Figure 7. Exogenous paerucumarin enhances the expression of different genes within the roc and cup systems.

The relative level of expression for each gene in each pair of strains was determined by RT-qPCR. The quantity of cDNA in different samples was normalized using 30S ribosomal RNA (rplS) as an internal standard. A. Over-expression of pvc by pJAC7-1 leads to accumulation of paerucumarin within the supernatant of MPAO1 but not those of PW4830 (ΔpvcA) and PW4832 (ΔpvcB). Strains carrying p18.230 (V) or pJAC7-1 (PtxR) were grown for 16 h at 37°C. Cells were pelleted and the supernatants isolated by centrifugation. Paerucumarin was extracted from the supernatant fractions by ethyl acetate and detected using TLC as described in Materials and Methods. Asterisk indicates position of paerucumarin. Purified paerucumarin served as a positive control (C). B. Exogenously added paerucumarin enhances the expression of rocS1, rocS2, cupB2, and cupC2 in PW4830 (ΔpvcA). An overnight culture of PW4830 was subcultured into fresh LB broth to an OD₆₀₀ of 0.02. A 15-µl aliquot of either ethyl acetate (mock) or purified paerucumarin 300 µM) was added at the time of subculturing. Cells were grown at 37°C for 16 h. The levels of gene expression in PW4830 plus paerucumarin were compared to the levels in mock-treated PW4830. Values in B represent the average of triplicate PCR experiments conducted on three independently obtained RNA preparations ± SEM (n = 3); P<0.05 (*); P<0.01 (**); P<0.001 (***).