Pip, a Novel Activator of Phenazine Biosynthesis in Pseudomonas chlororaphis PCL1391

Abstract

Secondary metabolites are important factors for interactions between bacteria and other organisms. Pseudomonas chlororaphis PCL1391 produces the antifungal secondary metabolite phenazine-1-carboxamide (PCN) that inhibits growth of Fusarium oxysporum f. sp. radius lycopersici the causative agent of tomato foot and root rot. Our previous work unraveled a cascade of genes regulating the PCN biosynthesis operon, phzABCDEFGH. Via a genetic screen, we identify in this study a novel TetR/AcrR regulator, named Pip (phenazine inducing protein), which is essential for PCN biosynthesis. A combination of a phenotypical characterization of a pip mutant, in trans complementation assays of various mutant strains, and electrophoretic mobility shift assays identified Pip as the fifth DNA-binding protein so far involved in regulation of PCN biosynthesis. In this regulatory pathway, Pip is positioned downstream of PsrA (Pseudomonas sigma factor regulator) and the stationary-phase sigma factor RpoS, while it is upstream of the quorum-sensing system PhzI/PhzR. These findings provide further evidence that the path leading to the expression of secondary metabolism gene clusters in Pseudomonas species is highly complex.

Differential regulation of phenazine biosynthesis by RpeA and RpeB in Pseudomonas chlororaphis 30-84

“…We hypothesize that the phosphatase/kinase activity of RpeA may be dependent on environmental conditions, including growth media or temperature, which then influences the ratio of phosphorylated to non-phosphorylated RpeB. Phosphorylated RpeB promotes the expression of pip at the transcriptional level, which in turn enhances phenazine production through the QS system, similar to pip regulation of quorum sensing in PCL1391 (Girard et al, 2006a). In minimal media, RpeA modulates the expression of pip, phzR and the phenazine biosynthetic genes.…”

“…However, neither the mechanism of RsmZ control of phenazine production nor of the potential interaction between GacS/GacA and RpeA/RpeB has been determined. One possible mechanism of interaction could be through control of pip, since it has been proposed that GacS/GacA regulates pip expression via RpoS (Girard et al, 2006a).…”

“…biosynthetic genes (Girard et al, 2006a, b). The RNAbinding protein Hfq also positively regulates phenazine production by activating the quorum sensing system (E. Pierson and D. Wang, unpublished data).…”

“…CsaI/CsaR, a second QS system in 30-84 shown to be important in exoprotease production and cell aggregation also plays a minor role in phenazine production (Zhang & Pierson, 2001). Another gene essential for phenazine production in P. chlororaphis PCL1391 is that encoding phenazine inducing protein (pip) (Girard et al, 2006a), a homologue of the TetR family of transcriptional regulators (Ramos et al, 2005). Although the exact targets of Pip remain unclear, Pip has been shown to promote phenazine production by enhancing phzI and phzR expression.…”

Disruption of MiaA provides insights into the regulation of phenazine biosynthesis under suboptimal growth conditions in Pseudomonas chlororaphis 30-84

“…Previous studies with P. chlororaphis 30-84 established that a hierarchical network of genes regulates the PhzR/PhzI QS system that controls the phenazine biosynthetic operon [16,17]. This includes regulation by the transcriptional regulator Pip, the two-component signal transduction system RpeA/ RpeB, the stationary sigma factor RpoS and the Gac/Rsm system [16][17][18][19][20]. Pip is an important positive regulator of QS in P. chlororaphis 30-84 and PCL1391 [16,18].…”