Introduction

The genus Streptomyces was proposed by Waksman and Henrici (Williams 1989), and belongs to phylum Actinobacteria, one of the major taxa among the lineages presently documented within the domain Bacteria (Gao and Gupta 2012). According to latest updates, the phylum Actinobacteria comprises of 6 classes (Acidimicrobiia, Actinobacteria, Coriobacteriia, Nitriliruptoria, Rubrobacteria and Thermophilia), 50 families and 221 genera. Recently more than 30 new genera have been added (Euzéby 2016; Ludwig et al. 2012; Kämpfer 2012) and 787 species as well as 38 sub-species have been listed in Streptomyces files (http://www.bacterio.net/streptomycesa.html). The members of this phylum are well known for their high DNA G + C content and secondary metabolites (Gao and Gupta 2012; Verma et al. 2013). The DNA G + C content ranges from 42 % (Gardnerella vaginalis, Greenwood and Pickett 1980) to 74.4 % (Kineococcus radiotolerans, Phillips et al. 2002). The type species of the genus Streptomyces, Streptomyces albus, also possess high DNA G + C content, i.e. 73.3 mol % (Zaburannyi et al. 2014). The technical challenges related to the classification of the genus Streptomyces at sub-generic level have been addressed by the advancement of genotypic and phenotypic methods (Goodfellow et al. 2007; Rong and Huang 2010), which resulted in reassignment of misclassified species of the genus Streptomyces for example Streptomyces hygroscopicus strains as Streptomyces aldersoniae sp. nov., Streptomyces angustmyceticus sp. nov., comb. nov., Streptomyces ascomycinicus sp. nov., Streptomyces decoyicus sp. nov., comb. nov., Streptomyces milbemycinicus sp. nov., Streptomyces wellingtoniae sp. nov. (Kumar and Goodfellow 2010).

Recently, several new species of the genus Streptomyces have been described, such as Streptomyces indoligenes (Luo et al. 2016), Streptomyces andamanensis (Sripreechasak et al. 2016), Streptomyces rhizosphaerihabitans, Streptomyces adustus (Lee and Whang 2016) Streptomyces verrucosisporus (Phongsopitanun et al. 2016), Streptomyces scabiei (Labeda 2016), Streptomyces bryophytorum (Li et al. 2016) and Streptomyces xinjiangensis (Cheng et al. 2016), but these are still not valid names. During the course of a study on microbial diversity of Tatta Pani hot water spring, Kotli, Azad Jammu and Kashmir, Pakistan, a strain, designated NCCP-1331T, was isolated. The results of polyphasic taxonomic studies indicated that strain NCCP-1331T represents a novel species of genus Streptomyces, for which the name Streptomyces caldifontis sp. nov. is proposed.

Materials and methods

Isolation, morphology and phenotypic characterization

Strain NCCP-1331T was isolated from soil sample collected from hot water spring of Tatta Pani, Kotli (Azad Jammu and Kashmir), Pakistan (74°32′46.10″E, 35°28′30.40″N, ASL 3841 ft). About 2 g of soil sample, pre-heated at 55 °C for 20 min, was incubated at 28 °C for 21 days and then diluted with distilled water to 10−3 and 10−4 dilution. The supernatant was spread on starch casein agar medium supplemented with 25 µg mL−1 nystatin. The inoculated agar plates were incubated at 28 °C for 2 weeks (Williams and Davies 1965). Colonies were picked and further purified by subculturing several times on same medium. The purified strain was preserved in glycerol suspensions (25 % (w/v) at −80 °C and was also maintained on oatmeal agar slopes (International Streptomyces Project—ISP medium 3; Shirling and Gottlieb 1966) at 4 °C and as mixtures of mycelial fragments and spores in 20 % (w/v) glycerol at −80 °C. The closely related strains Streptomyces brevispora KACC 21093T and Streptomyces drosdowiczii CBMAI 0498T were used as reference strains. For optimization of growth on various media, cells were streaked on media such as XYL, CMC, MSC, T5, R2A, ISP 2, Starch/casein (Composition of these are given in supplementary Table S1). Sodium carboxymethyl cellulose (CMC) and microcrystalline cellulose (MSC) were added to check cellulase activity. Cell morphology of strain NCCP-1331T was observed under a light microscope (model BH2; Olympus, Japan) and further detailed with scanning electron microscopy (ESEM-TMP, Philips) using cells grown at 30 °C for 3 days on ISP 2 medium. Gram staining was performed according to standard Gram staining procedure and was confirmed by KOH lysis test (Gregersen 1978). Cell motility was studied using the hanging-drop technique (Bernardet et al. 2002). The temperature range for growth was investigated on ISP 2 agar at different temperatures (4, 10, 15, 18, 20, 28, 30, 37, 40, 45, 50, 55 and 60 °C). NaCl tolerance at various concentrations (0–10 % (w/v) at intervals of 1 %) were tested on ISP 2 agar at pH 7.5 at optimum growth temperature 30 °C for NCCP-1331T. Growth at pH 4.0-10.0 (in 0.5 pH unit intervals) was examined at 30 °C on ISP 2 agar medium with pH adjusted as described by Xu et al. (2005). Spores were examined under scanning electron microscope according to the procedure described by O’Donnell et al. (1993). Cellulolytic activity was determined according to protocol described by Semêdo et al. (2000, 2004). Growth under anaerobic conditions was determined on ISP 2 agar supplemented with or without 0.1 % nitrate by using the GasPak Anaerobic Systems (BBL) according to the manufacturer’s instructions.

