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The basic assay was as described in the previous post. She tested four strains: the wildtype parent, which expresses T4P genes and becomes moderately competent at the onset of stationary phase, a strain unable to induce its competence genes (including the T4P genes), a strain whose type 4 pilin gene is deleted, and a hypercompetent strain that expresses all the competence genes very strongly at all stages of growth.
Cultures were grown for one and two days in 2 ml of rich medium in new glass tubes, either stationary in a rack or being gently mixed on a roller wheel. Here's a photo of two of the Day 2 culture tubes, inverted to dry after staining. Most of the stationary-culture tubes had a film of cells, mainly at the bottom of the tube (exception explained below). All of the rolling-culture tubes had a bright film at the air-medium interface.
And here are the results. (Each bar is the mean of three replicate cultures.) With the exception of the stationary culture of the 'no pilin' strain, which failed to grow, all cultures gave equivalent staining intensity. There was no effect of expression of competence genes or deletion of the pilin gene.
Now I need to go back and look at the H. influenzae T4P literature, to see if this is a new result or an entirely predictable outcome.
Later: I looked through the H. influenzae pilus/biofilm literature. Other types of pili are needed for biofilm formation. A knockout of the T4P pilin induced in competent cells causes biofilms (grown in a flow-through chamber and observed microscopically) to be thinner and less 'organized', and reduces biofilm formation in the inner ears of chinchillas, so we might have expected our mutants to show altered biofilm staining.
Maybe it is worth having the summer student repeat her experiment, so we can describe this in the toxin/antitoxin paper. What improvements should we include?
- Including no-cells control tubes
- Measuring the OD600 of each culture? But would this require that the tubes be vortexed to resuspend the cells? Maybe just do it for the 'rolling' cultures (removing 100 µl to 900 µl blank), which won't need to be vortexed.
- Anything else?
For #2, perhaps save the culture when you decant the tube prior to staining, vortex that in another tube and take its OD600. This would underestimate actual OD600 (since some cells would remain at the bottom of the tube) but would at least provide a minimum constraint. Alternatively (perhaps more informatively), the number of tubes in each condition might be duplicated, with half used to established the OD600 after vortexing in the tube.
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