Pavel Hrouzek

Metabolomics enables the simultaneous monitoring of hundreds to thousands of metabolites in complex biological matrices; however, confident identification of low-abundance and unknown compounds remains challenging. Gas chromatography–mass spectrometry (GC–MS) workflows based on electron ionization (EI) often provide limited molecular-ion information because of extensive fragmentation. Here, we present a novel rapid screening workflow combining 1 min heptafluorobutyl chloroformate (HFBCF) derivatization of protic metabolites and concurrent liquid–liquid microextraction with gas chromatography–atmospheric pressure chemical ionization mass spectrometry (GC–APCI–MS). The proven HFBCF–mediated reaction produces stable heptafluorobutyl derivatives that yield abundant protonated molecular ions in the APCI mass spectra, predictable class-specific fragmentation, and characteristic fluorine-specific mass signatures. Compared with conventional full-scan GC–EI–MS, full-scan GC–APCI–MS provided up to 10–1000-fold higher sensitivity, enabling metabolite screening from extremely limited sample amounts. In low-input HeLa cell extracts, at least 100 metabolites were consistently detected from as few as 15,000 cells. Specific heptafluorobutyl-derived mass shifts and fluorine-based elemental constraints facilitated determination of the number and, in selected cases, the type of derivatized functional groups and supported elemental-composition assignment of unknown metabolites, even at mass accuracies of several tens of ppm. To aid compound annotation, we established an openly accessible database comprising more than 620 derivatizable metabolites and experimentally characterized retention and mass-spectral data for 240 reference standards. Together, these results establish HFBCF–GC–APCI–MS as a sensitive molecular-ion-centric workflow for exploratory metabolomics, enabling annotation of unknown metabolites across defined confidence levels, from standard-confirmed identifications to database-supported candidate assignments, suitable for limited biological samples.

Siderophore transport is central to microbial competition, because it determines access to iron, frequently a limiting nutrient. While siderophore-mediated iron uptake via TonB-dependent transporters (TBDTs) has been extensively studied in heterotrophic bacteria, little is known about the functionality and specificity of TBDTs in cyanobacteria. In the present study we functionally characterise the import system of cyanochelin B, a photolytic β -hydroxy aspartate siderophore produced by several filamentous cyanobacteria, including Leptolyngbya sp. NIES-3755. We have identified a cyanochelin B putative transport cassete localized in the vicinity of the cyanochelin biosynthetic gene cluster in Leptolyngbya genome. By expressing the import genes heterologously in a model unicellular cyanobacterium Synechocystis sp. PCC 6803, we established that the transport cassette reconstitutes cyanochelin B-dependent growth, consistent with cyanochelin-mediated iron acquisition. Systematic gene dissection showed that the TBDT (CctA) and the substrate-binding protein (CctB), responsible for binding the siderophore in the periplasm, are alone sufficient for cyanochelin import into Synechocystis cells, with the permeases, ATPase and a cassette-associated ferredoxin supplied in trans by the host. CctA carries an N-terminal AMIN domain, a fusion found only in cyanobacterial TBDTs. The cassette accepts the structurally similar cyanochelin A but not cyanochelin C, enterobactin or pyoverdine, indicating limited promiscuity. The phylogenetic placement of cyanochelin receptors within a broader clade containing citrate-hydroxamate-type siderophore receptors suggests an evolutionary link between transport systems for chemically distinct cyanobacterial siderophores. Our study reports the first functional heterologous expression of a cyanobacterial TonB-dependent transporter and establishes Synechocystis as a promising platform for cyanobacterial xenosiderophore-uptake studies.