Utilization of different carbon sources was examined by using GNIII MicroPlates (Biolog, USA). Catalase activity was detected by production of bubbles on addition of 3 % (v/v) H2O2, whereas oxidase activity was tested using 1 % N,N,N′,N’-tetramethyl-p-phenylenediamine. Hydrolysis of starch, gelatin, casein, urea and Tweens (20, 40, 60, 80) were determined as described by Cowan and Steel (1974). Nitrite reduction and H2S production were performed as recommended by Tindall et al. (2007). Other physiological and biochemical properties were analysed using API 50CH/B, API 20E, API ZYM, API-ATB Vet strips (bioMérieux, France) and Biolog GN III microplates™ according to the manufacturers’ instructions.

16S rRNA gene amplification, sequencing and phylogenetic analysis

PCR amplification of 16S rRNA gene was carried out as described by Li et al. (2007). The 16S rRNA gene sequence was purified by using a PCR purification kit (Sangon Biotech, China) and cloned by pEASY-T1 cloning kit (Transgen Biotechnology) to obtain a complete sequence. The almost complete sequence of 16S rRNA gene was determined using commercial service of Sangon Biotech (Shanghai, China). The sequences of closely related validly named species were retrieved from on EzTaxon-e server (Kim et al. 2012). Multiple alignment of 16S rRNA gene sequences was carried out using the CLUSTAL X program (Thompson et al. 1997) and evolutionary distances were calculated using Kimura’s two-parameter model (Kimura 1980). Phylogenetic analyses were performed using neighbour-joining (Saitou and Nei 1987), maximum-likelihood (Felsenstein 1981) and maximum-parsimony (Fitch 1971) methods with MEGA version 6.0 (Tamura et al. 2013) software. Bootstrap analysis with (Felsenstein 1985) was performed using 1000 replications to assess topology of phylogenetic trees.

DNA–DNA hybridizations and DNA base composition

To determine DNA–DNA relatedness of strain NCCP-1331T with the reference strains, S. brevispora KACC 21093T and S. drosdowiczii CBMAI 0498T, total genomic DNA was extracted using protocols of Marmur (1963) and Pitcher et al. (1989) as described previously (Goris et al. 1998). DNA–DNA hybridization was performed with biotin-labelled probes in 96-micro-well plate (NUNC) and fluorescence measurements were carried out using an HTS7000 Bio Assay Reader (Perkin Elmer) according to Ezaki et al. (1989) and Christensen et al. (2000) with some modifications by Goris et al. (1998). The DNA–DNA hybridization was performed at 40 °C with eight replications. The G + C content of genomic DNA was determined using reversed phase HPLC and DNA of Escherichia coli DH5α was used as the reference (Mesbah et al. 1989).

Chemotaxonomic characterisation

For chemotaxonomic analyses, freeze-dried cells of strain NCCP-1331T and the reference strains were prepared by growing biomass in tryptone yeast extract broth (ISP medium 1, Shirling and Gottlieb 1966) for 7 days at 37 °C. Fatty acid methyl esters were analysed using freeze-dried cells and were identified using the Microbial Identification System (MIDI Sherlock Version 6.0, ACTINO) as described by Sasser (1990). Menaquinones were extracted according to method of Collins et al. (1977) and were analysed by HPLC (LC-10AD; Shimadzu, Japan). Analysis of the diaminopimelic acid isomers was performed as described by Lechevalier and Lechevalier (1970) and Lechevalier and Lechevalier (1980). Polar lipids were extracted and separated by two-dimensional thin-layer chromatography (TLC) and identified using procedures described earlier (Collins and Jones 1980; Minnikin et al. 1979, 1984).

