UV-vis absorbance spectra, molar extinction coefficients and circular dichroism spectra for the two cyanobacterial metabolites anabaenopeptin A and anabaenopeptin B.

The UV-vis absorbance spectra, molar extinction coefficients and circular dichroism spectra, as well as NMR and high resolution tandem mass spectrometry spectra were determined for two prominent secondary metabolites from cyanobacteria, namely anabaenopeptin A and anabaenopeptin B. The compounds were extracted from the cyanobacterium Planktothrix rubescens CBT929 and purified by flash chromatography and HPLC. Exact amounts of isolated compounds were assessed by quantitative 1H-NMR with internal calibrant ethyl 4-(dimethylamino)benzoate in DMSO­d6 at 298 K with a recycle delay (d1) of 120 s. UV-vis absorbance spectra were recorded in methanol at room temperature. Molar extinction coefficients were determined at 278 nm as 4190 M-1 cm-1 and 2300 M-1 cm-1 in methanol for anabaenopeptin A and anabaenopeptin B, respectively. Circular dichroism spectra and secondary fragmentation mass spectra are also reported.

Tracking Extensive Portfolio of Cyanotoxins in Five-Year Lake Survey and Identifying Indicator Metabolites of Cyanobacterial Taxa.

Cyanobacterial blooms require monitoring, as they pose a threat to ecosystems and human health, especially by the release of toxins. Along with widely reported microcystins, cyanobacteria coproduce other bioactive metabolites; however, information about their dynamics in surface waters is sparse. We investigated dynamics across full bloom successions throughout a five-year lake monitoring campaign (Greifensee, Switzerland) spanning 150 sampling dates. We conducted extensive suspect screening of cyanobacterial metabolites using the database CyanoMetDB. Across all 850 samples, 35 metabolites regularly co-occurred. Microcystins were present in 70% of samples, with [d-Asp3,(E)-Dhb7]MC-RR reaching concentrations of 70 ng/L. Anabaenopeptins, meanwhile, were detected in 95% of all samples with concentrations of Oscillamide Y up to 100-fold higher than microcystins. Based on LC-MS response and frequency, we identified indicator metabolites exclusively produced by one of three cyanobacteria isolated from the lake, these being [d-Asp3,(E)-Dhb7]MC-RR from Planktothrix sp. G2020, Microginin 761B from Microcystis sp. G2011, and Ferintoic acid B from Microcystis sp. G2020. These indicators showed distinct temporal trends and peaking seasons that reflect the variance in either the abundance of the producing cyanobacteria or their toxin production dynamics. Our approach demonstrates that selecting high LC-MS response and frequent and species-specific indicator metabolites can be advantageous for cyanobacterial monitoring.

Reactivity of Cyanobacteria Metabolites with Ozone: Multicompound Competition Kinetics.

Cyanobacterial blooms occur at increasing frequency and intensity, notably in freshwater. This leads to the introduction of complex mixtures of their products, i.e., cyano-metabolites, to drinking water treatment plants. To assess the fate of cyano-metabolite mixtures during ozonation, a novel multicompound ozone (O3) competition kinetics method was developed. Sixteen competitors with known second-order rate constants for their reaction with O3 ranging between 1 and 108 M-1 s-1 were applied to cover a wide range of the O3 reactivity. The apparent second-order rate constants (kapp,O3) at pH 7 were simultaneously determined for 31 cyano-metabolites. kapp,O3 for olefin- and phenol-containing cyano-metabolites were consistent with their expected reactivity (0.4-1.7 × 106 M-1 s-1) while kapp,O3 for tryptophan- and thioether-containing cyano-metabolites were significantly higher than expected (3.4-7.3 × 107 M-1 s-1). Cyano-metabolites containing these moieties are predicted to be well abated during ozonation. For cyano-metabolites containing heterocycles, kapp,O3 varied from <102 to 5.0 × 103 M-1 s-1, giving first insights into the O3 reactivity of this class of compounds. Due to lower O3 reactivities, heterocycle- and aliphatic amine-containing cyano-metabolites may be only partially degraded by a direct O3 reaction near circumneutral pH. Hydroxyl radicals, which are formed during ozonation, may be more important for their abatement. This novel multicompound kinetic method allows a high-throughput screening of ozonation kinetics.

Lethal and behavioral effects of semi-purified microcystins, Micropeptin and apolar compounds from cyanobacteria on freshwater microcrustacean Thamnocephalus platyurus.

