On Thursday I did a competence time course for wildtype cells growing in a defined medium with and without a purine source. The medium was a 50:50 mix of the RPMI-based medium and the MMB medium with glutathione, because that's the only medium where growth has been reproducible. I'll call it RM medium for now.
The first graph shows growth of the cells; C is medium without inosine, D is medium with inosine. There aren't many time points for two reasons. First, the cells initially didn't grow from an overnight-culture inoculum - I'm going to solve this for future experiments by freezing vials of cells already in log phase in this medium. Second, I was planning to also test cells growing in plain MMB medium ± inosine, but they didn't grow at all. Time zero on the graph is actually 4 hours after the cultures were inoculated.
The red and blue lines are the cells in RM medium with and without inosine respectively, and the purple and green lines are cells transferred from these cultures into the competence-inducing starvation medium MIV. There's not much to learn from this graph on its own - the cells are growing in the RM medium, but slower and to lower densities than they would in the rich medium sBHI, and they continue to divide once or twice in MIV, as we see with sBHI-grown cells.
The third surprise was that the cells growing in RM + inosine became much more competent after being transferred to MIV. Cells grown in sBHI are expected to do this, but competence development hasn't been examined in either the RPMI-based medium or in MMB. Many years ago (1970) Roger Herriott reported that H. influenzae cells grown in a defined medium called MI wouldn't become competent when transferred to MIV, and he developed a modified defined medium called MIc that did allow subsequent competence development in MIV.
More surprising was the high competence induced by MIV in cells that had been growing in RM without inosine (green line). MIc and the other published defined media for H. influenzae include a high concentration of inosine (even though we now know it's not required for growth), because Ranhand and Herriott had previously found that adding inosine and lactate to a defined medium promoted subsequent competence development. And our thinking about the regulation of competence has been influenced by this. I don't think we've ever tested how inosine in the growth medium affects competence in MIV, but I expected to see little or no competence induction, rather than the 3-4 logs the graph shows.
Tomorrow I'll repeat this time course, including cells in sBHI as a control, and sampling more time points, especially early in growth. I'll start these new cultures with my frozen RM-grown cells.
11 hours ago in Variety of Life