Results and discussion

Morphology and phenotypic characterization

Strain NCCP-1331T was observed to be aerobic, Gram-stain positive, catalase positive and oxidase negative. Cells were observed to be non-motile, formed a branched substrate mycelium and rectiflexibilis aerial hyphae, white coloured smooth-surfaced spores appear on oatmeal agar (Supplementary Figure S1). Strain NCCP-1331T efficiently degraded cellulose at 45 °C and was observed to show cellulolytic activity at high temperature condition (up to 60 °C). The organism grew well on ISP 7, but did not grow on GYM agar and grew poorly on all other tested media. Growth occurred at temperatures ranging from 18 to 40 °C (optimum 30 °C). The pH range for growth was observed to be 6.0–9.0 (optimum pH 7.0). Strain NCCP-1331T was able to grow in presence of 0–8 % (optimum 0–2 %) (w/v) NaCl. Strain NCCP-1331T could be distinguished from closely related reference species, S. brevispora KACC 21093T and S. drosdowiczii CBMAI 0498T by having negative reaction for nitrate reduction, melanin production, and positive for catalase activity. In contrast to reference species, strain NCCP-1331T can utilize p-hydroxy-phenylacetic acid, l-alanine, glycyl-l-proline and γ-amino-butryric acid as carbon source and hydroxy-l-proline and l-histidine as nitrogen source. The detailed differentiating characteristics are listed in Table 1 and further described in the species description.

Table 1 Differential physiological characteristics of strain NCCP-1331T and its close phylogenetic neighbours in the genus Streptomyces
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Phylogenetic analysis, DNA–DNA hybridization and DNA base composition

The DDBJ/EMBL/GenBank accession number for the 16S rRNA gene sequence of strain NCCP-1331T is LC065358. The nearly complete 16S rRNA gene sequence showed that strain NCCP-1331T (1541 bp) belongs to the genus Streptomyces and shared 97.9 and 97.8 % sequence similarities with S. brevispora BK160T and S. drosdowiczii NRRL B-24297T, respectively. Neighbour-joining phylogenetic tree (Fig. 1) delineated that strain NCCP-1331T clustered with S. brevispora BK160T and S. drosdowiczii NRRL B-24297T at high bootstrap value (87 and 72 %, respectively). This phylogenetic relationship was also confirmed in phylogenetic trees generated with maximum-parsimony and maximum-likelihood algorithms (Fig. 1), suggesting that strain NCCP-1331T is a member of genus Streptomyces.

Fig. 1
figure 1

Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences showing the phylogenetic relationship of strain NCCP-1331T and related species of the genus Streptomyces. Filled circles at nodes indicate generic branches that were also recovered by using neighbour joining, maximum-parsimony and maximum-likelihood algorithms. Empty circle represents branches which were recovered by any two of the algorithms. Kitasatospora setae DSM 43861T (U93332) was selected as an outgroup. Bootstrap values (>60 %) based on 1000 replications shown at branch nodes. Bar represents 0.2 % nucleotide substitutions per site

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To confirm a separate taxonomic position of strain NCCP-1331T, DNA–DNA hybridization (DDH) was performed with closely related reference species. The results showed that strain NCCP-1331T had 42.7 ± 1.6 % DDH value with S. brevispora KACC 21093T and 34.7 ± 2.3 % with S. drosdowiczii CBMAI 0498T. These values are less than the threshold of 70 % used to define a strain as a member of a new species (Stackebrandt and Goebel 1994). DNA base composition is an important indicator at the species level. The results showed that strain NCCP-1331T was found to have 69.8 mol % of genomic DNA G + C content, which is within the range reported for members of the genus Streptomyces (69–78 mol %) (Kämpfer 2012).

Chemotaxonomic analyses

Strain NCCP-1331T was found to have a cellular fatty acid profile predominantly comprised of iso-C16:0 (19.6 %), summed feature 8 (18:1 ω7c/18:1 ω6c, 22.1 %), anteiso-C15:0 (12.7 %) and C16:0 (10.8 %) and some other minor components (Table 2). This profile is similar to other members of the genus Streptomyces, especially with closely related reference strains having the same components although in slightly different amounts, except summed feature 3, which was present in higher amount in strain NCCP-1331T than the reference strains. Members of the genus Streptomyces have straight chain, iso- and anteiso-branched chain fatty acids (Kämpfer 2012). The phylogenetic position of strain NCCP-1331T together with similarity of cellular fatty acids profile clearly indicated that the isolate belongs to genus Streptomyces (Ludwig et al. 2012; Semêdo et al. 2004; Zucchi et al. 2012; Kämpfer 2012).

