I've been doing a lot of digging around and reading about Hfq's possible involvement in post-transcriptional regulation of sxy. Now I need to write it all down here or I'll forget what I've done.
1. H. influenzae Sxy is a great in vivo system for investigating Hfq activity: A commenter on the previous post (thanks, Mike!) recommended doing in vitro assays for Hfq's ability to bind to sxy mRNA. But, although we've done in vitro RNA work in the past, it certainly isn't our strength. Fortunately, our in vivo transformation assays let us detect changes in Sxy activity over at least 6 orders of magnitude, because fully competent cells have a transformation frequency of ~10^-2 (higher if we use a pure marker fragment rather than chromosomal DNA), and the threshold of detection is <10^-8. The secondary structure has been confirmed experimentally, and we also have mutations that affect the secondary structure of sxy mRNA, some that destabilize the main stem and increase transformation frequency dramatically and some that stabilize the stem and reduce transformation frequency dramatically.
2. In Vibrio cholerae, post-transcriptional activation of the sxy homolog tfoX depends on both a small RNA (tfoR) and Hfq (Yamamoto et al. 2011. J. Bacteriol. 193:1953-1965, doi:10.1128/JB.01340-10). This is consistent with Hfq's usual role in facilitating adaptation to stress, and predicts that loss of Hfq in H. influenzae will decrease (or even eliminate) transformation. This couldn't be tested directly in V. cholerae because of other harmful effects of the Hfq knockout. I'm hopeful that this won't be a big problem in H. influenzae, since Hfq is unlikely to affect as many processes. H. influenzae has a much smaller genome; it doesn't do quorum sensing and doesn't regulate catabolite repression with a Hfq-dependent spot42 small as E. coli and V. cholerae do.
I've gone through the list of H. influenzae RNA sequences on the Rfam server (thanks, Paul, for the link!). There are only about 100 of these, and most are rRNAs and tRNAs. The rest all have predicted functions that are unlikely to affect competence. We don't have any RNA-seq results yet (though this is planned to happen soon), but there's a published report of RNA-seq analysis in the related Haemophilus somnus (renamed Histophilus somni by the evil Danish group) which includes a list of small RNAs, tagged with their distributions. I grabbed the sequences of the eleven of these that are present in other Pasteurellaceae, and tested them for complementarity to H. somnus sxy mRNA using the IntaRNA server from U. Freiburg. One of them (HS29) showed quite strong pairing. HS29 has homologs in most Pasteurellaceae but not in H. influenzae, which is a drag, but at least this analysis suggests that a similar small RNA might interact with sxy mRNA in H. influenzae.
So the RA has just ordered the oligos she'll need to make a knockout mutant of hfq (HI0411). I'll transform the mutation into wild type cells and into cells that are hypercompetent due to mutations that destabilize the main stem of sxy mRNA. The V. cholerae precedent predicts a decrease in competence; if I don't see this, Hfq probably doesn't play any role. If competence is down I may be able to rule out effects just due to changes in growth properties by comparing the effects in wildtype and hypercompetent backgrounds. I might use the murE hypercompetent background as a control, though it's also possible that a murE-associated small RNA mediates the Hfq effect. Just be sure I'll also check for increases in competence using one of our low-expression sxy mutants.
Pause while I spend two hours trying to see whether part of the murE mRNA, or an antisense RNA to it, could pair with the regulatory part of sxy mRNA. Hmm, tantalizingly. it does. This would be more exciting if the pairing segment included the site of one of the mutations known to cause hypercompetence... Anyway, here's a figure:
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1. H. influenzae Sxy is a great in vivo system for investigating Hfq activity: A commenter on the previous post (thanks, Mike!) recommended doing in vitro assays for Hfq's ability to bind to sxy mRNA. But, although we've done in vitro RNA work in the past, it certainly isn't our strength. Fortunately, our in vivo transformation assays let us detect changes in Sxy activity over at least 6 orders of magnitude, because fully competent cells have a transformation frequency of ~10^-2 (higher if we use a pure marker fragment rather than chromosomal DNA), and the threshold of detection is <10^-8. The secondary structure has been confirmed experimentally, and we also have mutations that affect the secondary structure of sxy mRNA, some that destabilize the main stem and increase transformation frequency dramatically and some that stabilize the stem and reduce transformation frequency dramatically.
2. In Vibrio cholerae, post-transcriptional activation of the sxy homolog tfoX depends on both a small RNA (tfoR) and Hfq (Yamamoto et al. 2011. J. Bacteriol. 193:1953-1965, doi:10.1128/JB.01340-10). This is consistent with Hfq's usual role in facilitating adaptation to stress, and predicts that loss of Hfq in H. influenzae will decrease (or even eliminate) transformation. This couldn't be tested directly in V. cholerae because of other harmful effects of the Hfq knockout. I'm hopeful that this won't be a big problem in H. influenzae, since Hfq is unlikely to affect as many processes. H. influenzae has a much smaller genome; it doesn't do quorum sensing and doesn't regulate catabolite repression with a Hfq-dependent spot42 small as E. coli and V. cholerae do.
I've gone through the list of H. influenzae RNA sequences on the Rfam server (thanks, Paul, for the link!). There are only about 100 of these, and most are rRNAs and tRNAs. The rest all have predicted functions that are unlikely to affect competence. We don't have any RNA-seq results yet (though this is planned to happen soon), but there's a published report of RNA-seq analysis in the related Haemophilus somnus (renamed Histophilus somni by the evil Danish group) which includes a list of small RNAs, tagged with their distributions. I grabbed the sequences of the eleven of these that are present in other Pasteurellaceae, and tested them for complementarity to H. somnus sxy mRNA using the IntaRNA server from U. Freiburg. One of them (HS29) showed quite strong pairing. HS29 has homologs in most Pasteurellaceae but not in H. influenzae, which is a drag, but at least this analysis suggests that a similar small RNA might interact with sxy mRNA in H. influenzae.
So the RA has just ordered the oligos she'll need to make a knockout mutant of hfq (HI0411). I'll transform the mutation into wild type cells and into cells that are hypercompetent due to mutations that destabilize the main stem of sxy mRNA. The V. cholerae precedent predicts a decrease in competence; if I don't see this, Hfq probably doesn't play any role. If competence is down I may be able to rule out effects just due to changes in growth properties by comparing the effects in wildtype and hypercompetent backgrounds. I might use the murE hypercompetent background as a control, though it's also possible that a murE-associated small RNA mediates the Hfq effect. Just be sure I'll also check for increases in competence using one of our low-expression sxy mutants.
Pause while I spend two hours trying to see whether part of the murE mRNA, or an antisense RNA to it, could pair with the regulatory part of sxy mRNA. Hmm, tantalizingly. it does. This would be more exciting if the pairing segment included the site of one of the mutations known to cause hypercompetence... Anyway, here's a figure:
