Figure 4

Overproduction of RibM enhanced roseoflavin sensitivity of a Δ ribU ::Kan^r B. subtilis strain. (A) Streaks (top) and drops (bottom, about 50,000 cells) of B. subtilis ΔribU::Kan^R cells expressing ribM from plasmid pHT01ribM_opt were applied to LB plates containing the indicated amounts of the toxic riboflavin analog roseoflavin and 100 μM IPTG. Growth was recorded after incubation for 36 h at 37°C. As controls, strains were transformed with the empty vector pHT01. At 10 μM roseoflavin it was most obvious that the presence of RibM increased roseoflavin sensitivity due to import of the toxic compound. (B) B. subtilis ΔribU::Kan^R expressing ribM from plasmid pHT01ribM_opt was grown in LB broth in the presence of the indicated amounts of roseoflavin and 100 μM IPTG. Growth was recorded at μ = 600 nm. As controls, ΔribU::Kan^R strains containing empty pHT01 were analyzed. At 50 μM roseoflavin, the effect of RibM was most obvious (see bracket). Cells (black triangles) producing RibM imported toxic roseoflavin and consequently grew to an OD₆₀₀ of about 2.5 only. The control strain (open triangles) was less affected by roseoflavin and grew to an OD₆₀₀ of 4.2.