Ecology

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General experimental proceduresAll chemicals were purchased from Sigma-Aldrich, Acros Organics or Iris BIOTECH. Isotope-labelled chemicals were purchased from Cambridge Isotope Laboratories. Genomic DNA of selected Xenorhabdus and Photorhabdus strains was isolated using the Qiagen Gentra Puregene Yeast/Bact Kit. DNA polymerases (Taq, Phusion and Q5) and restriction enzymes were purchased from New England Biolabs or Thermo Fisher Scientific. DNA primers were purchased from Eurofins MWG Operon. PCR amplifications were carried out on thermocyclers (SensoQuest). Polymerases were used according to the manufacturers’ instructions. DNA purification was performed from 1% Tris-acetate-EDTA (TAE) agarose gel using an Invisorb Spin DNA Extraction Kit (STRATEC Biomedical AG). Plasmids in E. coli were isolated by alkaline lysis. HPLC-UV-MS analysis was conducted on an UltiMate 3000 system (Thermo Fisher) coupled to an AmaZonX mass spectrometer (Bruker) with an ACQUITY UPLC BEH C18 column (130 Å, 2.1 mm × 100 mm, 1.7-μm particle size, Waters) at a flow rate of 0.6 ml min−1 (5–95% acetonitrile/water with 0.1% formic acid, vol/vol, 16 min, UV detection wavelength 190–800 nm). HPLC-UV-HRMS analysis was conducted on an UltiMate 3000 system (Thermo Fisher) coupled to an Impact II qTof mass spectrometer (Bruker) with an ACQUITY UPLC BEH C18 column (130 Å, 2.1 mm × 100 mm, 1.7-μm particle size, Waters) at a flow rate of 0.4 ml min−1 (5–95% acetonitrile/water with 0.1% formic acid, vol/vol, 16 min, UV detection wavelength 190–800 nm). Flash purification was performed on a Biotage SP1 flash purification system (Biotage) by a C18 main column (Interchim, PF50C18HP-F0080, 120 g) with a self-packed pre-column (Interchim, PF-DLE-F0012, Puriflash dry-load empty F0012 Flash column) coupled with a UV detector. HPLC purification was performed on preparative and semipreparative Agilent 1260 systems coupled to a diode array detector (DAD) and a single quadrupole detector with a C18 ZORBAX Eclipse XDB column (9.4 mm × 250 mm, 5 μm, 3 ml min−1; 21.2 mm × 250 mm, 5 μm, 20 ml min−1; 50 mm × 250 mm, 10 μm, 40 ml min−1). Freeze drying was performed using a BUCHI Lyovapor L-300 Continuous system. NMR experiments were carried out on a Bruker AVANCE 500-, 600- or 700-MHz spectrometer equipped with a 5-mm cryoprobe. 2R,3S-IOC (1) and GameXPeptide A (16) were synthesized by WuXi App Tec following the literature (ref. 73 for 2R,3S-1 and ref. 74 for 16).Genome sequencing, assembly and annotationIsolated DNA was sequenced on the Illumina NextSeq 500 platform. DNA libraries were constructed using the Nextera XT DNA preparation kit (Illumina) and whole-genome sequencing was performed using 2× 150-bp paired-end chemistry. A sequencing depth of >50× was targeted for each sample. Adapters and low-quality ends were trimmed with Trimmomatic 0.39 (ref. 75) and the parameters [2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:10 MINLEN:12] using a database of adapter sequences as provided by Illumina. All genomes were assembled using SPAdes v. 3.10.1 (ref. 76) executed with the following parameters: –cov-cutoff auto –careful in paired-end mode plus mate pairs (in cases where accompanying mate-pair libraries were available). Genome annotation was performed using Prokka v. 1.12 (ref. 77) with the following parameters: –usegenus–genus GENUS–addgenes–evalue 0.0001–rfam–kingdom Bacteria–gcode 11–gram –mincontiglen 200. Geneious Prime 2021 was used in genome visualization and analysis.antiSMASH annotations and BiG-FAM preliminary classificationThe antiSMASH 5.0 (ref. 23) web server was employed to mine all the genome sequences for the presence of putative natural-product BGCs. The annotations were conducted using default settings with the extended parameters of ClusterBlast, Cluster Pfam analysis and Pfam-based GO term annotation. The annotated BGCs were summarized for each strain (Supplementary Fig. 1) and visualized in the anvi’o 6.1 (refs. 28,78) layers (Fig. 1 and Supplementary Figs. 3 and 5). We then submitted the antiSMASH job IDs to the biosynthetic gene cluster families database (BiG-FAM 1.0.0)25 for preliminary GCF explorations and classifications of annotated BGCs (Supplementary Table 3), followed by BiG-SCAPE 1.0.0 (ref. 42) refinement with a cutoff of 0.65 (Source Data Fig. 3). The GCFs were double-checked manually via the interactive network (Fig. 3), and corrections were made if necessary. A putative thiopeptide BGC (Xszus_1.region006, Xsze_2.region003, Xsto_4.region001, Xpb_30.3_21.region001, Xmir_10.region001, Xmau_6.region001, Xkoz_3.region001, Xjap_NZ_FOVO01000011.region001, Xish_1.region003, Xhom_ANU1.region005, Xhom_2.region003, Xets_11.region001, XenKK7.region002, XenDL20_c00108_NODE_12.region001, Xekj_19.region001, Xehl_28.region001, Xe30TX1_c0031_NODE_38.region001, Xdo_HBLC131_1.region001, Xdo_FRM16.1.region005, Xbov_NC_013892.1.region004, Ptem_HBLC135_17.region001, Ppb6_4.region001, Plum_TT01_1.region008, Pthr_PT1.1_23.region001, Plau_IT4.1_12.region001, Plum_IL9_35_scf0001.region001, Pbod_HU2.3_20.region001, Plau_HB1.3_105.region001, Plum_EN01_24_scf0009.region001, Pbod_DE6.1_24.region001, Plau_DE2.2_108.region001, Phpb_1.region001, Pbod_LJ_007.region001, Pbod_CN4_25_scf0020.region001, Paeg_BKT4.5_19.region001, P_tem_1.region017 and so on) that exists throughout 45 XP genomes was excluded in the analysis, because it turned out that its annotation by antiSMASH 5.0 is a false positive and early reports suggest that this cluster is responsible for ribosomal methylthiolation79,80. Two BGCs, Xdo_HBLC131_4.region001 encoding the biosynthesis of glidobactins in X. doucetiae HBLC131 and Ptem_HBLC135_2.region002 encoding the biosynthesis of ririwpeptides in P. temperata HBLC135, were artificially integrated into their respective genome by CRAGE39 previously, and thus the two BGCs were also excluded in our analysis.Pangenome analysisBiosynthetic gene cluster boundary definitionThe cluster boundary was defined by antiSMASH with the start nucleotide of the first biosynthetic gene (5′ end) and the stop nucleotide of the last biosynthetic gene (3′ end), and was manually corrected if necessary. Non-structural genes (such as transporters, regulators, transposases and so on) on the outer periphery of an operon were excluded. We compiled a table with contigs of all BGCs encoded by a given genome, BGC start and stop nucleotide positions, BGC classifications by antiSMASH and BiG-SCAPE (see the BiG-SCAPE analysis section), and possible biosynthetic pathways that the BGCs encode (Source Data Fig. 1). These tables would be integrated into the contigs databases of the pangenome for filtering the biosynthetic genes and monitoring distributions of biosynthetic gene homology groups.Interface generationAll genomes were obtained from the National Center for Biotechnology Information (NCBI). Supplementary Table 1 reports their accession numbers. The pangenome analysis herein mainly followed the anvi’o 6.1 pangenomic workflow28,78. After simplifying the header lines of 45 FASTA files for genomes using ‘anvi-script-reformat-fasta’, we converted FASTA files into anvi’o contigs databases by the ‘anvi-gen-contigs-database’ and then decorated the contigs database with hits from HMM models by ‘anvi-run-hmms’. The program ‘anvi-run-ncbi-cogs’ was run to annotate genes in the contigs databases with functions from the NCBI’s Clusters of Orthologous Groups (COGs). Tables of gene caller IDs with start and stop nucleotide positions were exported by ‘anvi-export-table’. By linking the gene caller IDs with BGCs via the start and stop nucleotide positions, genes that fell within a given BGC boundary were considered to be natural product biosynthetic genes (Source Data Fig. 1). Thereafter, the biosynthetic genes were furnished with a classification and a possible compound name, both of which were derived from the BGC that the biosynthetic