ORIGINAL PAPER

Streptomyces tritolerans sp. nov., a novel actinomyceteisolated from soil in Karnataka, India

Dastager G. Syed Æ Dayanand Agasar Æ Chang-Jin Kim Æ Wen-Jun Li ÆJae-Chan Lee Æ Dong-Jin Park Æ Li-Hua Xu Æ Xin-Peng Tian ÆCheng-Lin Jiang

Received: 10 January 2007 / Accepted: 12 March 2007 / Published online: 9 June 2007� Springer Science+Business Media B.V. 2007

Abstract A Gram-positive, nonmotile, moder-

ately halophilic, alkali and thermotolerant strain

designated DAS 165T, was isolated from a dry

land soil sample from the Gulbarga region,

Karnataka province, India. The isolate produced

yellow substrate mycelia and gray aerial mycelia

on most tested media. Strain DAS 165T showed

growth in the presence of 5 to 7% NaCl and at

45�C. The DNA G + C content was 69.7%. 16S

rRNA gene sequence analysis together with these

characteristics consistently assigned strain DAS

165T to the genus Streptomyces. The 16S rRNA

gene sequence analysis revealed that strain DAS

165T was most closely related to S. tendae ATCC

19812T (D 63873) with a sequence similarity of

99.6% (three nucleotide differences out of 1,517).

Strain DAS 165T formed a distinct clade based on

analysis of the almost complete sequence and

120-nucleotide variable c region of the 16S rRNA

gene. Despite the high sequence similarity, strain

DAS 165T was phenotypically different from

S. tendae ATCC 19812T. DNA–DNA hybridi-

zation between these strains was 47% showing

that strain DAS 165T is a distinct genomic species.

Phenetic and genetic results support the classifi-

cation of strain DAS 165T as a new species, for

which the name S. tritolerans is proposed, with

strain DAS 165T as the type strain (=DSM

41899T = CCTCCAA 206013T).

Keywords Streptomyces tritolerans sp. nov �Polyphasic taxonomy � 16S rRNA

Introduction

Streptomyces systematics has become increasingly

objective due to the application of chemotaxo-

nomic, molecular systematic and numerical taxo-

nomic methods (Kim et al.1999). The species

concept is still a difficult issue in Streptomyces

systematics due to the taxonomic complexity of

the genus and the absence of accepted minimal

D. G. Syed � C.-J. Kim � J.-C. Lee � D.-J. ParkKorea Research Institute of Bioscience andBiotechnology (KRIBB), Daejeon 305-333, SouthKorea

W.-J. Li � L.-H. Xu � X.-P. Tian � C.-L. JiangThe Key Laboratory for Microbial Resources ofMinistry of Education, Yunnan Institute ofMicrobiology, Yunnan University, Kunming, Yunnan650 091, P.R. China

D. G. Syed � D. Agasar (&)Department of Studies and Research in Microbiology,Gulbarga University, Gulbarga 585 106, Indiae-mail: iamdaya62@rediffmail.com

X.-P. TianGuangdong Key Laboratory of Marine MateriaMedica, South China Sea Institute of Oceanology,Chinese Academy of Sciences, Guangzhou 510301,P.R. China

123

Antonie van Leeuwenhoek (2007) 92:391–397

DOI 10.1007/s10482-007-9166-2

standards for the delineation of species. However,

it is evident that Streptomyces species should be

circumscribed using judicious combinations of

genotypic and phenotypic data (Labeda 1993;

Labeda and Lyons 1991a, b; Goodfellow et al.

1992). Comparative taxonomic studies are needed

to find rapid and reliable phenotypic methods for

delineating potentially novel species of Strepto-

myces. Alkalitolerant actinomycetes that thrive in

alkaline environments have specific nutrient

requirements, cultural conditions and physiolog-

ical properties (Collins 1985; Lechevalier and

Lechevalier 1980). There have been previous

reports on the physiology and energetics of

alkalitolerant bacteria (Kim et al. 1999). Strain

DAS 165T was recovered and characterized by a

combination of genotypic and phenotypic meth-

ods during an investigation of the extremophilic

microbial flora of the Gulbarga region, India. It

was evident that the actinomycete isolate was

sufficiently distinct from the closely related spe-

cies of Streptomyces to warrant the description of

a novel species, for which the name Streptomyces

tritolerans sp. nov., is proposed.

Material and methods

Strain DAS 165T was isolated using a standard

serial dilution plating method, from an alkaline

soil sample (pH 9–10) collected from Gulbarga,

northern Karnataka, southern India. The isola-

tion medium used was starch casein agar and

cultures were incubated at 28�C for about

2 weeks. The strain was maintained on ISP 2

[International Streptomyces project (ISP; Shirling

and Gottlieb 1966)] and ISP 5 agar slants at 4�C,

and as glycerol suspensions (20% v/v) at -20�C.

