The RA's heroic project to create knockout mutants of every gene in the H. influenzae competence regulon has turned up one big surprise - the HI0659 gene. This small cytoplasmic protein, whose mRNA was induced about 25-fold in competent cells in our old microarray experiments, turns out to be essential for competence. The knockout mutant doesn't detectably take up DNA and produces no transformants.
The first question to consider (and maybe to answer) is whether this protein plays an essential mechanistic role in DNA uptake or has a regulatory function that's needed for effective expression of the other genes. It's only 98 amino acids long, and most of that is a single helix-turn-helix (HTH) domain (see figure). HTH domains typically regulate gene expression by binding to specific DNA sequences, but they usually are only part of larger proteins whose activities are in turn regulated by other effectors such as sugars and amino acids. But HI0659 doesn't have much room for other interactions, decreasing the likelihood of a regulatory function. On the other hand, the protein doesn't have much room for DNA uptake functions either. And it doesn't have any targeting signals that would send it into the cell envelope.
The postdoc speculates that it might bind RNA rather than DNA, perhaps interacting with sxy mRNA. Apparently some HTH motifs do bind RNA. But ssRNA has a very different structure than dsDNA, so I wonder if what these motifs bind is actually dsRNA.
One thing the microarray summary doesn't tell us is the basal level of HI0659 mRNA expression. This is of interest because, if it's a regulator of competence that's needed for expression of the other genes in the CRP-S regulon, it should be active before they come on. Maybe it's active constitutively at a moderate level, and induced even higher in competent cells.
It's just downstream of HI0660, another tiny protein with no known function. HI0660 is even less conserved than I0659, and knockouts of it have normal competence. Surprisingly, the 'marked' knockout of HI0659 (the same deletion with an inserted SpcR cassette) retains some competence.
Here's a graphic of HI0660 and HI0659 aligned with homologs. I asked the database to find homologs in other Pasturellacean species (H. ducreyi, Mannheimia succinoproducens, Pasteurella multocida) but none were found even though I lowered the % similarity cutoff to 30% from the default 40%. This is a bit surprising, because A. pleuropneumoniae is a more distant relative than M. succinoproducens and P. multocida. The operon is also in M. haemolytica, a close relative of A. pleuropneumoniae not shown in the figure, and I think the grad student who did the analysis also found that it has a CRP-S promoter in these species.
For the CIHR proposal we're going to propose to do 'RNA-seq' of the HI0659 knockout and controls, to look for changes in the mRNA population. RNA-seq is the shorthand term for measuring the abundances of all a cell's transcripts by doing deep sequencing of a cDNA prep. It's pretty straightforward; the only big problem is avoiding wasteful sequencing of ribosomal RNAs, which are by far the most abundant RNAs in bacterial cells, but the postdoc says there's a good kit for that.
Before doing this we should confirm that the HI0659 mutation we've made is responsible for the competence defect we see by 'backcrossing' the unmarked mutation into a clean genetic background,. This would be much easier if the mutation is closely linked to a marker we can select for. We could instead transform it into the marked HI0660 knockout and screen for loss of SpcR; in principle this wouldn't be as efficient as selection but it might be easier.
The RNA-seq experiment will be useful in other ways. Because the genome (and transcriptome) are small, we can afford to include lots of controls. At a minimum we'll do wildtype and mutant cells in log phase and after competence induction, but we might also include crp and sxy mutants, and maybe even one or more of the mysterious hypercompetence mutants of HI1133 (murE). This analysis will complement our previous microarray analysis, putting our identification of competence-regulated genes on a very solid foundation.