The mass proliferation of cyanobacteria, episodes known as blooms, is a concern worldwide. One of the most critical aspects during these blooms is the production of toxic secondary metabolites that are not limited to the four cyanotoxins recognized by the World Health Organization. These metabolites comprise a wide range of structurally diverse compounds that possess bioactive functions. Potential human and ecosystem health risks posed by these metabolites and co-produced mixtures remain largely unknown. We studied acute lethal and sublethal effects measured as impaired mobility on the freshwater microcrustaceans Thamnocephalus platyurus for metabolite mixtures from two cyanobacterial strains, a microcystin (MC) producer and a non-MC producer. Both cyanobacterial extracts, from the MC-producer and non-MC-producer, caused acute toxicity with LC50 (24 h) values of 0.50 and 2.55 mgdw_biomass/mL, respectively, and decreased locomotor activity. Evaluating the contribution of different cyanopeptides revealed that the Micropeptin-K139-dominated fraction from the MC-producer extract contributed significantly to mortality and locomotor impairment of the microcrustaceans, with potential mixture effect with other cyanopeptolins present in this fraction. In the non-MC-producer extract, compounds present in the apolar fraction contributed mainly to mortality, locomotor impairment, and morphological changes in the antennae of the microcrustacean. No lethal or sublethal effects were observed in the fractions dominated by other cyanopetides (Cyanopeptolin 959, Nostoginin BN741). Our findings contribute to the growing body of research indicating that cyanobacterial metabolites beyond traditional cyanotoxins cause detrimental effects. This underscores the importance of toxicological assessments of such compounds, also at sublethal levels.

Lethal and sublethal effects towards zebrafish larvae of microcystins and other cyanopeptides produced by cyanobacteria.

Cyanobacterial blooms affect aquatic ecosystems across the globe and one major concern relates to their toxins such as microcystins (MC). Yet, the ecotoxicological risks, particularly non-lethal effects, associated with other co-produced secondary metabolites remain mostly unknown. Here, we assessed survival, morphological alterations, swimming behaviour and cardiovascular functions of zebrafish (Danio rerio) upon exposure to cyanobacterial extracts of two Brazilian Microcystis strains. We verified that only MIRS-04 produced MCs and identified other co-produced cyanopeptides also for the MC non-producer NPCD-01 by LC-HRMS/MS analysis. Both cyanobacterial extracts, from the MC-producer and non-producer, caused acute toxicity in zebrafish with LC50 values of 0.49 and 0.98 mgdw_biomass/mL, respectively. After exposure to MC-producer extract, additional decreased locomotor activity was observed. The cyanopeptolin (micropeptin K139) contributed 52% of the overall mortality and caused oedemas of the pericardial region. Oedemas of the pericardial area and prevented hatching were also observed upon exposure to the fraction with high abundance of a microginin (Nostoginin BN741) in the extract of the MC non-producer. Our results further add to the yet sparse understanding of lethal and sublethal effects caused by cyanobacterial metabolites other than MCs and the need to better understand the underlying mechanisms of the toxicity. We emphasize the importance of considering mixture toxicity of co-produced metabolites in the ecotoxicological risk assessment of cyanobacterial bloom events, given the importance for predicting adverse outcomes in fish and other organisms.

The NORMAN Suspect List Exchange (NORMAN-SLE): facilitating European and worldwide collaboration on suspect screening in high resolution mass spectrometry.

