Biosynthesis of Antibacterial Iron-Chelating Tropolones in Aspergillus nidulans as Response to Glycopeptide-Producing Streptomycetes

Abstract

The soil microbiome comprises numerous filamentous fungi and bacteria that mutually react and challenge each other by the production of bioactive secondary metabolites. Herein, we show in liquid co-cultures that the presence of filamentous Streptomycetes producing antifungal glycopeptide antibiotics induces the production of the antibacterial and iron-chelating tropolones anhydrosepedonin (1) and antibiotic C (2) in the mold Aspergillus nidulans. Additionally, the biosynthesis of the related polyketide tripyrnidone (5) was induced, whose novel tricyclic scaffold we elucidated by NMR and HRESIMS data. The corresponding biosynthetic polyketide synthase-encoding gene cluster responsible for the production of these compounds was identified. The tropolones as well as tripyrnidone (5) are produced by genes that belong to the broad reservoir of the fungal genome for the synthesis of different secondary metabolites, which are usually silenced under standard laboratory conditions. These molecules might be part of the bacterium-fungus competition in the complex soil environment, with the bacterial glycopeptide antibiotic as specific environmental trigger for fungal induction of this cluster.

Keywords: Aspergillus nidulans; Streptomyces; fungal-bacterial co-cultivation; glycopeptide antibiotics; structure elucidation; tropolones.

Figure 1

A growth inhibition zone is formed between colonies of S. mobaraensis and A. nidulans during co-cultivation. Confrontation assay of A. nidulans strain AGB552 with different Streptomycete strains (S. mobaraensis, S. coelicolor and S. cellulosae). 10⁴ spores of A. nidulans (left colony) were point inoculated with 3 cm distance to 10⁴ spores of Streptomycete strains (right colony) on GYM medium and incubated at 30°C for 6 days.

Figure 2

Co-cultivation of A. nidulans with S. mobaraensis induces the production of anhydrosepedonin (1), antibiotic C (2) and 2,4-dihydroxy-3-methyl-6-(2-oxopropyl)benzaldehyde (DHMBA, 3). (A) A. nidulans reference strain AGB552 (rf) was co-cultivated with S. mobaraensis, S. coelicolor and S. cellulosae in liquid medium for 2 days. SMs were extracted from medium and analyzed with LC-MS/MS equipped with a DAD. The relative absorbance at 366 nm is shown. As control, only A. nidulans was cultivated. (B) Extracted ion chromatograms (EIC) for DHMBA (3).

Figure 3

The presence of the glycopeptide antibiotics bleomycin and phleomycin induces the production of the dbaI-dependent metabolites 1, 2, 4, and 5. A. nidulans reference strain AGB552 (rf) was cultivated in liquid medium with or without the presence of 0.1 μg/ml bleomycin (A) or 1 μg/ml phleomycin (B) for 2 days. SMs were extracted from medium and analyzed with LC-MS/MS equipped with a DAD. The relative absorbance at 366 nm is shown.

Figure 4

Overview of identified metabolites and their structures. (A) Chemical structures of anhydrosepedonin (1), antibiotic C (2), DHMBA (3), azanidulone (4), tripyrnidone (5), sepedonin (6) and TAL, (7). The stereochemistry of 6 is not known. (B) Selected 1,1 ADEQUATE (red arrows) and ¹H,¹³C HMBC (black arrows) correlations of compound 5.

Figure 5

Expression of genes from AN7893 (troA) to AN7903 (dbaI) is induced in presence of phleomycin. (A) Scheme of dba gene cluster (orange) and neighbored genes (blue) on chromosome II of A. nidulans. (B) Relative gene expression levels of AN7890-AN7904 in reference strain AGB552 (rf), ΔdbaA and ΔdbaG after cultivation with (+) and without (–) the addition of 1 μg/ml phleomycin. Expression of the housekeeping genes h2A, gpdA and rps15 were used for normalization. Error bars represent the standard error of two biological replicates.

