Ending the semester on a high note!

Before winter break laziness (and hopefully some reading/writing energy) fully take hold, I wanted to report on a discovery from the end of the semester that will definitely be a focus of my project in the spring. The J75 cag negative strain of HP has never been the most cooperative. After going through several rounds of mutagenesis reactions and several primer sets, I finally altered the oipA locus to “phase on” and transformed this plasmid into HP. But when June and I started using these strains in adherence assays with AGS cells, the results were, frankly, boring. Unlike the other cag negative strain J68 where there were clear differences in AGS cell attachment between the cells with oipA phase on and off, there was no difference in J75 across several experiments. After confirming that each strain was indeed what it was supposed to be with sequencing, I was perplexed.

Of course it was possible for the changes to simply have no effect, however it seemed strange for the difference between two similar strains to be so stark. After all, one of the key reasons for this study is the fact that the oipA gene is so highly conserved in all strains of HP. Typically, the promoters for these types of genes show little variation. But after being so fixated on the coding region, we looked a few hundred base pairs upstream to see if anything stood out in the promoter region. To our surprise, J68 (cag negative), 26695 (cag positive), and J75 were all almost exactly identical, except for an 11 base pair insertion in the promoter of J75. This was a very exciting revelation, if only to explain the strange experimental results. Hindsight is 20/20 and it would have been nice to have figured this out prior to all of those AGS experiments. But now there is much work to be done to try to figure out why this strain has evolved to have this insertion in the promoter region such that, even with phase variation in the actual oipA gene, it could never be transcribed.

We know that it takes about 5 base pairs to make a full turn of the DNA helix, so this insertion adds in two full turns. Studies from our lab (Harvey, Acio et al. 2014) and others show that changes like this caused by insertions or phase variation in repeats can greatly impact RNAP’s ability to bind. This can be seen in the figure below from a 2014 study by Aberg et al. I am excited to use this knowledge to further pursue J75’s unique insertion next semester, and I am thankful to the SRG for making this research possible!

Figure-8-T-or-A-tracts-adjacent-to-2-35-elements-regulate-gene-expression-by-a

 

Harvey, V. C., C. R. Acio, A. K. Bredehoft, L. Zhu, D. R. Hallinger, V. Quinlivan-Repasi, S. E. Harvey, and M. H. Forsyth. “Repetitive Sequence Variations in the Promoter Region of the Adhesin-Encoding Gene SabA of Helicobacter Pylori Affect Transcription.” Journal of Bacteriology 196.19 (2014): 3421-429. Web.

Aberg, Anna, P. Gideonsson, A. Vallstram, Annelie Olofsson, Carina A–hman, Lena Rakhimova, Thomas Boran, Lars Engstrand, Kristoffer Brannstrom, and Anna Arnqvist. “A Repetitive DNA Element Regulates Expression of the Helicobacter Pylori Sialic Acid Binding Adhesin by a Rheostat-like Mechanism.” PLoS Pathogens 10.7 (2014): n. pag. Web.