Natural products, including a new caboxamycin, from Streptomyces and other Actinobacteria isolated in Spain from storm clouds transported by Northern winds of Arctic origin

Introduction

Members of the Phylum Actinobacteria, also known as actinomycetes, are major producers of secondary metabolites of medical and biotechnological use, being the Streptomyces genus the most prolific source of natural products of pharmaceutical interest. At present, there is an urgent need of new secondary metabolites, essentially antibiotics to combat pathogenic resistant bacteria, but also compounds with applications as anticancer agents, antivirals, antiparasitics and immunosuppresants, among others (Ramirez-Rendon et al., 2022).

New trends in natural products discovery are now focused in the search for novel producers in the less explored environments of our planet, including deep-sea habitats and the atmosphere. Regarding the biogeography of the Streptomyces genus, it is known that they are ubiquitous since they are widespread not only in terrestrial habitats, as was established in the last century, but also in oceanic and atmospheric environments (Braña et al., 2015; Sarmiento-Vizcaíno et al., 2017a). An atmospheric dispersal model, coupled with the main Earth hydrological cycle, was proposed to explain the biogeography of Streptomyces species (Sarmiento-Vizcaíno et al., 2016).

Highly diverse bioactive Streptomyces and Nocardiopsis species were isolated from multiple precipitation events happened in the Cantabrian Sea Coast, Northern Spain, with prevalent Western and North Western winds (Sarmiento-Vizcaíno et al., 2021, 2018). It has been suggested that during different precipitation events and by changing the latitude of the sampling place, it is possible to isolate a great diversity of Actinobacteria producing a remarkable reservoir of natural products with relevant biological activities, thus revealing the pharmaceutical and biotechnological potential of the atmosphere as a relevant source for natural products discovery (Sarmiento-Vizcaíno et al., 2021).

As a result of a multidisciplinary approach, we explored here the phylogenetic and biosynthetic diversity of Actinobacteria obtained in the Cantabrian Sea Cost, Spain, over 3 years’ time in precipitation events with Northern winds, the still remaining unexplored source of precipitations in this geographical region. This culture-dependent approach involved taxonomical and phylogenetic analyses as well as meteorological analyses. Antimicrobial assays, metabolic profiling by LC-UV-MS, followed by identification by comparison to natural products databases, purification and structural elucidation, were used to uncover the biosynthetic diversity of these atmospheric-derived Actinobacteria.

Materials and methods

Sampling of atmospheric precipitations

Rainwater, hailstone and snow samples were collected within the years 2013–2016 in Northern Spain, at the Asturias Cantabrian Sea coastal region, a very wet and rainy region whose climate is influenced by the Atlantic Ocean (Figure 1). Samples of 2–3 ml were taken in sterile recipients mainly at the coastal locality of Gijón (43° 32′ N, 5° 39′ W), but also in Oviedo (43° 21′ N, 5° 52′ W), and plated on selective media as has been described (Braña et al., 2015; Sarmiento-Vizcaíno et al., 2016). During most of the precipitation events sampled the prevailing wind direction was Northern.

FIGURE 1

FIGURE 1. Sampling locations. Overview of the Western Atlantic Ocean and the sampling locations in Northern Spain indicated by stars.

Actinobacteria strains isolation and culture

Actinobacteria strains were isolated after plating of atmospheric samples on selective agar media, prepared with cycloheximide (80 μg mL–1) as antifungal and nalidixic acid (20 μg mL–1) as anti-Gram negative bacteria, using MOPS BLEB 1/6 (Oxoid) basal medium as previously reported (Sarmiento-Vizcaíno et al., 2016). The selection plates were prepared either with distilled water or with a supplement of 3.5% NaCl. Colonies were selected based on different morphological features and pigment production on R5A agar plates after incubation for 2–3 weeks at 28 °C. Pure cultures were conserved in 20% glycerol at −20°. MOPS BLEB 1/6 was also used as the basal medium for halotolerance studies, adding NaCl at 0, 3.5, 7.0, and 10.5% (w/v) final concentrations. For secondary metabolite production R5A medium was used as previously described (Sarmiento-Vizcaíno et al., 2018).

