The addition of PQS partially recovered MV production in ΔpqsR. When both indole and PQS were added in ΔpqsR, MV production was not decreased to the level Natural Product Library cell assay of ΔpqsR, suggesting that indole inhibits MV production by regulating PQS

and does not inhibit the interaction between PQS and lipopolysaccharide. PQS acts as a ligand for PqsR, the transcriptional activator that controls the pqsABCDE operon (Xiao et al., 2006). The gene products of pqsABCD catalyze the synthesis of HHQ from anthranilic acid and the expression of pqsABCDE is autoregulated positively (Déziel et al., 2004). To determine whether the decreased PQS levels in cultures grown in the presence of indole were due to decreased expression of the PQS biosynthetic operon, the expression of the pqsA promoter was examined. A reporter gene xylE, which encodes C23O,

was introduced downstream of PAO1 chromosomal pqsE (10 bp behind the stop codon of pqsE), and C23O activity was measured at several growth points. The addition of 500 μM indole resulted in a reduction of pqsABCDE expression (Fig. 2e). Furthermore, we examined the effect of exogenous PQS on pqsABCDE expression in the presence and absence of indole to investigate how indole affects it. We used the pqsH deletion mutant, which synthesized HHQ, but not PQS, to avoid the effect of self-produced PQS. Indole and/or PQS were added at the point of the late exponential phase to avoid the effect of the indole degradation

selleck kinase inhibitor because it has been reported that indole was degraded by P. aeruginosa PAO1 (Lee et al., 2009). The addition of PQS to the culture increased pqsABCDE expression (Fig. 2f), indicating that the expression of the chromosomal Meloxicam xylE fusion responds to PQS. When both PQS and indole were added, the pqsABCDE expression showed a tendency similar to that of the only PQS-added culture (Fig. 2f), suggesting that indole does not inhibit the autoregulation of pqsABCDE by PQS. In addition, to examine whether indole inhibits the expression of PqsH, which plays a role in the final reaction of PQS synthesis and converts HHQ into PQS, a reporter gene xylE was inserted directly behind the stop codon of the chromosomal pqsH, and C23O activity was measured in the presence and absence of indole at several growth points. The results showed that the addition of indole did not affect pqsH transcription (data not shown), suggesting that pqsH is not involved in the inhibition of PQS synthesis by indole. Given these results, two hypotheses can be considered regarding how indole inhibits PQS synthesis. One is that indole is related to the synthesis or the degradation of anthranilic acid. The other is that indole inhibits the activation of any enzymes, which are related to PQS synthesis, after transcription. Detailed analysis is needed to understand the mechanism for the inhibition of PQS by indole. It is well known that MVs released by P.