Biomass for chemical and molecular systematic

studies was obtained by growing in shake flasks in

ISP 2 broth (28�C, 1 week, 150 rpm).

Cultural characteristics were determined after

2 weeks growth at 28�C as per the methods

prescribed in the ISP (Shirling and Gottlieb

1966). Morphological properties were examined

by light microscopy (Olympus microscope) and

scanning electron microscopy following dessica-

tion of the cells and gold coating, using a JEOL

model JSM 5600LV. A range of phenotypic

properties was examined using standard proce-

dures (Williams et al. 1983). In addition, acid

production from carbohydrates was carried out

using the media and methods described by Shir-

ling and Gottlieb (1966). The utilization of sole

carbon and sole nitrogen sources was investigated

according to Shirling and Gottlieb (1966) and

Tsukamura (1966). Tolerance to temperature (10,

27, 37 and 45�C) and sodium chloride (0, 1, 3, 5, 7

and 10%) were tested using modified Bennett’s

agar (Williams et al. 1983). Resistance to antibi-

otics was examined using novobiocin (30 lg/ml),

ampicillin (10 lg/ml), amoxicillin (10 lg/ml), nali-

dixic acid (30 lg/ml), penicillin G (10 lg/ml),

norfloxacin (10 lg/ml), rifampicin (5 lg/ml) and

carbenicillin (100 lg/ml) incorporated into glu-

cose yeast extract agar (Lechevalier and Leche-

valier 1970a, b) as a basal medium. The results

were recorded following incubation at 28�C up to

14 days. Color determination was done with color

chips from the ISCC-NBS COLOR CHARTS

standard sample No. 2106 (Kelly 1964).

Chemotaxonomy

The cell wall fraction of strain DAS 165T was

purified and analyzed by the method of Lecheva-

lier and Lechevalier (1980). The procedure of

Becker et al. (1965) and Lechevalier and Leche-

valier (1980) were used to determine the whole-

cell composition. Menaquinones were determined

according to the Collins (1985) method. Biomass

for quantitative fatty acid analysis was prepared

by scraping growth from trypticase soy broth agar

plates that had been incubated for 3 days at 28�C.

The fatty acids were extracted, methylated and

analyzed using the standard MIDI (Microbial

identification) system (Kampfer and Kroppen-

stedt 1996; Sasser 1990).

16S rRNA gene sequence

The genomic DNA of strain DAS 165T was

isolated as described by Hoopwood et al. (1985).

The 16S rRNA gene was amplified by PCR, using

procedures described by Xu et al. (2003). Multi-

ple alignments with a selection of 16S rRNA gene

sequences were used and calculation of levels

of sequence similarity were carried out using

392 Antonie van Leeuwenhoek (2007) 92:391–397

123

CLUSTAL X (Thompson et al.1997). A phylo-

genetic tree was reconstructed using the neigh-

bor-joining method of Saitou and Nei (1987) from

Knuc values (Kimura 1980,1983). The topology of

the phylogenetic tree was evaluated by the

bootstrap resampling method of Felsentein

(1985) with 1,000 replicates. S. almquistii NBRC

13015T (AB184258) was used as an out group.

DNA for determining the G + C content was

obtained by the method of Marmur (1962). The

DNA G + C content was determined by the

thermal denaturation method (Mandel and Mar-

mur 1968). DNA–DNA hybridization was carried

out by applying the optical renaturation method

(De Ley et al. 1970; Huss et al. 1983; Jahnke

1992) under optimal hybridization conditions,

using Escherichia coli as control.

Nucleotide sequence accession number

The 16S rRNA gene sequence of strain DAS 165T

determined in this study has been deposited in

GenBank under the accession number DQ

345779.

Results and discussion

Strain DAS 165T was Gram-positive. The isolate

developed well on several media including ISP 2,

ISP 3, ISP 4, ISP 5, ISP 7, starch casein agar,

czapek’s agar and glucose asparagine agar. Mor-

phological observation of a 14-day-old culture of

strain DAS 165T on ISP 2 showed good growth of

aerial and vegetative hyphae which were well

developed and did not exhibit fragmentation.

Smooth, oval-rod shaped spores were borne in

long straight to flexous chains (Fig. 1). DAS 165T

developed gray to yellow aerial hyphae on all

media tested. The substrate mycelium differed

from light yellow to dark yellow. Yellow colored

substrate mycelium was produced on ISP2, ISP3,

ISP4, ISP5 and Czapek’s agar; darkish yellow

substrate mycelium was produced on starch case-

in and glucose asparagine agar; greenish yellow

substrate mycelium was produced on tyrosine

agar. No diffusible pigments were produced. The

detailed physiological and biochemical properties

are given in Table 1 and the species description.