Air mass backward trajectories analyses

Backward trajectories of the air masses were generated using the HYSPLIT model (Hybrid Single Particle Lagrangian Integrated Trajectory) from the Global Data Assimilation System of National Oceanic and Atmospheric Administration, USA (Stein et al., 2015) to estimate the long-range transport journey of air masses that originated the precipitation events herein studied. Five-day backward trajectories were obtained using the NOAA model (http://ready.arl.noaa.gov/hypub-bin/trajtype.pl?runtype=archive) to track the transport pathways of air masses and determine the origin of diverse air parcels. Sampling locations were used as the backward trajectory start point with altitudes over the sea level of 30, 1,000 and 3,000 m (Gijón), and 300, 1,000 and 3,000 m (Oviedo) as previously reported (Sarmiento-Vizcaíno et al., 2021).

Antimicrobial bioassays

To determine the antimicrobial activities of isolates, agar diffusion methods were used against a panel of the following indicator microorganisms: the Gram-positive bacteria Micrococcus luteus ATCC 14452 and Streptomyces 85E ATCC 55824, the Gram-negative Escherichia coli ESS, and the yeast Saccharomyces cerevisiae var. Carlsbergensis. Analyses were performed in TSA1/2 (Merck) against bacteria and in Sabouraud 1/2 (Pronadisa) against yeast, and bioassays were carried out both with agar plugs (7 mm diameter) and also with 6-mm-diameter AA Discs (Whatman), loaded with ethyl acetate extracts of bioactive isolates, as previously reported (Sarmiento-Vizcaíno et al., 2021). Agar plugs assays detect all diffusible compounds produced by actinobacterial strains, both polar and apolar, whereas the AA discs bioassays only detect diffusible apolar molecules extracted with ethyl acetate.

16S RNA analysis identification and phylogenetic analysis

DNA was extracted with a microbial isolation kit (Ultra Clean, MoBio Laboratories, Inc.) for taxonomic identification of the strains, using standard methods for checking the purity (Russell and Sambrook, 2001). Partial 16S rRNA gene sequences of the bacterial strains were obtained by PCR amplification using the 616V (forward) and 699R (reverse) primers (Arahal et al., 2008) (Braña et al., 2015), using the BLAST program (Basic Local Alignment Search Tool) against the NCBI (National Centre for Biotechnology Information). The nucleotide sequences were compared to sequences in databases, submitted and deposited in the EMBL database with accession numbers OL587568-OL587585. Phylogenetic analysis of the strains based on 16S rRNA gene sequences was performed as previously reported (Sarmiento-Vizcaíno et al., 2021).

Chromatographic analysis

Plugs of R5A plates (about 7 ml) were extracted using ethyl acetate in neutral and acidic (with 1% formic acid) conditions. After evaporation, the organic fraction residue was redissolved in 100 µL of a mixture of DMSO and methanol (50:50). The analyses of the samples were performed by reversed phase liquid chromatography as previously described (Braña et al., 2015; Sarmiento-Vizcaíno et al., 2016).

Identification of compounds by LC-UV-Vis and LC-UV-HRMS analyses

Samples were first analyzed and evaluated using an in-house HPLC-UV-Vis database. LC-UV-HRMS analyses were carried out as has been described (Pérez-Victoria et al., 2016; Sarmiento Vizcaíno et al., 2018) and major peaks in each chromatogram were searched against the MEDINA’s internal database and also against the Dictionary of Natural Products (DNP) (Chapman and Hall, 2015).

Purification of novel compounds

For purification of the secondary metabolites produced, Streptomyces sp. A-177 was cultured in 40 Erlenmeyer flasks (250 ml), each containing 50 ml of R5A medium supplemented with 3%. DMSO inoculated with spores and incubated in an orbital shaker at 28°C and 250 rpm during 7 days. The cultures were centrifuged and extracted with ethyl acetate acidified with 1% formic acid. The extracts were dried and the residue was subsequently redissolved in a small volume of acetonitrile and DMSO (1:1). The same peaks were also found in the organic extract of the culture pellets, which were dried and redissolved in the same way. The desired compounds were purified by preparative HPLC using a SunFire C18 column (10 μm, 10 × 250 mm, Waters). The purification was performed in two steps. The mobile phase was a mixture of 90% acetonitrile and TFA 0.1% in the first step and 80% acetonitrile and TFA 0.1% in the second one, in isocratic conditions at 5 ml/min. In both cases, the solutions containing the collected peaks were evaporated in rotavapor and finally lyophilized, resulting in 3.9 mg of caboxamycin B (1).

