Field of Science

Planning more RNAseq experiments

The postdoc and former RA generated some great RNAseq data, which I'll write about in another post.  But we have some money that needs to be spent on sequencing in the next couple of months, so we need to decide which additional RNA seq runs we should do.  And then I'm going to grow the cultures and prep the RNAs.

We have data sets showing how RNA levels change after transfer to competence-inducing MIV medium for several Haemophilus influenzae strains: wildtype (2 expts), sxy- (2 expts), HI0659- (1 (antitoxin?, 1 expt) and HI0660- (toxin?, 1 expt).  For each we have samples at t=0, t=10, t=30 and t=100 minutes.  (The figure shows a comparison between wildtype and sxy- at t=0 and t=10; the red circles are CRP-regulated genes and the blue ones are competence genes.)  We need to do at least one replicate of the HI0659 and HI0660 cultures.  If we also did another replicate of everything, that would be a full 24 sample run (one lane?) for the sequencer, and enough data that we could do proper statistical analyses.

But I also want to get RNAseq data for strains with other mutations, especially the hypercompetence-causing mutation murE749 in exponential growth.  This would be a single condition, replicated once or twice, so 2 or 3 samples total.  I might be able to squeeze this in with the run described above; depending on what other experiments we plan to do with these strains, two replicates of some might be enough.  Or it might be better to do a second run since we have the funds, doing a more comprehensive analysis of other conditions and other mutants too.

One condition I'd like to examine is 'late-log' growth, where wildtype cells develop moderate levels of competence.  I want to see if these levels are comparable to those in the HI0659 antitoxin mutant, which shows no competence at all although it appears to have (compared to wildtype cells) only a slight decrease in competence gene expression at t=10, no decrease at t=30, and a moderate decrease at t=100.  It's possible that the toxin acts only by decreasing mRNA levels of other competence genes, but the disproportion between its absolute competence defect and modest RNA defect makes me wonder if it also does something else.

We have three other hypercompetence mutants with mutations in murE - I don't know if it would be worth doing one sample of each of these in exponential growth.  We also have a hypercompetent mutant of unknown genotype - RNAseq might find the mutation as well as show us the RNA changes.  Might it be worth testing a crp mutant or cya mutant, to confirm our understanding of cAMP/CRP regulation?  Or the purR or purH mutant, or cells whose MIV-induced competence development has been blocked by adding AMP?  Or cells whose DNA replication has been blocked by hydroxyurea (depletes dNTP pools).  Or the hfq mutant, which has 10-fold lower competence.


  1. For selfish reasons the hfq mutant is my choice (but I think in some later posts you've already decided on the strains). My real question is does your RNA isolation method also collect the small RNAs? I think to fully rule out an sRNA role in competence development that information on sRNA expression is key. For some reason I still stubbornly like the idea of an sRNA influencing competence.

    -Estevan Santana
    Munson Lab grad student

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  3. We'll definitely be looking for small RNAs affecting sxy expression. There's also the toxin-antitoxin genes, which are the kind that affect RNAs.

    Do you have a 'selfish' interest in this because it's part of your thesis project? If so, might we collaborate rather than stepping on your toes?

  4. Sorry for the late reply, I've been "wrapping up" in the lab and am now furiously writing my thesis. No, Hfq is not a direct component of my thesis project. I have focused on Fur-regulated sRNAs (and discovered it, soon to be published).

    So I have only tangentially looked at Hfq in H. flu. To be honest, we have not found a true role for Hfq in NTHi 86-028NP. We cannot find a testable phenotype in an hfq mutant. Terry Stull published a paper on Hfq's role in virulence. Their lab was also unable to find any testable phenotypes in an hfq mutant like those seen for E. coli etc.

    However, I have evidence that Hfq is important in the regulation of a few genes suspected of being regulated by small RNAs. So I am a little skeptical on how big a role Hfq plays in H. flu gene regulation compared to other well-studied bacteria. Unfortunately, we have no data about sxy and sRNAs. Our lab's initial screen for small RNAs by RNA-seq was fairly short-sighted and only was able to identify a small RNA directly regulated by Fur (similar to RyhB in E. coli). However, we did grow NTHi 86-028NP in sBHI to mid-log and performed RNA-seq. We then annotated all potential small RNA loci. That data might complement whatever RNA-seq you have done as far as identifying potential sRNAs. That list will be published as suppl. table in my paper.

    My "selfish" reason for liking the idea of Hfq and sRNAs regulating competence comes from my project's focus on sRNAs in general. We once attempted an Hfq pulldown in NTHi 86-028NP but only recovered a very small list of RNAs bound to Hfq (but our result may have just been due to uz being novices at performing Hfq pulldowns). Im pretty positive after I graduate in August our lab will not be pursing sRNA/Hfq/competence work at all.

    Good luck! I am really interested in your work and the idea that (in the simplest terms) nutritional need drives many phenotypes in H. flu and bacteria in general.

    It's been great reading your blog the last 4 years or so. After I graduate I will be starting a fellowship in Sacramento, CA designed to get PhD scientists into science policy careers to advise policy makers on the science behind their policy issues.

    Im not sure how you get your RNA-seq data anaylzed, but Bob has a great collaboration with a bioinformatics group out of IUPUI in Indianapolis. They are smart and have a very fast turnaround.

    -Estevan Santana
    Munson Lab


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