Figure 3. Differential colony formation of essential gene knockout mutant strains.

We attempted to knock out the essential genes in strains for which we isolated an HCS. We were successful in the four cases shown here. For each essential gene, the top row illustrates the growth that is observed when the native promoter is replaced by P_ara, under permissive (0.1% arabinose) or restrictive (0.4% glucose) growth conditions. The bottom row indicates the growth that is observed when the essential gene is knocked out, under conditions in which the suppressor is not induced (0 mM IPTG) or induced (1 mM IPTG). In the absence of the HCS, almost no growth is observed under restrictive conditions. However, when the HCS is induced, robust growth is almost always observed, even in the cases in which the essential gene is knocked out. Note that the ΔspoT strain is complemented by the mutT plasmid without induction of mutT expression. To grow the spot plates, cultures were grown overnight in 0.1% arabinose with 15 µg/ml chloramphenicol and 1 mM IPTG, diluted into LB medium, and 5 µl of the indicated dilution were spotted onto plates containing 0.1% arabinose with 15 µg/ml chloramphenicol, or 0.4% glucose with 1 mM IPTG. Arabinose plates were incubated for 14 hours at 37°, while glucose plates were incubated for 40 hours.