genes made up. The obtained tables were imported back to contigs databases by ‘anvi-import-functions’. External genome storage was created by ‘anvi-gen-genomes-storage’ to store DNA and amino-acid sequences, as well as functional annotations of each gene. With the genome storage in hand, we used the program ‘anvi-pan-genome’ with the genomes storage database, the flag ‘–use-ncbi-blast’ and the parameter ‘–mcl-inflation 8’. The results were displayed in an interface by ‘anvi-display-pan’. The organization of the pangenome interface as shown in the dendrogram in the centre was represented by ‘presence/absence’ patterns. The core gene bin was characterized by searching the gene homology group (gene homology group represents amino-acid sequences from one or more genomes aligned by muscle81) using filters with ‘Min number of genomes gene homology group occurs, value = 45’. The singleton bin was identified by ‘Max number of genomes gene homology group occurs, value = 1’. The rest of the gene clusters that were neither sorted into the core gene bin nor the singleton bin were appended to the accessory bin. The single-copy-core-gene (scg) bin was found by ‘Min number of genomes gene homology group occurs, value = 45’ and ‘Max number of genes from each genome, value = 1’. The scg bin was refined by ‘Max functional homogeneity index 0.9’ and ‘Min geometric homogeneity index 1’. The resulting protein sequences were exported by ‘anvi-get-sequences-for-gene-clusters’ and aligned using ClustalW 1.2.2, which is incorporated in Geneious Prime 2021. Phylogenetic trees were generated using the Geneious tree builder utilizing the Jukes–Cantor distance model and the unweighted pair group method with arithmetic mean (UPGMA), and subsequently imported back to anvi’o by ‘anvi-import-misc-data’ and visualized by the interface. The statistical data of BGCs obtained from antiSMASH 5.0 (ref. 23) and BiG-SCAPE42 were imported to the layers of the interface by ‘anvi-import-misc-data’ for visualization.Biosynthetic gene and biosynthetic gene cluster filteringThe bin summary (scg, core, accessory and singleton) with BGC classifications was exported by ‘anvi-summarize’ to monitor the distributions of the biosynthetic gene homology group in the pangenomes (Source Data Fig. 1 and Supplementary Data). In the Excel sheets, ‘core’ and ‘scg’ filters were selected from the ‘bin_name’ column, and the ‘(Blank)’ filter from the ‘BGC_classification’ column was unselected. The table was then sorted by ‘genome_name’ and ‘gene_callers_id’ columns in ascending order. This then displayed consecutive core biosynthetic genes that could possibly make up a BGC. The same procedure was used to filter BGCs in the accessory or singleton region.BiG-SCAPE analysisBGCs in all genome sequences obtained from antiSMASH 5.0 (ref. 23) analyses were compared to reference BGCs from MIBiG repository 2.0 (refs. 41,82) using BiG-SCAPE 1.0.0 (ref. 42) with the PFAM database 32.0 (ref. 83). The analysis was conducted using default settings with the mode ‘auto’, mixing all classes and retaining singletons. Networks were computed for raw distance cutoffs of 0.30–0.95 in increments of 0.05. Results were visualized as a network using Cytoscape 3.7.2 (ref. 84) for a cutoff of 0.65 (Fig. 3 and Source Data Fig. 3). Statistical data for the BGCs were analysed and evaluated using Origin 2020b and Excel from Microsoft Office 365.Strain and culture conditionsWild-type strains and the mutants thereof and E. coli (Supplementary Table 14) were cultivated on lysogeny broth (LB) agar plates at 30 °C overnight, and subsequently inoculated into liquid LB culture at 30 °C with shaking at 200 r.p.m. For compound production, the overnight LB culture was transferred into 5 ml of LB, XPP19 or Sf-900 II SFM medium (1:100, vol/vol) with 2% (vol/vol) Amberlite XAD-16 resins, 0.1% l-arabinose as the inducer for mutants with a PBAD promoter, and selective antibiotics such as ampicillin (Am, 100 µg ml−1), kanamycin (Km, 50 µg ml−1) or chloramphenicol (Cm, 34 µg ml−1) at 30 °C, with shaking at 200 r.p.m.Culture extraction and HPLC-UV-MS analysisThe XAD-16 resins were collected after 72 h and extracted with 5 ml of methanol or ethyl acetate. The solvent was dried under rotary evaporators, and the dried extract was resuspended in 500 μl of methanol or acetonitrile/water (1:1 vol/vol for photoxenobactins), of which 5 μl was injected and analysed by HPLC-UV-MS or HPLC-UV-HRMS. Unless otherwise specified, HPLC-UV-MS and HPLC-UV-HRMS chromatograms in the figures are shown on the same scale. Bruker Compass DataAnalysis 4.3 was used for data collection and analysis of chromatography and MS. MetabolicDetec 2.1 was utilized to differentiate MS profiles between induced and non-induced promoter insertion mutants for identifying possible metabolites produced by targeted BGCs.Construction of PBAD promoter insertion mutantsA 500–800-bp section upstream of the target gene (lpcS, pxbF, rdb1A and xvbA) was amplified with a corresponding primer pair as listed in Supplementary Table 15. The resulting fragments were cloned using Hot Fusion85 into a pCEP_kan or pCEP_cm backbone that was amplified by pCEP_Fw and pCEP_Rv. After transformation of a constructed plasmid into E. coli S17-1 λ pir, clones were verified by PCR with primers pCEP-Ve-Fw and pDS132-Ve-Rv. A wild-type strain (X. bovienii SS-2004, X. szentirmaii DSM 16338, X. budapestensis DSM 16342 or X. vietnamensis DSM 22392) or a deletion mutant (X. szentirmaii ∆hfq, X. budapestensis ∆rdb1P or X. budapestensis ∆rdb1P ∆hfq) was used as a recipient strain. The recipient strain was mated with E. coli S17-1 λ pir (donor) carrying a constructed plasmid (Supplementary Table 16). Both strains were grown in the LB medium to an optical density at 600 nm (OD600) of 0.6 to 0.7, and the cells were washed once with fresh LB medium. Subsequently, the donor and recipient strains were mixed on an LB agar plate in ratios of 1:3 and 3:1, and incubated at 37 °C for 3 h followed by incubation at 30 °C for 21 h. After that, the bacterial cell layer was collected with an inoculating loop and resuspended in 2 ml of fresh LB medium. A 200-μl sample of the resuspended culture was spread out on an LB agar plate with Am/Km or Am/Cm and incubated at 30 °C for two days. Individual insertion clones were cultivated and analysed by HPLC-UV-HRMS, and the genotype of all mutants was verified by plasmid- and genome-specific primers.Construction of deletion mutantsA ~1,000-bp upstream and a ~1,000-bp downstream fragment of hfq in X. budapestensis DSM 16342 were amplified using the primer pairs listed in Supplementary Table 15. The amplified fragments were fused using the complementary overhangs introduced by primers and cloned into the pEB17 vector that was linearized with PstI and BglII by Hot Fusion85. Transformation of E. coli S17-1 λ pir with the resulting plasmid (Supplementary Table 16) and conjugation with X. budapestensis DSM 16342, as well as the generation of double crossover mutants via counterselection on LB plates containing 6% sucrose, were carried out as previously described86. The deletion mutant was verified via PCR using the primer pairs listed in Supplementary Table 15, which yielded a ~2,000-bp fragment for mutants genetically equal to the WT strain and a ~1,000-bp fragment for the desired deletion mutant. The same procedure was used to generate Δrdb1P mutants, during which E. coli S17-1 λ pir carrying pEB17 rdb1P was mated with the X. budapestensis DSM 16342 wild-type and X. budapestensis ∆hfq mutant.Labelling experiments for structural elucidation of photoxenobactins C and D by MSThe cultivation of strains for labelling experiments was carried out as described above. For photoxenobactin C (6) labelling experiments, the overnight culture was transferred into LB medium additionally fed with 4-fluorosalicylate-SNAC, l-methionine-(methyl-d3), l-[U-13C,15N]cysteine and l-[U-34S]cysteine at a final