Plasmids carrying the sxy gene often acquire mutations; we have learned (painfully) that we need to recheck their sequences before using them in experiments. Our assumption has been that the Sxy protein is harmful to cells, at least when inappropriately expressed, and that the mutations are selected because they make this expression less harmful. I’ve always just treated this as a nuisance (a major nuisance), an obstacle to be tolerated because I haven’t seen any way to overcome it.
But last night I realized that we might be able to use it as a probe into what Sxy does. Although it's possible that Sxy’s toxic effects on cells have nothing to do with how Sxy induces expression of CRP-S genes, it’s more likely that the two effects are connected.
One obvious candidate connection is that Sxy affects how CRP acts, and perturbs CRP’s normal contributions to maintaining the cell's carbon and energy balance. An even more obvious candidate would be that inappropriate expression of CRP-S genes is toxic. (However the hypercompetence mutations cause such expression without being detectably toxic...) A less obvious but more exciting candidate is that Sxy activates transcription by interacting with RNA polymerase (or a general transcription factor), and that inappropriate expression interferes with transcription at other genes.
So the simple experiment is to propagate a sxy-expression plasmid in H. influenzae (or E. coli) without population bottlenecks (i.e. in a large culture grown for many generations), plate for single colonies, and isolate plasmids and sequence inserts from multiple colonies.
This will probably give a mix of obvious loss-of-function mutations (stop codons, deletions) and amino acid substitution mutations. My recollection of the mutations we’ve seen in the past is that they were mostly substitutions, which is good as these will be the interesting ones. If the majority are loss-of-function mutations we might want a way to screen these out before sequencing. We could do this if we started with a sxy- mutant, although this would need to be a complete deletion so that it wouldn’t recombine with the sxy gene on the plasmid. How would we screen them? Would screening for function be more trouble than it’s worth? I guess this would depend on how common the loss-of-function mutations turned out to be.
Mutations creating stop codons are expected to arise less frequently than simple substitutions (only three of the 64 codons specify STOP). Deletions are also expected to be relatively rare, at least in the absence of predisposing short repeats. So if we found that the majority are loss-of-function mutations, this might itself be our answer. This would tell us that Sxy is intrinsically harmful, and that getting rid of Sxy entirely is much more effective than changing its sequence.
So the first approach would be to sequence every plasmid that was isolated from a reasonably large colony. (Choosing large colonies will reduce the frequency of unchanged inserts.) Then we would compare changes, looking for clustering of substitution mutations. [This sounds like a good project for an undergraduate.] And we would use our anti-Sxy antibody to confirm that the plasmids with substitution mutations still produce full-length Sxy protein.
Then we would characterize the effects of the mutations on Sxy’s ability to induce CRP-S genes, probably by transforming the mutant version into wildtype cells and doing competence assays. We'd also look for effects on any other properties of Sxy we have a handle on, such as pull-down of complexed proteins, or two-hybrid interactions.
20 hours ago in The Phytophactor