International Journal of Systematic and Evolutionary Microbiology (2014), 64, 1365–1372
DOI 10.1099/ijs.0.056614-0
Streptomyces barkulensis sp. nov., isolated from an estuarine lake Lopamudra Ray,1 Samir Ranjan Mishra,1 Ananta Narayan Panda,1 Gurdeep Rastogi,2 Ajit Kumar Pattanaik,2 Tapan Kumar Adhya,1 Mrutyunjay Suar1 and Vishakha Raina1 Correspondence
1
Vishakha Raina
2
School of Biotechnology, KIIT University, Bhubaneswar – 751024, Odisha, India Chilika Development Authority, Plot No. C-11, BJB Nagar, Bhubaneswar – 751014, Odisha, India
[email protected]
The taxonomic position of a novel actinomycete, strain RC 1831T, isolated from the sediment of a fish dumping yard at Barkul village near Chilika Lake, Odisha, India, was determined by a polyphasic approach. Based on morphological and chemotaxonomic characteristics the isolate was determined to belong to the genus Streptomyces. The phylogenetic tree based on its nearly complete 16S rRNA gene sequence (1428 nt) with representative strains showed that the strain consistently falls into a distinct phyletic line together with Streptomyces glaucosporus DSM 41689T (98.22 % similarity) and a subclade consisting of Streptomyces atacamensis DSM 42065T (98.40 %), Streptomyces radiopugnans R97 DSM 41901T (98.27 %), Streptomyces fenghuangensis GIMN4.003T (98.33 %), Streptomyces nanhaiensis DSM 41926T (98.13 %), Streptomyces megasporus NBRC 14749T (97.37 %) and Streptomyces macrosporus NBRC 14748T (98.22 %). However, the levels of DNA–DNA relatedness between strain RC 1831T and phylogenetically related strains Streptomyces atacamensis DSM 42065T (28.75±3.25 %) and Streptomyces glaucosporus DSM 41689T (15±2.40 %) were significantly lower than the 70 % threshold value for delineation of genomic species. Furthermore, the isolate could be distinguished phenotypically on the basis of physiological, morphological and biochemical differences from its closest phylogenetic neighbours and other related reference strains. Strain RC 1831T is therefore considered to represent a novel species of the genus Streptomyces, for which the name Streptomyces barkulensis sp. nov. is proposed. The type strain is RC 1831T (5JCM 18754T5DSM 42082T).
Streptomyces is the largest genus of the phylum Actinobacteria (Ka¨mpfer & Labeda 2006; Lodders & Ka¨mpfer, 2007), comprising, at the time of writing, more than 600 recognized species (Hain et al., 1997). They are typically aerobic, Gram-positive and with a high DNA G+C content, showing presence of spore-bearing aerial and substrate mycelia and LL-diaminopimelic acid (LL-DAP) with no characteristic sugars in the cell wall (Williams et al., 1983; Embley & Stackebrandt, 1994). They are widely known for their ability to produce a broad range of secondary metabolites, bioactive compounds and industrially important enzymes, which makes them an important genus for scientific exploration of many useful products (Be´rdy, 2005; Watve et al., 2001; Chater et al., 2010). The conventional systematics of the genus Streptomyces based Abbreviation:
LL-DAP, LL-diaminopimelic
acid.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain RC 1831T is JQ862602. Three supplementary figures are available with the online version of this paper.
056614 G 2014 IUMS
Printed in Great Britain
on phenotypic characteristics (Williams et al., 1983; Ka¨mpfer et al., 1991) has undergone a radical change with the application of molecular systematics (Goodfellow et al., 1992; Embley & Stackebrandt, 1994; Lanoot et al., 2002, 2004; Hatano et al., 2003; Ka¨mpfer, 2012). Although comparison of 16S rRNA gene sequences for classification at the genus level has been widely used for Streptomyces taxonomy, differences at the species level are difficult to establish when 16S rRNA gene sequence similarities are .97 % (Stackebrandt & Goebel, 1994). Moreover, high 16S rRNA gene sequence similarities between type strains often pose problems for the description of novel species within the genus Streptomyces. These species can be delineated through a polyphasic approach, i.e. study of the combination of phenotypic and genotypic features (Ka¨mpfer et al., 2008). Polyphasic approaches hence continue to present us with large numbers of novel species and has shown that the genus Streptomyces is largely underspeciated (Goodfellow et al., 1992; Kim and Goodfellow, 2002; Saintpierre et al., 2003). Species description with recently developed techniques, such as numerical classification for taxonomic 1365
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evaluation and multilocus sequence analysis (Ka¨mpfer & Labeda, 2006; Goodfellow et al., 2007; Rong & Huang, 2010; Labeda et al., 2012), have also been found to be valuable in the classification of new affiliates to the taxon isolated from diverse geographical environments. While screening for chitin-degrading bacteria, strain RC 1831T was isolated from a fish dumping yard sediment (pH 8.5 and salinity 9.6 p.p.t.) near the shoreline of the brackish Chilika Lake (19u 719 N 85u 199 E) in Barkul village, Khurda district, Odisha, India. Taxonomic investigation using a polyphasic approach confirmed that the new isolate represented a novel species within the genus Streptomyces. Strain RC 1831T was isolated after 7 days of incubation at 30 uC with the appearance of a zone of clearance on a colloidal chitin agar medium (Hsu & Lockwood, 1975) supplemented with nystatin (50 mg l21), when inoculated with a sediment sample suspension. Strain RC 1831T showed the characteristic presence of spore-bearing aerial and substrate mycelia, which is a morphological attribute of the genus Streptomyces (Ka¨mpfer, 2012). The pure strain was maintained routinely on agar plates of modified Luria– Bertini (LB) media fortified with seawater (SWLB; 10.0 g tryptone, 5.0 g yeast extract, 5.0 g NaCl, 500 ml seawater, 500 ml distilled water, 18 g agar, pH 7.0) at 4 uC and as a suspension of mycelia and spores in 50 % (v/v) glycerol at 280 uC. Biomass required for chemotaxonomic and