concentration of 1 mM. In terms of inverse feeding experiments, cell pellets of the 100-μl overnight culture were washed once with ISOGRO 13C or 13C,15N medium (100 μl) and resuspended in the corresponding isotope labelling medium (100 μl). The feeding culture in the isotope labelling medium (5 ml) was inoculated with a washed overnight culture (50 μl) and additional l-cysteine was added at a final concentration of 1 mM.For photoxenobactin D (7) labelling experiments, the cell pellets of the 100-μl overnight culture were washed once with ISOGRO 13C or 15N medium (100 μl) and then resuspended in the corresponding isotope labelling medium (100 μl). A 5-ml isotope labelling medium was inoculated with a washed overnight culture (50 μl).Isolation and purificationFor photoxenobactin isolation, 10 ml of LB medium was inoculated with a colony of the X. szentirmaii PBAD pxbF ∆hfq mutant from an LB agar plate and cultivated overnight. A 10-ml culture was taken to inoculate 2 × 100 ml of LB medium (OD600 ≈ 0.1). The 2 × 100-ml cultures were incubated overnight and the whole culture volume (200 ml) was used to inoculate a 20-l LB fermenter (Braun) supplemented with 2% XAD-16 and 0.2% arabinose (antifoam was added when required). Fermenter settings were as follows: 30 °C without pH control, three six-blade impellers 150 r.p.m. After 24 h, 10 l of the culture was collected from the fermenter, and the XAD resins were separated from the cells by filtration. (1) The XAD resins were extracted with 2 × 1 l of ethyl acetate with 1% formic acid, and the combined organic phase was dried under reduced pressure. (2) The culture without XAD was centrifuged and the supernatant was extracted with 3 × 5 l of ethyl acetate with 1% formic acid, and the combined organic layers were dried under reduced pressure. (3) The cell pellet was extracted with 2 × 1 l of ethyl acetate with 1% formic acid, and the organic supernatant was dried under reduced pressure. After 48 h, the remaining 10 l of bacterial culture were extracted as described in steps (1) to (3). The combined extracts from 20 l of culture were fractionated by a flash purification system with a C18 column with a gradient elution of acetonitrile/water 20–100% at 20 ml min−1 (every 10% gradient step was performed with five column volumes, except the 60–70% step, which was performed with ten column volumes). Fractions containing photoxenobactins were combined and dried under reduced pressure. Final purification was achieved via preparative and semipreparative HPLCs with a gradient of 30% acetonitrile/water (0–30 min) and 30–100% acetonitrile/water (30–40 min). The fractions were combined in brown flasks and were immediately freeze-dried to afford photoxenobactin A (4, 0.8 mg), photoxenobactin B (5, 0.6 mg), photoxenobactin C (6, 1.2 mg) and photoxenobactin E (8, 2.2 mg).For the isolation and purification of lipocitides A and B, 2% of XAD-16 resins from a 6-l LB culture of the X. bovienii PBAD lpcS mutant induced by l-arabinose were collected after 72 h of incubation at 30 °C with shaking at 120 r.p.m., and were washed with water and extracted with methanol (3 × 1 l) to yield a crude extract (5.3 g after evaporation). The extract was dissolved in methanol and was subjected to preparative HPLC with a C18 column using an acetonitrile/water gradient (0.1% formic acid) for 0–32 min, 55–80%, 40 ml min−1 to afford lipocitides A (17, 4.8 mg) and B (18, 9.0 mg).Two percent of XAD-16 resins from a 12-l LB culture of the X. budapestensis PBAD rdb1A ∆rdb1P ∆hfq mutant induced by l-arabinose were collected after 72 h of incubation at 30 °C with shaking at 120 r.p.m. and washed with water and extracted with methanol (3 × 2 l) to yield a crude extract (15.3 g after evaporation). The extract was subject to a Sephadex LH-20 column eluted with methanol. The fraction (2.8 g) containing pre-rhabdobranins was subjected to preparative HPLC with a C18 column using an acetonitrile/water gradient (0.1% formic acid) for 0–20 min, 15–35%, 40 ml min−1 to afford a fraction (206 mg) mainly containing pre-rhabdobranin D, which was further purified by semipreparative HPLC with a C18 column using an acetonitrile/water gradient (0.1% formic acid) for 0–24 min, 5–53%, 3 ml min−1 to afford pre-rhabdobranin D (27, 59.1 mg).Benzobactin A (28) and its methyl ester (29), which were detected in X. vietnamensis PBAD xvbA, were also produced by Pseudomonas chlororaphis subsp. piscium DSM 21509 (unpublished). Owing to the high production level in Pseudomonas chlororaphis subsp. piscium DSM 21509, 28 and 29 were isolated from the Pseudomonas strain. Four percent of XAD-16 resins from a 12-l XPP culture of Pseudomonas chlororaphis subsp. piscium DSM 21509 PBAD pbzA mutant induced by l-arabinose were collected after 72 h of incubation at 30 °C with shaking at 120 r.p.m., and washed with water and extracted with methanol (3 × 2 l) to yield a crude extract (95.4 g after evaporation). The extract was dissolved in methanol and subjected to preparative HPLC with a C18 column using an acetonitrile/water gradient (0.1% formic acid) for 0–18 min, 5–59%, 20 ml min−1 to afford ten fractions. Fractions 2 (95.6 mg) and 3 (50.7 mg) were further purified by semipreparative HPLC with a C18 column using an acetonitrile/water gradient (0.1% formic acid) for 0–35 min, 5–95%, 3 ml min−1 to afford benzobactin A (28, 3.2 mg) and its methyl ester (29, 0.9 mg), respectively.NMR spectroscopyMeasurements were carried out using 1H and 13C NMR, 1H-13C heteronuclear single quantum coherence (HSQC), 1H-13C heteronuclear multiple bond correlation (HMBC), 1H-1H correlation spectroscopy (COSY), 1H-13C heteronuclear multiple quantum correlation/1H-1H correlation spectroscopy (HMQC-COSY) and 1H-13C heteronuclear single quantum coherence/1H-1H total correlation spectroscopy (HSQC-TOCSY). Chemical shifts (δ) were reported in parts per million (ppm) and referenced to the solvent signals. Data are reported as follows: chemical shift, multiplicity (br = broad, s = singlet, d = doublet, t = triplet, dd = doublet of doublet, m = multiplet and ov = overlapped) and coupling constants (in hertz). Bruker TopSpin 4.0 was used for NMR data collection and spectral interpretation.General synthetic proceduresThe Fmoc protecting group was removed with 2 ml of 40% piperidine/dimethylformamide (DMF; 5 min) followed by 2 ml of 20% piperidine/DMF (10 min). Washings between coupling and deprotection steps were performed with DMF (five syringe volumes) and dichloromethane (DCM) (five syringe volumes). Resin loadings were determined by Fmoc cleavage from a weighted resin sample87. The combined filtrates containing Fmoc cleavage products were quantified spectrophotometrically at 301 nm using a UV–vis spectrophotometer with Hellma absorption cuvettes with a path length of 1 cm. Loadings were calculated (in mmol resin) using Lambert–Beer’s law with ɛ = 7,800 M−1 cm−1: loading (mmol) = ({frac{{{rm{Abs}},{({rm{sample}})}}}{{varepsilon l}}} times V), where ɛ is the molar extinction coefficient, V is the sample volume in liter and l is the optical path length in cm. Final cleavage was achieved by shaking the resin in 2 ml of a mixture of TFA/TIPS/H2O (95:2.5:2.5) for 1 h. The filtrate was then collected and the resin washed three times (2 ml each) with DCM, and the combined filtrates were dried under reduced pressure.Syntheses of lipocitide AFmoc-protected Rink Amide resin (192 mg, 0.52 mmol g−1, 0.1 mmol) was placed in a polypropylene 6-ml syringe vessel fitted with polyethylene porous filter disks and swollen in 3 ml of DMF for 10 min. Subsequently, the Fmoc-protected resin was deprotected and then washed as described in the general synthetic procedures. Fmoc-d-Cit-OH (198.0 mg, 0.5 mmol, 5 equiv.), 1-hydroxy-7-azabenzotriazole (HOAT, 0.83 ml, 0.5 mmol, 5 equiv.), hexafluorophosphate azabenzotriazole tetramethyl uronium (HATU, 190.5 mg, 0.5 mmol, 5 equiv.) and N,N-diisopropylethylamine (DIPEA, 170 