Biosynthesis of fragin is controlled by a novel quorum sensing signal

Abstract

Members of the diazeniumdiolate class of natural compounds show potential for drug development because of their antifungal, antibacterial, antiviral, and antitumor activities. Yet, their biosynthesis has remained elusive to date. Here, we identify a gene cluster directing the biosynthesis of the diazeniumdiolate compound fragin in Burkholderia cenocepacia H111. We provide evidence that fragin is a metallophore and that metal chelation is the molecular basis of its antifungal activity. A subset of the fragin biosynthetic genes is involved in the synthesis of a previously undescribed cell-to-cell signal molecule, valdiazen. RNA-Seq analyses reveal that valdiazen controls fragin biosynthesis and affects the expression of more than 100 genes. Homologs of the valdiazen biosynthesis genes are found in various bacteria, suggesting that valdiazen-like compounds may constitute a new class of signal molecules. We use structural information, in silico prediction of enzymatic functions and biochemical data to propose a biosynthesis route for fragin and valdiazen.

Biosynthesis and Structure–Activity Relationship Investigations of the Diazeniumdiolate Antifungal Agent Fragin

“…This compound was shown to possess antifungal, antitumour, antibacterial, and plant growth inhibitory activities . More recently, the previously unknown signaling compound rac ‐valdiazen ( 2 ) was isolated from Burkholderia cenocepacia H111 (H111), and in the same year Hertweck and co‐workers discovered a new family of diazeniumdiolate siderophores …”

“…The (−)‐fragin gene cluster consists of seven genes separated into two oppositely orientated operons (Scheme ). The predicted functions of the corresponding proteins include those of a haem oxygenase for HamA, of a cupin domain superfamily protein for HamB, of a p ‐aminobenzoate N ‐oxygenase for HamC, of a non‐ribosomal‐peptide‐synthetase (NRPS)‐like protein for HamD, of a polyketide cyclase/dehydratase for HamE, of a starter condensation domain for HamF, and of an aminotransferase for HamG …”

“…Furthermore, our group investigated the p ‐aminobenzoate N ‐oxygenase enzyme HamC, a homologue of AurF . HamC was produced, isolated and shown, similarly to AurF, to oxidize p ‐aminobenzoic acid to p ‐nitrobenzoic acid . Furthermore, preliminary experiments explored the origin of the terminal N atom of the diazeniumdiolate group and chromatographic evidence suggested that it originated from nitrite.…”

“…They included the amine 6 , the hydroxylamine 7 , the nitroso compound 8 , the oxime 9 , the nitro compound 10 , the hydroxy compound 11 and the keto derivative 12 (Scheme ). The amine and hydroxylamine derivatives 6 and 7 were synthesized by a recently reported procedure …”

“…The supernatants of the wild type and the previously reported mutant strains Δ hamA , Δ hamB , Δ hamC , Δ hamD , Δ hamE , Δ hamF , and Δ hamG were extracted with CHCl 3 and analyzed by HPLC‐MS with use of the synthesized derivatives as analytical standards. Interestingly, the oxime 9 was present in small amounts in the extract of the wild‐type strain (see the Supporting Information), but this could also be formed by decomposition of fragin .…”