LC-HRMS and NMR analyses

HRMS spectra (ESI-TOF) were acquired using a Bruker maXis QTOF mass spectrometer coupled to an Agilent 1,200 Rapid Resolution HPLC. NMR spectra were recorded at 297 K on a Bruker Avance III spectrometer (500 and 125 MHz for 1H and 13C, respectively) equipped with a 1.7 mm TCI MicroCryoProbeTM. 1H and 13C chemical shifts were reported in ppm using the signals of the residual solvent as internal reference (δH 2.50 and δC 39.5 ppm for DMSO-d6).

Results

Selection of bioactive Actinobacteria from precipitation events in Northern Spain

The strains studied herein were isolated and characterized in Spain by sampling multiple precipitation events, as previously reported (Sarmiento-Vizcaíno et al., 2021). After a dereplication process, involving antimicrobial activity, phenotypical features, metabolic profiling and meteorological analyses, 18 morphologically different bioactive strains, isolated in precipitations events with prevalent Northern winds, were here selected. To analyze antibiotic production, the isolates were initially screened for antimicrobial activity against bacteria and fungi as indicator microorganisms (Table 1), using agar diffusion methods as recently reported (Sarmiento-Vizcaíno et al., 2021). Table 1A shows the results of bioassays with agar plugs and Table 1B displays the results of ethyl acetate extracts using agar diffusion with AA discs. Most of the strains display strong antibiotic activities against Gram-positive bacteria, such as M. luteus and Streptomyces 85E, whereas 10 strains were active against the Gram-negative E. coli ESS and seven strains against the yeast S. cerevisiae.

TABLE 1

TABLE 1. Antibiotic activities of Actinobacteria against a panel of Gram-negative, Gram-positive bacteria and fungi.

Air mass backward trajectories analyses

Selected bioactive strains were isolated in Northern Spain during years 2013, 2015 and 2016 in nine precipitation events in which the prevailing wind direction was Northern. Samples were mostly collected in Gijón, sampling place, during eight events of rainfall and hailstone, and one snow event was sampled in Oviedo.

NOAA meteorological analyses were addressed to estimate the origin and trajectories of the air masses that caused different precipitation events in which the isolate were obtained. As shown in Figure 2, 5 days HYSPLIT backward trajectories were performed in the sampling locations at three different arriving heights. In all events the air masses were transported by prevalent Northern Winds, and the backward trajectories showed that air masses were mainly originated in the Arctic Ocean, close to the North Pole, and traveled toward South Europe, such as the sampling place in North Spain, thus revealing a main oceanic route from the Arctic and the North Atlantic Oceans. In some events, the air masses trajectory also involves terrestrial routes in Europe, Canada and Greenland to downwind areas.

FIGURE 2

FIGURE 2. Five-day backward trajectories of air masses generating the storms that caused the precipitation events. They were calculated with the NOAA HYSPLIT Model with three different transects with different arriving height as previously reported (Sarmiento-Vizcaíno et al., 2018). The sampling locations were used as the backward trajectory start point with altitudes over sea level of 30, 1,000 and 3,000 m (Gijón), 300, 1,000 and 3,000 m (Oviedo). Black asterisks indicate the sampling places. The strains isolated and the dates of the precipitation events are shown on the top of the figures.

Taxonomic identification, phylogenetic analyses and distribution of bioactive isolates

The nucleotide sequences of the 16S rRNA gene fragments of bioactive strains were deposited in the EMBL database and accession numbers are displayed in Table 2, which also shows the closest phylogenetic relatives with indication of their isolation source. A generalized feature in all strains is their salt tolerance (Table 2). Most of the isolates tolerate NaCl concentrations, up to 7%, which is in agreement with previous reports (Sarmiento-Vizcaíno et al., 2021, 2018).

TABLE 2

TABLE 2. Phylogenetic diversity of Actinobacteria isolates.