μl, 1.0 mmol, 10 equiv.) were dissolved in 1.5 ml of dry DMF. After 5 min, the clear solution was added to the resin and shaken at room temperature overnight. The resin was washed and loading was calculated (79.2%) as described in the general synthetic procedures. Acylation of Fmoc-l-Ala-OH (74.1 mg, 0.24 mmol, 3 equiv.), Fmoc-d-Leu-OH (84.8 mg, 0.24 mmol, 3 equiv.) and myristic acid (54.8 mg, 0.24 mmol, 3 equiv.) were carried out using the abovementioned procedure. Final cleavage was performed as described in the general synthetic procedures, and the crude product (70.8 mg) was purified by HPLC to obtain lipocitide A (17, Supplementary Fig. 100; 24.3 mg, 54.0%) as a white solid.Syntheses of lipocitide B2-CTC resin (63 mg, 1.6 mmol g−1, 0.1 mmol) was placed in a polypropylene 6-ml syringe vessel fitted with polyethylene porous filter disks. The resin was incubated with Fmoc-d-Cit-OH (119.0 mg, 0.3 mmol, 3 equiv.) and DIPEA (153 μl, 0.9 mmol, 9 equiv.) in 1.5 ml of dry DCM at room temperature overnight. The resin was washed and loading was calculated (56.7%) as described in the general synthetic procedures. Acylations of Fmoc-l-Ala-OH (52.9 mg, 0.17 mmol, 3 equiv.), Fmoc-d-Leu-OH (60.1 mg, 0.17 mmol, 3 equiv.) and myristic acid (38.9 mg, 0.24 mmol, 3 equiv.) were performed with additional HOAT (0.47 ml, 0.28 mmol, 5 equiv.), HATU (108 mg, 0.28 mmol, 5 equiv.) and DIPEA (96 μl, 0.56 mmol, 10 equiv.). Final cleavage was carried out as described in the general synthetic procedures, and the crude (54.2 mg) was purified by HPLC to obtain lipocitide B (18, Supplementary Fig. 101; 18.6 mg, 57.6%) as a white solid.Synthesis of S-(2-acetamidoethyl)4-fluoro-2-hydroxybenzothioate (4-fluorosalicylate SNAC)To a solution of 4-fluorosalicylic acid (156 mg, 1.0 mmol, 1.0 equiv.) and hydroxybenzotriazole (HOBt, 162 mg, 1.2 mmol, 1.2 equiv.) in 45 ml of THF, N,N′-dicyclohexylcarbodiimide (DCC, 248 mg, 1.2 mmol, 1.2 equiv.) was added, followed by N-acetylcysteamine (112 µl, 1.0 mmol, 1.0 equiv.). After 1 h at room temperature, K2CO3 (138 mg, 1.0 mmol, 1.2 equiv.) was added and the reaction was stirred for an additional 2 h. The reaction mixture was then filtered and concentrated by rotary evaporation. The solid residue was dissolved in ethyl acetate and washed with sat. NaHCO3 (50 ml) and water (50 ml). The organic layer was dried over MgSO4, concentrated, and purified by flash chromatography (1–10% MeOH in CHCl3) to give 26 mg (10%) S-(2-acetamidoethyl)4-fluoro-2-hydroxybenzothioate (Supplementary Fig. 102).IC50 value determination with the purified yeast 20S proteasome core particleYeast 20S proteasome core particle (yCP) from Saccharomyces cerevisiae was purified according to previously described methods88,89. The concentration of purified yCP was determined spectrophotometrically at 280 nm. yCP (final concentration: 0.05 mg ml−1 in 100 mM Tris-HCl, pH 7.5) was mixed with dimethyl sulfoxide (DMSO) as a control or serial dilutions of IOC (1) in DMSO, thereby not surpassing a final concentration of 10% (vol/vol) DMSO. After an incubation time of 45 min at room temperature, fluorogenic substrates Boc-Leu-Arg-Arg-AMC (AMC, 7-amino-4-methylcoumarin), Z-Leu-Leu-Glu-AMC and Suc-Leu-Leu-Val-Tyr-AMC (final concentration of 200 µM) were added to measure the residual activity of caspase-like (C-L, β1 subunit), trypsin-like (T-L, β2 subunit) and chymotrypsin-like (ChT-L, β5 subunit), respectively. The assay mixture was incubated for another 60 min at room temperature, then diluted 1:10 in 20 mM Tris-HCl, pH 7.5. The AMC molecules released by hydrolysis were measured in triplicate with a Varian Cary Eclipse fluorescence spectrophotometer (Agilent Technologies) at λexc = 360 nm and λem = 460 nm. Relative fluorescence units were normalized to the DMSO-treated control. The calculated residual activities were plotted against the logarithm of the applied inhibitor concentration and fitted with GraphPad Prism 9.0.2. IC50 values were deduced from the fitted data. These depend on enzyme concentration and are comparable within the same experimental settings.Crystallization and structure determination of the yCP in complex with IOC (1)Crystals of the yCP were grown in hanging drops at 20 °C, as previously described88,89. The protein concentration used for crystallization was 40 mg ml−1 in Tris/HCl (20 mM, pH 7.5) and EDTA (1 mM). The drops contained 1 μl of protein and 1 μl of the reservoir solution (30 mM magnesium acetate, 100 mM 2-(N-morpholino)ethanesulfonic acid (pH 6.7) and 10% (wt/vol) 2-methyl-2,4-pentanediol). Crystals appeared after two days and were incubated with 1 at a final concentration of 10 mM for at least 24 h. Droplets were then complemented with a cryoprotecting buffer (30% (wt/vol) 2-methyl-2,4-pentanediol, 15 mM magnesium acetate, 100 mM 2-(N-morpholino)ethanesulfonic acid, pH 6.9) and vitrified in liquid nitrogen. The dataset from the yCP:IOC complex was collected using synchrotron radiation (λ = 1.0 Å) at the X06SA-beamline (Swiss Light Source). X-ray intensities and data reduction were evaluated using the XDS program package version 5 February 2021 (Supplementary Table 17)90. Conventional crystallographic rigid body, positional and temperature factor refinements were carried out with REFMAC5 5.0.32 (ref. 91) and the CCP4 Program Suite 7.1.016 (ref. 92) using coordinates of the yCP structure as the starting model (PDB 5CZ4)50. Model building was performed by the programs SYBYL-X and COOT 0.8.7 (ref. 93). The final coordinates yielded excellent residual factors, as well as geometric bond and angle values. Coordinates were confirmed to fulfil the Ramachandran plot and have been deposited in the RCSB (PDB 7O2L).Haemocyte-spreading assaysSpodoptera exigua larvae were collected from Welsh onion (Allium fistulsum L.) fields in Andong, Korea. Insects were reared in the laboratory under the following conditions: 25 ± 2 °C constant temperature, 16:8 h (light/dark) photoperiod and 60 ± 5% relative humidity. Larvae were reared on an artificial diet94 and 10% sucrose solutions were fed to adult insects. Fifth instar larvae were used in all experiments. For analysing haemocyte behaviours in vivo, fifth instar larvae of S. exigua were co-injected with 1 µl of heat-killed (95 °C for 10 min) E. coli TOP10 (2.4 × 104 cells per larva) with the test compound (0–1,000 ng per larva) by using a Hamilton microsyringe (Reno). At 1 h post-injection, 10 µl of haemolymph from each larva was collected on the glass slide and incubated for 5 min inside a dark wet chamber at room temperature. The medium was replaced with 3.7% of formaldehyde dissolved in phosphate buffered saline (PBS) and incubated for 10 min. After washing three times with PBS, cells were permeabilized with 0.2% Triton X-100 in PBS for 2 min at room temperature. After incubation, the slides were washed with PBS three times. Blocking was performed using 5% skimmed milk (Invitrogen) dissolved in PBS, followed by incubation for 10 min. After washing once with PBS, the cells were incubated with fluorescein isothiocyanate (FITC)-tagged phalloidin in PBS for 1 h at room temperature. After washing three times, the cells were incubated with 4′,6-diamidino-2-phenylindole (DAPI, 1 mg ml−1, Thermo Scientific) in PBS for nucleus staining. Finally, after washing twice in PBS, cells were observed under a fluorescence microscope (DM2500, Leica) at ×400 magnification. Haemocyte spreading was determined by the extension of F-actin out of the original cell boundary. For the in vitro assay, ~100 μl of haemolymph was collected into 400 μl of anticoagulation buffer (ACB; 186 mM NaCl, 17 mM Na2EDTA, 41 mM citric acid, pH 4.5). After adding ACB, the medium was incubated for 30 min on ice. After centrifugation at 300g for 5 min, 400 μl of supernatant was discarded. The rest of the suspension was gently mixed with 200 μl of TC100 insect tissue culture medium (Welgene). From this suspension, 10 µl of haemolymph was