The Polyextreme Ecosystem, Salar de Huasco at the Chilean Altiplano of the Atacama Desert Houses Diverse Streptomyces spp. with Promising Pharmaceutical Potentials

Salar de Huasco at the Chilean Altiplano of the Atacama Desert is considered a polyextreme environment, where solar radiation, salinity and aridity are extremely high and occur simultaneously. In this study, a total of 76 bacterial isolates were discovered from soil samples collected at two different sites in the east shoreline of Salar de Huasco, including H0 (base camp next to freshwater stream in the north part) and H6 (saline soils in the south part). All isolated bacteria were preliminarily identified using some of their phenotypic and genotypic data into the genera Streptomyces (86%), Nocardiopsis (9%), Micromonospora (3%), Bacillus (1%), and Pseudomonas (1%). Streptomyces was found dominantly in both sites (H0 = 19 isolates and H6 = 46 isolates), while the other genera were found only in site H0 (11 isolates). Based on the genotypic and phylogenetic analyses using the 16S rRNA gene sequences of all Streptomyces isolates, 18% (12 isolates) revealed <98.7% identity of the gene sequences compared to those in the publicly available databases and were determined as highly possibly novel species. Further studies suggested that many Streptomyces isolates possess the nonribosomal peptide synthetases-coding gene, and some of which could inhibit growth of at least two test microbes (i.e., Gram-positive and Gram-negative bacteria and fungi) and showed also the cytotoxicity against hepatocellular carcinoma and or mouse fibroblast cell lines. The antimicrobial activity and cytotoxicity of these Streptomyces isolates were highly dependent upon the nutrients used for their cultivation. Moreover, the HPLC-UV-MS profiles of metabolites produced by the selected Streptomyces isolates unveiled apparent differences when compared to the public database of existing natural products. With our findings, the polyextreme environments like Salar de Huasco are promising sources for exploring novel and valuable bacteria with pharmaceutical potentials.


Introduction
Actinomycetes are the most important bacteria that are capable of producing bioactive compounds.The majority of natural products for new pharmaceutical applications are derived from actinomycetes [1], while the notable member of actinomycetes, the genus Streptomyces is the major producer [2].Many of these specialized metabolites biosynthesized by actinomycetes correspond to polyketides and nonribosomal peptides, which may act as antibiotics, immunosuppressants, anticancer/antitumor agents, toxins, and siderophores [3].The members of the genus Streptomyces are widely distributed across various habitats and geographical locations [4].Streptomyces produces spores that are characteristically resistant, allowing this bacterium to persist in the extreme environments and to maintain its viability for many years [5].Previous studies showed that bacteria isolated from the extreme environments, such as the Mariana Trench [6], the polar and permafrost soils in the Arctic [7,8], and the extremely dry and saline soils of the Atacama Desert [9] are unique sources for the discovery of new bioactive compounds and many of them are possibly novel species [5,10,11].
The Atacama Desert is the driest place in the world located in South America, precisely in Chile, covering a 1000-km strip of land on the Pacific coast, west of the Andes Mountains.It is bordering Peru in the north and extending to the Copiapó River in the south.Nowadays, four novel Streptomyces species derive from this extreme desert, including Streptomyces atacamensis [12], Streptomyces desertii [13], Streptomyces bulli [14], and Streptomyces leeuwenhoekii [15].S. leeuwenhoekii is the producer of two newly bioactive compounds comprised of (i) chaxamycins, showing antagonism against Staphylococcus aureus ATCC 25923, inhibiting the heat shock protein 90, and degrading proteins involved in cell proliferation [9] and (ii) chaxalactins, the anti-Gram-positive bacterial agents [16].Two more novel compounds, atacamycins and chaxapeptin, each derive respectively from Streptomyces sp., C38 [17] and C58 [18] isolated from hyper-arid soil of the Laguna de Chaxa of Salar de Atacama in the north of Chile (a lagoon located in the commune of San Pedro de Atacama, province of El Loa).These two compounds are novel lasso-peptides produced by nonribosomal biosynthesis with post-translational modification and capable of inhibiting human lung cancer cell line A549.Another group of new compounds with antimicrobial activity, the abenquines A-D, was obtained from Streptomyces sp.DB634 isolated from the Salar de Tara in the Chilean Altiplano (>4000 m.a.s.l.) [19].
In Chile, the Chilean Altiplano of the Atacama Desert, is considered the highest plateau of the Andes Mountains (>3000 m.a.s.l.) with 14 • to 22 • S of latitude, in addition, it is subjected to strong climatic variation over different temporal scales [19].This site offers a unique ecosystem with highly extreme conditions, such as changes in daily temperature from −10 to +25 • C [19] and it divides into different sites, for instance, Salar de Tara (at 150 km east of the town of San Pedro de Atacama, in the Province of El Loa, region Antofagasta) and Salar de Huasco (in the south of the town of Parinacota in the Arica and Parinacota regions).The Salar de Huasco is known by its polyextreme conditions, i.e., salinity gradient (from freshwater to saturation), elevated levels of solar radiation (<1100 W/m 2 ), and negative water balance, which differs from the other locations previously studied in the Atacama Desert.Some areas of the Salar de Huasco have been found to house diverse microbes [20,21].However, there is no study regarding the cultivable actinomycetes from this polyextreme ecosystem and their pharmaceutical potentials.Here, we aim to isolate bacteria with a focus on actinomycetes from different sites of the Salar de Huasco and to assess their pharmaceutical potentials in possession of nonribosomal peptide synthases (NRPS)-coding gene, antimicrobial activity, and cytotoxicity.

