Frankobactin Metallophores Produced by Nitrogen-Fixing Frankia Actinobacteria Function in Toxic Metal Sequestration.

A series of new metallophores, referred to as frankobactins, were extracted from cultures of the symbiotic and nitrogen-fixing actinobacterium Frankia sp. CH37. Structure elucidation revealed a 2-hydroxyphenyl-substituted oxazoline core and a chain composed of five proteinogenic and nonproteinogenic amino acids, suggesting nonribosomal peptide synthesis as the biosynthetic origin. By whole-genome sequencing, bioinformatic analysis, and comparison with other Frankia strains, the genetic locus responsible for the biosynthesis was detected. Spectrophotometric titration of frankobactin with Fe(III) and Cu(II) and mass spectrometry established the 1:1 (metal:frankobactin) coordination. Uptake experiments suggested that frankobactin A1 (1) did not serve to recruit iron, but to detoxify Cu(II). As frankobactin A1 prevents the cellular entry of Cu(II), it could play a crucial role in the symbiosis of Frankia sp. and its host in the reclamation of copper-contaminated soil.

Macroalgal-bacterial interactions: identification and role of thallusin in morphogenesis of the seaweed Ulva (Chlorophyta).

Macroalgal microbiomes have core functions related to biofilm formation, growth, and morphogenesis of seaweeds. In particular, the growth and development of the sea lettuce Ulva spp. (Chlorophyta) depend on bacteria releasing morphogenetic compounds. Under axenic conditions, the macroalga Ulva mutabilis develops a callus-like phenotype with cell wall protrusions. However, co-culturing with Roseovarius sp. (MS2) and Maribacter sp. (MS6), which produce various stimulatory chemical mediators, completely recovers morphogenesis. This ecological reconstruction forms a tripartite community which can be further studied for its role in cross-kingdom interactions. Hence, our study sought to identify algal growth- and morphogenesis-promoting factors (AGMPFs) capable of phenocopying the activity of Maribacter spp. We performed bioassay-guided solid-phase extraction in water samples collected from U. mutabilis aquaculture systems. We uncovered novel ecophysiological functions of thallusin, a sesquiterpenoid morphogen, identified for the first time in algal aquaculture. Thallusin, released by Maribacter sp., induced rhizoid and cell wall formation at a concentration of 11 pmol l-1. We demonstrated that gametes acquired the iron complex of thallusin, thereby linking morphogenetic processes with intracellular iron homeostasis. Understanding macroalgae-bacteria interactions permits further elucidation of the evolution of multicellularity and cellular differentiation, and development of new applications in microbiome-mediated aquaculture systems.

Gramibactin is a bacterial siderophore with a diazeniumdiolate ligand system.

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 logß 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.

Metallophore mapping in complex matrices by metal isotope coded profiling of organic ligands.

Metal isotope coded profiling (MICP) introduces a universal discovery platform for metal chelating natural products that act as metallophores, ion buffers or sequestering agents. The detection of cation and oxoanion complexing ligands is facilitated by the identification of unique isotopic signatures created by the application of isotopically pure metals.

Hydroxypyridinones in nitrogen-fixing bacterial cultures: a metal buffer for molybdenum and simulation of natural conditions.

Organic matter regulates the availability of important trace elements in aquatic and terrestrial ecosystems by acting as a source and container for microbes. To overcome the limitation of trace elements, nitrogen-fixing bacteria, e.g. release low-molecular-weight chelators (metallophores), which scavenge the essential cofactors of the nitrogenase, iron, and molybdenum (Mo), via complexation and subsequent uptake. The formation of metallophores is triggered by limiting conditions, which must be replicated in the laboratory in order to study metallophores as a mediator in metal cycling. While ethylenediaminetetraacetic acid (EDTA)-based buffer systems for metal cations are well established, there is limited knowledge regarding the buffering of oxoanions such as molybdate in a bacterial growth medium. To mimic the availability of molybdenum in nature under laboratory conditions, this study created a Mo-buffer system for bacterial growth media of the model organisms Azotobacter vinelandii and Frankia sp. CH37. We investigated selected hydroxypyridinones (HPs) as potential molybdenum-chelating agents, determining the amount required for efficient molybdenum complexation by calculating speciation plots of the various candidate complexes in artificial growth media at various pH values. The Mo-maltol system was identified as an ideal, nontoxic molybdenum-buffer system. In the presence of the Mo-maltol system, the growth of Frankia sp. was limited under diazotrophic conditions, whereas A. vinelandii could acquire molybdenum through the release of protochelin and subsequent molybdenum uptake. The study paves the way for unravelling molybdenum recruitment and homeostasis under limiting conditions in bacteria.