Background: The NORMAN Association (https://www.norman-network.com/) initiated the NORMAN Suspect List Exchange (NORMAN-SLE; https://www.norman-network.com/nds/SLE/) in 2015, following the NORMAN collaborative trial on non-target screening of environmental water samples by mass spectrometry. Since then, this exchange of information on chemicals that are expected to occur in the environment, along with the accompanying expert knowledge and references, has become a valuable knowledge base for "suspect screening" lists. The NORMAN-SLE now serves as a FAIR (Findable, Accessible, Interoperable, Reusable) chemical information resource worldwide. Results: The NORMAN-SLE contains 99 separate suspect list collections (as of May 2022) from over 70 contributors around the world, totalling over 100,000 unique substances. The substance classes include per- and polyfluoroalkyl substances (PFAS), pharmaceuticals, pesticides, natural toxins, high production volume substances covered under the European REACH regulation (EC: 1272/2008), priority contaminants of emerging concern (CECs) and regulatory lists from NORMAN partners. Several lists focus on transformation products (TPs) and complex features detected in the environment with various levels of provenance and structural information. Each list is available for separate download. The merged, curated collection is also available as the NORMAN Substance Database (NORMAN SusDat). Both the NORMAN-SLE and NORMAN SusDat are integrated within the NORMAN Database System (NDS). The individual NORMAN-SLE lists receive digital object identifiers (DOIs) and traceable versioning via a Zenodo community (https://zenodo.org/communities/norman-sle), with a total of > 40,000 unique views, > 50,000 unique downloads and 40 citations (May 2022). NORMAN-SLE content is progressively integrated into large open chemical databases such as PubChem (https://pubchem.ncbi.nlm.nih.gov/) and the US EPA's CompTox Chemicals Dashboard (https://comptox.epa.gov/dashboard/), enabling further access to these lists, along with the additional functionality and calculated properties these resources offer. PubChem has also integrated significant annotation content from the NORMAN-SLE, including a classification browser (https://pubchem.ncbi.nlm.nih.gov/classification/#hid=101). Conclusions: The NORMAN-SLE offers a specialized service for hosting suspect screening lists of relevance for the environmental community in an open, FAIR manner that allows integration with other major chemical resources. These efforts foster the exchange of information between scientists and regulators, supporting the paradigm shift to the "one substance, one assessment" approach. New submissions are welcome via the contacts provided on the NORMAN-SLE website (https://www.norman-network.com/nds/SLE/). Supplementary Information: The online version contains supplementary material available at 10.1186/s12302-022-00680-6.

Quantification of Multi-class Cyanopeptides in Swiss Lakes with Automated Extraction, Enrichment and Analysis by Online-SPE HPLC-HRMS/MS.

The frequency and intensity of cyanobacterial blooms continue to increase in freshwater systems across the globe. Cyanobacteria can release toxins and several bioactive secondary metabolites and analytical methods are needed to effectively assess their concentrations in surface waters. Since booms can evolve rapidly in parts of a lake, high resolution of spatial and temporal sampling increases the complexity of monitoring efforts. Here, we present the validation of an automated, online-solid phase extraction (SPE) high performance liquid chromatography (HPLC)-high resolution tandem mass spectrometry (HRMS/MS) method. This online-SPE HPLC-HRMS/MS methods enables quantitative monitoring of surface waters for 17 cyanobacterial peptides (cyanopeptides), spanning 5 distinct cyanopeptide classes, including: microcystins, anabaenopeptins, nodularins, cyclamides and cyanopeptolins. The method can quantify these cyanopeptides in the low ng/L-range with high accuracy (85-116%) and low relative matrix effects (<25%). We demonstrated its application to Swiss lake waters (Zürichsee, Hallwilersee, Greifensee), which also highlighted the value of adding cyanopeptides beyond common microcystins when monitoring surface waters for cyanobacteria.

The Natural Products Atlas 2.0: a database of microbially-derived natural products.

Within the natural products field there is an increasing emphasis on the study of compounds from microbial sources. This has been fuelled by interest in the central role that microorganisms play in mediating both interspecies interactions and host-microbe relationships. To support the study of natural products chemistry produced by microorganisms we released the Natural Products Atlas, a database of known microbial natural products structures, in 2019. This paper reports the release of a new version of the database which includes a full RESTful application programming interface (API), a new website framework, and an expanded database that includes 8128 new compounds, bringing the total to 32 552. In addition to these structural and content changes we have added full taxonomic descriptions for all microbial taxa and have added chemical ontology terms from both NP Classifier and ClassyFire. We have also performed manual curation to review all entries with incomplete configurational assignments and have integrated data from external resources, including CyanoMetDB. Finally, we have improved the user experience by updating the Overview dashboard and creating a dashboard for taxonomic origin. The database can be accessed via the new interactive website at https://www.npatlas.org.

Cyanobacterial Toxins and Cyanopeptide Transformation Kinetics by Singlet Oxygen and pH-Dependence in Sunlit Surface Waters.