The results of the phylogenetic analyses based on 16S rRNA gene alignments, revealed that all 18 strains share 99.4–100% identity with known actinobacterial species, belonging to three different genera among the Phylum Actinobacteria. Among 18 studied strains, 15 belonged to the Streptomyces genus, having their closest homologues in species previously isolated from terrestrial, marine and atmospheric environments (Table 2), in association with lichens, plants, terrestrial invertebrates (honeybees, beetles), marine macroalgae and marine invertebrates. In addition, two strains belonging to the Micromonospora genus were obtained in two precipitation events (Table 2). This genus has been proposed as a source for bioactive natural products discovery (Hifnawy M. S. et al., 2020; Hifnawy M. et al., 2020). Also a strain belonging to the genus Nocardiopsis was identified in one precipitation event. A neighbour-joining phylogenetic tree (Figure 3) was built to estimate the relationship between the strains and their nearest phylogenetic relatives.

FIGURE 3

FIGURE 3. Neighbor-joining phylogenetic tree generated by distance matrix analysis of 16S rRNA gene sequences from atmospheric Actinobacteria, Streptomyces, Micromonospora and Nocardiopsis (highlighted) and their nearest phylogenetic relatives. The numbers on branch nodes indicate bootstrap values (1,000 resamplings; only values > 50% are shown). Bar represents 1% sequence divergence.

Identification of bioactive secondary metabolites

The biosynthetic potential of the strains studied herein was uncovered by metabolite profiling analyses of ethyl acetate extracts of strains. Extracts were analyzed by LC-UV and LC/HRMS in combination with searches in UV and MS databases or the Dictionary of Natural Products (DNP), after generation of a molecular formula of each peek based on HRMS results. Complex metabolic profiles were obtained, with most of the strains producing multiple natural products in R5A medium (Supplementary Material S1). As an example, Figure 4 displays UV_{210 nm} chromatograms corresponding to three samples.

FIGURE 4

FIGURE 4. UV_210nmchromatogram of ethyl acetate extracts from Streptomyces sp A-163, Streptomyces sp A-177 and Micromonospora sp. A-199. Dereplicated components in sample A-163: 1) Hydroxystreptazolin, 2) Streptazolin, 3) Cyclo (leucylprolyl), 4) C₁₂H₂₄O₄ (molecular formula not found in the Dictionary of Natural Products for prokaryotes), 5) C₁₈H₁₆O₄ (no UV coincidence in the Dictionary of Natural Products for prokaryotes), 6) C₁₁H₁₃NO₂ (no UV coincidence in the Dictionary of Natural Products for prokaryotes), 7) C₁₂H₂₀O₃ (no UV coincidence in the Dictionary of Natural Products for prokaryotes) (8 and 9) BE 14348B (10) BE 14348D/E (11) WS 9761B (12) Antibiotic W 007. Dereplicated components in sample A-177: 1) Cyclo (phenylalanylprolyl), 2) C₁₃H₁₄O₄ (no UV coincidence in the Dictionary of Natural Products for prokaryotes), 3) Alteramide A, 4) Clifednamide A, 5) C₁₅H₁₀ClNO₄ new caboxamycin derivative, 6) Caboxamycin B (1). Dereplicated components in sample A-199: (1) Cyclo (4-hydroxyprolylphenylalanyl), (2) N-[2-(1H-Indol-3-yl)-2-oxoethyl]acetamide, (3) C₁₄H₁₂N₂O₅ (molecular formula not found in the Dictionary of Natural Products for prokaryotes), (4) Macrolactin N, (5) Diazepinomicin.

Among a total of 94 secondary metabolites detected after LC/MS dereplication and comparative analyses of ethyl acetate extracts of all strains, 69 were identified as having matches in DNP, as shown in Table 3. The remaining 25 compounds had molecular formulae determined by HRMS not previously reported for any molecule included in natural product databases (see Table 4) and are of great pharmaceutical interest since they might be new natural products and thus candidates for novel drugs discovery. Regarding the previously reported biological activities of known natural products identified, 36 display biological activities as antibiotics, both antibacterial and antifungal, 22 as antitumor/cytotoxic, four anti-inflammatory, three immunosuppresant, two antiviral, two antiparasitic, two neuroprotective, one hepatoprotective one immunostimulator, and other compounds of pharmacological interest (Table 3). They belong to remarkably diverse structural families, such as anthracyclines, angucyclinones, ansamycins, macrolactams, polienes, polyketides, alkaloids, macrolides, phenazines and others.