Cyanobacteria are key prokaryotic primary producers in diverse ecosystems, yet the role of cyanobacterial siderophores in shaping their associated microbiomes remains unexplored. Our study demonstrates the benefits provided to the heterotrophic co-habitants of filamentous cyanobacteria in terrestrial microbial biofilms, focusing on the recently discovered widespread siderophores cyanochelins. To address the acceptance of cyanochelin B (CychB) across multiple bacterial classes, we first investigated its role in providing iron to a model siderophore producer P. aeruginosa PAO1 and selected Pseudomonas natural isolates, which were found to utilize CychB under iron limiting conditions while downregulating endogenous siderophore production. In response to CychB, PAO1 expresses a siderophore internalization cluster, which is localized in multiple Pseudomonas natural isolates. Using metagenome analysis, we characterized the bacterial community recruited along with CychB producing Phormidesmis cyanobacteria under long-term iron starvation. Potential CychB acceptor bacteria associated with the CychB producer were predominantly lacking endogenous siderophore machineries. Using siderophore selective pressure, we isolated a genuine CychB acceptor, gram-negative bacterium Methyloversatilis sp. S146 and demonstrated that its genome hosts an iron processing cluster overexpressed after CychB feeding, recognizing Methyloversatilis as a candidate for further mechanistic investigation of iron acquisition-driven microbial interactions. Our results indicate that CychB supports a specific subset of co-habiting heterotrophic bacteria during iron starvation, further emphasizing the role of cyanobacteria as key drivers of nutrient flows within globally important microbial soil crust ecosystems, supporting microbial life in nutrient-limited environments. These findings provide a mechanistic foundation to elucidate the role of cyanochelins as a public good in these communities.

Iron is a key micronutrient that constrains microbial growth and productivity in many aquatic and terrestrial environments due to its limited bioavailability. Microorganisms evolved sophisticated acquisition strategies, including the production of siderophores, high-affinity iron-chelating molecules that facilitate iron solubilisation and uptake. Cyanobacteria, photosynthetic prokaryotes and major contributors to global primary production, also depend on iron as a cofactor to their core metabolic enzymes. However, very few cyanobacterial siderophores were described so far, and cyanobacteria remain an underxplored source of possibly novel siderophores. Here we report a novel cyanobacterial siderophore, cyanochelin C, that employs two β-hydroxyaspartate residues for iron chelation. We provide extensive nuclear magnetic resonance (NMR) and mass spectrometry (MS) evidence on the molecular structure and identify the corresponding biosynthetic gene cluster (BGC). Bioinformatic analysis of the BGC further revealed the presence of an acylase CcsQ clustering with a broader cyanobacteria-specific family of acylases associated with predicted siderophore-encoding BGCs. Discovery of cyanochelin C and its deacylation by CcsQ expands the known structural diversity of cyanobacterial siderophores and improves the understanding of important enzymatic reactions.

Synura sphagnicola, a peat bog chrysophyte, forms conspicuous red cytoplasmic droplets. These droplets previously showed an unidentified pigment, prompting a detailed chemical investigation. Large-scale cultivation and chromatographic purification yielded three structurally related orange-to-red compounds, previously undescribed chlorinated naphthoquinone pigments─synurin A (1), synurin B (2), and synurin C (3)─whose identities were established using HPLC-HRMS, FT-ICR MS, NMR spectroscopy, FTIR, fluorescence spectroscopy, and Raman microscopy. The compounds were identified as 2,3-dichloro-5,8-dihydroxy-6-propylnaphthalene-1,4-dione (1) and its two O-methylated regioisomers (2, 3). Raman spectra revealed strong environment-dependent vibrational differences, particularly for 1 and 2. In vitro assays demonstrated potent but nonselective cytotoxicity toward cancerous and nontransformed cell lines (IC₅₀ in the low micromolar range), while only 1 exhibited ferric-reducing antioxidant activity.