collected on the glass slide. The slides were co-injected with 1 µl of E. coli TOP10 (2.4 × 104 cells per larva) with the test compound (0–1,000 ng per larva), followed by the procedure described above. Means were compared by a least squared difference (LSD) test of one-way analysis of variance (ANOVA) using POC GLM of the SAS program (SAS Institute, 1989) and discriminated at type I error = 0.05.Nodulation assaysE. coli TOP10 was heat-killed by incubating at 95 °C for 10 min. Fifth-instar larvae of S. exigua were injected with 1 µl of bacteria (2.4 × 104 cells per larva) using a Hamilton microsyringe along with 1 µl of different concentrations (10, 50, 100, 500 and 1,000 ppm) of inhibitors. Control larvae were injected with bacteria and DMSO. At 8 h after bacterial injection, nodules were counted by dissecting larvae under a stereomicroscope (Stemi SV 11, Zeiss) at ×50 magnification.Phenoloxidase activity assaysThe PO activity from plasma was estimated as previously described95. Briefly, DOPA (l-3,4-dihydroxyphenylalanine) was used as a substrate for determining PO activity from treated larvae plasma. For PO activation, each fifth-instar larva of S. exigua was challenged with 2.4 × 104 cells of heat-killed E. coli TOP10. Different inhibitors were co-injected (1 µg per larva) along with E. coli TOP10. After 8 h of bacterial challenge, haemolymph was collected from treated larvae in a 1.5-ml tube containing a few granules of phenylthiocarbamide (Sigma-Aldrich) to prevent melanization. Haemocytes were separated from plasma by centrifuging at 4 °C for 5 min at 300g. A reaction volume of 200 µl consisted of 180 µl of 10 mM DOPA in PBS (pH 7.4) and 20 µl of plasma. Absorbance was measured using a VICTOR multi-label plate reader (PerkinElmer) at 490 nm. PO activity was expressed as ΔABS per min per µl of plasma. Each treatment was replicated three times with independent samples.Measurement of nitric oxideThe NO was indirectly quantified by measuring its oxidized form, nitrate (NO3−), using the Griess reagent of a Nitrate/Nitrite Colorimetric Assay Kit (Cayman Chemical). Fifth-instar larvae were injected with 1 µl of heat-killed E. coli TOP10 (2.4 × 104 cells per larva) using a Hamilton microsyringe along with 1 µl of the test compound. Haemolymph was collected from each sample 1 h post infection. A 150-μl volume of haemolymph from three L5 larvae was collected and homogenized in 350 μl of 100 mM PBS pH 7.4 with a homogenizer (Ultra-Turrax T8, Ika Laboratory). After centrifugation at 14,000g for 20 min at 4 °C, the supernatant was used to measure the nitrate amounts, and the total protein was measured in each sample by a Bradford assay. The samples were analysed in a 200-μl final reaction volume. Briefly, 80 μl of samples were added to the wells, then 10 μl of enzyme cofactor mixture and 10 μl of nitrate reductase mixture were added. After incubation at room temperature for 1 h, 50 μl of Griess reagent R1 and immediately 50 μl of Griess reagent R2 were added to each well. The plate was left at room temperature for 10 min for colour development. For a standard curve to quantify the nitrate concentrations of the samples, nitrates with final concentrations of 0, 5, 10, 15, 20, 25, 30 and 35 μM in a 200-μl reaction volume were used. The absorbance was recorded at 540 nm on a VICTOR multi-label plate reader. Our measurements used three larvae per sample, and we repeated the treatment with three biological samples.Galleria injection assaysPrecultures of X. szentirmaii DSM wild-type strain and the mutants thereof were grown in LB medium and inoculated into fresh cultures at an OD600 of 0.1. Cells were grown to exponential phase (OD600 ≈ 1) and then diluted to an OD600 of 0.00025. A 5-µl volume of the diluted bacterial culture was injected into the last left pro-leg of the larvae (LB medium as a negative control). G. mellonella larvae were kept at 4 °C for 10 min before injection. After infection, the larvae were incubated at 25 °C. Dead Galleria larvae were frozen at −20 °C, then at −80 °C, and freeze-dried for one day. Freeze-dried larvae were ground. Every injection experiment was aliquoted into two portions, one of which was extracted with 25 ml of acetone/ethyl acetate (vol/vol, 1:1) while the other one was extracted with acetone/methanol. Extracts were dried and resuspended in 3 ml of acetonitrile/water (1:1, vol/vol) with a tenfold dilution for HPLC-MS-UV analysis. To compare the survival percentage of G. mellonella larvae infected with the WT strain and mutants and to determine median lethal time (LT50) values, Kaplan–Meier curves were generated by GraphPad PRISM 8.4.3.Cytotoxicity assaysHepG2 cells (hepatoblastoma cell line; ACC 180, DSMZ) were cultured under conditions recommended by the depositor, and cells were propagated in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum. To determine the cytotoxicity of test compounds, cells were seeded at 6 × 103 cells per well of 96-well plates in 120 μl of complete medium. After 2 h of equilibration, compounds were added in serial dilution in 60 µl of complete medium. Compounds as well as the solvent control and doxorubicin as an in-assay positive control (IC50 of 0.06 ± 0.01 µg ml−1) were tested as duplicates in two independent experiments. After 5 days of incubation, 20 μl of 5 mg ml−1 MTT (thiazolyl blue tetrazolium bromide) in PBS was added per well, and the cells were further incubated for 2 h at 37 °C. The medium was then discarded and cells were washed with 100 μl of PBS before adding 100 μl of 2-propanol/10 N HCl (250:1) to dissolve the formazan granules. The absorbance at 570 nm was measured using a microplate reader (Tecan Infinite M200Pro with Tecan iControl 2.0), and cell viability was expressed as a percentage relative to the respective solvent control. IC50 values were determined by sigmoidal curve fitting using GraphPad PRISM 8.4.3.Statistical analysisIn Fig. 5d,e,g,j,k, means were compared using an LSD test of one-way ANOVA using POC GLM of the SAS program (SAS Institute, 1989) for continuous variables and discriminated at type I error = 0.05. The results were plotted using Sigma Plot 12.0.Reporting SummaryFurther information on research design is available in the Nature Research Reporting Summary linked to this Article. More

Description of test samples and theirs preparation84 samples of recycled rubber granules with a particle size of 0.5 to 4 mm, produced for the construction of sport field surfaces, were tested. The samples of rubber granules were collected from 17 sport fields and 67 samples rubber granules were supplied by recyclers. Research included 57 samples of SBR granules and 27 samples of EPDM granules. The numbers of samples in relation to their sources of origin are shown in Fig. 1.Figure 1Number of samples of the tested SBR and EPDM granules in relation to their sources of origin.Full size imageThe samples were taken from the surface of sport fields with artificial turf in accordance with the laboratory instructions or delivered to the laboratory by recyclers. The mass of the granulate samples delivered for testing was approx. 0.5 kg. Sampling from sport fields was carried out using a scheme based on 6 sampling points, shown in Fig. 2, in accordance with point 4 of the FIFA guidelines: “Quality Programme for Football Turf. Handbook of Test Methods for Football Turf”. The number and weight of granular samples and the locations of the granular sampling points on the field indicated in the aforementioned guidelines are indicated in order to obtain a representative homogenized granular sample for the tested field42.Figure 2Scheme of distribution of granulate sampling points on a sport field. Designation:
