Introduction

Streptomycetaceae is the one of largest families within Actinobacterium, containing the genus Streptomyces as proposed by Waksman and Henrici,1 and later emended to separate the genera Streptomyces, Kitasatospora2 and Streptacidiphilus.3 Members of the family Streptomycetaceae have attracted great attention because of their production of various natural products of considerable commercial value.4 Streptomycetes are predominantly found in soil and decaying vegetation, most produce chains of spores and some are noted for their distinct ‘earthy’ odor that results from production of a volatile metabolite, geosmin. Up to now, there have been several reports on the physiology and energetics of alkaliphilic bacteria,5, 6 whereas there are very few reports on alkaliphilic actinobacteria. Thus, studies on the physiology of alkaliphilic actinobacteria are urgently required to exploit this microbial resource with great biotechnological potential. The soils from an open pit limestone mine in the Gulbarga region, Karnataka, India, seems to be great sources for a rich biodiversity of actinobacteria. Streptomyces gulbargensis,7 Streptomyces tritolerans8 and Streptomyces deccanensis9 have been already reported as novel isolates from these soils. A study was undertaken to discover novel actinomycetes from this limestone quarry during which an actinobacterium strain, YIM 75704T, was isolated and characterize by a polyphasic study designed to establish the taxonomic status of this strain. On the basis of the data obtained from this study, isolate YIM 75704T represents a novel species of recently proposed genus Allostreptomyces of the family Streptomycetaceae, for which we propose the name A. indica sp. nov.,

Materials and methods

Organisms, maintenance and culture conditions

Strain YIM 75704T was isolated by standard serial dilution technique from soil collected from an open pit limestone mine. Aliquots of serial dilutions were spread onto starch-casein agar (1% soluble starch, 0.03% casein, 0.2% KNO3, 0.2% NaCl, 0.2% KH2PO4, 0.002% CaCO3, 0.005% MgSO4·7H2O, 0.001% FeSO4·7H2O and 1.8% agar; pH 7.2) and the plates were incubated at 28 °C for 2–3 weeks. A single colony was selected and further streaked on ISP 3 medium (Hi-media, Mumbai, India) at least three times to obtain a pure culture. The pure culture was maintained as agar slopes and as suspensions of hyphal fragments and spores in 20% (v/v) glycerol at −80 °C. Further, for phenotypic and genetic comparison, strain YIM 75704T was grown on ISP 3 medium for 5 days at 28 °C.

Morphological, physiological and biochemical characterization

The temperature and pH range for growth were determined at temperatures 4, 10, 20, 30, 37, 45, 50 and 55 °C and pH 4.0–12.0 (at intervals of pH 1.0 unit), respectively. NaCl tolerance for growth was tested on ISP 3 medium supplemented with 0–9% (w/v) NaCl. A range of phenotypic properties was examined using standard procedures,10, 11, 12 Gram stain, oxidase and catalase activities, degradation ability and utilization of carbohydrates were determined using previously described methods.13 In addition, acid production from carbohydrates was tested using the media and methods described by Gordon et al.14 Other physiological and biochemical properties were tested using the API- 50CH, API-20E and API-ZYM kits (bioMérieux, New Delhi, India) according to the recommendations of the manufacturer. The arrangements of hyphae and spore chains were detected on ISP 3 after 14 days at 28 °C, using the coverslip technique of Kawato and Shinobu.15, 16 Spore chain arrangement and spore surface ornamentation were observed by examining gold coated dehydrated specimens, taken from the ISP 3 plate and under a scanning electron microscope (Philips XL30; ESEM-TMP, MD Eindhoven, The Netherlands) as described by O’Donnell et al.17 Cultural characteristics were determined using ISP media after incubation at 28 °C for 14 days.

