The requirement for the LysR-type regulator PtrA for Pseudomonas chlororaphis PA23 biocontrol revealed through proteomic and phenotypic analysis

Background: Pseudomonas chlororaphis strain PA23 is a biocontrol agent capable of suppressing the fungal pathogen Sclerotinia sclerotiorum. This bacterium produces the antibiotics phenazine and pyrrolnitrin together with other metabolites believed to contribute to biocontrol. A mutant no longer capable of inhibiting fungal growth was identified harboring a transposon insertion in a gene encoding a LysR-type transcriptional regulator (LTTR), designated ptrA (Pseudomonas transcriptional regulator). Isobaric tag for relative and absolute quantitation (iTRAQ) based protein analysis was used to reveal changes in protein expression patterns in the ptrA mutant compared to the PA23 wild type. Results: Relative abundance profiles showed 59 differentially-expressed proteins in the ptrA mutant, which could be classified into 16 clusters of orthologous groups (COGs) based on their predicted functions. The largest COG category was the unknown function group, suggesting that many yet-to-be identified proteins are involved in the loss of fungal activity. In the secondary metabolite biosynthesis, transport and catabolism COG, seven proteins associated with phenazine biosynthesis and chitinase production were downregulated in the mutant. Phenotypic assays confirmed the loss of phenazines and chitinase activity. Upregulated proteins included a lipoprotein involved in iron transport, a flagellin and hook-associated protein and four proteins categorized into the translation, ribosome structure and biogenesis COG. Phenotypic analysis revealed that the mutant exhibited increased siderophore production and flagellar motility and an altered growth profile, supporting the proteomic findings. Conclusion: PtrA is a novel LTTR that is essential for PA23 fungal antagonism. Differential protein expression was observed across 16 COG categories suggesting PtrA is functioning as a global transcriptional regulator. Changes in protein expression were confirmed by phenotypic assays that showed reduced phenazine and chitinase expression, elevated flagellar motility and siderophore production, as well as early entrance into log phase growth.