location and numbers of granulate sampling points.Full size imageAt the designated points (1 ÷ 6), 6 samples of granulate were collected. The collected and secured samples were stabilized in the laboratory conditions of natural drying, in which the moisture of the sample was in equilibrium with the ambient moisture. After stabilization, the samples were purified and homogenized to give a pooled sample. Images of exemplary SBR and EPDM granules used in the study are shown in Fig. 3. The average values of the physical parameters of the tested rubber granules are given in Table 1.Figure 3Samples taken from sport fields: (a) SBR granules, (b) EPDM granules.Full size imageTable 1 Some physical parameters of the tested SBR and EPDM granules (data from the Technical Data Sheets provided by the recyclers).Full size tableSamples weighing at least 100 g were taken from the granulate samples using the quartering method. This way allowed to ensure full qualitative and quantitative compliance of the sample composition with the composition of the analyzed material. Samples for testing the content of PAHs were grounded by grinding in a cryogenic mill 6770 Freezer/Mill, by SPEX SamplePrep LLC. Samples for testing other substances were not crushed.The scope and methods of testing rubber granulesThe scope of the research on rubber granules included: content determination of the PAHs, leached elements, organotin compounds and PAHs. In all samples of rubber granules, the content of 8 polycyclic aromatic hydrocarbons, resulting from the REACH Regulation, was determined: benzo[a]pyrene (BaP), dibenz[a,h]anthracene (DBAhA), benzo[e]pyrene (BeP), benz[a]anthracene (BaA), chrysene (CHR), benzo[b]fluoranthene (BbFA), benzo[j]fluoranthene (BjFA) and benzo[k]fluoranthene (BkFA). The content of indeno[1,2,3-cd]pyrene (IcdP), benzo[ghi]perylene (BghiP), phenanthrene, anthracene, fluoranthene, pyrene and naphthalene was determined for 38 samples from recyclers, additionally, that the number of PAHs covered by the requirements of the document43 was increased by 7.The leaching tests of elements and organotin compounds were carried out for 18 samples and the leachability of PAHs and elements were carried out for 4 samples. The tests were carried out with the methods listed below, using the following apparatus.The content and leachability of PAHs from rubber granules was determined by gas chromatography with tandem mass spectrometry (GC–MS/MS) using a gas chromatograph coupled with a mass detector GCMS/MS/7890B/7000C. The method was chosen because of the high sensitivity and selectivity obtained for low PAHs levels when used GC–MS/MS, compared to other commonly used analytical techniques such as high-performance liquid chromatography (HPLC) combined with UV, fluorescence or diode array detector (DAD). In studies carried out with the use of the above-mentioned techniques trace amount of PAHs identification is easily interfered by sample matrix and other components if only based on retention44.Determination of leaching of elements: Al, Sb, As, Ba, B, Cd, Co, Cu, Pb, Mn, Hg, Cr, Ni, Se, Sr, Sn, Zn and elution of the Cd, total Cr, Pb, Sn, Zn from rubber granules was carried out by the inductively coupled plasma mass spectrometry (ICP-MS) method with the use of Agilent 7900 ICP-MS (Agilent Technology, Santa Clara, CA, USA). The selected method is characterized by a low limit of quantification, which stands out among other instrumental methods used in elemental analysis, such as ICP-OES or AAS (Inductively coupled plasma–optical emission spectrometry or atomic absorption spectrometry). It is also characterized by high sensitivity and precision, selectivity enabling the simultaneous determination of many elements in complex matrices in a wide range of concentrations.Leachability of Cr (III) and Cr (VI) and elution of Cr (VI) from rubber granules were determined by high-performance liquid chromatography with inductively coupled plasma mass spectrometry (HPLC-ICP-MS) using Agilent 7700 Series ICP-MS with Agilent 1260 Infinity series HPLC (Agilent Technology, Santa Clara, CA, USA). The decision to use HPLC in conjunction with ICP-MS was dictated by the need to determine chromium in two oxidation states. In the case of the selected method, the speciation separation of Cr (III) and Cr (VI) takes place on the HPLC column, where Cr (III) and Cr (VI) are adsorbed. In the next step it allows for the separation and determination of Cr (III) and Cr (VI) in the ICP-MS spectrometer. The HPLC-ICP-MS method is characterized by a short analysis time and a low detection limit compared to the other spectrophotometric methods used for determination of Cr (VI). The leaching of organotin compounds was assessed on the basis of the results of total Sn leaching.Cold-vapor atomic absorption spectroscopy (CV-AAS) with the PerkinElmer FIMS 100 mercury analyser was selected for the Hg leaching study due to the use of a unique technique of mercury vapour measurement at room temperature. Among other alternative methods of Hg determination in aqueous solutions (ICP-MS or GF-AAS (graphite furnace atomic absorption spectrometry)), the selected method is distinguished by a low limit of quantification, simple preparation of samples for analysis, easy elimination of interference and short analysis time.Tests of the content of PAHsShredded samples of rubber granules were subjected to the ultrasonic extraction process for 1 h with the use of toluene as a solvent. Samples were taken from the obtained extract for chromatographic analysis. The analysis was carried out for the following conditions: dispenser operation mode: splitless, carrier gas: Helium: 1.8 ml/min, DB-EUPAH column with dimensions: 20 m × 180 µm × 0.14 µm (the 20 m column is in the form of a coiled wire), injection temperature: 275 °C. The PAHs were identified on the basis of mass spectra and retention times—Table 2.Table 2 Target and identification ions and retention times for the determined PAHs.Full size tableTests of the leaching of elements and organotin compoundsSamples of rubber granules for the study of the leaching of organotin elements and compounds were extracted in a solution of hydrochloric acid (HCl), with concentration 0.07 ± 0.005 mol/dm3 in temperature 37 ± 2 °C. Solutions for the determination the Cr(VI) and Cr(III) prepared by diluting the extraction solution to obtain the pH equal to 7.0 ± 0.5 by adding 1 ml of 0.07 mol/dm3 ammonia and 60 µL of 0.1 mol/dm3 EDTA solution. In parallel, a reagent blank was prepared, as the test samples were. The obtained extracts were analyzed by ICP-MS and HPLC-ICP-MS. The analyzes were performed for the isotopes of the elements: Al—27, Sb—121, As—75, Ba—137, B—11, Cd—111, 112, Cr—52, 53, Co—59, Cu—63, Pb—206, 207, 208, Mn—55, Hg—201, Ni—60, Se—78, Sr—88, Sn—118, 120, Zn—64, 66.Tests of the leachability of polycyclic aromatic hydrocarbons (PAHs) and elementsDetermination of the dry mass of the rubber granulate samples for the leachability tests was carried out in accordance with ISO 11465:199945, using a drying oven (Pol-Eco-Apparatus SLW-115 Top, Wodzisław Śląski, Poland) and analytical balances (SARTORIUS, Kostrzyn Wlkp. i Radwag, Radom, Poland).The rubber granulate samples were dynamically washed with deionized water according to EN 12457-4:200246 providing a ratio of 1 ml of liquid to 1 g of rubber granulate. The pH value of the water used for dynamic leaching did not exceed 6.7. Elution was performed using a bottle/tube roller mixer (Thermo scientific model, Thermo Fisher Scientific (China) Co., Ltd., Shanghai China). After washing, the effluents were left for 15 min and then filtered through 0.45 mm membrane filters using a pressure filtration device.The leachate obtained from dynamic leaching was subjected to the process of transferring PAHs from the water phase to the organic phase using the algorithm:

SPE column: C18 bed—6 ml/1000 mg;

activation: 10 ml of methanol, 10 ml of methanol:water (40:60) (v:v), flow: 1 ml/min;

sample:eluting solution of methanol (100 ml:10 ml), flow: 0.5 ml/min;

drying: minimum 15 min, maximum flow;

elution: 3 × 3 ml of dichloromethane, flow: 0.5 ml/min.