Growth of strain DAS 165T was observed at a

wide range of temperature (28–45�C), at pH 7–10

and in the presence of 7% NaCl. Whole organism

hydrolysates of strain DAS 165T were rich in

LL-diaminopimelic acid (DAP) and glycine. No

diagnostic sugars were found in the cell wall

constituents. The diagnostic phospholipid is

phosphatidylethanolamine. The predominant

menaquinones were MK-9 (H4)-8%, MK-9 (H6)-

78% and MK-9 (H8)-14%. The major cellular

fatty acids were i-C15:0(13.6%), ai-C15:0 (17.8%),

C16:0(9.6%) and i-C16:0 (28.1%).

An almost complete 16S rRNA gene sequence

of strain DAS 165T (1,517 nucleotides) was

determined. BLAST search results of the strain

DAS 165T sequence against the non-redundant

GenBank database was used to select sequence as

reference sequences. Comparison of this

sequence with those of related organisms showed

clearly that it contained all the signature nucle-

otides designated for the genus Streptomyces

(Christova et al. 1995). The value for the 16S

rRNA gene sequence similarity to closely related

members of the genus Streptomyces were 99.4–

99.7% (three nucleotide differences out of 1,517).

The phylogenetic tree (Fig. 2) from representa-

tive strains of the related species indicated that

strain DAS 165T should be placed in the genus

Streptomyces and forms a separate lineage to-

gether with its closest neighbour S. tendae (ATCC

19812T) with 99.6% 16S rRNA gene sequence

similarity. Despite this very high sequence simi-

larity, strain DAS 165T was phenetically distinct

from S. tendae ATCC 19812T (Table 1).

The DNA G + C content of strain DAS 165T

was 69.7%. DNA–DNA hybridization between

strains DAS 165T and S. tendae ATCC 19812T

was carried out and the determined DNA relat-

edness value of 47% was significantly lower than

70%, which is considered to be the threshold

value for the delineation of genomic species

(Wayne et al. 1987).

Description of Streptomyces tritolerans

Streptomyces tritolerans (tri’to.le.rans.L.pref. tri-

ple. three. L. pres. part. tolerans. tolerating; N.L.

part. adj. tritolerans referring to the ability of the

Antonie van Leeuwenhoek (2007) 92:391–397 393

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Table 1 Phenotypic properties separating strain DAS 165T from its closest phylogenetic neighbors

Characteristics DAS 165T Streptomyces tendaeATCC 19812T

S. thermoalcalitoleransDSM 41741T

Spore mass White to gray Gray GraySpore chain morphology Straight-flexous Retinaculiperti-spirals SpiralsSpore surface Smooth Smooth WartyReverse side pigments Dark yellow Yellow Not distinctiveDiffusible pigments – + –Melanin pigments + – –Resistance toRifampicin + ND +Penicillin G + – +Growth at15�C _ – +45�C + – –NaCl (7%) + – –pH range 7.0–10.0 7.0–8.0 6.0–11.5Optimum pH 7.5 7.0 NDHydrolysis ofCasein + – +Cellulose + – +Pectin – + NDStarch + + +Utilization ofD-mannitol + + +meso-inositol _ + +Sucrose + + +L-rhamnose – – +Raffinose – – –L-phenylalanine + + NDL-histidine – – NDAntagonistic toB. subtilis _ _ NDS. aureus + + NDM. luteus – – NDA. niger + + NDC. albicans – – ND

+ positive, – negative, ND not determined

Data for reference strains were taken from Shirling and Gottlieb (1966) and Kim et al. (1999)

Fig. 1 Scanning electronmicroscopic view of sporearrangement in strainDAS 165T

394 Antonie van Leeuwenhoek (2007) 92:391–397

123

organism to tolerate high salinity, alkalinity and

temperature).

The strain is Gram-positive, non motile, alka-

litolerant and thermotolerant with good growth

without fragmentation. Aerial mycelium is white

to gray. Diffusible pigments are not formed.

Spores are smooth, 0.6–1.0 lm in diameter

arranged in straight to flexous chains of 10–15

spores. Arabinose, fructose, galactose, glucose,

D-mannitol, rhamnose, raffinose, sucrose and

xylose are utilized as sole carbon and energy

sources but not inositol. Acid is not formed from

these carbon sources. L-asparagine, L-alanine,

L-cysteine (weak), L-histidine and L-phenylala-

nine are used as sole nitrogen sources, but not

L-arginine. Starch, casein and cellulose are

hydrolyzed. Urea is not hydrolyzed. Nitrate

reduction and H2S production are negative.

