My last experiment showed that HI0660 encodes a 'toxin' of some sort, which prevents transformation when induced in the absence of the antitoxin encoded by HI0659. So yesterday I did detailed growth curves for cultures carrying the HI0659 knockout and either of our hypercompetence mutations.
The logic is that neither HI0660 or HI0659 will be expressed during growth in wildtype cultures, because the competence genes are not induced then. They're weakly induced at the end of growth, but strongly induced only when cells are starved or in the presence of the hypercompetence mutations. This means that, if HI0660 does kill cells or prevent growth, this effect will best be seen in the presence of the hypercompetence mutations.
The results show no evidence of significant growth or survival differences due to unopposed expression of HI0660. Each line in the two graphs below shows the mean for 7 replicate wells of the same inoculum. The upper graph used inocula that were of single small colonies diluted into 10 ml medium. The lower graph used inocula that were 1/1000 dilutions of overnight cultures into medium.
This is an interesting result, because it suggests that HI0660 acts directly on DNA uptake, not by killing cells or interfering with their growth. Because homologs of HI0660 are known to act by inactivating specific mRNAs, we may have to use RNA-seq to identify its mode of action.
End of the semester, at last!
8 hours ago in The Phytophactor