Introduction

Limestone habitats usually have high deposits of CaCO3 salts and therefore may be considered as unique habitats. Although microbes have been reported to play a major role in the formation of caves by dissolution of limestone and other calcareous rocks, and through extracellular precipitation of calcium carbonate (Danielli and Edington 1983; Engel et al. 2001; Riding 2000), relatively few studies have been performed on microbial diversity, including actinobacterial diversity, in limestone habitats (Groth et al. 1999a; Jurado et al. 2009; Kim et al. 1998; Nakaew et al. 2009; Niyomvong et al. 2012). To date, three new genera, Beutenbergia, Hoyosella and Knoellia, have been reported from limestone habitats and related limestone ecosystems, such as cave biofilms (Groth et al. 1999b, 2002; Jurado et al. 2009).

The genus Streptomyces is the currently most diverse known group of bacteria with more than 650 species reported (http://www.bacterio.net/streptomycesa.html; Parte 2014). Streptomyces species have been found to have diverse roles in nature, from being antibiotic producers to being pathogens (Keiser et al. 2000). The genus is characterized by high G+C content in DNA, formation of extensively branched substrate and aerial mycelia, presence of LL-diaminopimelic acid (LL-DAP) and absence of characteristic sugars in the cell wall (cell wall type I) (Kämpfer 2012). The present study reports the polyphasic characterization of a novel strain isolated from a limestone environment, MBRL 172T, which is proposed as representative of a novel species of the genus Streptomyces.

Materials and methods

Strain and culture conditions

Strain MBRL 172T was isolated from a soil sample collected from a limestone quarry site at Hundung, Manipur, India (25.05°N, 94.33°E). Isolation was performed on Gauze’s Medium No. 1 using a procedure described earlier (Nimaichand et al. 2012). Strain MRBL 172T was preserved as lyophilized spore suspensions in skim milk at room temperature and as glycerol suspensions (20 %, v/v) at −80 °C.

The related type strains Streptomyces coeruleofuscus NBRC 12757T and Streptomyces nogalater NBRC 13445T were obtained from Biological Resource Center (NBRC), National Institute of Technology and Evaluation, Japan and cultured under comparable conditions as reference strains.

Phenotypic characterization

To observe its morphological characteristics, strain MBRL 172T was cultivated aerobically in Gauze’s Medium No. 1 (28 °C) for 2 weeks. Morphology of spores and mycelia was observed by light microscopy (Olympus BH2) and scanning electron microscopy (SEM) (Quanta 200, FEI) (Williams and Davies 1967). Growth on various International Streptomyces Project media (ISP, Shirling and Gottlieb 1966), Tryptic Soy Agar (TSA, Difco), Starch Casein Nitrate Agar (SCNA), Gauze’s Medium No. 1, Czapek’s Dox agar and Nutrient agar (NA) were observed. The colony colour was determined using the ISCC-NBS colour chart (Kelly 1964).

Utilization of sole carbon and nitrogen sources was determined as described by Shirling and Gottlieb (1966). Tests for decomposition of casein and acid production from carbohydrates were performed following the methods of Gordon et al. (1974). Hydrolysis of gelatin and starch was determined as described by Collins et al. (2004) and that of Tweens 20, 40, 60 and 80 according to Sierra (1957). Growth at different temperatures (5, 15, 20, 28, 37, 42, 50 and 60 °C), pH (4, 5, 6, 7, 8, 9 and 10) and NaCl concentrations (0, 2, 5, 7 and 10 % w/v) was determined on TSA as described by Goodfellow (1986). Catalase activity was observed by assessing bubble production in 3 % (v/v) H2O2 and oxidase activity was determined by using a 1 % (w/v) solution of tetramethyl-p-phenylenediamine (Kovacs 1956). Other biochemical tests including Voges-Proskauer, methyl red and indole tests were performed according to Goodfellow (1986).

Chemotaxonomy

The amino acid content of the cell wall was determined according to Staneck and Roberts (1974) and the sugars in the whole cell hydrolysates were analyzed as described by Tang et al. (2009). For other chemotaxonomic analyses, cell biomass from a two week old culture on Tryptic Soy Broth (TSB, Difco) was harvested by centrifugation, washed with distilled water and lyophilized. Polar lipids were extracted and analyzed by two-dimensional TLC as described by (Minnikin et al. 1984). The extraction of menaquinones was performed according to Collins et al. (1977) and analyzed by HPLC (Tamaoka et al. 1983). Cellular fatty acids were extracted, methylated and analyzed by using Sherlock Microbial Identification System (MIDI) according to the method of Sasser (1990) and the manufacturer’s instructions. Fatty acid methyl esters were analysed by GC (Agilent Technologies 7890A GC System) and identified using the Microbial Identification Software Package (Sherlock Version 6.1; MIDI database: TSBA6).