Бұл жұмыста Балқаш көлінің тұзды бөлігінің альгофлорасының түрлік құрамы анықталып, келешегі мол микробалдырлар мен цианобактериялардың жаңа дақылдары бөлініп алынып, олардың морфологиялық, дақылдық және физиологиялық қасиеттері зерттелді. Талдау нәтижесінде Балқаштың шығыс жағалауынан алынған су сынамаларының альгофлора құрамында диатомды балдырлар (Bacillariophyta) 40,33%, жасыл балдырлар (Chlorophyta) 32%, көк-жасыл балдырлар (Cyanophyta) 19,33% және эвгленалық балдырлар (Euglenophyta) 8,33% құрады. Олар 4 бөлімге, 6 классқа, 6 қатарға, 11 тұқымдасқа және 22 туысқа жататындығы анықталды. Микробиологиялық әдістері негізінде микробалдырлар мен цианобактериялардың 8 аксеникалық таза дақылдары бөлініп алынып, олар Oscillatoria sp, Nostoc sp, Arthrospira sp, Anabaena sp, Chlorella vulgaris, Parachlorella kessleri, Dunaliella salina, Navicula sp. болып анықталды. Әр дақыл үшін өсірудің оңтайлы жағдайлары анықталды. Бөлініп алынған цианобактерия дақылдарының өсу қарқындылығы BG-11, Заррука қоректік орталарында рН 7 - 9 кезінде 28-32°С оптималды температура аралығында байқалды. Ал, бөлініп алынған микробалдыр дақылдары, Тамия, Артария және BG-11 қоректік орталарында рН 6,5–7,5 кезінде 27°C -30°C температура аралығында тиімді өсу көрсеткішін көрсетті. Бұл таза дақылдар өндірістік биотехнологияда, атап айтқанда биологиялық белсенді қоспалар әзірлеуде ерекше қызығушылық тудыратын объект болып табылады. Кілт сөздер: альгофлора, аксеникалық таза дақылдарды бөлу, микробалдырлар мен цианобактерия дақылдары, тұзды көл.

Siderophores are low-molecular-weight compounds excreted by microorganisms to acquire iron and possibly to monopolize iron resource to achieve competitive advantage over other strains, or to trade for other substrates in mutualistic relationships. Siderophores that employ β-hydroxy-aspartate (β-OH-Asp) for iron chelation can undergo UV-mediated photolytic cleavage, simultaneously reducing Fe ³⁺ to Fe ²⁺ . Photolytic siderophores can promote algal-bacterial mutualism, where the bacteria provide iron in exchange for dissolved organic carbon. We present a comprehensive characterization of cyanochelin B, a photolytic β-OH-Asp-containing siderophore produced by the filamentous cyanobacterium Leptolyngbya sp. NIES-3755. Combining nuclear magnetic resonance, high-resolution mass spectrometry, bioinformatic analyses, and Marfey’s and Murata’s method, we elucidated the structure of cyanochelin B, including the configuration of its stereocenters. Cyanochelin B-iron complexes rapidly photolyse under UV light (t _1/2 = 2.3 min; 19.6 µmol m ⁻² s ⁻¹ UV-A) and release Fe ²⁺ . Using a coculture setup with Leptolyngbya and Synechocystis sp. PCC 6803 (a non-siderophore producer) in membrane-separated compartments and alginate-embedded FeCl ₃ to simulate poorly accessible precipitated iron, we demonstrate cyanochelin B mode of actions. Our results show that in the absence of UV light, cyanochelin B efficiently monopolizes iron, favoring Leptolyngbya . However, UV light eliminates this monopolization, making iron available to any cohabiting, also possibly competing, organisms. We further report isolating novel cyanochelin B-producing Phormidesmis strains from field material and discuss the broader implications of photolytic siderophores. In conclusion, our interdisciplinary approach led to the discovery of a novel photolytic siderophore, cyanochelin B, and highlighted its possible role in distributing iron in microbial communities. IMPORTANCE: Iron is an essential micronutrient that is required by all living organisms as a cofactor of indispensable enzymes. Due to its specific properties, it is mostly precipitated and is biologically unavailable. Microbes produce siderophores, low-molecular-weight compounds that bind iron, to facilitate iron uptake. Siderophores are mediators of microbial interactions and facilitate competitive exclusion of non-compatible strains or support mutualistic partners and cheater strains. Here, we adopt an interdisciplinary strategy and report a complete structural elucidation of cyanochelin B, a photolytic cyanobacterial siderophore that contains β-hydroxy-aspartate (β-OH-Asp). Our coculture experiments show that cyanochelin B can either monopolize iron to its producer or make it accessible to other strains, depending on the presence of UV light. Moreover, our data suggest that the benefits from production of photolytic siderophores are not restricted to the producer or cohabiting bacteria but are rather available to the entire irradiated community. Out of the known siderophores, 17.5% contain the photoreactive β-OH-Asp and therefore may play a similar role.