molecular analyses was obtained by culturing isolate RC 1831T in SWLB for 48 h at 30 uC and cells were harvested by centrifugation and resuspended in water/2-propanol (1 : 1, v/v) (Ray et al., 2013). The genomic DNA of strain RC 1831T was extracted by the method of Kieser et al. (2000) and the 16S rRNA gene was amplified by PCR (Gene Amp PCR machine; Perkin Elmer) using primers as described by Lane (1991). Amplified product was purified with a spin column (QIAquick PCR Purification kit; Qiagen) and sequenced using an ABI prism dye terminator cycle sequencing kit (ABI prism model 3700; Applied Biosystems) as described by Edwards et al. (1989). The nearly complete 16S rRNA gene sequence (1428 nt) was compared with sequences of representative species of the genus Streptomyces deposited in the GenBank database (Altschul et al., 1997) using the BLAST program and EzTaxone server version 2.1 (Kim et al., 2012). The 20 sequences showing the highest scores were selected for the calculation of pairwise sequence similarity using a global alignment algorithm, which was implemented at the EzTaxon-e server (http://eztaxon-e.ezbiocloud.net/). The recovered sequences were aligned with the sequence of strain RC 1831T using CLUSTAL W (Larkin et al., 2007) and edited using BioEdit Sequence Alignment Editor, version 7.1.3 (Hall, 1999). The phylogenetic relationships were established using the maximum-likelihood (Felsenstein, 1985), maximum-parsimony (Fitch, 1971) and minimum-evolution (Rzhetsky & Nei, 1992) algorithms of MEGA version 5.1 software (Tamura et al., 2011). An evolutionary distance matrix was calculated using the method of Kimura’s two-parameter model (Kimura, 1980) and a phylogenetic tree was reconstructed 1366
using the neighbour-joining (Saitou & Nei, 1987) method with Actinomadura hibisca JCM 9627T as an outgroup. The topology of the phylogenetic tree was evaluated by MEGA v. 5 using the bootstrap resampling method with 1000 replicates (Felsenstein, 1985). Culture traits of strain RC 1831T were recorded after 21 days of incubation at 30 uC on several International Streptomyces Project (ISP; Shirling & Gottlieb, 1966) media. Colour evaluations on ISP media for diffusible pigments were assessed by comparing the cultures with colour chips from ISCC-NBS Color Charts standards no. 2 2106 (Kelly, 1964). Investigation of morphological and chemotaxonomic attributes which are representative of the genus Streptomyces were carried out for strain RC 1831T (Williams et al., 1989; Embley & Stackebrandt, 1994; Manfio et al., 1995; Stackebrandt & Ebers, 2006). Strain RC 1831T was incubated on SWLB agar for 3–4 weeks at 30 uC to observe spore chain morphology, spore surface and spore chain ornamentation by light microscopy (DM2000, LAS v. 4.2; Leica) and scanning electron microscopy (EVO40; Zeiss). Several biochemical and physiological characteristics of strain RC 1831T and its phylogenetically closest neighbours were tested and compared using the well-established procedures of Shirling and Gottlieb (1966), Williams et al. (1983) and Ka¨mpfer (2012). Enzyme hydrolysis, and urea and citrate utilization tests were performed on LB agar media and carbohydrate utilization tests were carried out on basal salt media supplemented with 1 % (v/v) of the individual carbon source. Catalase activity was determined by oxygen bubble formation on addition of H2O2 (4 %) to colonies and oxidase activity was checked by a change in colour of an oxidase disc (HiMedia). Estimation of melanin production and nitrate reduction were tested on ISP media according to the methods described by Gordon et al. (1974), Williams et al. (1989) and Goodfellow (1971). Physiological tolerance to temperature (20–50 uC) and pH (6.0–10.0) was tested on LB agar media and NaCl tolerance (0–18 % w/v) was tested in LB broth media incubated for 14–21 days at 30 uC. Antibiotic susceptibility was examined using antibiotic discs (HiMedia) as described by Williams et al. (1983) on LB agar plates. Antimicrobial activity of strain RC 1831T was analysed on LB agar for 7 days against indicator strains Escherichia coli MTCC 82, Staphylococcus aureus MTCC96, Bacillus cereus IP 4061 and Salmonella typhi MTCC734, procured from the Microbial Type Culture Collection (MTCC, Chandigarh, India), and B. cereus IP 4061 and Micrococcus luteus (a kind gift from Dr. Peter Luethy, Institute of Microbiology, ETH, Zurich), by observing the formation of a zone of inhibition (Madigan et al., 1997). The diagnostic isomer LL-DAP and whole-cell wall sugars were analysed as described by Rhuland et al. (1955) and Staneck & Roberts (1974), respectively. Polar lipid analysis was carried out according to Bligh and Dyer (1959) and Tindall et al. (2007). Respiratory quinones were extracted using the two-stage method and separated into their different classes (menaquinones, ubiquinones, etc.) as described by Tindall (1990a, b) and further analysed by HPLC International Journal of Systematic and Evolutionary Microbiology 64
Streptomyces barkulensis sp. nov.
(Kroppenstedt, 1985). Fatty acid analysis was performed according to standard procedures as described by Sasser (1990) and comparison of the results was done with fatty acid databases included in the Microbial Identification Software package (Sherlock, Version 6.1). The G+C content of the genomic DNA of strain RC 1831T was determined by using the thermal denaturation method of Mandel and Marmur (1968) on a Peltier system spectrophotometer (Perkin Elmer). Microscopic studies for morphological observations revealed the presence of branching aerial and substrate mycelia which differentiate into well-developed aerial spore chains. At maturity, aerial hyphae produced rounded spores (161 mm) in spore chains which are straight to flexuous (Rectiflexibles) with a warty spore surface (Figs 1 and S1, available in the online Supplementary Material). The isolate exhibited good growth on ISP 3 and ISP 6, moderate growth on ISP 4, ISP 5 and ISP 7, but no growth on ISP 2. No