To assess the risks associated with cyanobacterial blooms, the persistence and fate processes of cyanotoxins and other bioactive cyanobacterial metabolites need to be evaluated. Here, we investigated the reaction with photochemically produced singlet oxygen (1O2) for 30 cyanopeptides synthesized by Dolichospermum flos aquae, including 9 anabaenopeptins, 18 microcystins, 2 cyanopeptolins, and 1 cyclamide. All compounds were stable in UVA light alone but in the presence of a photosensitizer we observed compound-specific degradation. A strong pH effect on the decay was observed for 18 cyanopeptides that all contained tyrosine or structurally related moieties. We can attribute this effect to the reaction with 1O2 and triplet sensitizer that preferentially react with the deprotonated form of tyrosine moieties. The contribution of 1O2 to indirect phototransformation ranged from 12 to 39% and second-order rate constants for 9 tyrosine-containing cyanopeptides were assessed. Including the pH dependence of the reaction and system-independent second-order rate constants with 1O2 will improve the estimation of half-lives for multiclass cyanopeptide in surface waters. Our data further indicates that naturally occurring triplet sensitizers are likely to oxidize deprotonated tyrosine moieties of cyanopeptides and the specific reactivity and its pH dependence needs to be investigated in future studies.

CyanoMetDB, a comprehensive public database of secondary metabolites from cyanobacteria.

Harmful cyanobacterial blooms, which frequently contain toxic secondary metabolites, are reported in aquatic environments around the world. More than two thousand cyanobacterial secondary metabolites have been reported from diverse sources over the past fifty years. A comprehensive, publically-accessible database detailing these secondary metabolites would facilitate research into their occurrence, functions and toxicological risks. To address this need we created CyanoMetDB, a highly curated, flat-file, openly-accessible database of cyanobacterial secondary metabolites collated from 850 peer-reviewed articles published between 1967 and 2020. CyanoMetDB contains 2010 cyanobacterial metabolites and 99 structurally related compounds. This has nearly doubled the number of entries with complete literature metadata and structural composition information compared to previously available open access databases. The dataset includes microcytsins, cyanopeptolins, other depsipeptides, anabaenopeptins, microginins, aeruginosins, cyclamides, cryptophycins, saxitoxins, spumigins, microviridins, and anatoxins among other metabolite classes. A comprehensive database dedicated to cyanobacterial secondary metabolites facilitates: (1) the detection and dereplication of known cyanobacterial toxins and secondary metabolites; (2) the identification of novel natural products from cyanobacteria; (3) research on biosynthesis of cyanobacterial secondary metabolites, including substructure searches; and (4) the investigation of their abundance, persistence, and toxicity in natural environments.

Inactivation and Site-specific Oxidation of Aquatic Extracellular Bacterial Leucine Aminopeptidase by Singlet Oxygen.

Extracellular enzymes are master recyclers of organic matter, and to predict their functional lifetime, we need to understand their environmental transformation processes. In surface waters, direct and indirect photochemical transformation is a known driver of inactivation. We investigated molecular changes that occur along with inactivation in aminopeptidase, an abundant class of extracellular enzymes. We studied the inactivation kinetics and localized oxidation caused by singlet oxygen, 1O2, a major photochemically derived oxidant toward amino acids. Aminopeptidase showed second-order inactivation rate constants with 1O2 comparable to those of free amino acids. We then visualized site-specific oxidation kinetics within the three-dimensional protein and demonstrated that fastest oxidation occurred around the active site and at other reactive amino acids. However, second-order oxidation rate constants did not correlate strictly with the 1O2-accessible surface areas of those amino acids. We inspected site-specific processes by a comprehensive suspect screening for 723,288 possible transformation products. We concluded that histidine involved in zinc coordination at the active site reacted slower than what was expected by its accessibility, and we differentiated between two competing reaction pathways of 1O2 with tryptophan residues. This systematic analysis can be directly applied to other proteins and transformation reactions.

Chlorothalonil transformation products in drinking water resources: Widespread and challenging to abate.

