Streptomyces aridus sp. nov., isolated from a high altitude Atacama Desert soil and emended description of Streptomyces noboritoensis Isono et al. 1957

A polyphasic study was undertaken to determine the taxonomic status of a Streptomyces strain which had been isolated from a high altitude Atacama Desert soil and shown to have bioactive properties. The strain, isolate H9T, was found to have chemotaxonomic, cultural and morphological properties that place it in the genus Streptomyces. 16S rRNA gene sequence analyses showed that the isolate forms a distinct branch at the periphery of a well-delineated subclade in the Streptomyces 16S rRNA gene tree together with the type strains of Streptomyces crystallinus, Streptomyces melanogenes and Streptomyces noboritoensis. Multi-locus sequence analysis (MLSA) based on five house-keeping gene alleles showed that isolate H9T is closely related to the latter two type strains and to Streptomyces polyantibioticus NRRL B-24448T. The isolate was distinguished readily from the type strains of S. melanogenes, S. noboritoensis and S. polyantibioticus using a combination of phenotypic properties. Consequently, the isolate is considered to represent a new species of Streptomyces for which the name Streptomyces aridus sp. nov. is proposed; the type strain is H9T (=NCIMB 14965T=NRRL B65268T). In addition, the MLSA and phenotypic data show that the S. melanogenes and S. noboritoensis type strains belong to a single species, it is proposed that S. melanogenes be recognised as a heterotypic synonym of S. noboritoensis for which an emended description is given.


Introduction
Streptomycetes remain a unique source of novel pharmaceutically important products with antibacterial, anti-inflammatory and antitumor activities (Bérdy 2005;Demain 2014; Barka et al. 2016), hence the continued interest in these organisms as a source of new specialised metabolites (Goodfellow Electronic supplementary material The online version of this article (doi:10.1007/s10482-017-0838-2) contains supplementary material, which is available to authorized users. and Fiedler 2010; Chaudhary et al. 2013). However, the continued search for new bioactive compounds against drug-resistant microorganisms needs to be focused on novel streptomycetes to avoid the rediscovery of known compounds from common Streptomyces species. Given this imperative, novel Streptomyces species are being sought from unusual and neglected habitats (Hong et al. 2009;Tiwari and Gupta 2012), notably from extreme biomes (Bull 2011;Hamedi et al. 2013;Goodfellow 2013). Previous work from our group has shown that novel Streptomyces species abound in arid Atacama Desert soils (Okoro et al. 2009;Busarakam 2014;Busarakam et al. 2014), some of which synthesise new specialised metabolites with encouraging bioactivities (Bull et al. 2016), thereby underpinning the premise that extreme environmental conditions promote unique actinobacterial diversity which is the basis of novel chemistry (Bull and Stach 2007;Bull 2011;Gomez-Escribano et al. 2015).
Streptomyces, the type genus of the family Streptomycetaceae, was proposed by Waksman and Henrici (1943) and the description of the taxon emended by Witt and Stackebrandt (1990) and Wellington et al. (1992). The genus encompasses aerobic, Gram-stain positive actinobacteria with a high DNA G?C content, which form extensively branched substrate mycelia supporting aerial hyphae that typically differentiate into chains of spores, have a wall peptidoglycan rich in LL-diaminopimelic acid, contain major amounts of saturated, iso-and anteiso-fatty acids, usually have either hexa-or octa-hydrogenated menaquinones with nine isoprene units as predominant isoprenologues and complex polar lipid patterns which tend to include diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and phosphatidylinositol mannosides (Kämpfer 2012). The genus includes over 700 validly named species (http://www.bacterio.net/streptomyces.html) which can be assigned to many multi-and single-membered subclades in the Streptomyces 16S rRNA gene tree (Kämpfer 2012;Labeda et al. 2012) and based on multi-locus sequence analysis (MLSA; Labeda et al. 2017). Despite being the largest genus in the domain Bacteria the taxon remains underspeciated (Okoro et al. 2009;Antony-Babu et al. 2010;Busarakam 2014). New species are assigned to the genus using a combination of genotype and phenotype properties, and some recent studies have featured MLSA Huang 2012, 2014;Labeda et al. 2014Labeda et al. , 2017Labeda 2016).
The aim of the present study was to establish the taxonomic position of a Streptomyces strain, isolate H9 T , which had been isolated from a high altitude Atacama Desert soil and shown to produce novel specialised metabolites (Idris 2016). The results of a polyphasic taxonomic study showed that isolate H9 T belongs to a novel Streptomyces species for which we propose the name Streptomyces aridus sp. nov.