diffusible pigments were detected on any of the ISP media tested. No aerial mycelia were recorded on ISP 4, ISP 5 or ISP 7 (Table 1). Strain RC 1831T was positive for hydrolysis of starch, cellulose and pectin, gelatin liquefaction, milk coagulation, degradation of Tween 80, lecithin, tributyrin and chitin, and presence of catalase and oxidase; it was negative for urea and citrate utilization, nitrate reduction and hydrolysis of xylan. Growth occurred at pH 8–10 (optimum, pH 8), at 40 uC (optimum, 30 uC) and with 0–10 % (w/v) NaCl (optimum, 8 % NaCl); it was able to tolerate up to 18 % NaCl. Cellobiose, D-fructose, D-galactose, D-glucose, D-mannose, maltose, raffinose, ribose, L-rhamnose and D-xylose could be utilized as sole carbon and energy source, but not arabinose, inositol or lactose (Table 1). The chemotaxonomic features of strain RC 1831T were consistent with the genus Streptomyces. It showed the diagnostic presence of LL-DAP in the cell-wall peptidoglycan with no characteristic sugar pattern, which is indicative of cell-wall type I (Lechevalier & Lechevalier, 1970;
Williams et al., 1989). The whole-cell hydrolysate contained glucose and ribose and the most prominent type II polar lipids were diphosphatidylglycerol and phosphatidylethanolamine. Phosphatidylinositol, phosphatidylinositolmannoside and an unknown phospholipid were also observed (Fig. S2). The major menaquinones were MK-9 (H4) (12 %), MK-9 (H6) (53 %) and MK-9 (H8) (16 %), with minor traces of MK-9 (H2) (2 %), MK-9 (1 %), MK-8 (H4) (1 %), MK-10 (H4) (3 %), MK-10 (H6) (7 %) and MK-10 (H8) (3 %). The cellular fatty acids consisted of iso-C14 : 0 (7.03 %), C14 : 0 (0.56 %) iso-C15 : 0 (3.83 %), antiso-C15 : 0 (25.91 %), C15 : 1 B (0.37 %), C15 : 0 (1.94 %), iso-C16 : 1 H (1.04 %), iso-C16 : 0 (25.24 %), C16 : 1v9c (3.12 %), C16 : 0 methyl (2.31 %), C16 : 0 (8.72 %), antisoC17 : 1 (3.83 %), iso-C17 : 0 (2.89 %), C17 : 1v9c (0.81 %), antiso-C17 : 0 (8.89 %), iso-C18 : 1 H (0.38 %), iso-C18 : 0 (0.38 %), C18 : 1v9c (0.18 %), C18 : 0 (0.64 %), C14 : 1v9t/ 9c (0.13 %) and C18 : 1v11c/9t/6t (0.24 %). The G+C content of the genomic DNA was 66.2 mol% (mean of two replicates). All the above features confirmed the classification of strain RC 1831T to the genus Streptomyces. Comparison of the almost-complete 16S rRNA gene sequence of strain RC 1831T (1428 nt) with those of closely related type strains clearly revealed its phylogenetic affiliation to the genus Streptomyces. Based on 16S rRNA gene sequences, the closest neighbour to strain RC 1831T was Streptomyces atacamensis DSM 42065T (Santhanam et al., 2012), at a similarity of 98.4 %, a value which corresponds to 23 nt differences over 1401 locations. This was supported by all tree-making algorithms (neighbourjoining, minimum-evolution, maximum-likelihood, maximum-parsimony) with a high bootstrap value (.90 %) (Fig. 2). However, strain RC 1831T also formed a distinct phyletic line with Streptomyces glaucosporus R97 DSM 41689T (98.22 % 16S rRNA gene sequence similarity) and a close subclade showed a corresponding branch composed
Fig. 1. Scanning electron micrograph of cells of strain RC 1831T grown on SWLB (50 %, v/v) for 3 weeks (21 days) at 30 6C. Bar, 1 mm. http://ijs.sgmjournals.org
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Table 1. Differential phenotypic characteristics between strain RC 1831T and its closest phylogenetic relatives in the genus Streptomyces Strains: 1, RC 1831T; 2, Streptomyces atacamensis DSM 42065T; 3, Streptomyces glaucosporus DSM 41689T; 4, Streptomyces fenghuangensis GIMN4.003T; 5, Streptomyces radiopugnans R97 DSM 41901T; 6, Streptomyces nanhaiensis DSM 41926T. All data represented here have been generated in this study except for Streptomyces fenghuangensis GIMN4.003T (Zhu et al., 2011). All strains were grown at pH 7 and utilized Dfructose, D-glucose and D-galactose as sole carbon sources. +, Positive; 2, negative; OW, off-white; BR, brownish red; PY, pale yellow; PO, pale orange; YW, yellowish white; GW, greenish white; BL, black; OB, off-brown; PB, pale brown; B, brown; ND, not determined. Characteristic Morphology* Spore chain morphology Aerial spore mass colour Spore surface ornamentation Colour and growth of substrate mycelia on ISP mediaD Yeast extract malt extract agar (ISP 2) Oatmeal agar (ISP 3) Inorganic salt starch agar (ISP 4) Glycerol asparagine agar (ISP 5) Peptone yeast extract iron agar (ISP 6) Tyrosine agar (ISP 7) Diffusible pigment Enzyme hydrolysis§ Starch Gelatin Caesin Tributyrin Tween 80 Lecithin Chitin Pectin Cellulose CM-cellulose Utilization of:§ Urea Citrate Growth on sole carbon source [1 % (w/v)]|| L-Arabinose Cellobiose D-Fructose D-Galactose D-Glucose Inositol Lactose D-Mannose Maltose Raffinose Ribose L-Rhamnose D-Xylose Growth with NaCl (%, w/v) 3, 5, 6 7, 9 11 12, 15, 18
1368
1
2
3
4
5
6
Rectiflexibles White Warty
Straight Grey Smooth
Spiral Pale grey Rough to warty
Rectiflexibles White Rough to warty
Spiral Pale yellow Smooth
Rectiflexibles White Smooth to rough
2
OW+++
2
YW+++
PY+
PY+
OW+++ OW++ OW++ BR+++
PY++ OW++ PY++ PO++
PY++ OW+ OW++ PY++
G++ GW+ YW++ 2
BL+++ OW++ OB+++ PB+
PY+ PY+ PY++ B+++
OW++ 2
OW++ 2
OW++ 2
2 Blued
OW++ 2
PY+ 2
+ + + + + + + + + +
+ + 2 + + 2 2 2 + +
+ + 2 2 + 2 + 2 + +
2
+ + 2 2 + 2 + 2 + +
+ + 2 2 + + + 2 + +
2 2
+ +
+ 2
2 2
2 2
2 + + + + 2 2 + + + + + +
2 2 + + + + + + + + + + +
+ + + + + 2 2 + + + 2 + +
2 2 + + + 2 2 2 + + 2 2 +
2 2 + + + + + + + + + + +
+ + + +
+ + + 2
+ + 2 2
+ 2 2 2
+ + 2 2
ND
+ ND ND ND ND ND ND ND ND ND
+ + + + + + ND ND
2 2 2 2 2 ND ND ND ND
International Journal of Systematic and Evolutionary Microbiology 64
Streptomyces barkulensis sp. nov.
Table 1. cont. Characteristic Growth at pH:§ 8, 9, 10 Growth at:§ 20 uC 50 uC
1
2
3
4
5
6
+
2
2
ND
+
2
2 2
2 +
2 +
ND
2 +
+ 2
ND
*Growth on modified seawater (50 %; v/v) LB. D+++, Abundant growth; ++, moderate growth; +, poor growth; 2, no growth. dISP 5. §LB agar. ||Basal salt media (Shirling and Gottlieb, 1966). LB broth.