Molecular analysis

Genomic DNA extraction and PCR amplification of the 16S rRNA gene was performed as described by Li et al. (2007) with a slight modification. The strain was given a brief ultrasonic wave shock (53 kHz, 30 s) prior to lysozyme treatment. The almost complete 16S rRNA gene sequence (1,534 bp) of the strain was identified using the EzTaxon-e server database (Kim et al. 2012) and aligned with the 16S rRNA gene sequences of other Streptomyces species using CLUSTAL X version 2.1 (Larkin et al. 2007). Phylogenetic analyses were performed using the software package MEGA version 6.0 (Tamura et al. 2013). Phylogenetic distances were calculated with the Kimura two-parameter model (Kimura 1983) and tree topologies were inferred using the maximum-likelihood (Felsenstein 1981), maximum-parsimony (Fitch 1971) and neighbour-joining (Saitou and Nei 1987) methods. To determine the support of each clade, bootstrap analysis was performed with 1,000 resamplings (Felsenstein 1985).

The G + C content of the genomic DNA prepared by the method of Marmur (1961) was determined as described by Mesbah et al. (1989). DNA–DNA relatedness was studied by a fluorimetric method (Ezaki et al. 1988, 1989). One of the two DNA molecules for hybridization was labeled while the other was immobilized and vice versa. Six replications were done for each sample and the two extreme values (highest and lowest) for each sample were excluded. The relatedness values are expressed by calculating the means of the remaining four values and the DNA–DNA hybridization was taken from the two means of relatedness values.

Results and discussion

Strain MBRL 172T was observed to form extensive substrate and white coloured aerial mycelia on Gauze’s Medium No. 1. The strain was observed to form spiralis spore chains with warty spores (approximately 50 spores per chain, each spore measuring ~600 × 900 nm in dimensions, Fig. 1). The strain was found to grow well in all the media tested and formed brown and blue diffusible pigments on ISP 7 and Gauze’s Medium No. 1, respectively (Table 1). The differentiating properties of strain MBRL 172T relative to the related type strains Streptomyces coeruleofuscus NBRC 12757T and Streptomyces nogalater NBRC 13445T are listed in Table 2 and the detailed phenotypic characteristics are mentioned in the species description.

Fig. 1
figure 1

Scanning electron micrograph for strain MBRL 172T grown on Gauze’s medium no. 1 for 2 weeks at 28 °C, Bar 10 μm

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Table 1 Culture characteristics of strain MBRL172T on different ISP and other media as observed using ISCC-NBS Colour Chart (Kelly 1964)
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Table 2 Differential characteristics between strains MBRL 172T, S. coeruleofuscus NBRC 12757T and S. nogalater NBRC 13445T
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Strain MBRL 172T was found to have LL-DAP as the diagnostic cell wall diamino acid and xylose (61 %), glucose (23 %) and mannose (13 %) were detected as the major sugars in whole cell hydrolysates, along with small amounts of ribose (4 %). The major polar lipids detected were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol and phosphatidylinositolmannoside, along with other unknown phospholipids and lipids (see Supplementary Fig. S1). MK-9(H8) (47.5 %) and MK-9(H6) (41.2 %) were identified as the predominant menaquinones, along with small amounts of MK-9(H4) (9.8 %) and MK-9(H2) (1.5 %). The fatty acid methyl ester profile (>1 %) was found to contain anteiso-C15:0 (19.7 %), iso-C16:0 (19.5 %), iso-C15:0 (14.9 %), anteiso-C17:0 (8.5 %), Summed Feature 9 (as defined by MIDI) containing iso-C17:1ω9c and/or 10-methyl C16:0 (6.5 %), iso-C16:1- H (5.9 %), anteiso-C17:1ω9c (5.8 %), C16:0 (4.8 %), iso-C17:0 (4.2 %), Summed Feature 3 containing C16:1ω7c and/or C16:1ω6c (3.6 %) and iso-C14:0 (3.1 %). The major fatty acids in S. coeruleofuscus NBRC 12757T are unsaturated fatty acids iso-C15:0 (26.0 %), iso-C16:0 (24.8 %) and anteiso-C15:0 (15.7 %), and in the case of S. nogalater NBRC 13445T are iso-C15:0 (34.4 %), anteiso-C15:0 (32.3 %) and anteiso-C17:0 (14.9 %). The fatty acids C16:0 (7.6 %) and iso-C14:0 (5.3 %) in S. coeruleofuscus NBRC 12757T were not detected or detected in lesser quantities (<5 %) in the other two strains, indicating the variability in the fatty acids profile (Table 2).