Chlorothalonil, a fungicide applied for decades worldwide, has recently been banned in the European Union (EU) and Switzerland due to its carcinogenicity and the presence of potentially toxic transformation products (TPs) in groundwater. The spread and concentration range of chlorothalonil TPs in different drinking water resources was examined (73 groundwater and four surface water samples mainly from Switzerland). The chlorothalonil sulfonic acid TPs (R471811, R419492, R417888) occurred more frequently and at higher concentrations (detected in 65-100% of the samples, ≤2200 ngL-1) than the phenolic TPs (SYN507900, SYN548580, R611968; detected in 10-30% of the samples, ≤130 ngL-1). The TP R471811 was found in all samples and even in 52% of the samples above 100 ngL-1, the drinking water standard in Switzerland and other European countries. Therefore, the abatement of chlorothalonil TPs was investigated in laboratory and pilot-scale experiments and along the treatment train of various water works, comprising aquifer recharge, UV disinfection, ozonation, advanced oxidation processes (AOPs), activated carbon treatment, and reverse osmosis. The phenolic TPs can be abated during ozonation (second order rate constant kO3 ∼104 M-1s-1) and by reaction with hydroxyl radicals (OH) in AOPs (kOH ∼109 M-1s-1). In contrast, the sulfonic acid TPs, which occurred in higher concentrations in drinking water resources, react only very slowly with ozone (kO3 <0.04 M-1s-1) and OH (kOH <5.0 × 107 M-1s-1) and therefore persist in ozonation and OH-based AOPs. Activated carbon retained the very polar TP R471811 only up to a specific throughput of 25 m3kg-1 (20% breakthrough), similarly to the X-ray contrast agent diatrizoic acid. Reverse osmosis was capable of removing all chlorothalonil TPs by ≥98%.

Cyanopeptide Co-Production Dynamics beyond Mirocystins and Effects of Growth Stages and Nutrient Availability.

Intensified cyanobacterial bloom events are of increasing global concern because of adverse effects associated with the release of bioactive compounds, including toxic cyanopeptides. Cyanobacteria can produce a variety of cyanopeptides, yet our knowledge about their abundance and co-production remains limited. We applied a suspect-screening approach, including 700 structurally known cyanopeptides, and identified 11 cyanopeptides in Microcystis aeruginosa and 17 in Dolichospermum flos-aquae. Total cyanopeptide concentrations ranged from high nmol to µmol gdry-1 with slightly higher cell quotas in the mid-exponential growth phase. Relative cyanopeptide profiles were unchanged throughout the growth cycle. We demonstrate that quantification based on microcystin-LR equivalents can introduce an error of up to 6-fold and recommend a class-equivalent approach instead. In M. aeruginosa, rarely studied cyclamides dominated (>80%) over cyanopeptolins and microcystins. While all nutrient reductions caused less growth, only lowering phosphorous and micronutrients reduced cyanopeptide production by M. aeruginosa. Similar trends were observed for D. flos-aquae and only lowering nitrogen decreased cyanopeptide production while the relative abundance of individual cyanopeptides remained stable. The synchronized production of other cyanopeptides along with microcystins emphasizes the need to make them available as reference standards to encourage more studies on their occurrence in blooms, persistence, and potential toxicity.

Inhibition of Extracellular Enzymes Exposed to Cyanopeptides.

Harmful cyanobacterial blooms in freshwater ecosystems produce bioactive secondary metabolites including cyanopeptides that pose ecological and human health risks. Only adverse effects of one class of cyanopeptides, microcystins, have been studied extensively and have consequently been included in water quality assessments. Inhibition is a commonly observed effect for enzymes exposed to cyanopeptides and has mostly been investigated for human biologically relevant model enzymes. Here, we investigated the inhibition of ubiquitous aquatic enzymes by cyanobacterial metabolites. Hydrolytic enzymes are utilized in the metabolism of aquatic organisms and extracellularly by heterotrophic bacteria to obtain assimilable substrates. The ubiquitous occurrence of hydrolytic enzymes leads to the co-occurrence with cyanopeptides especially during cyanobacterial blooms. Bacterial leucine aminopeptidase and alkaline phosphatase were exposed to cyanopeptide extracts of different cyanobacterial strains ( Microcystis aeruginosa wild type and microcystin-free mutant, Planktothrix rubescens) and purified cyanopeptides. We observed inhibition of aminopeptidase and phosphatase upon exposure, especially to the apolar fractions of the cyanobacterial extracts. Exposure to the dominant cyanopeptides in these extracts confirmed that purified microcystins, aerucyclamide A and cyanopeptolin A inhibit the aminopeptidase in the low mg L-1 range while the phosphatase was less affected. Inhibition of aquatic enzymes can reduce the turnover of nutrients and carbon substrates and may also impair metabolic functions of grazing organisms.

Environmental fate processes of antimicrobial peptides daptomycin, bacitracins, and polymyxins.