S.glaucosporus DSM 41689T (AB184664)
* 52 0.01 67
* 99
* 93
*
S.barkulensis RC1831T DSM 42082T (JQ862602) S.atacamensis DSM 42065T (HE 577171) S. radiopugnans R97 DSM 41901T (DQ912930)
* 62
S.fenghuangensis GIMN4.003T (GU356598) 86
* 63
* * 93
S.nanhaiensis SCSIO 01248 DSM 41926T (GQ871748) S.megasporus NBRC 14749T (AB184617) S.macrosporus NBRC 14748T (AB184616)
S.fimbriatus NBRC 15411T (AB184659) S.cacaoi subsp. cacaoi NBRC 12748T (AB184115) * 87
S.albus DSM 40313T (AJ621602) * *
65
73
S.platensis JCM 4662T (AB045882)
89
53 *
S. libani subsp. rufus LMG 20087T (AJ781351)
S.hygroscopicus NBRC 13786T (AB184479) S.caniferus NBRC 15389T (AB184640) S.nigrescens NBRC 12894T (AB184225) S.angustmyceticus NBRC 3934T (AB184817) S.catenulae ISP 5258T (AY999778)
66 * *
S.albiaxialis NRRL B-24327T (AY999901) S.tateyamensis DSM 41969T (AB473555) S.chrestomyceticus DSM 40545T (AJ621609)
* 100
S. rimosus subsp. paromomycinus DSM 41429T (AJ621610) S.abikoensis NBRC 13860T (AB184537) A.hibisca JCM 9627T (AF163115)
Fig. 2. Neighbour-joining tree (Saitou & Nei, 1987) based on analysis of the nearly complete (1428 nt) 16S rRNA gene sequence showing the position and phylogenetic relationship between strain RC 1831T and other related members of the genus Streptomyces. Asterisks indicate conserved branches recovered when the minimum-evolution (Kimura, 1980) method was used to reconstruct the phylogenetic tree. Actinomadura hibisca JCM 9627T was taken as an outgroup. GenBank accession numbers have been included in parentheses. Only bootstrap values (expressed as a percentage of 1000 replicates) .50 % are shown at branch points. Bar, 0.01 substitutions per nucleotide position. http://ijs.sgmjournals.org
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of Streptomyces atacamensis DSM 42065T (98.40 %), Streptomyces radiopugnans R97 DSM 41901T (98.27 %), Streptomyces fenghuangensis GIMN4.003T (98.33 %), Streptomyces nanhaiensis DSM 41926T (98.13 %), Streptomyces megasporus NBRC 14749T (97.37 %) and Streptomyces macrosporus NBRC 14748T (98.22 %), as supported by all four tree-making algorithms (Figs 2 and S3). DNA–DNA relatedness tests were carried between strain RC 1831T and Streptomyces atacamensis DSM 42065T and Streptomyces glaucosporus DSM 41689T using the optical renaturation method (De Ley et al., 1970; Huss et al., 1983). Levels of relatedness (mean±SD of triplicate determinations) between strain RC 1831T, Streptomyces atacamensis DSM 42065T and Streptomyces glaucosporus DSM 41689T were 28.75±3.25 and 15±2.40 %, respectively, significantly below the 70 % threshold value for delineation of genomic species (Wayne et al., 1987). Strain RC 1831T could be distinguished morphologically, physiologically and biochemically from its phylogenetically closest neighbours, Streptomyces atacamensis DSM 42065T, Streptomyces glaucosporus DSM 41689T, Streptomyces radiopugnans R97 DSM 41901T, Streptomyces fenghuangensis GIMN4.003T and Streptomyces nanhaiensis DSM 41926T, based on a broad range of phenotypic characteristics as shown in Table 1. The spore chain arrangement, colour of aerial spore mass (ISP 3), spore shape and spore surface ornamentation for strain RC 1831T were straight to flexuous, white, round and warty, as compared with straight, grey, cylindrical and smooth for Streptomyces atacamensis DSM 42065T. The results also showed differences between strain RC 1831T and Streptomyces atacamensis in the colour of substrate mycelium on several ISP media (Table 1). Hence, a combination of the phenotypic and genotypic data presented clearly demonstrates that strain RC 1831T merits recognition as the type strain of a novel species within the genus Streptomyces, for which the name Streptomyces barkulensis sp. nov. is proposed.
pH 7–10 (optimum, pH 8) and with 0–10 % (w/v) NaCl (optimum, 8 % NaCl); tolerates up to 18 % NaCl. No growth is seen below 30 uC and at pH 6. The cell wall is type I and contains LL-DAP. Cellobiose, D-fructose, Dgalactose, D-glucose, D-mannose, maltose, raffinose, ribose, L-rhamnose and D-xylose are utilized as sole source of carbon, but not arabinose, inositol or lactose. Positive for hydrolysis of starch, chitin, cellulose, CM-cellulose, pectin, Tween 80, tributyrin and lecithin, gelatin liquefaction, milk coagulation, and presence of catalase and oxidase. Negative for utilization of urea and citrate, nitrate reduction and hydrolysis of xylan. Sensitive to (mg per disc) amoxicillin (50), co-trimoxazole (25), cloxacillin (30), cephalexin (30), ciprofloxacin (30), erythromycin (10), furazolidone (50), nalidixic acid (30), oxytetracyclin (30) and norfloxacin (10). No antimicrobial activities are observed against Escherichia coli MTCC82, Staphylococcus aureus MTCC96, Micrococcus luteus, Salmonella typhi MTCC734 and Bacillus cereus IP 4061. The type strain, RC 1831T (5JCM 18754T5DSM 42082T), was isolated from estuarine brackish water sediment samples collected from a fish dumping yard in Barkul village, Chilika Lake, Odisha, India. The DNA G+C content of the type strain is 66.2 mol%.
Acknowledgements We are grateful to the Chilika Development Authority (CDA), Bhubaneswar, for providing financial assistance through the World Bank-supported Integrated Coastal Zone Management Project of Odisha State. Staff of the CDA are acknowledged for their help and providing necessary facilities in collection of sediment samples. We thank Dr Rup Lal, University of Delhi, for his valuable suggestions and providing laboratory help for G+C content analysis. We acknowledge Dr Devadhyan Behera (IMMT, Bhubaneswar) for providing microscopy facilities. We are grateful to Dr Cathrine Spro¨er and Dr Susanne Verbarg of the Deutsche Sammlung von Mikroorganismen und Zellkulturen for their assistance with DNA– DNA hybridization and chemotaxonomic analysis.
Description of Streptomyces barkulensis sp. nov.