Chemotaxonomy and morphology

The isolate was examined for chemotaxonomic properties typical of the genus Streptomyces18 with cell biomass obtained from culture grown in ISP 3 broth for 3 days at 28 °C for the isomer of diaminopimelic acid in cell wall, respiratory quinones, polar lipids and DNA G+C content according to standard procedure.19, 20, 21, 22 Strain YIM 75704T was grown on tryptone soy broth agar for 5 days at 28 °C for fatty acid analysis. The cellular fatty acid methyl esters were prepared and analyzed according to the standard protocol of the Microbial Identification System (version 6; MIDI, Hewlett-Packard Co., Palo Alto, CA, USA) ACTINO database.23

16S rRNA gene sequencing, phylogenetic analyses and DNA–DNA hybridization

Extraction of genomic DNA and PCR amplification of 16S rRNA gene followed the methods of Li et al.24 Comparisons with sequences of most closely related Streptomycetaceae and calculations of levels of sequence similarity were carried out using MEGA 6.0 (www.megasoftware.net) with sequences downloaded from EzTaxon server 2.0.25 Phylogenetic analyses were performed using the neighbour-joining,26 maximum-likelihood27 and maximum-parsimony28 methods. A phylogenetic tree was constructed using the neighbor-joining method of Saitou and Nei26 from Knuc values,29 using MEGA version 6.0.30 The topology of the phylogenetic tree was evaluated by the bootstrap resampling method of Felsenstein31 with 1,000 replicates. The genomic DNA of strain YIM 75704T for the determination of G+C content was prepared according to the method of Marmur.32 The G+C content of the DNA was determined by reverse-phase HPLC of nucleosides according to Mesbah et al.22 For DNA–DNA hybridization experiments, genomic DNA was extracted and purified according to the method of Marmur.32 Hybridization was carried out based on the principles and equations described by De Ley et al.33 under the consideration of modifications carried out by Huss et al.34 and the optimized fluorimetric procedure Loveland-Curtze et al.35 was evaluated by using a Step One Plus Real-Time PCR system (Applied Biosystems, Thermo-fisher Scientific, Carlsbad CA, USA) fitted with 96 well thermal cycling blocks. DNA suspended in the 2 × saline sodium citrate (SSC) is used for the analysis in three independent samples. The reassociation was carried out at an optimum renaturation temperature of 75 °C.36, 37

Nucleotide sequence accession number

The 16S rRNA gene sequence of strain YIM 75704T determined in this study has been deposited in GenBank under the accession number GQ357647.

Results and Discussion

An almost complete 16S rRNA gene sequence (1430 nt) of strain YIM 75704T determined in this study was compared with those of representatives species of the family Streptomycetaceae. The results of 16S rRNA gene sequence comparison clearly demonstrated that strain YIM 75704T is a novel member of the genus Allostreptomyces, with 99.0% similarity with A. psammosilenae DSM 42178T. All other type strains of the genus Streptomyces were below 95% sequence similarity. In the phylogenetic tree based on the neighbour-joining algorithm, strain YIM 75704T formed a separate clade with the Allostreptomyces species with a higher bootstrap value of 100% (Figure 1). Topologies of phylogenetic trees built using the maximum-likelihood and maximum-parsimony algorithms were similar to those of the tree constructed by neighbour-joining analysis. The determined DNA–DNA relatedness values between strain YIM 75704T and A. psammosilenae DSM 42178T, which is the only strain shared above 99% 16S rRNA gene sequence similarity, was 54.5% (3.0) (reading are the mean of triplicates), thereby indicating that the whole-genome DNA–DNA relatedness values with the isolate’s closest phylogenetic neighbors are well below the delineating 70% cutoff point for species identification,38 thus suggesting that the strain YIM 75704T should be compared to the members of the genus Allostreptomyces. The genomic DNA G+C content of strain YIM 75704T was 75.0 mol%, which was similar to related reference species of Allostreptomyces genera.

Figure 1
figure 1

Neighbor-joining tree based on 16S rRNA gene sequences, showing the relationships between strain YIM 75704T and other type strains of Streptomyces species. Bifidobacterium bifidum ATCC 29521T (KE993182) was used as the out-group. Asterisks indicate branches that were also recovered using the maximum parsimony and maximum likelihood tree. Numbers at nodes are levels of bootstrap support (>50%) for branch points (1000 resampling’s). Scale bar=0.02 substitutions per nucleotide position.

