A TetR/AcrR family regulator Pip induces phenazine biosynthesis but represses pyrrolnitrin biosynthesis in biocontrol agent Pseudomonas chlororaphis G05

Abstract Pseudomonas chlororaphis G05 produces both phenazine-1-carboxylic acid as well as pyrrolnitrin. Both metabolites play a key role in inhibiting some phytopathogenic fungi growth and bioprotecting plants against some diseases in modern agriculture. Though a few regulatory genes critical for pyrrolnitrin production were identified, the regulatory pathways involving pyrrolnitrin biosynthesis were not well understood. By employing random transposon mutagenesis, we isolated a dark blue colony G05D02 in which a pip gene was identified to be disrupted with the insertion of a transposon mini-Tn5Kan. Like in other Pseudomonas spp., Pip positively activates phenazine production in P. chlororaphis G05, but Pip may act as a repressor for pyrrolnitrin biosynthesis in our isolate. To verify its regulatory function, we constructed the pip-negative mutants, G05Δpip, G05Δphz::lacZΔpip and G05Δprn::lacZΔpip. By quantifying β-galactosidase activity, we found that absence of pip dramatically upregulates the prn operon expression. With quantification of pyrrolnitrin produced by the mutant G05Δpip, moreover, we confirmed that Pip deficiency caused remarkable increase of pyrrolnitrin production. As expected, phenazine-1-carboxylic acid biosynthesized by the mutant G05Δpip was much less than that in the wild-type strain. Further analysis of fusion constructs and RT-qPCRs also confirmed these results. Taken together, Pip induces phenazine-1-carboxylic acid production but represses pyrrolnitrin production in P. chlororaphis G05.