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by using SDS-PAGE of whole-cell proteins. Emendation of the species Streptomyces aurantiacus, Streptomyces cacaoi subsp. cacaoi, Streptomyces caeruleus and Streptomyces violaceus. Int J Syst Evol Microbiol 52, 823–829. Lanoot, B., Vancanneyt, M., Dawyndt, P., Cnockaert, M., Zhang, J., Huang, Y., Liu, Z. & Swings, J. (2004). BOX-pCR fingerprinting as a
powerful tool to reveal synonymous names in the genus Streptomyces. Emended descriptions are proposed for the species Streptomyces cinereorectus, S. fradiae, S. tricolor, S. colombiensis, S. filamentosus, S. vinaceus and S. phaeopurpureus. Syst Appl Microbiol 27, 84–92. Larkin, M. A., Blackshields, G., Brown, N. P., Chenna, R., McGettigan, P. A., McWilliam, H., Valentin, F., Wallace, I. M., Wilm, A. & other authors (2007). CLUSTAL W and CLUSTAL_X version 2. Bioinformatics
23, 2947–2948. Lechevalier, M. P. & Lechevalier, H. A. (1970). Chemical composition
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chilikensis sp. nov., a halophilic streptomycete isolated from brackish water sediment. Int J Syst Evol Microbiol 63, 2757–2764. Rhuland, L. E., Work, E., Denman, R. F. & Hoare, D. S. (1955). The
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International Journal of Systematic and Evolutionary Microbiology 64
DOI 10.1099/ijs.0.056614-0
Streptomyces barkulensis sp. nov., isolated from an estuarine lake Lopamudra Ray,1 Samir Ranjan Mishra,1 Ananta Narayan Panda,1 Gurdeep Rastogi,2 Ajit Kumar Pattanaik,2 Tapan Kumar Adhya,1 Mrutyunjay Suar1 and Vishakha Raina1 Correspondence
1
Vishakha Raina
2
School of Biotechnology, KIIT University, Bhubaneswar – 751024, Odisha, India Chilika Development Authority, Plot No. C-11, BJB Nagar, Bhubaneswar – 751014, Odisha, India
[email protected]
The taxonomic position of a novel actinomycete, strain RC 1831T, isolated from the sediment of a fish dumping yard at Barkul village near Chilika Lake, Odisha, India, was determined by a polyphasic approach. Based on morphological and chemotaxonomic characteristics the isolate was determined to belong to the genus Streptomyces. The phylogenetic tree based on its nearly complete 16S rRNA gene sequence (1428 nt) with representative strains showed that the strain consistently falls into a distinct phyletic line together with Streptomyces glaucosporus DSM 41689T (98.22 % similarity) and a subclade consisting of Streptomyces atacamensis DSM 42065T (98.40 %), Streptomyces radiopugnans R97 DSM 41901T (98.27 %), Streptomyces fenghuangensis GIMN4.003T (98.33 %), Streptomyces nanhaiensis DSM 41926T (98.13 %), Streptomyces megasporus NBRC 14749T (97.37 %) and Streptomyces macrosporus NBRC 14748T (98.22 %). However, the levels of DNA–DNA relatedness between strain RC 1831T and phylogenetically related strains Streptomyces atacamensis DSM 42065T (28.75±3.25 %) and Streptomyces glaucosporus DSM 41689T (15±2.40 %) were significantly lower than the 70 % threshold value for delineation of genomic species. Furthermore, the isolate could be distinguished phenotypically on the basis of physiological, morphological and biochemical differences from its closest phylogenetic neighbours and other related reference strains. Strain RC 1831T is therefore considered to represent a novel species of the genus Streptomyces, for which the name Streptomyces barkulensis sp. nov. is proposed. The type strain is RC 1831T (5JCM 18754T5DSM 42082T).
Streptomyces is the largest genus of the phylum Actinobacteria (Ka¨mpfer & Labeda 2006; Lodders & Ka¨mpfer, 2007), comprising, at the time of writing, more than 600 recognized species (Hain et al., 1997). They are typically aerobic, Gram-positive and with a high DNA G+C content, showing presence of spore-bearing aerial and substrate mycelia and LL-diaminopimelic acid (LL-DAP) with no characteristic sugars in the cell wall (Williams et al., 1983; Embley & Stackebrandt, 1994). They are widely known for their ability to produce a broad range of secondary metabolites, bioactive compounds and industrially important enzymes, which makes them an important genus for scientific exploration of many useful products (Be´rdy, 2005; Watve et al., 2001; Chater et al., 2010). The conventional systematics of the genus Streptomyces based Abbreviation:
LL-DAP, LL-diaminopimelic
acid.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain RC 1831T is JQ862602. Three supplementary figures are available with the online version of this paper.
056614 G 2014 IUMS
Printed in Great Britain
on phenotypic characteristics (Williams et al., 1983; Ka¨mpfer et al., 1991) has undergone a radical change with the application of molecular systematics (Goodfellow et al., 1992; Embley & Stackebrandt, 1994; Lanoot et al., 2002, 2004; Hatano et al., 2003; Ka¨mpfer, 2012). Although comparison of 16S rRNA gene sequences for classification at the genus level has been widely used for Streptomyces taxonomy, differences at the species level are difficult to establish when 16S rRNA gene sequence similarities are .97 % (Stackebrandt & Goebel, 1994). Moreover, high 16S rRNA gene sequence similarities between type strains often pose problems for the description of novel species within the genus Streptomyces. These species can be delineated through a polyphasic approach, i.e. study of the combination of phenotypic and genotypic features (Ka¨mpfer et al., 2008). Polyphasic approaches hence continue to present us with large numbers of novel species and has shown that the genus Streptomyces is largely underspeciated (Goodfellow et al., 1992; Kim and Goodfellow, 2002; Saintpierre et al., 2003). Species description with recently developed techniques, such as numerical classification for taxonomic 1365
L. Ray and others
