I've (at last) gotten back into working on our review article about the regulation of competence. This morning I was reading about competence regulation in Vibrio, and found out that that the 5'-end of the sxy (tfoX) mRNA has a riboswitch secondary structure that responds to cyclic-di-GMP (a 'GEMM' riboswitch).
The H. influenzae sxy mRNA has a long untranslated 5' leader whose secondary structure limits translation - might this be because it's also a GEMM riboswitch? A few years ago we checked it for similarities to the then-known riboswitches, and it didn't fit the pattern at all. But I found a useful genome-survey paper by Michael Galperin which found that H. influenzae (and the other sequenced Pasteurellaceae) have no homologs of the proteins that synthesize and break down c-di-GMP. So they're very unlikely to have any riboswitches that recognize this molecule.
However there are several reasons to suspect that c-di-GMP might regulate sxy expression and competence in E. coli. Like Vibrio speciess, E. coli strains have multiple proteins predicted to synthesize c-di-GMP. E. coli sxy mRNA also has a long leader. In many bacteria, increased levels of c-di-GMP repress flagellar genes, as does sxy overexpression in E. coli.
In principle we could check for regulatory effects by adding c-di-GMP to cultures of E. coli (or H. influenzae) and look for changes in expression of sxy or of genes it regulates. BUT, very few of the papers I've been reading today did this. Instead the researchers went to a lot of work to genetically engineer cells to produce abnormally high or low levels of c-di-GMP, which makes my suspect that cells may not be permeable to c-di-GMP. Even the few papers that did add it to cultures didn't directly measure changes in gene expression, but just described phenotypic changes such as alterations in biofilm formation. But the papers don't come out and say that exogenous c-di-GMP can (or can't) enter cells. Perhaps I should email some authors about this.
I also should check the E. coli sxy mRNA leader sequence to see if it has the properties expected of a GEMM riboswitch. The RA, always ahead of the game, has already gone to a lot of effort to map the 5' end of this mRNA, so we can sit down with the sequence tomorrow.
Experiments with inertia
8 hours ago in Doc Madhattan