|PHILMUS, BENJAMIN - Oregon State University
|KIDARSA, TERESA - Former ARS Employee
|YAN, QING - Oregon State University
|RAAIJMAKERS, JOS - Wageningen University
|BEGLEY, TADHG - Texas A&M University
Submitted to: ChemBioChem
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/9/2015
Publication Date: 7/3/2015
Citation: Philmus, B.J., Shaffer, B.T., Kidarsa, T.A., Yan, Q., Raaijmakers, J.M., Begley, T.P., Loper, J.E. 2015. Investigations into the biosynthesis, regulation and self-resistance of toxoflavin in Pseudomonas protegens Pf-5. ChemBioChem. doi: 10.1002/cbic.201500247.
Interpretive Summary: This manuscript reports that toxoflavin, a broad-spectrum antibiotic and phytotoxin, is produced by a soil bacterium called Pseudomonas protegens strain Pf-5. This is the first report of toxoflavin production by Pseudomonas spp.; it was known previously to be produced only by Burkholderia and Streptomyces spp. We describe the toxoflavin biosynthesis gene cluster in Pseudomonas protegens Pf-5, which has ten genes, nine of which are also present in the previously-described described toxoflavin biosynthesis gene cluster of Burkholderia spp. One gene in the Pf-5 cluster is unique, and we isolated the protein encoded by this gene and showed that it degrades toxoflavin, possibly functioning in self-protection of the bacterium against this antibiotic. We generated mutants in many of the toxoflavin biosynthesis genes of Pf-5 and demonstrated their functions in toxoflavin biosynthesis. From these experiments, we disproved the current hypothesis for the pathway for toxoflavin biosynthesis and propose a new toxoflavin biosynthetic pathway that is consistent with our results. We also show that toxoflavin production contributes to the inhibition of a number of plant pathogenic bacteria by Pf-5. These results extend our knowledge of antibiotics produced P. protegens strain Pf-5, a model biological control strain that can suppress plant diseases by producing antibiotics toxic against plant pathogens.
Technical Abstract: The pseudomonads are prolific producers of natural products from a variety of structural classes. Here we show that the plant commensal Psuedomonas protegens Pf-5 is capable of producing the triazine natural product toxoflavin (1). Using a strain of P. protegens that overproduces toxoflavin, we investigate the biosynthesis of toxoflavin using genetic knockouts. We propose that the first two steps in toxoflavin biosynthesis are identical to riboflavin biosynthesis and propose a new biosynthetic pathway. We also identify a self-resistance gene in the toxoflavin biosynthetic cluster (PFL_1030, toxM) that acts as a monooxygenase and detoxifies toxoflavin in an identical manner to toxoflavin lyase, TflA, from Paenicillum polymixa. We show that homologs of toxM are found in multiple strains of Pseudomonas and that introduction toxM into the toxoflavin-sensitive strain P. syringae DC3000 results in resistance to toxoflavin.