evaluation and multilocus sequence analysis (Ka¨mpfer & Labeda, 2006; Goodfellow et al., 2007; Rong & Huang, 2010; Labeda et al., 2012), have also been found to be valuable in the classification of new affiliates to the taxon isolated from diverse geographical environments. While screening for chitin-degrading bacteria, strain RC 1831T was isolated from a fish dumping yard sediment (pH 8.5 and salinity 9.6 p.p.t.) near the shoreline of the brackish Chilika Lake (19u 719 N 85u 199 E) in Barkul village, Khurda district, Odisha, India. Taxonomic investigation using a polyphasic approach confirmed that the new isolate represented a novel species within the genus Streptomyces. Strain RC 1831T was isolated after 7 days of incubation at 30 uC with the appearance of a zone of clearance on a colloidal chitin agar medium (Hsu & Lockwood, 1975) supplemented with nystatin (50 mg l21), when inoculated with a sediment sample suspension. Strain RC 1831T showed the characteristic presence of spore-bearing aerial and substrate mycelia, which is a morphological attribute of the genus Streptomyces (Ka¨mpfer, 2012). The pure strain was maintained routinely on agar plates of modified Luria– Bertini (LB) media fortified with seawater (SWLB; 10.0 g tryptone, 5.0 g yeast extract, 5.0 g NaCl, 500 ml seawater, 500 ml distilled water, 18 g agar, pH 7.0) at 4 uC and as a suspension of mycelia and spores in 50 % (v/v) glycerol at 280 uC. Biomass required for chemotaxonomic and molecular analyses was obtained by culturing isolate RC 1831T in SWLB for 48 h at 30 uC and cells were harvested by centrifugation and resuspended in water/2-propanol (1 : 1, v/v) (Ray et al., 2013). The genomic DNA of strain RC 1831T was extracted by the method of Kieser et al. (2000) and the 16S rRNA gene was amplified by PCR (Gene Amp PCR machine; Perkin Elmer) using primers as described by Lane (1991). Amplified product was purified with a spin column (QIAquick PCR Purification kit; Qiagen) and sequenced using an ABI prism dye terminator cycle sequencing kit (ABI prism model 3700; Applied Biosystems) as described by Edwards et al. (1989). The nearly complete 16S rRNA gene sequence (1428 nt) was compared with sequences of representative species of the genus Streptomyces deposited in the GenBank database (Altschul et al., 1997) using the BLAST program and EzTaxone server version 2.1 (Kim et al., 2012). The 20 sequences showing the highest scores were selected for the calculation of pairwise sequence similarity using a global alignment algorithm, which was implemented at the EzTaxon-e server (http://eztaxon-e.ezbiocloud.net/). The recovered sequences were aligned with the sequence of strain RC 1831T using CLUSTAL W (Larkin et al., 2007) and edited using BioEdit Sequence Alignment Editor, version 7.1.3 (Hall, 1999). The phylogenetic relationships were established using the maximum-likelihood (Felsenstein, 1985), maximum-parsimony (Fitch, 1971) and minimum-evolution (Rzhetsky & Nei, 1992) algorithms of MEGA version 5.1 software (Tamura et al., 2011). An evolutionary distance matrix was calculated using the method of Kimura’s two-parameter model (Kimura, 1980) and a phylogenetic tree was reconstructed 1366
using the neighbour-joining (Saitou & Nei, 1987) method with Actinomadura hibisca JCM 9627T as an outgroup. The topology of the phylogenetic tree was evaluated by MEGA v. 5 using the bootstrap resampling method with 1000 replicates (Felsenstein, 1985). Culture traits of strain RC 1831T were recorded after 21 days of incubation at 30 uC on several International Streptomyces Project (ISP; Shirling & Gottlieb, 1966) media. Colour evaluations on ISP media for diffusible pigments were assessed by comparing the cultures with colour chips from ISCC-NBS Color Charts standards no. 2 2106 (Kelly, 1964). Investigation of morphological and chemotaxonomic attributes which are representative of the genus Streptomyces were carried out for strain RC 1831T (Williams et al., 1989; Embley & Stackebrandt, 1994; Manfio et al., 1995; Stackebrandt & Ebers, 2006). Strain RC 1831T was incubated on SWLB agar for 3–4 weeks at 30 uC to observe spore chain morphology, spore surface and spore chain ornamentation by light microscopy (DM2000, LAS v. 4.2; Leica) and scanning electron microscopy (EVO40; Zeiss). Several biochemical and physiological characteristics of strain RC 1831T and its phylogenetically closest neighbours were tested and compared using the well-established procedures of Shirling and Gottlieb (1966), Williams et al. (1983) and Ka¨mpfer (2012). Enzyme hydrolysis, and urea and citrate utilization tests were performed on LB agar media and carbohydrate utilization tests were carried out on basal salt media supplemented with 1 % (v/v) of the individual carbon source. Catalase activity was determined by oxygen bubble formation on addition of H2O2 (4 %) to colonies and oxidase activity was checked by a change in colour of an oxidase disc (HiMedia). Estimation of melanin production and nitrate reduction were tested on ISP media according to the methods described by Gordon et al. (1974), Williams et al. (1989) and Goodfellow (1971). Physiological tolerance to temperature (20–50 uC) and pH (6.0–10.0) was tested on LB agar media and NaCl tolerance (0–18 % w/v) was tested in LB broth media incubated for 14–21 days at 30 uC. Antibiotic susceptibility was examined using antibiotic discs (HiMedia) as described by Williams et al. (1983) on LB agar plates. Antimicrobial activity of strain RC 1831T was analysed on LB agar for 7 days against indicator strains Escherichia coli MTCC 82, Staphylococcus aureus MTCC96, Bacillus cereus IP 4061 and Salmonella typhi MTCC734, procured from the Microbial Type Culture Collection (MTCC, Chandigarh, India), and B. cereus IP 4061 and Micrococcus luteus (a kind gift from Dr. Peter Luethy, Institute of Microbiology, ETH, Zurich), by observing the formation of a zone of inhibition (Madigan et al., 1997). The diagnostic isomer LL-DAP and whole-cell wall sugars were analysed as described by Rhuland et al. (1955) and Staneck & Roberts (1974), respectively. Polar lipid analysis was carried out according to Bligh and Dyer (1959) and Tindall et al. (2007). Respiratory quinones were extracted using the two-stage method and separated into their different classes (menaquinones, ubiquinones, etc.) as described by Tindall (1990a, b) and further analysed by HPLC International Journal of Systematic and Evolutionary Microbiology 64
Streptomyces barkulensis sp. nov.
