Peter, Peter, DNA eater

During her research into the distribution of the competence phenotype in H. influenzae strains, Heather has discovered a strain, PittAA (which I call Peter), that demonstrates transformation frequencies 1000 fold greater than the reference strain (KW20) when cultured in rich medium. Specific mutations in the sxy and murE genes (and possibly also in crp) can cause similarly elevated transformation frequency in KW20, a phenotype we call hypercompetence. If we can track down the genetic cause(s) of PittAA’s hypercompetence, it may help us better understand competence gene regulation in our preferred organism, KW20. Can we track down a region of the PittAA genome that confers hypercompetence when transformed into KW20?

1. Is PittAA sxy responsible for hypercompetence?
Heather has described the amount of difference between the PittAA and KW20 sxy genes: http://heathermaughan.blogspot.com/. Unfortunately, we don’t know anything yet about how the Sxy protein works, so we don’t know if certain amino acid differences between PittAA and KW20 Sxy are functionally significant.

In KW20, mutations that destabilize the secondary structure of sxy mRNA cause hypercompetence. Examination of the homologous sequence in PittAA revealed three base differences compared to KW20: one in Loop C, one in Stem 3, and one in Stem 1A (see figure of KW20 structure). A base difference in Loop C is not expected to have an effect on 2º structure, and the differences in the stems conserve the base pairing potential seen in KW20 (C:G→U:G and A:U→G:U; differences between KW20 and PittAA are in bold). Therefore, non of these 3 sequence differences are expected to destabilize the mRNA 2º structure and thus do not readily explain the hypercompetence phenotype. Though unlikely, these base differences in PittAA may favour the formation of a different 2º structure than what we have characterized in KW20. I will see if the RNA-folding program Mfold predicts an alternate, but thermodynamically favourable, folding structure for PittAA sxy mRNA. One caveat is that we don’t know for sure whether transcription of the PittAA sxy gene initiates at the same base as in KW20; I will check for the position of the CRP site and RNAP binding sites in the PittAA sxy gene promoter and this should help us predict where transcription initiates in PittAA.

2. Is PittAA crp responsible for hypercompetence?
One possibility is that the PittAA CRP protein is constitutively active (ie. cAMP independent); in E. coli, multiple mutations have been identified in crp that make CRP constitutively active. To test whether PittAA CRP responds to cAMP, cAMP can be added to exponentially growing PittAA cells. Addition of cAMP is expected to increase transformation frequency, and if it does, we know that CRP activity is still limiting to competence gene induction during exponential growth.

The most exciting possibility is that PittAA has gain-of-function mutations in Sxy or CRP that allow one protein to act at CRP-S promoters in the absence of the other. If we could identify such a mutation(s), it would tell us a lot about how CRP and/or Sxy activate transcription at CRP-S promoters. To test whether PittAA has GOF mutations in CRP or Sxy, we can transform PittAA with KW20 crp- and sxy- constructs, but would have to use PCR amplified knockout genes in order to avoid transforming other parts of the PittAA genome. We predict that clean knockout mutations in PittAA crp or sxy will abolish transformation, as they do in KW20. If not, than a GOF mutation is the best explanation.

It is tempting to hope that only one genetic difference can explain the “non-KW20” phenotype in each H. influenzae strain that is non-competent, non-transformable, or hypercompetent. However, it seems likely that non-competent strains will have accumulated mutations in unused competence genes. On the other hand, a hypercompetence phenotype and a non-transformable phenotype can each be generated in KW20 with single mutations. Thus, it should be tested whether KW20 genes can "restore" the KW20 phenotype in the non-transformable strains.