Materials and methods
Selective isolation, maintenance and cultural conditions Strain H9 T was recovered from an arid subsurface soil sample (30 cm depth) at 4000 metres above sea level on Cerro Chajnantor (23 o 63 0 31 00 S, 67 o 52 0 27 00 W) east of San Pedro de Atacama, Chile. The strain was isolated on glucose-yeast extract agar (Athalye et al. 1981) supplemented with cycloheximide and nystatin (each at 25 lg/ml) after incubation at 28°C for 14 days following inoculation with a suspension of one gram of soil in 5 ml strength Ringer's solution. The isolate together with S. melanogenes NRRL B-2072 T , S. noboritoensis NRRL B-12152 T and S. polyantibioticus NRRL B-24448 T were maintained on yeast extract-malt extract agar (International Streptomyces Project [ISP medium 2], Shirling and Gottlieb 1966). All of the reference strains were obtained from the NRRL culture collection as indicated.
Biomass for the chemotaxonomic and molecular systematic analyses was prepared in shake flasks (180 revolutions per minute) of ISP2 broth following incubation at 28°C for 14 days and washed twice in distilled water; cells for the chemotaxonomic studies were freeze-dried and those for the molecular systematic analyses stored at room temperature.

Chemotaxonomy
Strain H9 T was examined for chemotaxonomic properties known to be characteristic of Streptomyces strains (Kämpfer 2012). Standard procedures were used to detect isomers of diaminopimelic acid (A 2 pm) (Staneck and Roberts 1974), menaquinones (Collins et al. 1985), polar lipids (Minnikin et al. 1984) and whole cell sugar composition (Lechevalier and Lechevalier 1970), using appropriate controls. The type strains of S. melanogenes and S. noboritoensis were also included in the analyses for A 2 pm isomers, cellular sugars and polar lipids. Fatty acid methyl esters (FAMEs) were prepared from isolate H9 T and the type strains of S. melanogenes, S. noboritoensis and polyantibioticus by saponification, methylation and extraction following protocols developed by Miller (1982) with minor modifications from Kuykendall et al. (1988). The FAMEs were separated by gas chromatography (Agilent 6890 N instrument) and the resultant peaks automatically integrated. Fatty acid names and properties were determined using the standard microbial identification (MIDI) system Version 4.5 and the ACTIN 6 database (Sasser 1990).

16S rRNA gene sequencing
Genomic DNA was extracted from isolate H9 T biomass and PCR-mediated amplification of a 16S rRNA purified gene product obtained, as described by Kim and Goodfellow (2002). Identification of phylogenetic neighbours and calculation of pairwise 16S rRNA gene sequence similarities were realised using the EzTaxon-e server (http://eztaxon-e.ezbiocloud.net/; Kim et al. 2012). The CLUSTAL W algorithm from MEGA 6 (Tamura et al. 2013) was used to align the sequences. Phylogenetic trees were generated using the maximumlikelihood, maximum-parsimony and neighbour-joining algorithms drawn from the MEGA 6 software package. Evolutionary distances were calculated using the Kimura two-parameter (Kimura 1980) and the topologies of the resultant trees evaluated by bootstrap analyses (Felsenstein 1985) based on 1000 replicates. The trees were rooted using the 16S rRNA gene sequence of Streptomyces albus subsp. albus DSM 40313 T (GenBank accession number AJ 621602).