(Kroppenstedt, 1985). Fatty acid analysis was performed according to standard procedures as described by Sasser (1990) and comparison of the results was done with fatty acid databases included in the Microbial Identification Software package (Sherlock, Version 6.1). The G+C content of the genomic DNA of strain RC 1831T was determined by using the thermal denaturation method of Mandel and Marmur (1968) on a Peltier system spectrophotometer (Perkin Elmer). Microscopic studies for morphological observations revealed the presence of branching aerial and substrate mycelia which differentiate into well-developed aerial spore chains. At maturity, aerial hyphae produced rounded spores (161 mm) in spore chains which are straight to flexuous (Rectiflexibles) with a warty spore surface (Figs 1 and S1, available in the online Supplementary Material). The isolate exhibited good growth on ISP 3 and ISP 6, moderate growth on ISP 4, ISP 5 and ISP 7, but no growth on ISP 2. No diffusible pigments were detected on any of the ISP media tested. No aerial mycelia were recorded on ISP 4, ISP 5 or ISP 7 (Table 1). Strain RC 1831T was positive for hydrolysis of starch, cellulose and pectin, gelatin liquefaction, milk coagulation, degradation of Tween 80, lecithin, tributyrin and chitin, and presence of catalase and oxidase; it was negative for urea and citrate utilization, nitrate reduction and hydrolysis of xylan. Growth occurred at pH 8–10 (optimum, pH 8), at 40 uC (optimum, 30 uC) and with 0–10 % (w/v) NaCl (optimum, 8 % NaCl); it was able to tolerate up to 18 % NaCl. Cellobiose, D-fructose, D-galactose, D-glucose, D-mannose, maltose, raffinose, ribose, L-rhamnose and D-xylose could be utilized as sole carbon and energy source, but not arabinose, inositol or lactose (Table 1). The chemotaxonomic features of strain RC 1831T were consistent with the genus Streptomyces. It showed the diagnostic presence of LL-DAP in the cell-wall peptidoglycan with no characteristic sugar pattern, which is indicative of cell-wall type I (Lechevalier & Lechevalier, 1970;
Williams et al., 1989). The whole-cell hydrolysate contained glucose and ribose and the most prominent type II polar lipids were diphosphatidylglycerol and phosphatidylethanolamine. Phosphatidylinositol, phosphatidylinositolmannoside and an unknown phospholipid were also observed (Fig. S2). The major menaquinones were MK-9 (H4) (12 %), MK-9 (H6) (53 %) and MK-9 (H8) (16 %), with minor traces of MK-9 (H2) (2 %), MK-9 (1 %), MK-8 (H4) (1 %), MK-10 (H4) (3 %), MK-10 (H6) (7 %) and MK-10 (H8) (3 %). The cellular fatty acids consisted of iso-C14 : 0 (7.03 %), C14 : 0 (0.56 %) iso-C15 : 0 (3.83 %), antiso-C15 : 0 (25.91 %), C15 : 1 B (0.37 %), C15 : 0 (1.94 %), iso-C16 : 1 H (1.04 %), iso-C16 : 0 (25.24 %), C16 : 1v9c (3.12 %), C16 : 0 methyl (2.31 %), C16 : 0 (8.72 %), antisoC17 : 1 (3.83 %), iso-C17 : 0 (2.89 %), C17 : 1v9c (0.81 %), antiso-C17 : 0 (8.89 %), iso-C18 : 1 H (0.38 %), iso-C18 : 0 (0.38 %), C18 : 1v9c (0.18 %), C18 : 0 (0.64 %), C14 : 1v9t/ 9c (0.13 %) and C18 : 1v11c/9t/6t (0.24 %). The G+C content of the genomic DNA was 66.2 mol% (mean of two replicates). All the above features confirmed the classification of strain RC 1831T to the genus Streptomyces. Comparison of the almost-complete 16S rRNA gene sequence of strain RC 1831T (1428 nt) with those of closely related type strains clearly revealed its phylogenetic affiliation to the genus Streptomyces. Based on 16S rRNA gene sequences, the closest neighbour to strain RC 1831T was Streptomyces atacamensis DSM 42065T (Santhanam et al., 2012), at a similarity of 98.4 %, a value which corresponds to 23 nt differences over 1401 locations. This was supported by all tree-making algorithms (neighbourjoining, minimum-evolution, maximum-likelihood, maximum-parsimony) with a high bootstrap value (.90 %) (Fig. 2). However, strain RC 1831T also formed a distinct phyletic line with Streptomyces glaucosporus R97 DSM 41689T (98.22 % 16S rRNA gene sequence similarity) and a close subclade showed a corresponding branch composed
Fig. 1. Scanning electron micrograph of cells of strain RC 1831T grown on SWLB (50 %, v/v) for 3 weeks (21 days) at 30 6C. Bar, 1 mm. http://ijs.sgmjournals.org
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Table 1. Differential phenotypic characteristics between strain RC 1831T and its closest phylogenetic relatives in the genus Streptomyces Strains: 1, RC 1831T; 2, Streptomyces atacamensis DSM 42065T; 3, Streptomyces glaucosporus DSM 41689T; 4, Streptomyces fenghuangensis GIMN4.003T; 5, Streptomyces radiopugnans R97 DSM 41901T; 6, Streptomyces nanhaiensis DSM 41926T. All data represented here have been generated in this study except for Streptomyces fenghuangensis GIMN4.003T (Zhu et al., 2011). All strains were grown at pH 7 and utilized Dfructose, D-glucose and D-galactose as sole carbon sources. +, Positive; 2, negative; OW, off-white; BR, brownish red; PY, pale yellow; PO, pale orange; YW, yellowish white; GW, greenish white; BL, black; OB, off-brown; PB, pale brown; B, brown; ND, not determined. Characteristic Morphology* Spore chain morphology Aerial spore mass colour Spore surface ornamentation Colour and growth of substrate mycelia on ISP mediaD Yeast extract malt extract agar (ISP 2) Oatmeal agar (ISP 3) Inorganic salt starch agar (ISP 4) Glycerol asparagine agar (ISP 5) Peptone yeast extract iron agar (ISP 6) Tyrosine agar (ISP 7) Diffusible pigment Enzyme hydrolysis§ Starch Gelatin Caesin Tributyrin Tween 80 Lecithin Chitin Pectin Cellulose CM-cellulose Utilization of:§ Urea Citrate Growth on sole carbon source [1 % (w/v)]|| L-Arabinose Cellobiose D-Fructose D-Galactose D-Glucose Inositol Lactose D-Mannose Maltose Raffinose Ribose L-Rhamnose D-Xylose Growth with NaCl (%, w/v) 3, 5, 6 7, 9 11 12, 15, 18
1368
1
2
3
4
5
6
Rectiflexibles White Warty
Straight Grey Smooth
Spiral Pale grey Rough to warty
Rectiflexibles White Rough to warty
Spiral Pale yellow Smooth
Rectiflexibles White Smooth to rough
2
OW+++
2
YW+++
PY+
PY+
OW+++ OW++ OW++ BR+++
PY++ OW++ PY++ PO++
PY++ OW+ OW++ PY++
G++ GW+ YW++ 2
BL+++ OW++ OB+++ PB+
PY+ PY+ PY++ B+++
OW++ 2
OW++ 2
OW++ 2
2 Blued
OW++ 2
PY+ 2
+ + + + + + + + + +
+ + 2 + + 2 2 2 + +
+ + 2 2 + 2 + 2 + +
2
+ + 2 2 + 2 + 2 + +
+ + 2 2 + + + 2 + +
2 2
+ +
+ 2
2 2
2 2
2 + + + + 2 2 + + + + + +
2 2 + + + + + + + + + + +
+ + + + + 2 2 + + + 2 + +
2 2 + + + 2 2 2 + + 2 2 +
2 2 + + + + + + + + + + +
+ + + +
+ + + 2
+ + 2 2
+ 2 2 2
+ + 2 2
ND
+ ND ND ND ND ND ND ND ND ND
+ + + + + + ND ND
2 2 2 2 2 ND ND ND ND
International Journal of Systematic and Evolutionary Microbiology 64
Streptomyces barkulensis sp. nov.
Table 1. cont. Characteristic Growth at pH:§ 8, 9, 10 Growth at:§ 20 uC 50 uC
1
2
3
4
5
6
+
2
2
ND
+
2
2 2
2 +
2 +
ND
2 +
+ 2
ND
*Growth on modified seawater (50 %; v/v) LB. D+++, Abundant growth; ++, moderate growth; +, poor growth; 2, no growth. dISP 5. §LB agar. ||Basal salt media (Shirling and Gottlieb, 1966). LB broth.