Table 2 Cellular fatty acid profile (%) of strain NCCP-1331T in comparison to type strains of closely related species of genus Streptomyces
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MK-9(H8) (84 %) was determined to be the predominant respiratory quinone system in the novel strain; however, MK-9(H6) (15 %); was also detected as a minor component. The presence of MK-9(H8) is common to the members of the genus Streptomyces. Strain NCCP-1331T contained LL-diaminopimelic acid (DAP), along with alanine, glycine, leucine and glutamic acid as the diagnostic amino acids, representing peptidoglycan type LL-DAP-Gly (which seems very likely to be cross-linkage of LL-DAP by glycine residues at position 3; Schleifer and Kandler 1972). Our results also showed that characteristic whole cell sugars were not detectable in a whole-organism hydrolysate of strain NCCP-1331T (wall chemotype I sensu Lechevalier and Lechevalier (1970). The presence of LL-diaminopimelic acid, glycine and lack of distinguishing sugars are characteristic of this cell wall type (Williams 1989). The polar lipids profile of strain NCCP-1331T contained phosphatidylethanolamine, phosphatidylinositol and unidentified phospholipids (Supplementary Figure S2). The presence of phosphatidylethanolamine is reported to be the major polar lipid in all the members of Streptomyces (Kämpfer, 2012). On the basis of phylogenetic, phenotypic and chemotaxonomic analysis, the isolated strain is distinct from other related species within the genus Streptomyces. It is concluded that strain NCCP-1331T represents a novel species of the genus Streptomyces, for which the name Streptomyces caldifontis sp. nov. is proposed.

Description of Streptomyces caldifontis sp. nov

Streptomyces caldifontis (cal.di.fon’tis. L. adj. caldus, hot; L. masc. n. fons, fontis, a spring; N.L. gen. n. caldifontis, of a hot spring).

Cells are Gram-stain positive, aerobic, non-motile and exhibit branched substrate mycelium that bears aerial hyphae. Colonies on T5 agar are pointed, white-coloured. Growth occurs at 18–40 °C (optimum 30 °C), at pH 6.0–9.0 (optimum 7.0) and in presence of 0–8 % (w/v) NaCl (optimum 0–2 %). Does not produce any diffusible pigment (melanin) and forms white colored spore mass. Negative for nitrate reduction. Positive for lysine- and ornithine-decarboxylases, hydrolysis of gelatin, glycerol and urea, indole production and catalase. Positive for acid production with d-fructose, l-arabitol, d-saccharose (sucrose), salicin, d-galactose (weak), but negative from l-arabinose, starch, inulin, d-tagatose, Positive for oxidation of d-mannitol and d-mannose (weak) but negative for d-maltose, d-melibiose, inositol and D-sorbitol (as determined with API 50CH and API20E bioMérieux, France). Strong enzyme activity for, acid phosphatase, β-glucosidase, napthol-As-BI- phosphohydrolase, β-glucoronidase and trypsin (API ZYM, bioMérieux, France). Can utilize hydroxy-l-proline and l-histidine as nitrogen source. Positive for utilization of p-hydroxy-phenylacetic acid, l-alanine, glycyl-l-proline, γ-amino-butyric acid, L-malic acid, N-acetyl-d-galactosamine, d-aspartic acid, l-serine, d-glucuronic acid, glucuronamide, mucic acid, l-pyroglutamic acid, d-saccharic acid, formic acid and l-fucose as carbon sources but does not utilize β-methyl-d-glucoside, l-arginine, bromo-succinic acid as carbon source (Biolog GN III microplates™, API-ATB Vet). The predominant menaquinone is MK-9(H8) with a minor amount of MK-9 (H6). Major fatty acids are iso-C16:0, summed feature 8 (18:1 ω7c/18:1 ω6c), anteiso-C15:0 and C16:0. Cell wall peptidoglycan contains LL-diaminopimelic acid. The peptidoglycan also contains glycine, alanine, leucine and glutamic acid. Major polar lipids are phosphatidylethanolamine, phosphatidylinositol and unidentified phospholipids. The genomic DNA G + C content of the type strain is 69.8 mol %.

The type strain NCCP-1331T (=KCTC 39537T = CPCC 204147T) was isolated from a soil sample collected from a hot water spring, located at Tatta Pani, Kotli, Azad Jammu and Kashmir, Pakistan.