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

Actinobacteria, mostly Streptomyces species, are the main source of natural products essential in medicine. While the majority of producer microorganisms of secondary metabolite are reported from terrestrial or marine environments, there are limited reports of their isolation from atmospheric precipitations. Clouds are considered as atmospheric oases for microorganisms and there is a recent paradigm shift whereby atmospheric-derived Actinobacteria emerge as an alternative source for drug discovery. In this context, we studied a total of 18 bioactive Actinobacteria strains, isolated by sampling nine precipitation events with prevailing Northern winds in the Cantabrian Sea coast, Northern Spain. Backward trajectories meteorological analyses indicate that air masses were originated mostly in the Arctic Ocean, and their trajectory to downwind areas involved the Atlantic Ocean and also terrestrial sources from continental Europe, and in some events from Canada, Greenland, Mauritania and Canary Islands. Taxonomic identification of the isolates, by 16S rRNA gene sequencing and phylogenetic analyses, revealed that they are members of three Actinobacteria genera. Fifteen of the isolates are Streptomyces species, thus increasing the number of bioactive species of this genus in the atmosphere to a 6.8% of the total currently validated species. In addition, two of the strains belong to the genus Micromonospora and one to genus Nocardiopsis. These findings reinforce a previous atmospheric dispersal model, extended herein to the genus Micromonospora. Production of bioactive secondary metabolites was screened in ethyl acetate extracts of the strains by LC-UV-MS and a total of 94 secondary metabolites were detected after LC/MS dereplication. Comparative analyses with natural products databases allowed the identification of 69 structurally diverse natural products with contrasted biological activities, mostly as antibiotics and antitumor agents, but also anti-inflammatory, antiviral, antiparasitic, immunosuppressant and neuroprotective among others. The molecular formulae of the 25 remaining compounds were determined by HRMS. None of these molecules had been previously reported in natural product databases indicating potentially novel metabolites. As a proof of concept, a new metabolite caboxamycin B (1) was isolated from the culture broth of Streptomyces sp. A-177 and its structure was determined by various spectrometric methods. To the best of our knowledge, this is the first novel natural product obtained from an atmospheric Streptomyces, thus pointing out precipitations as an innovative source for discovering new pharmaceutical natural products.


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 .
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.

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.

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 . 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 .

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 Gramnegative 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 Frontiers in Chemistry frontiersin.org 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; 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 A-199 -----10/9 -Antimicrobial activities were estimated as the zones of complete inhibition (diameter in mm). Table 1A. Assays performed with agar plugs from solid cultures. Table 1B. Antibiotic activities of ethyl acetate extracts of the cultures. 7 ml of culture, in neutral and acidic conditions, resuspended in 50μL of DMSO-methanol (1:1) from which 15 μl were loaded onto AA discs. Before applying to the indicator strain culture, the discs were allowed to fully dry.
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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 . 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.
Frontiers in Chemistry frontiersin.org 05 (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).

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.

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.

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. 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.
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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.
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.
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  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.

Compound LC/MS Strain Biological activities
Divergolide M A-106 Antibiotic, cytotoxic Ding et al. (2015) Espicufolin A A-145 Antitumor Tietze et al. (2007) Geldanamycin b A-120 Antifungal, anticancer, neuroprotective Tadtong et al. (2007) Germicidin A b A-182 Spore germination (hypha elongation Aoki et al. (2011) Germicidin B b /C A-182 Spore germination (hypha elongation Aoki et al. (2011) Glycinocin A A-28 Antibiotic Corcilius et al. (2018) Herbimycin A (Antibiotic TAN 420F) A-28 Antibiotic, antitumor Garcia et al. (1991) Hydroxystreptazolin   Figure S2). The 1 H-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 3 J H,H = 8.7 Hz) and two broad singlets (δ H 7.96 and 8.13 ppm), as well as one singlet methyl at δ H 2.66 ppm and one additional singlet at δ H 11.66 ppm. Also, a weak signal as a broad singlet is observed at δ H 13.51 ppm, suggesting the presence of a carboxylic acid group. Interpretation of the 13 C-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 5chloro-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 ,

WS 9761B
A-163 Androgen-receptor antagonist Hori et al. (1993) a The asterisk means that more than one compound was identified.The highlightened strains correspond to Micromonospora sp. and the underlined to Nocardiopsis sp., the rest are all Streptomyces species.
A Frontiers in Chemistry frontiersin.org 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 , 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
Frontiers in Chemistry frontiersin.org 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 , 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.