Isolation of Actinomyces
Six soil samples were collected from sites H0 (base camp; freshwater stream) and H6 (saline soils) located in the northern and southern parts of the eastern shoreline of the Salar de Huasco, respectively [22] (Figure 1).Sites H0 and H6 were characterized as meso-saline and hyper saline sites with pH 7.6 and pH 8.6, respectively [23].The samples were taken at 5 cm depth from the ground surface using sterile polypropylene tubes and transferred to the laboratory.These samples were stored at ambient temperature for a period not exceeding 5 days.One g of soil sample was suspended in 9 mL of 1  4 strength Ringer's solution, and 1 mL of the soil suspension was diluted 100-fold and mixed in an orbital shaker at 150 rpm for 1 h.The diluted soil suspension was pretreated by heating at 55 • C for 6 min in a thermo-regulated bath [4].The heated aliquot (0.1 mL) was spread over the surface of starch casein agar medium (SCA), supplemented with 50 µg/mL nystatin and 50 µg/mL cycloheximide to prevent fungal contamination [24].All seeded agar plates were prepared in triplicate and incubated at 28 • C for 14-21 days [25].Colonies with rough appearance, powdery or tough texture and branching filaments with and without aerial mycelia were subcultured on SCA plates and incubated at 28 • C for 14 days.A subculture of each isolate was prepared on International Streptomyces Project III (ISP3) medium [26] under the same condition cited above.The purity of the each isolates was checked using light microscope.
Diversity 2018, 10, x FOR PEER REVIEW 3 of 12 surface using sterile polypropylene tubes and transferred to the laboratory.These samples were stored at ambient temperature for a period not exceeding 5 days.One g of soil sample was suspended in 9 mL of ¼ strength Ringer's solution, and 1ml of the soil suspension was diluted 100-fold and mixed in an orbital shaker at 150 rpm for 1 h.The diluted soil suspension was pretreated by heating at 55 °C for 6 min in a thermo-regulated bath [4].The heated aliquot (0.1 mL) was spread over the surface of starch casein agar medium (SCA), supplemented with 50 µg/ml nystatin and 50 µg/mL cycloheximide to prevent fungal contamination [24].All seeded agar plates were prepared in triplicate and incubated at 28 °C for 14-21 days [25].Colonies with rough appearance, powdery or tough texture and branching filaments with and without aerial mycelia were subcultured on SCA plates and incubated at 28 °C for 14 days.A subculture of each isolate was prepared on International Streptomyces Project III (ISP3) medium [26] under the same condition cited above.The purity of the each isolates was checked using light microscope.