Antimicrobial peptides (AMPs) are increasingly important as a last resort against multi-drug resistant bacteria due to resistance formation towards conventional antibiotics. However, many AMPs were introduced to the market before environmental risk assessment was required, e.g., by the European Medicines Agency (EMA) since 1998. While AMPs have been administered as antibiotics and growth promotors in feedstock since the 1960s and were reconsidered for human medicine by the EMA in 2013, details about their mobility and persistence in the environment remain unknown. This study investigated the environmental fate of three commonly used AMPs: bacitracins, daptomycin, and polymyxins B and E (Colistin). We observed moderate sorption affinity of daptomycin to standard European soils (Kd = 20.6-48.6), while polymyxins adsorbed irreversibly. Bacitracin variants sorbed slightly to sandy soil (Kd = 5.8-8) and significantly to clayey soil (Kd = 169-250). We further investigated photochemical and microbial transformation processes relevant in surface waters. We demonstrated that phototransformation of all AMPs was enhanced in the presence of dissolved organic matter and fast bimolecular reaction rate constant with singlet oxygen contributed largely to indirect phototransformation (15-41%). Phototransformation product analysis for daptomycin was consistent with expected modifications of the tryptophan and kynurenine moieties. Moreover, riverine biofilm communities demonstrated biotransformation potential for all AMPs. Our findings of sorption behaviour, photo- and biotransformation suggest that these processes play a critical role in the fate of bacitracins, daptomycin, and polymyxins in environmental systems.

Cyanobacterial peptides beyond microcystins - A review on co-occurrence, toxicity, and challenges for risk assessment.

Cyanobacterial bloom events that produce natural toxins occur in freshwaters across the globe, yet the potential risk of many cyanobacterial metabolites remains mostly unknown. Only microcystins, one class of cyanopeptides, have been studied intensively and the wealth of evidence regarding exposure concentrations and toxicity led to their inclusion in risk management frameworks for water quality. However, cyanobacteria produce an incredible diversity of hundreds of cyanopeptides beyond the class of microcystins. The question arises, whether the other cyanopeptides are in fact of no human and ecological concern or whether these compounds merely received (too) little attention thus far. Current observations suggest that an assessment of their (eco)toxicological risk is indeed relevant: First, other cyanopeptides, including cyanopeptolins and anabaenopeptins, can occur just as frequently and at similar nanomolar concentrations as microcystins in surface waters. Second, cyanopeptolins, anabaenopeptins, aeruginosins and microginins inhibit proteases in the nanomolar range, in contrast to protein phosphatase inhibition by microcystins. Cyanopeptolins, aeruginosins, and aerucyclamide also show toxicity against grazers in the micromolar range comparable to microcystins. The key challenge for a comprehensive risk assessment of cyanopeptides remains their large structural diversity, lack of reference standards, and high analytical requirements for identification and quantification. One way forward would be a prevalence study to identify the priority candidates of tentatively abundant, persistent, and toxic cyanopeptides to make comprehensive risk assessments more manageable.

Non-Singlet Oxygen Kinetic Solvent Isotope Effects in Aquatic Photochemistry.

The kinetic solvent isotope effect (KSIE) is typically utilized in environmental photochemistry to elucidate whether a compound is susceptible to photooxidation by singlet oxygen (1O2), due to its known difference in lifetime in water (H2O) versus heavy water (D2O). Here, the overall indirect photodegradation rates of diarylamines in the presence of dissolved organic matter (DOM) were enhanced in D2O to a greater extent than expected based on their reactivity with 1O2. For each diarylamine, the relative contribution of reaction with 1O2 to the observed KSIE was determined from high resolution data of 1O2 lifetimes by time-resolved infrared luminescence spectroscopy. The additional enhancement in D2O beyond reaction with 1O2 contributed significantly to the observed KSIE for diarylamines (8-65%) and diclofenac (100%). The enhancement was ascribed to slower reduction of transient radical species of the diarylamines due to H/D exchange at DOM's phenolic antioxidant moieties. A slower second-order reaction rate constant with a model antioxidant was verified for mefenamic acid radicals using transient absorption spectroscopy. Changes in lifetime and reactivity with triplet sensitizers were not responsible for the additional KSIE. Other pollutants with quenchable radical intermediates may also be susceptible to such an additional KSIE, which has to be considered when using the KSIE as a diagnostic tool.

