Genome mining and chemical analyses revealed that rhizosphere bacteria (Paraburkholderia graminis) produce a new type of siderophore, gramibactin, a lipodepsipeptide that efficiently binds iron with a logβ value of 27.6. Complexation-induced proton NMR chemical shifts show that the unusual N-nitrosohydroxylamine (diazeniumdiolate) moieties participate in metal binding. Gramibactin biosynthesis genes are conserved in numerous plant-associated bacteria associated with rice, wheat, and maize, which may utilize iron from the complex.

Siderophores are key players in bacteria–host interactions, with the main function to provide soluble iron for their producers. Gramibactin from rhizosphere bacteria expands siderophore function and diversity as it delivers iron to the host plant and features an unusual diazeniumdiolate moiety for iron chelation. By mutational analysis of the grb gene cluster, we identified genes (grbD and grbE) necessary for diazeniumdiolate formation. Genome mining using a GrbD‐based network revealed a broad range of orthologous gene clusters in mainly plant‐associated Burkholderia/Paraburkholderia species. Two new types of diazeniumdiolate siderophores, megapolibactins and plantaribactin were fully characterized. In vitro assays and in vivo monitoring experiments revealed that the iron chelators also liberate nitric oxide (NO) in plant roots. This finding is important since NO donors are considered as biofertilizers that maintain iron homeostasis and increase overall plant fitness.

Micacocidin is a promising natural product for the treatment of Mycoplasma pneumoniae infections. In the biosynthesis of this antibiotic, a fatty acid-AMP ligase (FAAL) activates the starter unit hexanoic acid as acyl-adenylate and forwards it to an iteratively acting polyketide synthase. Biochemical analysis of the FAAL revealed an extended substrate tolerance, thereby opening the door for the modification of a micacocidin residue that is barely accessible via semisynthesis. A total of six new analogues were generated by precursor-directed biosynthesis in this study and profiled against M. pneumoniae.

Gramibactin (GBT) is an archetype for the new class of diazeniumdiolate siderophores, produced by Paraburkholderia graminis, a cereal‐associated rhizosphere bacterium, for which a detailed solution thermodynamic study exploring the iron coordination properties is reported. The acid‐base behavior of gramibactin as well as its complexing ability toward Fe3+ was studied over a wide range of pH values (2≤pH≤11). For the latter the ligand‐competition method employing EDTA was used. Only two species are formed: [Fe(GBT)]− (pH 2 to 9) and [Fe(GBT)(OH)2]3− (pH≥9). The formation of [Fe(GBT)]− and its occurrence in real systems was confirmed by LC‐HRESIMS analysis of the bacteria culture broth extract. The sequestering ability of gramibactin was also evaluated in terms of the parameters pFe and pL0.5. Gramibactin exhibits a higher sequestering ability toward Fe3+ than EDTA and of the same order of magnitude as hydroxamate‐type microbial siderophores, but smaller than most of the catecholate‐type siderophores and much higher than the most known phytosiderophores.

The bacterial pathogen Pseudomonas tolaasii severely damages white button mushrooms by secretion of the pore-forming toxin tolaasin, the main virulence factor of brown blotch disease. Yet, fungus-associated helper bacteria of the genus Mycetocola (Mycetocola tolaasinivorans and Mycetocola lacteus) may protect their host by an unknown detoxification mechanism. By a combination of metabolic profiling, imaging mass spectrometry, structure elucidation, and bioassays, we found that the helper bacteria inactivate tolaasin by linearizing the lipocyclopeptide. Furthermore, we found that Mycetocola spp. impair the dissemination of the pathogen by cleavage of the lactone ring of pseudodesmin. The role of pseudodesmin as a major swarming factor was corroborated by identification and inactivation of the corresponding biosynthetic gene cluster. Activity-guided fractionation of the Mycetocola proteome, matrix-assisted laser desorption/ionization (MALDI) analyses, and heterologous enzyme production identified the lactonase responsible for toxin cleavage. We revealed an antivirulence strategy in the context of a tripartite interaction that has high ecological and agricultural relevance.