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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.

The global rise of antibiotic resistance calls for new drugs against bacterial pathogens. A common approach is to search for natural compounds deployed by microbes to inhibit competitors. Here, we show that the iron-chelating pyoverdines, siderophores produced by environmental Pseudomonas spp., have strong antibacterial properties by inducing iron starvation and growth arrest in pathogens. A screen of 320 natural Pseudomonas isolates used against 12 human pathogens uncovered several pyoverdines with particularly high antibacterial properties and distinct chemical characteristics. The most potent pyoverdine effectively reduced growth of the pathogens Acinetobacter baumannii , Klebsiella pneumoniae, and Staphylococcus aureus in a concentration- and iron-dependent manner. Pyoverdine increased survival of infected Galleria mellonella host larvae and showed low toxicity for the host, mammalian cell lines, and erythrocytes. Furthermore, experimental evolution of pathogens combined with whole-genome sequencing revealed limited resistance evolution compared to an antibiotic. Thus, pyoverdines from environmental strains have the potential to become a new class of sustainable antibacterials against specific human pathogens. Despite the wide range of available antibiotics, a majority work through a similar mechanism, which enables some bacteria to become resistant to medical treatment. This means that the effectiveness of these molecules goes down and many curable infections may no longer be treatable. Collectively, antibiotic resistance poses a similar threat as other major diseases, such as malaria. One promising approach for discovering new antibiotics is to explore natural microbial communities for their ability to produce secondary metabolites with antimicrobial properties. Such metabolites are typically secreted by bacteria to compete with other community members for essential resources including nutrients. A well-known group of secreted metabolites are siderophores, which tightly sequester iron, a critical nutrient for bacterial growth. Each bacterial species produces its own set of specific siderophores, thus leading to a severe competition for iron. Vollenweider et al. investigated whether a group of siderophores secreted by Pseudomonas bacteria, called pyoverdines, are an effective antimicrobial agent against harmful human pathogens. Pyoverdines have a high affinity to iron and prevent competing bacteria from accessing the critical nutrient. This can inhibit their growth by starving them of iron. Vollenweider et al. treated resistant pathogens like Acinetobacter , Klebsiella and Staphylococcus grown in the laboratory, and found that pyoverdines significantly reduce their growth without the bacteria acquiring resistance. To test whether this treatment would work in a living infected animal, the group administered pyoverdines to moth larvae infected with the same pathogens and observed increased survival rates in the host. As iron is also required for human metabolism and found in the haemoglobin of red blood cells, Vollenweider, et al. confirmed pyoverdines do not retrieve iron from haemoglobin. Finally, in laboratory settings, pyoverdines did not negatively affect the growth of a human and a mouse cell line at low concentrations strong enough to inhibit the growth of pathogens. This approach is a promising example of adopting natural mechanisms that can have antimicrobial properties, and siderophores, in particular pyoverdines, may become a useful tool to treat otherwise incurable infections. Further research is needed in living mammalian models to confirm efficacy and safety of this novel antimicrobial treatment.

Microbial secondary metabolites are a rich source for pharmaceutical discoveries and play crucial ecological functions. While tools exist to identify secondary metabolite clusters in genomes, precise sequence-to-function mapping remains challenging because neither function nor substrate specificity of biosynthesis enzymes can accurately be predicted. Here, we developed a knowledge-guided bioinformatic pipeline to solve these issues. We analyzed 1928 genomes of Pseudomonas bacteria and focused on iron-scavenging pyoverdines as model metabolites. Our pipeline predicted 188 chemically different pyoverdines with nearly 100% structural accuracy and the presence of 94 distinct receptor groups required for the uptake of iron-loaded pyoverdines. Our pipeline unveils an enormous yet overlooked diversity of siderophores (151 new structures) and receptors (91 new groups). Our approach, combining feature sequence with phylogenetic approaches, is extendable to other metabolites and microbial genera, and thus emerges as powerful tool to reconstruct bacterial secondary metabolism pathways based on sequence data.

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.