Morphological Characterization Based on Color Grouping
The morphological characteristics of all bacterial isolates grown on ISP3 medium, such as aerial spore mass color, pigmentation of vegetative or substrate mycelium, and the production of diffusible pigment were examined.The color of the aerial and substrate mycelium of the isolates were described referring to the National Bureau of Standards (NBS) Color Name Charts [27].Each isolate was maintained on glucose yeast extract and malt extract (GYM) medium and stored as spore suspensions and hyphae in 25% (v/v) glycerol at −80 °C.

16S rRNA Gene-Based Phylogenetic Analysis
Bacterial cultures were prepared in tryptone yeast extract (ISP1) broth at 28 °C for 7 days, in which 2 mL was transferred to vials containing 0.5 mm glass beads (BioSpec Products Inc, Bartlesville, OK, USA) to breakdown mechanically the bacterial cells (Mini Bead Beater, Bead Homogenizer, BioSpec Products Inc, Bartlesville, OK, USA).Total genomic DNA was extracted from the homogenized bacterial suspension using the AxyPrep Bacterial Genomic DNA Miniprep kit (Axygen Biosciences, Union city, NJ, USA) as recommended by the manufacturer.

Morphological Characterization Based on Color Grouping
The morphological characteristics of all bacterial isolates grown on ISP3 medium, such as aerial spore mass color, pigmentation of vegetative or substrate mycelium, and the production of diffusible pigment were examined.The color of the aerial and substrate mycelium of the isolates were described referring to the National Bureau of Standards (NBS) Color Name Charts [27].Each isolate was maintained on glucose yeast extract and malt extract (GYM) medium and stored as spore suspensions and hyphae in 25% (v/v) glycerol at −80 • C.

16S rRNA Gene-Based Phylogenetic Analysis
Bacterial cultures were prepared in tryptone yeast extract (ISP1) broth at 28 • C for 7 days, in which 2 mL was transferred to vials containing 0.5 mm glass beads (BioSpec Products Inc, Bartlesville, OK, USA) to breakdown mechanically the bacterial cells (Mini Bead Beater, Bead Homogenizer, BioSpec Products Inc, Bartlesville, OK, USA).Total genomic DNA was extracted from the homogenized bacterial suspension using the AxyPrep Bacterial Genomic DNA Miniprep kit (Axygen Biosciences, Union City, NJ, USA) as recommended by the manufacturer.The 16S rRNA gene was amplified by PCR using the universal primers for bacteria, Eub9-27F and Eub1542R [28].Every reaction was performed in a final volume of 50 µL, containing 50-100 ng of genomic DNA, 2.0 µM of each primer, 19 µL of nuclease-free MilliQ-H 2 O (Merck Millipore, Burlington, MA, USA), and 25 µL of SapphireAmp ® Fast PCR Master Mix (TaKaRa, Japan).PCR was carried out in a thermocycler under the following conditions: Initial denaturation at 94 • C for 5 min, 30 cycles of denaturation at 94 • C for 45 s, annealing at 54 • C for 45 s, and extension at 72 • C for 1.5 min, and a final extension at 72 • C for 5 min.The PCR (Polymerase Chain Reaction) products were checked by 1% (w/v) agarose gel electrophoresis and subsequently sequenced by capillary sequencing using an ABI Prism 3730XL automated DNA sequencer (Applied Biosystems, Macrogen Inc., Seoul, Korea).
The 16S rRNA gene sequences were analyzed and edited using the software DNA Baser Sequence Assembler version 3.5 (Heracle BioSoft SRL, 2014).The partial 16S rRNA gene sequences of the isolates were aligned using RDP II [29].EzTaxon [30] was used to retrieve the nearest phylogenetic neighbors of all bacterial isolates.The nearly completed 16S rRNA gene sequences (>1300 nt) of the isolates were deposited in the GenBank database with the accession numbers KX130868-KX130886.
The phylogenetic tree was constructed using Neighbor-Joining algorithms [31] with a Tamura-Nei substitution model using MEGA 7.0 software [32].The multiple alignments of all the 16S rRNA gene sequences were performed using MUSCLE (MUltiple Sequence Comparison by Log-Expectation) algorithm [33].The robustness of the tree was evaluated using 1000 Bootstrap [34].