TABLE 3

TABLE 3. Identified compounds produced by bioactive Streptomyces, Micromonospora and Nocardiopsis strains and their biological activities.

TABLE 4

TABLE 4. Molecular formulae of secondary metabolites not previously found in the Dictionary of natural products.

Concerning the sources of compounds, Table 5 displays the number of identified and unidentified compounds produced by each producing strain, and the results of meteorological analyses to know the sources and trajectories of the air masses causing the precipitation events in which they were isolated, estimated with a 5-day NOAA HYSPLIT Model, (Figure 2). According to backward transport trajectories analyses, the air masses mainly originated in the Arctic Ocean and travelled southward to downwind areas, such as the sampling places in Europe in a trajectory that also involves the Atlantic Ocean. As shown in Table 5, the unidentified molecules were produced by 12 strains, 11 Streptomyces and one Micromonospora, and were sampled in winter events mostly sourced around the North Pole. Nine unidentified molecules were obtained in events sourced in Svalbard archipelago, Norway (01/02/2015), four in the North Pole (31/01/2015), three in the White Sea (12/01/2016), two in Lofoten Islands, Norway (16/05/2013). The remaining events in which unidentified molecules were obtained were sourced in the Davies Strait, in the Arctic Ocean, three in the 21/01/2015 event, one in the 24/03/2015 and one in 14/02/2016. Also, 2 novel molecules were obtained in the 20/12/2015 event, in which the air mass at 3,000 m is sourced in the Davies Strait, whereas at 1,000 m originated in Mauritania and Canary Islands, and at 30 m were sourced in Spain (Iberian Peninsula).

TABLE 5

TABLE 5. Sources of atmospheric Actinobacteria producing new natural products. Number of compounds, air masses sources and trajectories of the precipitation events. Air masses backward trajectories analyses were estimated as indicated in Figure 2.

Structural elucidation of caboxamycin B

LC-UV-HRMS analyses of extracts from Streptomyces sp. A-177 revealed the presence of two new natural products structurally related to caboxamycin, an antibiotic belonging to the benzoxazole family produced by a deep-sea Streptomyces isolated in the Canary Basin (Hohmann et al., 2009), and whose biosynthetic pathway was elucidated (Losada et al., 2017) (Table 4). One of these compounds, caboxamycin B (1), was isolated by HPLC as a peak with a characteristic UV spectrum (SI, Fig SX and its structure was determined as shown in Figure 5, based on HRMS and 1D/2D NMR spectroscopy. The (+)ESI-TOF spectrum of 1 (Supplementary Figure S1) showed a [M + H]⁺ ion at m/z337.9980, with an isotopic pattern corresponding to the presence of two chlorine atoms, indicative of the molecular formula C₁₅H₉Cl₂NO₄ (Δ = -0.30 ppm) which was not found in any natural products database. Interestingly, another ion present in the spectrum at m/z727.9016 and its isotopic distribution fitted very well with the molecular formula C₃₀H₁₅Cl₄N₂O₈Fe ([2M-2H + Fe]⁺; calcd, 727.9005, Δ = +1.52 ppm), which suggests that one is able to complex iron, as previously reported for caboxamycin (Supplementary Figure S2).

FIGURE 5

FIGURE 5. Structure of caboxamycin B (1) and key COSY/HMBC correlations.

The ¹H-NMR spectrum of 1 (Supplementary Table S1, Supplementary Figure S3) showed four aromatic hydrogen signals, two doublets (δ_H 7.19 and 7.60 ppm; both with ³J_H,H = 8.7 Hz) and two broad singlets (δ_H7.96 and 8.13 ppm), as well as one singlet methyl at δ_H2.66 ppm and one additional singlet at δ_H11.66 ppm. Also, a weak signal as a broad singlet is observed at δ_H13.51 ppm, suggesting the presence of a carboxylic acid group. Interpretation of the ¹³C-NMR (Supplementary Figure S4) and HSQC (Supplementary Figure S6) spectra confirmed the presence of four aromatic methines and one methyl group and further revealed the presence often quaternary carbons in the structure. These data, along with the molecular formula, clarified that proton signal at 11.66 ppm should be bound to heteroatom and further supported the presence of a carboxylate group in 1.