The global rise of antibiotic resistance calls for new drugs against bacterial pathogens. A common approach is to search for natural compounds deployed by microbes to inhibit competitors. Here, we show that the iron-chelating pyoverdines, siderophores produced by environmental Pseudomonas spp., have strong antibacterial properties by inducing iron starvation and growth arrest in pathogens. A screen of 320 natural Pseudomonas isolates used against 12 human pathogens uncovered several pyoverdines with particularly high antibacterial properties and distinct chemical characteristics. The most potent pyoverdine effectively reduced growth of the pathogens Acinetobacter baumannii , Klebsiella pneumoniae, and Staphylococcus aureus in a concentration- and iron-dependent manner. Pyoverdine increased survival of infected Galleria mellonella host larvae and showed low toxicity for the host, mammalian cell lines, and erythrocytes. Furthermore, experimental evolution of pathogens combined with whole-genome sequencing revealed limited resistance evolution compared to an antibiotic. Thus, pyoverdines from environmental strains have the potential to become a new class of sustainable antibacterials against specific human pathogens. Despite the wide range of available antibiotics, a majority work through a similar mechanism, which enables some bacteria to become resistant to medical treatment. This means that the effectiveness of these molecules goes down and many curable infections may no longer be treatable. Collectively, antibiotic resistance poses a similar threat as other major diseases, such as malaria. One promising approach for discovering new antibiotics is to explore natural microbial communities for their ability to produce secondary metabolites with antimicrobial properties. Such metabolites are typically secreted by bacteria to compete with other community members for essential resources including nutrients. A well-known group of secreted metabolites are siderophores, which tightly sequester iron, a critical nutrient for bacterial growth. Each bacterial species produces its own set of specific siderophores, thus leading to a severe competition for iron. Vollenweider et al. investigated whether a group of siderophores secreted by Pseudomonas bacteria, called pyoverdines, are an effective antimicrobial agent against harmful human pathogens. Pyoverdines have a high affinity to iron and prevent competing bacteria from accessing the critical nutrient. This can inhibit their growth by starving them of iron. Vollenweider et al. treated resistant pathogens like Acinetobacter , Klebsiella and Staphylococcus grown in the laboratory, and found that pyoverdines significantly reduce their growth without the bacteria acquiring resistance. To test whether this treatment would work in a living infected animal, the group administered pyoverdines to moth larvae infected with the same pathogens and observed increased survival rates in the host. As iron is also required for human metabolism and found in the haemoglobin of red blood cells, Vollenweider, et al. confirmed pyoverdines do not retrieve iron from haemoglobin. Finally, in laboratory settings, pyoverdines did not negatively affect the growth of a human and a mouse cell line at low concentrations strong enough to inhibit the growth of pathogens. This approach is a promising example of adopting natural mechanisms that can have antimicrobial properties, and siderophores, in particular pyoverdines, may become a useful tool to treat otherwise incurable infections. Further research is needed in living mammalian models to confirm efficacy and safety of this novel antimicrobial treatment.

Microbial secondary metabolites are a rich source for pharmaceutical discoveries and play crucial ecological functions. While tools exist to identify secondary metabolite clusters in genomes, precise sequence-to-function mapping remains challenging because neither function nor substrate specificity of biosynthesis enzymes can accurately be predicted. Here, we developed a knowledge-guided bioinformatic pipeline to solve these issues. We analyzed 1928 genomes of Pseudomonas bacteria and focused on iron-scavenging pyoverdines as model metabolites. Our pipeline predicted 188 chemically different pyoverdines with nearly 100% structural accuracy and the presence of 94 distinct receptor groups required for the uptake of iron-loaded pyoverdines. Our pipeline unveils an enormous yet overlooked diversity of siderophores (151 new structures) and receptors (91 new groups). Our approach, combining feature sequence with phylogenetic approaches, is extendable to other metabolites and microbial genera, and thus emerges as powerful tool to reconstruct bacterial secondary metabolism pathways based on sequence data.

The global rise of antibiotic resistance calls for new drugs against bacterial pathogens. A common approach is to search for natural compounds deployed by microbes to inhibit competitors. Here we show that the iron chelating pyoverdines, siderophores produced by environmental Pseudomonas spp., have strong antibacterial properties by inducing iron starvation and growth arrest in pathogens. A screen of 320 natural Pseudomonas isolates used against 12 human pathogens uncovered several pyoverdines with particularly high antibacterial properties and distinct chemical characteristics. The most potent pyoverdine effectively reduced growth of the pathogens Acinetobacter baumannii, Klebsiella pneumoniae and Staphylococcus aureus in a concentration- and iron-dependent manner. Pyoverdine increased survival of infected Galleria mellonella host larvae, and showed low toxicity for the host, mammalian cell lines, and erythrocytes. Furthermore, experimental evolution combined with whole-genome sequencing revealed reduced potentials for resistance evolution compared to an antibiotic. Thus, pyoverdines from environmental strains have the potential to become a new class of sustainable antibacterials against specific human pathogens.

Microbial secondary metabolites are a rich source for pharmaceutical discoveries and play crucial ecological functions. While tools exist to identify secondary metabolite clusters in genomes, precise sequence-to-function mapping remains challenging because neither function nor substrate specificity of synthesis enzymes can accurately be predicted. Here we developed a knowledge-guided bioinformatic pipeline to solve these issues. We analyzed 1928 genomes of Pseudomonas bacteria and focused on iron-scavenging pyoverdines as model metabolites. Our pipeline predicted 188 chemically different pyoverdines with nearly 100% structural accuracy and the presence of 94 distinct receptor groups required for the uptake of iron-loaded pyoverdines. Our pipeline unveils an enormous yet overlooked diversity of siderophores (151 new structures) and receptors (91 new groups). Our approach, combining feature sequence with phylogenetic approaches, is extendable to other metabolites and microbial genera, and thus emerges as powerful tool to reconstruct bacterial secondary metabolism pathways based on sequence data.Competing Interest StatementThe authors have declared no competing interest.Footnotes* author affiliations updated; Supplemental files updated；