S.glaucosporus DSM 41689T (AB184664)
* 52 0.01 67
* 99
* 93
*
S.barkulensis RC1831T DSM 42082T (JQ862602) S.atacamensis DSM 42065T (HE 577171) S. radiopugnans R97 DSM 41901T (DQ912930)
* 62
S.fenghuangensis GIMN4.003T (GU356598) 86
* 63
* * 93
S.nanhaiensis SCSIO 01248 DSM 41926T (GQ871748) S.megasporus NBRC 14749T (AB184617) S.macrosporus NBRC 14748T (AB184616)
S.fimbriatus NBRC 15411T (AB184659) S.cacaoi subsp. cacaoi NBRC 12748T (AB184115) * 87
S.albus DSM 40313T (AJ621602) * *
65
73
S.platensis JCM 4662T (AB045882)
89
53 *
S. libani subsp. rufus LMG 20087T (AJ781351)
S.hygroscopicus NBRC 13786T (AB184479) S.caniferus NBRC 15389T (AB184640) S.nigrescens NBRC 12894T (AB184225) S.angustmyceticus NBRC 3934T (AB184817) S.catenulae ISP 5258T (AY999778)
66 * *
S.albiaxialis NRRL B-24327T (AY999901) S.tateyamensis DSM 41969T (AB473555) S.chrestomyceticus DSM 40545T (AJ621609)
* 100
S. rimosus subsp. paromomycinus DSM 41429T (AJ621610) S.abikoensis NBRC 13860T (AB184537) A.hibisca JCM 9627T (AF163115)
Fig. 2. Neighbour-joining tree (Saitou & Nei, 1987) based on analysis of the nearly complete (1428 nt) 16S rRNA gene sequence showing the position and phylogenetic relationship between strain RC 1831T and other related members of the genus Streptomyces. Asterisks indicate conserved branches recovered when the minimum-evolution (Kimura, 1980) method was used to reconstruct the phylogenetic tree. Actinomadura hibisca JCM 9627T was taken as an outgroup. GenBank accession numbers have been included in parentheses. Only bootstrap values (expressed as a percentage of 1000 replicates) .50 % are shown at branch points. Bar, 0.01 substitutions per nucleotide position. http://ijs.sgmjournals.org
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L. Ray and others
of Streptomyces atacamensis DSM 42065T (98.40 %), Streptomyces radiopugnans R97 DSM 41901T (98.27 %), Streptomyces fenghuangensis GIMN4.003T (98.33 %), Streptomyces nanhaiensis DSM 41926T (98.13 %), Streptomyces megasporus NBRC 14749T (97.37 %) and Streptomyces macrosporus NBRC 14748T (98.22 %), as supported by all four tree-making algorithms (Figs 2 and S3). DNA–DNA relatedness tests were carried between strain RC 1831T and Streptomyces atacamensis DSM 42065T and Streptomyces glaucosporus DSM 41689T using the optical renaturation method (De Ley et al., 1970; Huss et al., 1983). Levels of relatedness (mean±SD of triplicate determinations) between strain RC 1831T, Streptomyces atacamensis DSM 42065T and Streptomyces glaucosporus DSM 41689T were 28.75±3.25 and 15±2.40 %, respectively, significantly below the 70 % threshold value for delineation of genomic species (Wayne et al., 1987). Strain RC 1831T could be distinguished morphologically, physiologically and biochemically from its phylogenetically closest neighbours, Streptomyces atacamensis DSM 42065T, Streptomyces glaucosporus DSM 41689T, Streptomyces radiopugnans R97 DSM 41901T, Streptomyces fenghuangensis GIMN4.003T and Streptomyces nanhaiensis DSM 41926T, based on a broad range of phenotypic characteristics as shown in Table 1. The spore chain arrangement, colour of aerial spore mass (ISP 3), spore shape and spore surface ornamentation for strain RC 1831T were straight to flexuous, white, round and warty, as compared with straight, grey, cylindrical and smooth for Streptomyces atacamensis DSM 42065T. The results also showed differences between strain RC 1831T and Streptomyces atacamensis in the colour of substrate mycelium on several ISP media (Table 1). Hence, a combination of the phenotypic and genotypic data presented clearly demonstrates that strain RC 1831T merits recognition as the type strain of a novel species within the genus Streptomyces, for which the name Streptomyces barkulensis sp. nov. is proposed.
pH 7–10 (optimum, pH 8) and with 0–10 % (w/v) NaCl (optimum, 8 % NaCl); tolerates up to 18 % NaCl. No growth is seen below 30 uC and at pH 6. The cell wall is type I and contains LL-DAP. Cellobiose, D-fructose, Dgalactose, D-glucose, D-mannose, maltose, raffinose, ribose, L-rhamnose and D-xylose are utilized as sole source of carbon, but not arabinose, inositol or lactose. Positive for hydrolysis of starch, chitin, cellulose, CM-cellulose, pectin, Tween 80, tributyrin and lecithin, gelatin liquefaction, milk coagulation, and presence of catalase and oxidase. Negative for utilization of urea and citrate, nitrate reduction and hydrolysis of xylan. Sensitive to (mg per disc) amoxicillin (50), co-trimoxazole (25), cloxacillin (30), cephalexin (30), ciprofloxacin (30), erythromycin (10), furazolidone (50), nalidixic acid (30), oxytetracyclin (30) and norfloxacin (10). No antimicrobial activities are observed against Escherichia coli MTCC82, Staphylococcus aureus MTCC96, Micrococcus luteus, Salmonella typhi MTCC734 and Bacillus cereus IP 4061. The type strain, RC 1831T (5JCM 18754T5DSM 42082T), was isolated from estuarine brackish water sediment samples collected from a fish dumping yard in Barkul village, Chilika Lake, Odisha, India. The DNA G+C content of the type strain is 66.2 mol%.
Acknowledgements We are grateful to the Chilika Development Authority (CDA), Bhubaneswar, for providing financial assistance through the World Bank-supported Integrated Coastal Zone Management Project of Odisha State. Staff of the CDA are acknowledged for their help and providing necessary facilities in collection of sediment samples. We thank Dr Rup Lal, University of Delhi, for his valuable suggestions and providing laboratory help for G+C content analysis. We acknowledge Dr Devadhyan Behera (IMMT, Bhubaneswar) for providing microscopy facilities. We are grateful to Dr Cathrine Spro¨er and Dr Susanne Verbarg of the Deutsche Sammlung von Mikroorganismen und Zellkulturen for their assistance with DNA– DNA hybridization and chemotaxonomic analysis.
Description of Streptomyces barkulensis sp. nov.
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