Proteomics Approach To Trace Site-Specific Damage in Aquatic Extracellular Enzymes During Photoinactivation.

Extracellular enzymes are major drivers of biogeochemical nutrient and carbon cycling in surface water. While photoinactivation is regarded as a major inactivation process of these enzymes, the underlying molecular changes have received little attention. This study demonstrates how light exposure leads to a rapid loss of phosphatase, aminopeptidase, and glucosidase activities of biofilm samples and model enzymes. Here, an optimized proteomics approach allowed simultaneous observation of inactivation and molecular changes. Site-specific fingerprints of degradation kinetics have been generated and visualized in the three-dimensional proteins. Oxidation of tryptophan, the chromophoric target, initiated secondary reactions. Evidence was obtained that tyrosine residues act as intramolecular antioxidants, reflected in decelerated decay of tryptophan-containing peptides and enhanced decay of tyrosine-containing peptides. In addition, subsequent methionine oxidation and disulfide reduction contribute to heterogeneous photodamage. The proximity to tryptophan residues explains >95% of the photodamage across the protein structures. The presence of redox active organic matter or a model antioxidant in solution quenched not only photoinactivation and tryptophan oxidation but also all subsequent damage. The developed analytical approach can be applied to other research questions in environmental sciences where site-specific damage in a protein is essential.

Magnitude and Mechanism of Siderophore-Mediated Competition at Low Iron Solubility in the Pseudomonas aeruginosa Pyochelin System.

A central question in microbial ecology is whether microbial interactions are predominantly cooperative or competitive. The secretion of siderophores, microbial iron chelators, is a model system for cooperative interactions. However, siderophores have also been shown to mediate competition by sequestering available iron and making it unavailable to competitors. The details of how siderophores mediate competition are not well understood, especially considering the complex distribution of iron phases in the environment. One pertinent question is whether sequestering iron through siderophores can indeed be effective in natural conditions; many natural environments are characterized by large pools of precipitated iron, and it is conceivable that any soluble iron that is sequestered by siderophores is replenished by the dissolution of these precipitated iron sources. Our goal here was to address this issue, and investigate the magnitude and mechanism of siderophore-mediated competition in the presence of precipitated iron. We combined experimental work with thermodynamic modeling, using Pseudomonas aeruginosa as a model system and ferrihydrite precipitates as the iron source with low solubility. Our experiments show that competitive growth inhibition by the siderophore pyochelin is indeed efficient, and that inhibition of a competitor can even have a stronger growth-promoting effect than solubilization of precipitated iron. Based on the results of our thermodynamic models we conclude that the observed inhibition of a competitor is effective because sequestered iron is only very slowly replenished by the dissolution of precipitated iron. Our research highlights the importance of competitive benefits mediated by siderophores, and underlines that the dynamics of siderophore production and uptake in environmental communities could be a signature of competitive, not just cooperative, dynamics.

Environmental photochemistry of fenamate NSAIDs and their radical intermediates.

Fenamates are a class of nonsteroidal anti-inflammatory drugs (NSAIDs) that are not fully removed during wastewater treatment and can be released to surface waters. Here, near-surface photochemical half-lives were evaluated to range from minutes to hours of four fenamates and the closely related diclofenac. While quantum yields for direct photochemical reactions at the water surface vary widely from 0.071 for diclofenac to <0.001 for mefenamic acid, all fenamates showed significant reactivity towards singlet oxygen and hydroxyl radical with bimolecular reaction rate constants of 1.3-2.8 × 107 M-1 s-1 and 1.1-2.7 × 1010 M-1 s-1, respectively. Photodecay rates increased in the presence of dissolved organic matter (DOM) for diclofenac (+19%), tolfenamic acid (+9%), and mefenamic acid (+95%), but decreased for flufenamic acid (-2%) and meclofenamic acid (-14%) after accounting for light screening effects. Fast reaction rate constants of all NSAIDs with model triplet sensitizers were quantified by laser flash photolysis. Here, the direct observation of diphenylamine radical intermediates by transient absorption spectroscopy demonstrates one-electron oxidation of all fenamates. Quenching rate constants of these radical intermediates by ascorbic acid, a model antioxidant, were also quantified. These observations suggest that the balance of oxidation by photoexcited triplet DOM and quenching of the formed radical intermediates by antioxidant moieties determines whether net sensitization or net quenching by DOM occurs in the photochemical degradation of fenamates.