The G + C content of the genomic DNA of strain MBRL 172T was found to be 69.4 %. Based on the 16S rRNA gene sequence similarities and the neighbour-joining tree (Fig. 2), the strain forms a clade with S. coeruleofuscus NBRC 12757T (99.2 %; 12/1462 nucleotide differences), Streptomyces chromofuscus NBRC 12851T (99.0 %; 15/1460), Streptomyces cinereospinus NBRC 15397T (98.8 %; 17/1462), S. nogalater JCM 4799T (98.6 %; 21/1469) and Streptomyces pluripotens MUSC 135T (98.5 %; 22/1483). This indicated that the strain belongs to clade 114 as defined by Labeda et al. (2012). The maximum-likelihood tree and maximum-parsimony tree are shown in Supplementary Fig. S2 and Fig. S3, respectively. As Streptomyces strains sharing more than 99.5 % 16S rRNA gene sequence similarities with their closest homologues have been reported among novel species (Bouchek-Mechiche et al. 2000; Dastager et al. 2007; Goodfellow et al. 2007; Nimaichand et al. 2012, 2013), strains S. coeruleofuscus NBRC 12757T and S. nogalater NBRC 13445T were selected as the representative strains to perform DNA–DNA hybridization studies. The experiments showed that DNA–DNA relatedness values with S. coeruleofuscus NBRC 12757T and S. nogalater NBRC 13445T (Table 3) are well below the 70 % cut off point for species identification (Wayne et al. 1987).

Fig. 2
figure 2

Neighbour-joining tree, based on 16S rRNA gene sequences, showing the relationships between strain MBRL 172T and other type strains of the genus Streptomyces. Hash and asterisks indicate the branches that were also recovered in the maximum-likelihood analysis, and both maximum-likelihood and maximum parsimony trees, respectively. Numbers at nodes are levels of bootstrap support (%) for branch points (1,000 resamplings). Bar 0.002 substitutions per nucleotide position

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Table 3 DNA relatedness between strains MBRL 172T, S. coeruleofuscus NBRC 12757T and S. nogalater NBRC 13445T as determined by the fluorimetric method
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The chemotaxonomic data and the phylogenetic analysis clearly indicates the affiliation of strain MBRL 172T to the genus Streptomyces. The genotypic and phenotypic features suggest that strain MBRL 172T could be clearly distinguished from its closest phylogenetic relatives. Besides low DNA–DNA relatedness values, the strain is also distinguished from S. coeruleofuscus NBRC 12757T and S. nogalater NBRC 13445T by several phenotypic properties as listed in Table 2. Therefore, strain MBRL 172T, isolated from Hundung, is considered to represent a new species of the genus Streptomyces, for which the name Streptomyces canchipurensis sp. nov. is proposed.

Description of Streptomyces canchipurensis sp. nov

Streptomyces canchipurensis (can.chi.pur.en´sis. N.L. masc. adj. canchipurensis of or belonging to Canchipur, Manipur University, India, where the MBRL research group which isolated the type strain is located).

Gram-stain positive, aerobic, with Spiralis spore chains containing up to 50 spores per chain. Each spore on maturity measures ~600 × 900 nm. Growth occurs at 20–37 °C and pH 6–9, with optimum growth at 28 °C and pH 7. Growth occurs in presence of up to 2 % NaCl. Able to utilise dulcitol, galactose, inositol, inulin, lactose, maltose, mannose, melibiose, raffinose, sorbitol and sucrose as sole carbon sources; and l-alanine, l-arginine, l-asparagine, l-glutamine, l-hydroxyproline, l-leucine, l-lysine, dl-methionine, l-tryptophan, l-tyrosine and l-valine as sole nitrogen sources. Does not utilise arabinose, cellobiose, salicin and l-cysteine as either sole carbon or nitrogen sources. Acid is produced from lactose, maltose and sucrose but not from mannose. Hydrolyzes starch and Tweens 20, 40, 60 and 80 but not casein, gelatin and urea. Positive in catalase, oxidase and citrate utilization tests but negative in methyl red, Voges-Proskauer and indole production tests. Contains LL-diaminopimelic acid, xylose, glucose and mannose with small amount of ribose in the whole cell hydrolysates. MK-9(H6) and MK-9(H8) are the predominant menaquinones, while the polar lipids consist of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol and phosphatidylinositolmannoside along with other unknown phospholipids and lipids. The fatty acid profile (>1 %) contains anteiso-C15:0, iso-C16:0, iso-C15:0, anteiso-C17:0, Summed Feature 9 containing iso-C17:1ω9c and/or 10-methyl C16:0; iso-C16:1- H, anteiso-C17:1ω9c, C16:0, iso-C17:0, Summed Feature 3 containing C16:1ω7c and/or C16:1ω6c and iso-C14:0. The G + C content of the genomic DNA of the type strain is 69.4 %.

The type strain MBRL 172T (=JCM 17575T = KCTC 29105T) was isolated from a limestone quarry site at Hundung, Manipur, India. The 16S rRNA gene sequence of strain MBRL 172T has been deposited in GenBank under the accession number JN560154. The strain belongs to clade 114 of the genus Streptomyces as defined by Labeda et al. (2012).