Multi-locus sequence analyses
The experimental and data handling procedures used in the MLSA were based upon modifications of described procedures (Labeda et al. , 2017Labeda 2016). Genomic DNA was isolated from the strain using an UltraClean Ò Microbial DNA isolation kit (MoBio Labs, Carlsbad, CA) by following the manufacturer's instructions. Partial sequences of the house-keeping genes atpD (ATP synthase F1, beta subunit), gyrB (DNA gyrase B subunit), rpoB (RNA polymerase beta subunit), recA (recombinase A) and trpB (tryptophan synthetase, beta subunit) were amplified and sequenced using primers and protocols, as described previously by Labeda et al. 2014. The amplified products were purified using ExoSAP-IT (Affymetrix, Santa Clara, CA), sequenced using BigDye 3.1 on an ABI sequencer model 3730 and assembled using Sequencher version 5.2 (Gene Codes, Ann Arbor, MI).
The gene sequences for the 5 house-keeping loci of the strain were deposited in GenBank (see Supplemental  Table S1) and were also organised using the Bacterial Isolate Genomic Sequence Database (BIGSdb) version 1.12.3 (Jolley and Maiden 2010) on the ARS Microbial Genomic Sequence Database server (http://199.133.98. 43). Genome sequences, where available, were uploaded into the sequence bin for the respective isolates in the BIGSdb isolate database. The genome sequences were scanned within BIGSdb for house-keeping loci, the sequences of which were then tagged and the allele sequences and respective allele designations added to the sequence database when new alleles were found. The strain record was then updated with the matching allele identification for each locus held in the strain database. The sequences for the alleles of the loci of isolate H9 T were individually aligned with MAFFT (Katoh and Standley 2013), subsequently concatenated head to tail in-frame, and exported in FASTA format, providing a dataset of 706 Streptomyces strains and 2622 positions (Labeda 2016).
Phylogenetic relationships were constructed in IQ-Tree version 1.41 (Nguyen et al. 2015) using the maximum-likelihood algorithm based on the general time reversible model (Nei and Kumar 2000 with invariable sites plus a discrete Gamma-model based on 4 rate categories (Gu et al. 1995) which had been shown to be the optimal model for such data using iModelTest 2 (Darriba et al. 2012). The individual trees were the subject of 1000 ultrafast bootstrap replications (Minh et al. 2013) followed by 1000 replications of assessment of branch supports with single branch tests using the SH-like approximate likelihood ration test (Guindon et al. 2010). MLSA evolutionary distances were determined using MEGA 6 by calculating the Kimura 2-parameter distances (Kimura 1980). Strain pairs having B0.007 MLSA evolutionary distances were considered conspecific based on the guideline empirically determined by Rong and Huang (2012), namely that this MLSA distance (Kimura 2-parameter distance), computed from the partial sequences of these house-keeping loci, equates to the 70% DNA:DNA cut-off point recommended for the delineation of prokaryotic species by Wayne et al. (1987).

Cultural and morphological properties
The cultural features of isolate H9 T and the type strains of S. melanogenes, S. noboritoensis and S. polyantibioticus were recorded on tryptone-yeast extract, yeast extract-malt extract, oatmeal, inorganic salts-starch, glycerol-asparagine, peptone-yeast extract-iron and tyrosine agar plates (ISP media 1-7, Shirling and Gottlieb 1966) that had been incubated for 14 days at 28°C. Spore chain morphology and spore surface ornamentation were detected following growth on oatmeal agar (ISP medium 3; Shirling and Gottlieb 1966) for 14 days at 28°C, by scanning electron microscopy (Cambridge 240 instrument) after O'Donnell et al. (1993).