Figure 6

The dba/troA cluster produces the tropolones anhydrosepedonin and antibiotic C. (A) The indicated strains were cultivated in liquid medium supplemented with 1 μg/ml phleomycin for 2 days. Ethyl acetate extracts of culture filtrates were analyzed with LC-MS/MS equipped with a DAD. The relative absorbance at 366 nm is shown. (1) anhydrosepedonin, (2) antibiotic C, (3) DHMBA, (4) azanidulone, (5) tripyrnidone. (B) Extracted ion chromatograms of 1–5 in positive ionization mode. Extracted ion chromatograms of all compounds are shown in Supplementary Figure 12.

Figure 7

Proposed biosynthesis of compounds 1–6. Compounds shown with names have been detected, compounds without names are hypothetical. The stereochemistry of 6 is not known.

Figure 8

A. nidulans produces triacetic acid lactone (TAL, 7). The production is increased in the presence of glycopeptide antibiotics bleomycin and phleomycin but is independent from the PKS DbaI. Reference strain AGB552 (rf) and ΔdbaI were cultivated in liquid medium with or without the presence of 0.1 μg/ml bleomycin or 1 μg/ml phleomycin. SMs were extracted from medium and analyzed with LC-MS/MS. Extracted ion chromatograms (EIC) for m/z = 127.0390 in positive ionization mode are shown. TAL was identified by comparison with commercial TAL (Sigma-Aldrich, lower panel).

Figure 9

SMs of dba/troA cluster of A. nidulans are siderophores. Bioassay for the identification of siderophores with monoprotic keto-hydroxy bidentate ligands (KHBL). A. nidulans reference strain AGB552 (rf) and ΔdbaI were cultivated in liquid medium with phleomycin to induce the tropolone production. The metabolites were extracted, dissolved in methanol and spotted on filter disks. Pure methanol served as negative control. Solid LB medium without and with the iron-chelator 2,2'-dipyridyl (600 μM) were inoculated with M. morganii SBK3. The filter disks containing the metabolite extracts were placed on the agar plates, which were cultivated for 3 days at 37°C. The inhibition zone on LB without 2,2'-dipyridyl was 1.6 ± 0.1 cm (p ≤ 0.01, n = 4) for the reference strain AGB552 (rf) in comparison to 0.2 ± 0.4 cm for ΔdbaI. On LB with 2,2'-dipyridyl the rf extract induces an inhibition zone of 2.6 ± 0.4 cm (***p ≤ 0.001, n = 4) (blue bar) surrounded by a growth zone of 3.9 ± 0.5 cm (p ≤ 0.001, n = 4) (gray bar). No inhibition neither a growth zone was detected around the extract from ΔdbaI in presence of 2,2'-dipyridyl [n = 4, for significance testing a T-test (ri-rj) was used].

Figure 10

Anhydrosepedonin/antibiotic C inhibit the growth of Streptomyces spp. Bioactivity tests with 2 × 10⁶ spores of S. mobaraensis, S. coelicolor, and S. cellulosae distributed on whole GYM medium plates. (A) The SM extract of A. nidulans reference strain AGB552 (rf) and ΔdbaI was dissolved in methanol and 10 μl were spotted in a filter disk which was incubated with the inoculated plates for 2 days at 30°C. The extract of the reference strain induces a growth inhibition zone for all tested Streptomyces strains. Methanol served as control. (B) Anhydrosepedonin/antibiotic C were isolated from fungal extracts and 12.5 μl of the extract (stock concentration 2 mg/ml) resuspended in methanol was spotted on a filter disk and methanol served as control. The inhibition zone induced by anhydrosepedonin/antibiotic C is 1.9 cm (S. mobaraensis), 1.7 cm (S. coelicolor) and 1.5 cm (S. cellulosae) after 2 days of incubation at 30°C. (C) Anhydrosepedonin (1) and antibiotic C (2) were purified and tested separately. The compounds were dissolved in methanol to a final concentration of 2 mg/ml. Filter disc were coated with 12.5 μl extract and incubated with the Streptomyces inoculated GYM plates for 2 days at 30°C. Both metabolites induce an inhibition zone with a diameter of 1.4 cm (S. mobaraensis), 1 cm (S. coelicolor and S. cellulosae). Methanol served as control.