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The spore surface appears to be smooth. The strain grew well in all the media tested (Table 1). Cells of strain YIM 75704T were aerobic, Gram-positive, filamentous, non-motile (0.6-1.0 μm in diameter), spores appears as pairs and in clusters. Spores appears similar as compared to other Streptomyces species (Supplementary Figure S1). Colonies of the cells have brown to yellow substrate mycelia and olive green aerial hyphae, circular, convex with entire margins and reached 1.0–3.0 mm in diameter after incubation for 5 days at 28 °C. Strain YIM 75704T was positive for gelatin liquefaction, milk coagulation and peptonization activity. The strain was positive for starch hydrolysis, arginine decarboxylase activity, negative for nitrate reduction, gas production from nitrate, growth on cellulose and H2S production. Growth occurred at pH 6.0–10.0 (optimum, pH 7.2) and with 0–10% NaCl (optimum, 0% NaCl). Aesculin, citrate, cellobiose, glucose, inositol, mannose, melibiose, sorbitol, starch and sucrose were utilized, urease and ornithine decarboxylase activity were negative and acids were not produced from any of these carbon sources tested. Complete carbon utilization were mentioned in the species description as well as in Table 2.

Table 1 Growth and cultural characteristics of strain YIM 75704T on different growth media after incubation for 2 weeks at 28 °C
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Table 2 Morphotypic and phenotypic properties of A. indicaYIM 75704T
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Strain YIM 75704T contained LL-isomer of diaminopimelic acid as the diagnostic diamino acid in the cell wall peptidoglycan and the major quinones were MK-9 (H6 and H8) with 43.5% and 29.3%, respectively, whereas minor amount of MK-9 (H4) was also present at 4.0%. The polar lipids comprised diphosphatidylglycerol, phosphatidylinositolmannosides and three unknown phospholipids (Supplementary Figure S2). The fatty acid profile of strain YIM 75704T was characterized by large amounts of saturated and unsaturated fatty acids. The major fatty acids (>5% of the total) were i-C15:0 (35.2), ai-C15:0 (31.5), ai-C17:0 (12.0) and i-C17:0 (9.2); complete fatty acid profile has been given in Supplementary Table 1. The DNA G+C content of the type strain is 75.0 mol%. It is evident from the phenotypic, chemotaxonomic and phylogenetic data that strain YIM 75704T should be given novel species status in recently described novel genus Allostreptomyces of the family Streptomycetaceae, for which we propose the name A. indica sp. nov.

Description of A. indica sp. nov.

A. indica (in'di.ca. L. fem. adj. indica pertaining to India, Indian).

Gram-positive, aerobic actinobacteria forms brown to yellow substrate mycelia and olive green aerial hyphae, which differentiate into straight to spiral spore chains arranged in linear chains. The spores are 1–2 μm with smooth to rough surface. This organism grows well on medium ISP 2, ISP 3, ISP 4, ISP 5, Czapeks solution agar and Nutrient agar (Difco, BD, Franklin Lakes, NJ, USA) at 28 °C, whereas aerial mycelia are not easily observed on Potato agar. Soluble pigments are not produced. Aesculin, arabinose, cellobiose, D-glucose, inositol, D-sucrose, D-mannose, mannitol, D-sorbitol, L-rhamnose, melibiose and citrate are utilized as sole carbon source for growth, but not adipose, D-maltose, xylitol and D-raffinose. Positive for hydrolysis of starch, urea, oxidase activity, gelatin liquefaction, Tween 80, milk coagulation and peptonization, weak positive for hydrolysis of cellulose, but negative for nitrate reduction, catalase reaction, H2S and melanin production. Acid is not produced from any of the tested carbon sources. Temperature, pH value and NaCl concentration (w/v) for growth are 10–45 °C, 6.0–10.0% and 0–10%, respectively, with the optimal ranges of 28 °C –37 °C, 7.2 and without any NaCl concentration (w/v). Sensitive to erythromycin (15 μg per disk), gentamicin (10 μg per disk), vancomycin (30 μg per disk), novobiocin (30 μg per disk), rifampicin (5 μg per disk), netilmicin (30 μg per disk), ciprofloxacin (5 μg per disk), amikacin (30 μg per disk), tobramycin (10 μg per disk), chloramphenicol (30 μg per disk) and norfloxacin (10 μg per disk). The organism is resistance to penicillin G (10 μg per disk) and ampicillin (10 μg per disk). The major fatty acids (>5%) are i-C15:0 (35.2%), ai-C15:0 (31.5%), ai-C17:0 (12.0%) and i-C17:0 (9.2%). The G+C content of genomic DNA of type strain is 68.0 mol%. The type strain, YIM 75704T (DSM 41985T=CCTCC AA 209051T=NCIM 5485T), was isolated from lime stone open pit of Gulbarga region of Karnataka, India.