Genome Sequence of Frankia sp. Strain CH37, a Metallophore-Producing, Nitrogen-Fixing Actinobacterium Isolated from the Sea Buckthorn, Hippophae rhamnoides (Elaeagnaceae).

We report the genome sequence of Frankia sp. strain CH37, a filamentous nitrogen-fixing soil-dwelling Gram-positive bacterium and hyperproducer of metal-complexing organic ligands (metallophores) isolated from the sea buckthorn (Hippophae rhamnoides). The 9.7-Mbp sequence, obtained using PacBio technology, harbors 7,766 predicted coding sequences, including gene clusters for metallophore production.

Metallophore profiling of nitrogen-fixing Frankia spp. to understand metal management in the rhizosphere of actinorhizal plants.

Frankia spp. are widespread nitrogen-fixing soil bacteria, which often live in symbiosis with a broad range of hosts. Metal homeostasis plays a crucial role in the success of the symbiosis regarding the acquisition of essential trace metals and detoxification of potentially toxic elements. We have hypothesised that Frankia releases many organic ligands with a broad spectrum of affinity for essential and toxic metals. We coined the term 'ligandosphere' to describe the entirety of excreted metal complexing agents and ligands derived from the dissolved organic matter. Using metal isotope-coded profiling (MICP); metallophores of physiological important and toxic trace metals were identified by the addition of stable metal isotope pairs such as 54Fe/58Fe, 63Cu/65Cu, 66Zn/68Zn or 95Mo/98Mo. Liquid chromatography coupled to a mass spectrometer revealed strong variations of the metallophore profile in between the 14 test-strains. In total, about 83 organic ligands were identified as binding to one of the tested metals. The predicted sum formula of the major Fe binding ligands and MS/MS experiments suggested that several metallophore candidates have a similar molecular backbone. Growth experiments with a hyper-producer of metallophores revealed a positive relationship between metallophore production and the concentration of Cu in the growth medium. The present study provides the first comprehensive overview of the complexity of Frankia's ligandosphere. It opens a path to a deeper understanding of mechanisms that regulate metal homeostasis in frankiae. Deciphering these mechanisms is important since the fitness of actinorhizal plants and their potential in ecological restoration relies heavily on their symbiosis with frankiae.

Iron-organic matter complexes accelerate microbial iron cycling in an iron-rich fen.

The accessibility of iron (Fe) species for microbial processes is dependent on solubility and redox state, which are influenced by complexation with dissolved organic matter (DOM) and water-extractable organic matter (WEOM). We evaluated the complexation of these pools of organic matter to soluble Fe(II) and Fe(III) in the slightly acidic Schlöppnerbrunnen fen and subsequent effects on Fe(II) oxidation and Fe(III) reduction. We found the majority of soluble Fe(II) and Fe(III) is complexed to DOM. High-resolution mass spectrometry identified potential complexing partners in peat-derived water extracts (PWE), including compound classes known to function as ligands or electron shuttles, like tannins and sulfur-containing compounds. Furthermore, we observed clear differences in the stability of Fe(II)- and Fe(III)-DOM, with more labile complexes dominating the upper, oxic layers (0-10 cm) and more stable complexes in lower, anoxic layers (15-30 cm). Metal isotope-coded profiling identified a single potential chemical formula (C42H57O13N9Fe2) associated with a stable Fe-DOM complex. Fe(III) reduction and Fe(II) oxidation incubations with Geobacter sulfurreducens PCA and Shewanella oneidensis MR-1 or Sideroxydans CL-21, respectively, were used to determine the influence of Fe-DOM complexes on Fe cycling rates. The addition of PWE led to a 2.3-fold increase in Fe(III) reduction rates and 0.5-fold increase in Fe(II) oxidation rates, indicating Fe-DOM complexes greatly influence microbial Fe cycling by potentially serving as electron shuttles. Molecular analyses revealed Fe(III)-reducing and Fe(II)-oxidizing bacteria co-exist across all depths, in approximately equal proportions (representing 0.1-1.0% of the total microbial community), despite observed changes in redox potential. The activity of Fe(III)-reducing bacteria might explain the presence of the detected Fe(II) stabilized via complexation with DOM even under oxic conditions in upper peat layers. Therefore, these Fe(II)-DOM complexes can be recycled by microaerophilic Fe(II)-oxidizers. Taken together, these results suggest Fe-DOM complexation in the fen accelerates microbial-mediated redox processes across the entire redox continuum.