Collected filtrates were evaporated using a vacuum evaporator (IKA RV 05 basic, IKA WERKE GMBH & CO.KG, Staufen) up to 1 ml. Evaporated filtrates were subjected to the chromatographic analysis performed for the conditions as for the determination of PAHs content. The content of eluted PAHs and elements was related to dry mass of the rubber granulate in each sample.The devices were calibrated and checked on a current basis, including the analysis of control samples, before starting the measurements. Calibrations of the chromatograph, spectrometer and mercury analyzer were performed on solutions of certified reference materials and 2 control samples. The correlation coefficients obtained during the calibration were above 0.995 for all analyzed substances. The analysis of the control samples confirmed the accuracy of the calibration curves, which are the basis for the calculations. Measurements of the content/leachability of the tested substances were carried out for two parallel samples and a reagent blank sample, taking into account the results obtained from it in the analysis of analytical samples. The arithmetic mean of two parallel determinations was assumed as the result of the analytical measurement. Content/leachability conversions of test substances were performed using the GC–MS/MS MassHunter Workstation Software, LCP MHLauncher HPLC-ICP-MS and ACP-MS software and WinLab32 with an AA mercury analyzer FIMS100. More

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The data used here provides evidence that particular relationships may determine sex-specific patterns of witchcraft accusation. Cases where women were targeted frequently came from affinal kin, while those directed at men were often from unrelated individuals and blood relatives. Most previous research on factors that determine the sex of accused ‘witches’ has largely consisted of qualitative studies of a single society or a few societies48, or historical studies that have not tested for correlations49. Our findings, in support of the overarching hypothesis that accusations may be driven by various forms of competition, can be tentatively aligned with evolutionary literature on patterns of intrasexual and kin competition, intersexual conflict and polygamous mating30,31,50.Men were more often accused than women in our sample, although we did not have a prediction in relation to this. But the finding suggests how overall patterns of competition within relationships may contribute to societal ‘phenotypes’ of witches as male or female. The ethnography of the Ndembele perhaps indicates why women were less frequently targeted in Bantu societies: ‘in a case of witchcraft, the complainant is actuated by caprice, jealousy or pique; and the defendant is a person of wealth or popularity, and is always a man, for the women have neither wealth nor honor worth coveting’51.Our predictions about how the sex of accused ‘witches’ might be associated with particular relationship categories were supported. The majority of accusations targeting men came from unrelated individuals, which is unsurprising, as inclusive fitness52 would not mitigate the effects of competition between them. Blood relatives were the next most common relationship category directing accusations at men. This aligns with more recent studies indicating that witchcraft fears between family members are significant in parts of Africa, to the extent that they can be construed as ‘the dark side of kinship’53. In evolutionary terms, kin may compete with one another in environments where resources are limited30,31,50 and in societies with patrilineal inheritance related males, and particularly brothers, compete for resources in order to marry31. This aligns with an ethnographic observation that among the Banyoro witchcraft accusations often occurred between brothers over inheritance, but not between brothers and sisters, whose interests did not conflict21. The situations relating to accusations of men were also often connected to the acquisition of wealth and status, such as rivalry over village headmanships32, power struggles between a chief’s counsellors54 or disputes over inheritance55. These connections can be found in more recent contexts such as twentieth century Ghana, where notions of obtaining political power and wealth through occult means involving human sacrifice were pervasive56.Accusations of women were more likely to come from affines. Husbands were the largest category of affinal kin to accuse women (Supplementary Fig. 2). The higher rate of accusations from husbands to wives than wives to husbands aligns with evolutionary perspectives suggesting male coercion of females is a strategy to maximize male reproductive success39,41. Accusations of wives who were suspected of being unfaithful can be interpreted as a strategy for reducing investment in unrelated offspring35,41. In a case from the Shona a woman gave birth to a stillborn child. This was attributed to an affair before marriage, and was followed by divorce and the repayment of bridewealth to her husband, who commented she was ‘a witch, a woman who had killed her own child’48. Other ethnographic accounts suggest accusations of wives by husbands were an attempt to gain control within the marital relationship55.A significant number of accusations of women by affinal kin were from co-wives in polygynous marriages, and these were often notably associated with jealousy connected to a husband’s attention and investment32. Evolutionary models predict competition for reproductive resources would occur among co-resident breeding women57, as has been found to occur among the Mosuo of southwest China58. In the patrilocal social systems that are predominant in our sample, women disperse at marriage and are isolated from kin, so conflict may be more extreme30. This is consistent with ethnographic observations reporting that the relationship between co-wives in polygynous marriages was often (although not always) marked by conflict, and liable to produce witchcraft accusations38,59.There were accusations of women from other categories of their affinal kin (Supplementary Fig. 2). These again may result from competition for a husband’s time and resources between his kin and wife. New wives may be vulnerable in environments where they enter their husband’s families as unrelated strangers, and are potentially expendable, at least before the arrival of offspring. Some accounts of accusations indicate that accusations of wives by in-laws in patrilocal households are common29.Accusations directed at elderly individuals targeted women more often than men. This may form part of a broader pattern of geronticide: societies close to subsistence-level are documented as sometimes accepting the abandonment or killing of elderly people19,60. In modern Tanzania, ‘witches’ are mostly post-reproductive women, who are more likely to be murdered in periods of income shock19. This is also the case in contemporary Ghana, where accusations are frequently directed at middle-aged or elderly women, whose families may subsequently cease to provide them with financial or material assistance61. In our sample, elderly women may have been targeted more frequently as a result of longer female lifespans: in a polygynous society, men may marry younger women, so wives would be widowed at an earlier age than husbands. Among the Bantu, older men were accused, but some were possibly protected by their status.Accusers’ payoffs from accusations are not always explicit but they can be inferred. The most common outcome of accusations in our sample was that accused ‘witches’ were exiled from their communities or forced to move from where they were living. This would mean resources and cooperative assistance they would have used became available to their accusers or others nearby. Where the accused acquires a negative reputation, which was the second most common outcome, there may be a subtle removal of benefits, which may be preferred to direct ‘punishment’ as it is less costly62. Accusers’ gains need not be direct, as harming behaviours may reduce the overall pressure of competition in an environment28. 