Microbial secondary metabolites represent a rich source for drug discovery, plant protective agents, and biotechnologically relevant compounds. Among them are siderophores, iron-chelating molecules, that show a great influence on bacterial community assembly and the potential to control pathogen invasions. One of such a siderophore is pyoverdine that is produced by fluorescent Pseudomonas members and consists of different peptide chains specific to each bacterial species. The identification and structural elucidation of such suites of siderophores remain widely underexplored as general high-throughput analytical protocols are missing. Therefore, a dedicated method was established allowing a rapid localization and structural elucidation of pyoverdines. Liquid bacterial culture samples were purified by an easy small-scale solid-phase extraction (SPE). Ultra-high-performance liquid chromatography high-resolution tandem mass spectrometry (UHPLC-HR-MS/MS) separated highly polar pyoverdines and their derivatives. All ion fragmentation (AIF) generated mass spectra containing the characteristic fragments of the biological precursor of pyoverdine, ferribactin. This led to the revelation of the mass of secreted pyoverdines. Targeted MS/MS experiments at multiple collision energies accomplished the full structure elucidation of the pyoverdine peptide chain. A mass calculator and a fragmentation predictor facilitated greatly the interpretation of MS/MS spectra by providing accurate masses for a straightforward comparison of measured and theoretical values. The method was successfully validated using four well-known pyoverdines with various peptide chains. Finally, the applicability was proven by the analysis of 13 unknown pyoverdines secreted by sampled bacterial cultures. Among these, 4 novel pyoverdine peptide chains were discovered and are herein reported for the first time. Graphical abstract
Bacteria secrete siderophores whose function is to acquire iron. In recent years, the siderophores of several Chryseobacterium species were shown to promote the health and growth of various plants such as tomato or rice. However, the chemical nature of Chryseobacterium siderophores remained unexplored despite great interest. In this work, we present the purification and structure elucidation by NMR and MS/MS of chryseochelin A, a novel citrate-based siderophore secreted by three Chryseobacterium strains involved in plant protection. It contains the unusual building blocks 3-hydroxycadaverine and fumaric acid. Furthermore, the unstable structural isomer chryseochelin B and its stable derivative containing fatty acid chains, named chryseochelin C, were identified by mass spectrometric methods. The latter two incorporate an unusual ester connectivity to the citrate moiety showing similarities to achromobactin from the plant pathogen Dickeya dadantii. Finally, we show that chryseochelin A acts in a concentration-dependent manner against the plant-pathogenic Ralstonia solanacearum strain by reducing its access to iron. Thus, our study provides valuable knowledge about the siderophores of Chryseobacterium strains, which have great potential in various applications.
Siderophores are iron-chelating molecules produced by bacteria and other microbes. They are involved with virulence in infections and play key roles in bacterial community assembly and as plant protectants due to their pathogen control properties. Although assays exist to screen whether newly isolated bacteria can produce siderophores, the chemical structures of many of these bio-active molecules remain unidentified due to the lack of rapid analytical procedures. An important group of siderophores are pyoverdines. They consist of a structurally diverse group of chromopeptides, whose amino acid sequence is characteristic for the fluorescent Pseudomonas species that secrets them. Although over 60 pyoverdine structures have been described so far, their characterization is cumbersome and several methods (isoelectrofocusing, iron uptake measurement, mass determination) are typically combined as ambiguous results are often achieved by a single method. Those additional experiments consume valuable time and resources and prevent high-throughput analysis. In this work, we present a new pyoverdine characterisation option by recording their collision cross sections (CCS) using trapped ion mobility spectrometry. This can be done simultaneously in combination with UHPLC and high-resolution MS resulting in a rapid identification of pyoverdines. The high specificity of CCS values is presented for 17 pyoverdines secreted by different Pseudomonas strains. The pyoverdine mass determination by full scan MS was supported by fragments obtained from broadband collision induced dissociation (bbCID). As iron contaminations in laboratories are not uncommon, CCS values of ferripyoverdines were also evaluated. Thereby, unusual and highly characteristic ion mobility patterns were obtained that are suitable as an alternative identification marker.
Microorganisms produce iron chelators called siderophores that are a rich source for drug discovery or plant protective agents. Pyoverdines are a class of siderophores from fluorescent Pseudomonas members and consist of different peptide chains specific to each bacterial species. The structural elucidation and characterization of pyoverdines require comprehensive analytical methods as bacterial extracts are complex mixtures. Here, we present a high-throughput UHPLC-MS/MS pipeline and the application of ion mobility spectrometry to facilitate research in the field of medicine and agriculture.
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