Phenotypic tests
Strain H9 T and the type strains of the three reference Streptomyces species were examined for a range of standard biochemical, degradative and physiological properties using media and methods described by Williams et al. (1983). Enzyme profiles of the strains were determined using API ZYM kits (bioMérieux) following the manufacturer's instructions. A standard inoculum corresponding to 5 on the McFarland scale (Murray et al. 1999) was used to inoculate all of these tests. In addition, the ability of the strains to oxidise diverse carbon and nitrogen sources and to show resistance to inhibitory compounds were determined using GEN III microplates in an Omnilog device (BIOLOG Inc., Haywood, USA). The exported data were analysed using the opm package for R (Vaas et al. 2012(Vaas et al. , 2013) version 1.06. All of these tests were carried out in duplicate.

Results and discussion
The chemotaxonomic, cultural and morphological properties of strain H9 T were found to be consistent with its classification in the genus Streptomyces (Kämpfer 2012). The organism forms an extensively branched substrate mycelium which carries aerial hyphae that differentiate into spiral chains of smooth surfaced spores on all of the ISP media tested, as exemplified in Fig. 1. The strain forms brown to black substrate mycelia on most of the ISP media and either mild brown or brown black diffusible pigments on ISP media 5-7 (Table 1). Whole organism hydrolysates of the strain were found to be rich in LL-A 2 pm, glucose, mannose and ribose; the predominant fatty acids were identified as anteiso-C 15:0 (34.6%), iso-C 16:0 (19.4%), anteiso-C 17:0 (17.9%) and C 16:0 (10.9%) ( Table 2); the major isoprenologues were identified as tetra-, hexaand octa-hydrogenated menaquinones (16, 23 and 30%, respectively); and the polar lipid pattern was found to consist of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, glycophosphatidylinositol, one unidentified lipid and an aminolipid (Fig. S1).
Isolate H9 T was found to form a distinct branch at the periphery of a well delineated subclade in the Fig. 1 Scanning electron micrograph of isolate H9 T showing spiral chains of smooth surfaced spores following growth on oatmeal agar at 28°C for 10 days. Bar 1 lm Streptomyces 16S rRNA gene tree together with S. crystallinus NBRC 15401 T , S. melanogenes NBRC 12890 T and S. noboritoensis NRRL B-12152 T , a relationship that was supported by all of the treemaking algorithms but not by a high bootstrap value (Fig. 2). The isolate is closely related to the S. melanogenes and S. noboritoensis strains sharing a 16S rRNA gene sequence similarity with them of 98.6%, a value found to correspond to 20 nucleotide (nt) differences at 1424 and 1423 locations respectively; the S. melanogenes and S. noboritoensis strains were shown to have identical 16S rRNA gene sequences. A subclade consisting of S. melanogenes NBRC 12890 T and S. noboritoensis NRRL B-12152 T was identified as cluster 30 in the 16S rRNA gene analysis of Labeda et al. (2012), S. polyantibioticus NRRL B-24448 T was found adjacent to this taxon. The corresponding 16S rRNA gene sequence homologies between the isolate and the remaining phylogenetically close strains were found to fall within the range 97.7-98.5%, values shown to equate to 21 and 31 nt differences. MLSA have been found to clarify relationships between closely related streptomycetes because of the strong phylogenetic signal provided by partial sequences of single-copy house-keeping genes Huang 2012, 2014;Labeda 2011, 2017; In the present MLSA analysis, the relationships found between isolate H9 T and the type strains of closely related Streptomyces species are shown in Fig. 3 and Table 3. The isolate is closely related to S. melanogenes NRRL B-2072 T , S. noboritoensis NRRL B-1252 T and S. polyantibioticus NRRL B-24448 T , relationships that are supported by a 100% bootstrap value; S. crystallinus NRRL B-3629 T is loosely associated with this lineage. The relationship of S. crystallinus NBRC 15401 T , S. melanogenes NBRC 12890 T and S. noboritoensis NRRL B-12152 T was also identified in a recent MLSA study (Labeda et al. 2017). Isolate H9 T was shown to have MLSA distances greater than 0.007 with all of these strains indicating that it forms the nucleus of a novel Streptomyces species. In contrast, the type strains of S. melanogenes and S. noboritoensis share a MLSA evolutionary distance of only 0.004 indicating that they belong to the same genomic species.