Preparation of Crude Extracts and Bioactivity Assessments
The secondary metabolites were extracted from Streptomyces isolates possessing NRPS domains following a protocol described by Schneemann et al. [36] with some modifications.Each isolate was grown in 100 mL of GYM (pH 7.2), and in starch-soy peptone (SPM, pH 7.0) liquid media supplemented with 2% NaCl and incubated at 28 • C for one week, with shaking at 135 rpm, in an orbital shaking incubator (MaxQ 4000, Thermo Fisher Scientific, Waltham, MA, USA).Extraction of metabolites from the whole culture broths started with the addition of 150 mL of ethyl acetate to each flask, followed by stirring and sonication cycles and kept at 4 • C overnight for effective phase separation.The lower aqueous phase was discarded, and the ethyl acetate phase (supernatant) was dried in a rotary evaporator (Büchi, Flawil, Switzerland) at ambient temperature.
The antimicrobial activity of the crude extracts was evaluated by bioassays using stocks solutions with a concentration of 1% w/v (equivalent to 10 mg/mL in methanol).The bioassays of antibacterial and antifungal activities were performed following a procedure described by Schneemann et al. [36].Aliquot of 5 µL of each crude extract was added in each well of the 96-well microtiter plate and then the solvent was evaporated using vacuum centrifuge (Biotage SPE Dry, Uppsala, Sweden) before append 195 µL of the test microbial suspension.The final concentration of each crude extract in the bioassays was 250 µg/mL.The test organisms comprised of gram-positive bacteria: Staphylococcus epidermidis DSM 20044 T , methicillin-resistant Staphylococcus aureus DSM 18827, Pseudomonas aeruginosa DSM 50071 T , and Propionibacterium acnes DSM 1897 T , gram-negative bacteria: Xanthomonas campestris DSM 2405 and Erwinia amylovora DSM 50901, and fungi: Candida albicans DSM 1386, Trichophyton rubrum, Septoria tritici, and Phytophthora infestans.Trichophyton rubrum was obtained from F. Horter (Department of Dermatology, Allergology, and Venerology, University Hospital Schleswig-Holstein, Kiel, Germany while Septoria tritici and Phytophthora infestans were obtained from Dr. J. B. Speakman (BASF, Ludwigshafen, Germany).The positive control for the test bacteria was 100 µg/well of chloramphenicol and that for the test fungi was 200 µg/well of cycloheximide, while the negative control was no compound applied.
The cytotoxicity test was performed following a method described by Schulz et al. [19].Aliquot (1 µL) of each crude extract was added in a final assay volume of 100 µL of the mouse fibroblasts (NIH-3T3) and hepatocellular carcinoma (HepG2 ACC 180) cell lines.NIH-3T3 cell line was provided by G. Rimbach, University of Kiel, Germany while HepG2 ACC 180 was obtained from Leibniz Institute DSMZ-German collection of microorganisms and cell cultures, Braunschweig, Germany.The final concentration of each crude extract in the bioassays was 100 µg/mL.The positive control for these assays was tamoxifen with a final concentration of 40 µM.No compound was added for the negative control.

Profiling of Metabolites in Crude Extracts
The crude extracts dissolved in methanol were elucidated by analytical reversed-phase HPLC-UV-MS (High-Performance Liquid Chromatography with UV detector coupled to Mass Spectrometry), in a VWR-Hitachi La-Chrom Elite System, coupled to a diode array detector and a Phenomenex Onyx Monolithic column (C18, 100 × 3.00 mm) according to the conditions described by Silber et al. [37].For the mass detection, the HPLC system was coupled to an ion trap detector (Esquire4000, Bruker Daltonics, Billerica, MA, USA).The UV-Vis spectra of peaks obtained from each bacterial crude extract were compared with the Dictionary of Natural Products 2012 [38].