Tests for gelatin hydrolysis and melanin produc-

tion are positive. Growth occurs from 28 to

45�C, pH 7–10, and in presence of sodium

chloride (7% w/v). Resistance to novobiocin,

ampicillin, amoxycillin, nalidixic acid, penicillin

G, norfloxacin, rifampicin and carbencillin but

sensitive to ciprofloxacin, gentamicin, neomycin,

erythromycin, streptomycin, tetracycline, chl-

oramphenicol and vancomycin. The cell wall of

strain DAS 165T contains LL-DAP, but no

diagnostic sugars. MK-9 (H6)-78% is the major

menaquinone and minor amount of MK-9 (H4)-

DAS 16598

Streptomyces tendae ATCC 19812T (D63873)67

Streptomyces (coelicolor )violaceoruber DSM 41007T(X60514)Streptomyces pseudogriseolus NRRL 3985T(X80827)

Streptomyces ambofaciens ATCC 23877T(M27245) Streptomyces caelestis NRRL 2418T(X80824)64

Streptomyces glaucescens DSM40716T(X79322) Streptomyces thermocarboxydus DSM 44293T (U94490)

Streptomyces eurythermus ATCC 14975T (D63870)Streptomyces sampsonii ATCC 25495T (D63871)72

Streptomyces diastaticus subsp. Disataticus DSM 40496T (X53161)Streptomyces brasiliensis DSM 43159T (X53162)

Streptomyces bluensis DSM 40564T (X79324)Streptomyces espinosus NRRL 5729T (X80826)

Streptomyces thermocarboxydovorans DSM 44296T(U94489)Streptomyces. thermonitrificans DSM 40579T (Z68 98)0

Streptomycs thermovulgaris DSM 40444T (Z68094)100

100

Streptomyces thermoalkalitolerans DSM 41741T(AJ000284) Streptomyces. thermodiastaticus DSM 40573T (Z68101)7459

Streptomyces thermolineatus DSM 41451T (Z68097)Streptomyces thermoviolaceus subsp. apingens DSM 41392T (Z68095)

Streptomyces thermoviolaceus subsp. thermoviolaceus DSM 40443 (Z68096)Streptomyces mashuensis DSM 40221T (X79323)

Streptomyces rimosus DSM 40260T (X62884)Streptomyces albus DSM 40313T (X53163)

Streptoverticillium baldacii DSM 40039T (X53164)88

Streptoverticillium olivoreticuli subsp. cellulophilum DPDU 0278T (X53166)Streptoverticillium cinnamoneum subsp.cinnamoneus DPDU 0093T(X53171)Streptoverticillium salmonis DPDU 0098T(X53169)

Streptomyces mobaraensis DSM 40587T(X53167)Streptomyces griseocarneus DSM 40004T(X99943)

Streptoverticillium abikoense DSM 40831T (X53168)50

Streptomyces galbus DSM 40089T(X79852)61

Streptomyces lincolnensis NRRL 2936T (X79854)Streptomyces acidiscabies ATCC 49003T(D63865)

Streptomyces scabies ATCC 49173T (D63862)Streptomyces neyagawaensis ATCC 27449T (D63869)53

Streptomyces bottropensis ATCC 25435T(D63868)97

Streptomyces diastatochromogenes ATCC 12309T(D 3867)6Streptomyces seoulensis IMSNU 21266T(Z71365)

Streptomyces bikiniensis DSM 40581T(X79851)Streptomyces purpureus DSM 43460T(X53170)

Streptomyces griseus KCTC 9080T(X61478)100

Streptomyces setonii ATCC 25497T(D63872)Streptomyces subrutilus DSM 40445T(X80825)70

Streptomyces virginiae IFO 3729T(D85119)97

Streptomyces thermolineatus DSM 41451T(Z68097)Streptomyces macrosporus DSM 41449T(Z68099)84

Streptomyces megasporus DSM 41476T(Z68100)98

0.005

Fig. 2 Phylogeneticdendrogram obtained bydistance matrix analysis of16S rRNA gene sequence,showing the position ofstrain DAS 165T amongits phylogeneticneighbors. Numbers atthe branch nodes are bootstrap values, expressed asa percentage of 1,000replicates (only valuesabove 50% are shown).Bar 0.005 substitutionsper nucleotide

Antonie van Leeuwenhoek (2007) 92:391–397 395

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8% and MK-9(H8)-14% are also present. The

diagnostic phospholipid is phosphatidylethanol-

amine. The major cellular fatty acids are

isoC15:0(14%), anteisoC15:0(18%), C16:0(10%)

and isoC16:0(28%). The G + C content of the

genomic DNA is 69.7 mol%. The strain DAS

165T was isolated from alkaline soil in Gulbarga,

Karnataka province, India.

Acknowledgments This work was supported by the 21CFrontier Microbial Genomics and Application Centreprogram. Ministry of Science and Technology, Republicof Korea. And Wen-Jun Li was supported by Program forNew Century Excellent Talent in University

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