The benzoxazole-type structure of one was fully elucidated based on COSY, HSQC and HMBC correlations (Supplementary Figures S5–S8). The COSY (Supplementary Figure S5) spectrum revealed the presence of a single spin system comprising vicinal hydrogens H-3 and H-4 (δ_H 7.19 and 7.60 ppm, respectively). HMBC correlations from H-3 to C-1 and C-5, from H-4 to C-2 and C-6, from H-6 to C-2, C-4 and C-2´ (all of them weak but genuine cross-peaks; SI, Fig SX)and from 2-OH to C-1, C-2 and C-3, together with the chemical shift of C-5 (δ_C 123.6 ppm), jointly established the presence of a 5-chloro-salicyclic acid moiety (Figure 5).

On the other hand, the 4-methyl-5-chloro-3-hydroxy-anthranilic acid moiety was determined based on HMBC correlations form H-5′ to C-3′a, C-7′ and C-1´´ as well as from 7′-Me to C-6′, C-7′ and C-7′a, along with the characteristic chemical shifts of C-3′a and C-7′a (δ_C 137.2 and 149.4 ppm respectively) (Hohmann et al., 2009), confirming the structure of caboxamycin B as shown in Figure 5.

Discussion

We provide here further evidence of the potential of the atmosphere in the discovery of natural products of interest in medicine and biotechnology. Antibiotic producing members of three genera of the phylum Actinobacteria were herein isolated in Spain during precipitation events with prevalent Northern winds over a 3 years period. Taxonomic identification and phylogenetic analyses revealed that atmospheric isolates belong mainly to Streptomyces genus, the largest of the phylum, with 690 currently validated species, but also to the Actinobacteria genus Micromonospora, with 110 validated species, and genus Nocardiopsis with 46 validated species. Bearing in mind previous reports on precipitations from Western and NorthWestern winds (Sarmiento-Vizcaíno et al., 2018, 2021), the results provided here from events with Northern winds further increases the number of Streptomyces homologues producing bioactive natural products so far isolated from atmospheric precipitations to a total of 47, which represents a 6.8% of the of the total number of currently validated species in this genus (http://www.bacterio.net/streptomyces.html).

Since atmosphere is an extreme environment, very selective for microorganisms, isolation of Actinobacteria from precipitations resulted much easier than from traditional terrestrial sources. In this innovative approach sampling is a straightforward process, precipitations literally fall from the sky and can bring in a great diversity of novel strains from sources that are potentially out of reach. Although many samples were herein analyzed throughout the years to explore the diversity of Actinobacteria in atmospheric environments, for an efficient natural product discovery approach it would be more convenient to analyze a higher volume from a single precipitation event. This way of proceeding has been successfully reported when sampling a hailstone storm in the same geographical region, where 38 potential novel molecules were found (Sarmiento-Vizcaino et al., 2018).

Meteorological analyses revealed that the air masses involving 5 days backward trajectories point to an Arctic Ocean origin and their diverse downwind pathways. These Actinobacteria remain viable after their atmospheric transport by winds across oceans and continents and are dispersed via the atmosphere before they fall down by precipitation. These findings provide further support for the Streptomyces atmospheric dispersal model (Sarmiento-Vizcaíno et al., 2016, 2021), which is herein extended to the genus Micromonospora. This cycle may have been occurring over geological time scales being of relevance for the biogeography and evolutionary history of these Actinobacteria.

