The RA and I have summarized our various results about the E. coli CRP-S regulon and its competence (or lack of competence).
Although we (she, really) have done a lot of work, we still have no experimental evidence that the E. coli sxy gene is inducible at all. And we have no evidence that artificial induction of sxy causes competence. We need something positive for our paper, otherwise it's just a string of negative results that's not nearly comprehensive enough to warrant publication.
The only sxy-dependent phenotypes we have are (1) a pseudo-natural plasmid transformation, where the sxy+ frequency of 9x10-8 is reduced to less than 2x10-9 in sxy- cells, and (2) competitive fitness in long-term co-culture, where a sxy- mutant is outcompeted by a sxy+ strain (~10-fold difference in cfu after 6 days, ~100-fold difference after 15 days). Palchevskiy and Finkel have shown that Sxy-induced genes are needed for cells to use DNA as a nutrient, but we haven't been able to replicate this. And we have shown that artificial induction of sxy induces all of the genes in the CRP-S regulon, and that the pilin protein encoded by one of these, ppdD, is translated and correctly processed.
The strong conservation of the CRP-S regulon (including sxy) and the very strong parallels to the H. influenzae competence regulon justify our hypothesis that sxy expression is inducible, and that this induction causes E. coli cells to take up DNA from its environment. To make progress we need at least partial answers to these questions:
Question 1: What factors induce E. coli sxy expression? Another lab has already done extensive testing of culture conditions, assaying for pilin expression from ppdD, but found no induction. I've also tested some conditions for induction of other Sxy-regulated gene fusions, and the RA and former postdoc tested growth-condition dependence using quantitative PCR of sxy itself, again with no good evidence of induction. What we need to do now is to bring our molecular expertise to bear on this question.
We expect to find that E. coli sxy has both transcriptional and translational regulation, because such dual regulation has been demonstrated in both H. influenzae and V. cholerae, and because its transcript has a long untranslated leader (116 nt) like these species. Transcription may be regulated by CRP and cAMP, because the sxy promoter has a partial CRP site. This site looks like a reversed E. coli CRP-S site rather than like a CRP-N site, which might mean that sxy transcription is autoregulated by Sxy itself. Because the site is inverted relative to the sites found in the CRP-S promoters identified by our microarray analysis, this autoregulation may be negative (high Sxy may cause reduced transcription). We haven't yet directly tested either cAMP/CRP or Sxy for direct effects on sxy transcription. We can't do this with either of of our sxy-expression plasmids, because neither has an intact sxy promoter, but we could test wildtype cells for altered expression of the chromosomal sxy gene. We could also test whether cells with an internal insertion/deletion in sxy (but an intact promoter and 5' end) have more or less sxy transcript. Unfortunately we still don't have a Sxy antibody so we can't test for protein.
Question 2: Does Sxy induce competence? So far we have not found any evidence of genetic transformation in cells artificially induced to express moderate levels of Sxy. This could be because Sxy doesn't induce DNA uptake in E. coli, but it could also be because the level of Sxy is too low, or because the cells take up DNA but don't recombine it, or because Sxy doesn't induce DNA uptake in the K-12 strain but does in other strains.
The Sxy expression level we tested was low enough that it didn't interfere at all with growth. This was fully-induced expression from a low copy number plasmid with an IPTG-inducible lac promoter. The higher expression level we've used (for the microarray analysis) was very toxic and we couldn't test for transformation at all.
We can clarify some of these alternative. We can measure DNA uptake directly using radiolabelled DNA. In principle this is not nearly as sensitive as measuring transformation, but that's only true if transformation is very efficient. So maybe Sxy expression is making E. coli competent, and it's taking up lots of DNA, but just not producing any transformants.
We can also measure sxy transcript levels in cells artificially induced to different extents, and use this information to decide which conditions we should use to examine DNA uptake. The high copy plasmid gives massive induction and toxicity, and lower concentrations of the inducer still reduce the growth rate quite a bit. The low copy plasmid doesn't reduce the growth rate at all. I think we should examine an intermediate level, using a low concentration of inducer with the high copy plasmid. And we should add cAMP to these cultures as well as inducer - cAMP isn't needed for sxy induction from the plasmid but it may help with expression of some or all of the CRP-S genes.
There are lots of other E. coli strains we could test in addition to K-12. The RA has already screened the entire ECOR collection (~70 strains) for the level pilin expression in overnight cultures. She found no detectable expression in any strain. It would be good to test at least one strain more thoroughly, for transformation and DNA uptake, but how to decide which strain(s) to test?
The true Geology behind The X-Files: Darkness Falls
1 day ago in History of Geology