Identical results were obtained for nearly all of the duplicated phenotypic tests, the exceptions being a  Table 4 that the isolate can be distinguished from the type strains of S. melanogenes, S. noboritoensis and S. polyantibioticus, its close phylogenetic neighbours, by a broad range of phenotypic tests though all four strains have many properties in common. In particular, the isolate can be distinguished from the three reference type strains by its ability to form spiral chains of spores, grow at 40°C and oxidise D-fucose and D-raffinose. Conversely, the three reference type strains can be differentiated from isolate H9 T by their ability to form straight to flexuous spore chains and degrade arbutin, hypoxanthine and L-tyrosine. In turn, the isolate can be It is apparent that the type strains of S. melanogenes and S. noboritoensis have many phenotypic properties in common, notably morphological and cultural features found to be of particular value in the circumscription of Streptomyces species by Labeda et al. (2012) in their phylogenetic survey of Streptomycetaceae species. The present results are in good agreement with those of previous studies in which the type strains of S. melanogenes and S. noboritoensis were assigned to the same numerically defined phenotypic clusters (Williams et al. 1983;Kämpfer et al. 1991) and the same MLSA lineage (Labeda et al. 2017). In addition, all three strains were shown to have whole organism hydrolysates rich in LL-A 2 pm, glucose, mannose and ribose and similar polar lipid patterns (Fig S1). Similar chemotaxonomic markers have been recorded for the type strain of S. polyantibioticus, NRRL B-2443 T (Roes and Meyers 2009). The fatty acid profiles of the type strains of S. melanogenes, S. noboritoensis and S. polyantibioticus, like that of isolate H9 T , were shown to contain major proportions of anteiso-C 15:0 , iso-C 16:0 , C 16:0 and anteiso-C 17:0 though quantitative differences were apparent; such differences were also detected between other fatty acids though a few trace components were discontinuously distributed (Table 2).
In summary, isolate H9 T is only loosely associated with its near phylogenetic neighbours in the Streptomyces 16S rRNA gene tree and its distinctness is strongly supported by corresponding MLSA data based on concatenated sequences of five housekeeping genes. It can also be distinguished from the type strains of S. melanogenes and S. noboritoensis, its close phylogenetic neighbours, based on a combination of phenotypic properties. These data clearly show that isolate H9 T forms a new centre of taxonomic variation within the genus Streptomyces. The name proposed for this taxon is Streptomyces aridus sp. nov. It is also clear from the MLSA and associated chemotaxonomic and phenotypic data that the type strains of S. melanogenes and S. noboritoensis belong to the same species. It is, therefore, proposed that S.    melanogenes Suguwara and Onuma (1957) be seen as a heterotypic synonym of S. noboritoensis Isono et al. (1957). An emended description is given of the latter.
Description of Streptomyces aridus sp. nov.
Streptomyces aridus (a'ri. dus. L. masc. adj. aridus, dry, referring to the isolation of the strain from arid soil). Aerobic, Gram-stain positive, catalase positive actinobacterium that forms an extensively branched substrate mycelium that bears aerial hyphae that differentiate into spiral chains of smooth surfaced spores (1-1.5 lm 9 0.5 lm) on oatmeal agar. A brown-black diffusible pigment is produced on yeast extract-malt extract agar. Grows from 10 to 40°C, optimally *28°C, from pH 5-10, optimally *pH 7.0 and in the presence of up to 2.5% w/v NaCl.
Additional cultural and phenotypic features are cited in the text and in Tables 1 and 4. Chemotaxonomic characteristics are typical of the genus Streptomyces. The type strain, H9 T (=NCIMB 14965 T =NRRL B-65268 T ), was isolated from a subsurface soil sample collected at 4000 metres above sea level on Cerro Chajnantor, east of San Pedro de Atacama in north eastern Chile. The GenBank accession number for the 16S rRNA gene sequence of isolate H9 T is LT594571.