of the unmarked mutation has already been carefully checked by PCR and it appears exactly as it should, so we suspect a problem with the marked mutation.  The RA has now created new versions of the marked mutation, and yesterday I made four of these MIV-competent and transformed them.  I'll learn the results of this test later today - if they don't transform at all we'll conclude that all is well with our mutants.

But if the new marked mutants do transform, we'll have to suspect that something is instead wrong with the unmarked mutant.  The most likely problem is that this strain accidentally acquired a mutation elsewhere in its chromosome that prevents DNA uptake.  Testing for this is a bit tricky, but here's my plan, diagrammed below).

HI0659 is in the same operon as HI0660, whose mutants both transform normally.  If the new ∆HI0659::spc mutants transform, I'm going to transform the marked HI0660 mutant (∆HI0660::spc; SpcR) with a PCR fragment containing the normal HI0660 allele and the unmarked version of HI0659.  I have frozen ∆HI0660::spc competent cells ready to use, and the RA is making the PCR fragment for me using primers she already used for another experiment.  I'll plate the transformation mix without selection, using a control transformation with chromosomal NovR DNA to confirm that the cells were competent.  Then I'll screen the colonies for loss of SpcR by picking them onto plain and Spc agar plates.  Colonies that grow on the plain plate but not on the Spc plate will be ones that have lost their spc cassette by recombination with the ∆HI0659 fragment.  I'll test these for transformability - if those that have acquired the ∆HI0659 deletion (checked by PCR) have lost the ability to transform then we can be reasonably confident that the ∆HI0659 deletion prevents transformation.  If not then something is probably wrong with the ∆HI0659 mutant.

This would be a fair amount of work, probably too much to get done before the RA goes on a few months' leave at the end of April. so I very much hope that the transformations I did yesterday give no transformants.

Later:  All the new ∆HI0659::spc mutants are nontransformable, just like the ∆HI059 mutant.  Because I actually tested deletion mutants created in two independent experiments, this means we can be extra confident that the deletion is responsible for the loss of competence.