Chromera velia is photosynthetic alga that is evolutionary closest to the group of het- erotrophic apicomplexan parasites. The main light harvesting carotenoid of C. velia has been described as isofucoxanthin-like pigment, however its structure remained unknown. Here, we purified this pigment from a 5 L culture of C. velia using a two-step HPLC procedure and its structure determined using combination of tandem mass spectrometry and 2D- and 3D-NMR techniques. The data identified this pigment as a new allenic xanthophyll which we denoted chromeraxanthin. Although previously predicted to be structurally similar to isofucoxanthin, chromeraxanthin possess different arrangement of the terminal cycles and thus it likely not derived directly from fucoxanthin biosynthe- sis pathway. The six membered ring connected to the carotenoid backbone via allenic bond is identical to that of fucoxanthin including its O-acetylation. However, the second six-membered ring bearing epoxy group in fucoxanthin is replaced by a capsanthin-like cyclopentane ring in chromeraxanthin. Notably, similar structure lacking only the O- acetylation has been previously described as allenicmytiloxanthin isolated from Pacific oyster.

Siderophores are low-molecular-weight compounds excreted by microorganisms to facilitate iron uptake in times of its unavailability. Microbes may produce siderophores to monopolize iron and achieve competitive exclusion of other strains. Alternatively, siderophores may be exchanged for other substrates in mutualistic relationships. Siderophores that employ β -hydroxy-aspartate ( β -OH-Asp) for iron chelation were shown to undergo UV-mediated photolytic cleavage with simultaneous reduction of Fe ³⁺ to Fe ²⁺ . Photolytic siderophores can mediate algal-bacterial mutualism, where the bacteria provide iron in exchange for dissolved organic carbon. We use an interdisciplinary strategy to provide a complex characterization of cyanochelin B, a photolytic β-OH-Asp-containing siderophore produced by filamentous cyanobacterium Leptolyngbya sp. NIES-3755. A combination of nuclear magnetic resonance, high resolution mass spectrometry and bioinformatic analyses complemented with Marfey’s and Murata’s methods yielded the structure of cyanochelin B with the configuration of its stereocenters. Cyanochelin B-iron complexes exposed to UV- light photolyze within minutes (t _1/2 = 8.9 min; ∼3.5 uE UV-A) and release reduced Fe ²⁺ . We have co-cultured Leptolyngbya together with Synechocystis PCC6803 as a reporter strain lacking siderophore production. The cultivation setup was based on membrane-separated compartments accommodating individual strains and employed alginate-embedded FeCl ₃ to simulate poorly accessible precipitated iron. Our results demonstrate that in the absence of UV-light cyanochelin B can monopolize iron in favor of Leptolyngbya. However, UV-light eliminates any monopolization of iron and makes it available to competing organisms. Finally, we report the isolation of novel cyanochelin B-producing strains of Phormidesmis from field material and discuss the phenomenon of photolytic siderophores in a broader context. Importance: Iron is an essential micronutrient that is required by all living organisms as a cofactor of indispensable enzymes. Due to its specific properties it is mostly precipitated and biologically unavailable. To facilitate iron uptake, microbes produce siderophores - low-molecular-weight compounds that bind iron. Siderophores are mediators of microbial interactions and facilitate competitive exclusion of non-compatible strains or foster mutualistic partners and cheater strains. We report a full structural elucidation of cyanochelin B, a photolytic cyanobacterial siderophore that contains β-OH-Asp. Our co-culture experiments show that cyanochelin B may monopolize iron to its producer or make it accessible to other strains depending on the presence of UV light. Moreover, our data suggest that the benefits from production of photolytic siderophores are not restricted to symbiotic partners of the producer but rather available to the whole irradiated community. Of known siderophores, 17.5% contain the photoreactive β-OH-Asp and can function in a similar way.

Nostatin A is highly modified ribosomally synthesized and post-translationally modified peptide belonging among proteusins. It inhibits multiple cancer cell lines in nanomolar concentrations and acts via cell cycle arrest and subsequent apoptosis.