In oceanic environments, among Actinobacteria associated to marine organisms, it has been reported that the dominant genera of producers of secondary metabolites of pharmaceutical relevance are Streptomyces (68%), followed by Micromonospora (6%) and Nocardiopsis (3%) (Chen et al., 2021). The strains herein reported are closely related to bioactive oceanic species isolated in the Cantabrian Sea, North Atlantic Ocean. Among Streptomyces, of particular interest are S. cyaneofuscatus homolog strains (A-123, A-125 and A-159) which are homologues to highly producers of biologically active compounds, isolated during an oceanographic expedition to the submarine Avilés Canyon, Cantabrian Sea (Sarmiento-Vizcaíno et al., 2017b), where novel natural products with antibiotic and cytotoxic activities were discovered (Ortiz-López et al., 2018; Rodríguez et al., 2018). In addition, Micromonospora and Nocardiopsis species, known as a source for structurally diverse novel products (Subramani and Sipkema. 2019), were also isolated herein. Micromonospora species were previously found in snow from Svalbard, Norway, in the Arctic Circle at 78° N (Amato et al., 2007) and also more than 30 isolates with Micromonopora-like phenotype were obtained in other precipitation events (Sarmiento-Vizcaíno, 2016). Furthermore, bioactive species of this genus were also found to colonize deep sea environments, such as the Avilés Canyon up to 4,700 depth, in cold waters of Arctic origin (Sarmiento-Vizcaíno, 2016; Sarmiento-Vizcaíno et al., 2017a), where a novel compound displaying cytotoxic activities against human tumour cell lines was discovered (Sarmiento-Vizcaíno et al., 2017b). Concerning Nocardiopsis, bioactive members of this genus were previously isolated in atmospheric precipitation events with prevalent Western winds (Sarmiento-Vizcaíno et al., 2021).

Comparative analyses of secondary metabolites detected herein with natural products databases, led to the identification of a total of 69 structurally diverse compounds. Concerning their biological activity, after a literature search, 52% of the identified natural products are antibiotics (both against Gram-positive and Gram-negative bacteria and against fungi) and 32% are antitumor/cytotoxic agents. There also compounds with anti-inflamatory, antiviral, antiparasitic, immunosuppresant, immunostimulator, neurprotective, hepatoprotective and other pharmaceutical properties (Table 3). Of great relevance are 25 compounds with molecular formulae not previously reported in Natural product Databases (Table 4), since they are candidates for drugs discovery. Taken into account previous data (Sarmiento-Vizcaíno et al., 2018, 2021), the results here provided increase up to 93 the number of unidentified compounds so far reported to be produced by atmospheric Actinobacteria.

As a proof of concept a novel caboxamycin, caboxamycin B, the second of a family, was herein obtained from cultures of Streptomyces sp. A-177, and its structure established after HPLC purification and HRMS and NMR analysis. This new molecule is structurally related to caboxamycin, an antibiotic of the benzoxazole family initially obtained in the Canary Basin (Hohmann et al., 2009) and later on from Streptomyces halstedii M-204, asociated to an equinoderm in the Avilés Canyon (Sarmiento-Vizcaíno et al., 2017a). Remarkably, caboxamycin B constitutes the first halogenated caboxamycin reported in the literature. Our findings provide further support of the potential of atmospheric-derived Actinobacteria in the discovery of pharmaceutical natural products, particularly antibiotics, which are urgently needed for fighting against pathogenic bacteria resistant to antibiotics in currently in clinical use.

Conclusion

Identification of Actinobacteria in precipitation events with prevalent Northern winds in Spain reveals that, besides Streptomyces and Nocardiopsis species previously reported in samples from Western and North Western winds, bioactive members of the genus Micromonospora are also present in atmospheric environments. In total, almost a hundred of potential novel natural products have been establish in these Actinobacteria isolated from precipitation events with different winds so far. These findings confirm that atmospheric Actinobacteria deserve further research, since they produce a remarkable diversity of bioactive compounds of pharmacological and biotechnological relevance. In this line of thought, the discovery of a novel molecule structurally related to the antibiotic caboxamycin, such as caboxamycin B, is to the best of our knowledge the first novel natural product reported in a Streptomyces strain isolated from the atmosphere.

Data availability statement

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material.

Author contributions

AS-V and GB isolated the strains. AS-V performed the bioactivity assays, taxonomic identification, phylogenetic analyses of the strains, extraction of cultures and isolation of caboxamycin B. GB analyzed the air masses backward trajectories. AS-V analyzed the compounds by LC-UV. JM and FR performed the metabolite profiling analysis and identified the compounds produced by LC-MS. FJO-L performed the structural elucidation of caboxamycin B. LG and GB conceived and coordinated the project. GB wrote the manuscript which has been revised, and approved by all the authors.

Acknowledgments

The authors want to thank the Universidad de Oviedo for financial support, Ayuda PAPI-18-PUENTE-6. We are grateful to José L. Martínez and Daniel Serna (Servicios científico-técnicos, edificio Severo Ochoa, Universidad de Oviedo) for their help in strains identification.

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Publisher’s note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fchem.2022.948795/full#supplementary-material

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