8% of accusations in the sample resulted in the acquisition of either resources or political positions from the accused, or in preventing the accused from acquiring them. Where the accused were penalised in other ways, such performing ceremonies to reconcile with accusers, this is perhaps akin to classic cooperation models involving the punishment of defectors (although the accused may not actually be uncooperative)11, providing accusers with subordinate partners who offer fitness benefits to avoid more serious allegations63. Where an accusation does not ‘stick’, ethnographic accounts sometimes indicate it was reversed through divination or ordeal54. In other cases, for various reasons accusations are short-lived and forgotten about4. Finally, although not tested in this dataset, accusers may gain informal prestige and dominance, an outcome analogous to competitive punishment63.Not all of the cases in our dataset support the hypothesis that witchcraft accusations are a mechanism for competition. There is a significant proportion where the accusation of a particular individual appears to be incidental, or dependent a on circumstantial association between the ‘witch’ and a negative event. Such accusations are unlikely to provide accusers with a competitive advantage. There are several possible explanations for such cases. They are in line with the hypothesis that witchcraft belief arises from attempts to identify the cause of an impactful misfortune3,4. Cultural evolutionary explanations of witchcraft beliefs suggest that they are a maladaptive attempt to explain misfortune. Although it is inaccurate, belief in witches is maintained through bias and selective inattention to evidence that would otherwise counter it64. Alternatively this could be viewed under the contention that superstitious beliefs (or errors in attributing cause and effect) are broadly adaptive if they occasionally lead individuals to acts which provide them with fitness benefits65.Although witchcraft accusations may be a mechanism for mitigating the damage to accusers’ reputations in harmful competitive acts, as with any behavioural strategy it is not without risks. Accusers may suffer costs in the form subsequent reputational damage or counter-accusations, as with punishment63, depending on factors such the level of support for an accusation by other members of the community.One limitation of our dataset is that it contains realized allegations of witchcraft, that cannot be tested against baseline population measures. We could not examine the risk that a particular individual, such as an elderly woman, would be accused. Instead, the analysis shows the odds, given an accusation occurred, that the ‘witch’ was male or female, given certain predictors. For example, if the accused was elderly, there are increased odds they were female rather than male.A dataset using historic witchcraft cases is almost certainly affected by selection bias. Cases with sensational outcomes are more likely to be reported, and cases that are dismissed or where the accused removes themselves from their accusers are liable to be overlooked19. Most incidents in our sample were reported anecdotally. Obtaining a random sample of witchcraft accusations within a population is challenging, if not impossible1,66. Attempts to systematically collect cases within a given location and timeframe cannot guarantee that all are brought to the attention of researchers19. Comparative studies of this kind usually use all the data that is available and control for confounding effects. Our sensitivity analyses suggest the large number of accusations of men in the dataset probably reflects patterns of accusations in these societies, rather than male-focused bias from ethnographers. There are many accounts of cultures where witches are predominantly male33,34,49. But the accuracy of historic ethnographic accounts cannot be verified, especially in relation to one-off events such as witchcraft accusations, just as it is unclear how much uncertainty there is in the ethnographic record overall67. Ethnographers may not always have noted the characteristics of the individuals involved, or there may be times where they were mistaken in reporting the circumstances surrounding an accusation. There are several explanations for cases where the identities of accusers or purported victims of witchcraft were not reported. Not all cases had identifiable ‘victims’, for example when the accused was thought to have used witchcraft to promote their own success, or ethnographers could not denote the relationship between the accused and their accusers when suspicions of witchcraft were communicated through general gossip. In a small number of cases, ethnographer perspectives on accusations (and possible inability to access further information) are salient, as they may ascribe more importance to one relationship over another in reporting a case, such as a witch’s envy of their victim, or a witch’s argument with an accuser.However, it is likely that ethnographers were for the most part accurate in documenting variables of interest such as the sex of an accused individual and their relationships with accusers. There is less certainty in relation to the situation connected to an accusation, especially taking into recent research that indicates the prevalence of phenomena such as the misperception of causation68,69. Our attempts to account for such possibilities with sensitivity analyses and meta-data on the production of ethnographies cannot conclusively provide reassurance that bias has not affected results, and so this section of the analysis should be treated with caution and regarded as exploratory. The situations documented in our study do however align with accounts of accusations from more contemporary observers and studies from different geographic locations, suggesting that similar causes of accusations arise convergently in different societies. For example in modern contexts accusations have led to accusers gaining land or property in India6 and cessation of the obligation to provide material and financial assistance to elderly relatives in Ghana61. One advantage of our cross-cultural data being drawn from numerous ethnographies is that it is not reliant on the perspective of one individual, meaning that random perceptual error or individual (as opposed to cultural) bias is more likely to be mitigated in the results than would be the case in the study of a single culture by one ethnographer.As a further limitation, we were reliant on accessible ethnographic records from the best-documented societies. Although selection bias in favour of better described societies is present in our sample, this should not impact the main aim of this research, which is to understand the determinants of witchcraft accusations being directed at male or female targets.Overall our findings may indicate allegations of witchcraft stem from diverse forms of competition between individuals. This aligns with evolutionary approaches to competition and conflict. Accusations may provide fitness benefits by allowing individuals to target competitors, but the exact form and direction of competition is determined by aspects of socio-ecology. This in turn influences which sex is most likely to be accused and the overall portrayal of witches in a society. Accusations may be more likely to occur in some relationships rather than others, when there is a gain for the accuser, as in disputes over inheritance and property, or where another individual may pose a threat, or by simply reducing numbers of competitors. The success of witchcraft accusations in removing competitors and their flexibility as an adaptive strategy may explain their widespread distribution. More