Selective Isolation and Identification of the Isolates
A number of 30 and 46 isolates were obtained from soils samples of sites H0 and H6, respectively.These isolates were obtained from plates with 10 fold dilutions, and 97% (74 isolates) of them were found to be affiliated with the phylum Actinobacteria based on the 16S rRNA gene sequence similarity.Two isolates were classified as Bacillus and Pseudomonas, while the others belong to Nocardiopsis (7 isolates), Micromonospora (2 isolates), and Streptomyces (65 isolates) (Table 1).The eleven non-Streptomyces isolates were found only in the soil sample with neutral pH 7.6 collected from site H0, while the majority of the Streptomyces isolates (40) were obtained from the hypersaline site H6 with pH 8.6.All 65 Streptomyces isolates were subdivided into three color grouping after incubation on ISP3 agar medium at 28 • C for 14 days (Table 2).They showed the typical morphology of the genus Streptomyces in formation of aerial mycelium and spore chains.Most of these isolates produced diffusible pigments and excreted colored aqueous droplets on the hydrophobic surface of their colonies.Nineteen out of 65 Streptomyces isolates were selected for further study based on their nearly complete 16S rRNA gene sequences (>1300 bp).These 19 isolates widely distributed across the phylogenetic tree of the genus Streptomyces supported by high bootstrap values as shown in Figure 2. A total of 12 isolates showed a 16S rRNA gene sequence similarity below the 98.7% threshold for proposing as novel species [39] (Table 2), while the other seven (HST09, HST19, HST21, HST22, HST23, HST28, and HST61) found to occupy distinct phylogenetic positions from their closest relatives with no close relationship with any species and type strains of the genus Streptomyces from the Atacama Desert described so far [11][12][13][14][15] (Figure 2).
The length of the branch of all Streptomyces isolates in the phylogenetic tree and the assignation to these isolates to completely different clades from each other, except for isolates HST21 and HST22, highlight the divergence of them from their closely related neighbors.Further studies need to be performed to confirm the right affiliations of these isolates to the novel species within the evolutionary radiation of the genus Streptomyces.
Most isolates (except for isolates HST83, HST72, HST21, and HST22) showed divergent phylogenetic positions compared to the type species of the genus Streptomyces (Figure 2).Therefore, these strains should be designed for further taxonomic and analytical chemistry analyses to confirm their novelty at species rank and as a source of novel chemical entities (Table 3).a The average results derived from triplicate assays are shown with inhibition percentage (%) of growth of Se (Staphylococcus epidermis), MRSA (methicillin-resistant Staphylococcus aureus), Pa (Propionibacterium acnes), Xc (Xanthomonas campestris), Ea (Erwinia amylovora), Ca (Candida albicans), Tru (Trichophyton rubrum), Sep (Septoria tritici), Pha (Phytophthora infestans), NIH-3T3 (mouse fibroblasts), and HepG2 (hepatocellular carcinoma).GYM and SPM refer respectively to glucose yeast extract plus malt extract medium and starch-soy peptone medium, in which the Streptomyces isolates were grown before the preparation of their crude extracts.The negative results are shown with (-).

Figure 2 .
Figure 2. Neighbor-joining phylogenetic tree based on almost complete 16S RNA sequences (>1300 nt) of the 19 Streptomyces isolates derived from Salar de Huasco and their closely related species.The tree was constructed using the Neighbor-Joining algorithm and the Jukes-Cantor substitution model.

Figure 2 .
Figure 2. Neighbor-joining phylogenetic tree based on almost complete 16S RNA sequences (>1300 nt) of the 19 Streptomyces isolates derived from Salar de Huasco and their closely related species.The tree was constructed using the Neighbor-Joining algorithm and the Jukes-Cantor substitution model.The scale bar indicates 0.005 substitutions per nucleotide, and Streptomyces albus subsp.albus DSM 40313 T was used as an out-group.Bootstrap values above 50% are present.

Table 1 .
16S rRNA gene-based identification of all bacteria isolated from Salar de Huasco.

Table 2 .
Morphological and molecular characteristics of some Streptomyces spp. a isolated from Salar de Huasco.These Streptomyces isolates were selected based on the quality of their 16S rRNA gene sequences (>1300 nt).b The morphological characteristics were observed after growing Streptomyces isolates on ISP3 agar medium at 28 • C for 14 days. a

Table 3 .
Bioactivities of crude extracts derived from Streptomyces spp. of Salar de Huasco.