ORIGINAL PAPER

Diversity of culturable actinobacteria from Qinghai–Tibetplateau, China

Yu-Qin Zhang • Hong-Yu Liu • Jie Chen •

Li-Jie Yuan • Wei Sun • Li-Xin Zhang •

Yue-Qin Zhang • Li-Yan Yu • Wen-Jun Li

Received: 3 December 2009 / Accepted: 22 March 2010 / Published online: 2 April 2010

� Springer Science+Business Media B.V. 2010

Abstract To investigate the diversity of culturable

actinobacteria and further screen for microbial pharma-

ceutics, seven different media were chosen to isolate

actinobacteria from 87 soil samples collected from

Qinghai–Tibet plateau. A total of 1930 strains was

isolated and identified to belong to 11 suborders, i.e.,

Actinopolysporineae, Corynebacterineae, Frankineae,

Glycomycineae, Kineosporiineae, Micrococcineae,

Micromonosporineae, Propionibacterineae, Pseudono-

cardineae, Streptomycineae and Streptosporangineae,

and 16 families, i.e., Nocardioidaceae, Actinopolyspor-

aceae, Actinosynnemataceae, Dermacoccaceae, Geo-

dermatophilaceae, Glycomycetaceae, Kineosporiaceae,

Microbacteriaceae, Micromonosporaceae, Nocardia-

ceae, Promicromonosporaceae, Propionibacteriaceae,

Pseudonocardiaceae, Streptomycetaceae, Streptospo-

rangiaceae and Thermomonosporaceae. A primary

taxonomic study showed that at least 22 genera of

actinobacteria were identified from the soil samples,

among which ten isolates represented hitherto

unknown species. The results showed that there was

abundant actinobacterial species diversity in the soil

samples from the Qinghai–Tibet plateau.Electronic supplementary material The online version ofthis article (doi:10.1007/s10482-010-9434-4) contains supple-mentary material, which is available to authorized users.

Y.-Q. Zhang � H.-Y. Liu � J. Chen � L.-J. Yuan �W. Sun � Y.-Q. Zhang � L.-Y. Yu (&)

Institute of Medicinal Biotechnology, Chinese Academy

of Medical Sciences and Peking Union Medical College,

Beijing 100050, People’s Republic of China

e-mail: yuliyan_2000@yahoo.com

Y.-Q. Zhang

e-mail: zhyuqin@126.com

W.-J. Li (&)

The Key Laboratory for Microbial Resources of the

Ministry of Education, Kunming, People’s Republic of

China

e-mail: wjli@ynu.edu.cn

W.-J. Li

Laboratory for Conservation and Utilization of Bio-

Resources, Yunnan Institute of Microbiology, Yunnan

University, Kunming 650091, People’s Republic of China

L.-X. Zhang � W.-J. Li

Guangdong Key Laboratory of Marine Materia Medica,

South China Sea Institute of Oceanology, Chinese

Academy of Sciences, Guangzhou 510301, People’s

Republic of China

L.-X. Zhang � W.-J. Li

Institute of Microbiology, Chinese Academy of Sciences,

100101 Beijing, People’s Republic of China

123

Antonie van Leeuwenhoek (2010) 98:213–223

DOI 10.1007/s10482-010-9434-4

Keywords Actinobacteria � 16S rRNA �Isolation � Identification

Introduction

It is becoming increasingly clear that un- and under-

explored habitats are a rich source of novel actinobac-

teria which have the capacity to produce interesting new

bioactive compounds, including antibiotics (Bull et al.

2005; Bull and Stach 2007; Okoro et al. 2008). The

Qinghai–Tibet plateau is the largest plateau in China

(about 2,400,000 km2) and the highest plateau in the

world (the average altitude is above 4000 m), encom-

passing Qinghai province, the Tibet Autonomous

Region, the western part of Sichuan and parts of Gansu

and Yunnan provinces. The complex climatic factors

and geographical topography of the Qinghai–Tibet

plateau promote the formation of a unique ecological

environment. In recent studies, several new taxa of

bacteria (Li et al. 2005a, 2006; Zhang et al. 2008) and

actinobacteria (Li et al. 2005b; Zhang et al. 2005, 2007)

were reported to have been isolated from the Qingha–

Tibet plateau, which show that such habitats are a

promising source of microorganisms. This has encour-

aged further studies on the biodiversity of actinomycetes

from the Qinghai–Tibet plateau. The present study

focused on the isolation and identification of Actino-

bacteria to reveal the diversity of culturable actinobac-

teria from Qinghai–Tibet plateau.

Materials and methods

Soil samples

Soil samples collected from different locales on

Qinghai–Tibet plateau were taken from a depth of

10–15 cm and kept in sterilized paper bags at 4�C

before processing. The details of the 87 samples used

for the isolation of actinobacteria are: 58 samples (No.

1–58) were from Qinghai Province; most of the

sampling locations were in hyper-arid areas (latitude

36�350–37�360N, longitude 99�520–101�460E, eleva-

tion 3000–3900 m). Seven samples were collected

from the Xinjiang Uygur Autonomous Region (latitude

40�420–43�530N, longitude 88�080–89�220E, elevation

700–1900 m), 15 samples from Deqen in Yunnan

Province (latitude 28�080–28�280N, longitude 103�540–

104�280E, elevation 800–1920 m), and seven samples

were collected at Ganzi Tibetan Autonomous Prefecture

in Sichuan Province (latitude 27�300–29�580N, longi-

tude 102�180–108�110E, elevation 1200–2000 m). The

pH value for most soil samples is around 7.0–8.0

(neutral to alkalescent), and few samples are low to 6.5

or above to 9.0, thus the pH values of the isolation media

were designed to be as 7.2–7.5.

Pretreatment of soil samples

Soil samples were air-dried at room temperature for

about 2 weeks before isolation. They were subse-

quently ground and passed through a 2 mm-mesh

sieve and then dry-heated at 80�C for 1 h. After

cooling, 2 g soil samples were put into 18 ml mod-

ified Ringer’s solution (g/l; NaCl 7, KCl 0.1, CaCl21.1, NaHCO3 0.2, Na4P2O7 1.0, pH 7.0) and shaken

(about 150 rpm) for 1 h at 28�C. Then 1 ml of each

suspension was added to 9 ml of phenol (1% w/v),

pH 7.0, then shaken (about 100 rpm) for 30 min at

30�C. The samples were finally diluted to 1-in-10 in

0.1% (w/v) sodium pyrophosphate solution (pH 7.0).

Isolation and maintenance of putative

actinobacteria

Seven isolation media were employed in this study

(Table 1). M5 and M6 media preparation followed

the references (Hayakawa and Nonomura 1987;

Hsu and Lockwood 1975) with little modification

(Table 1). Gellan Gum was used as a setting agent

instead of agar in all media. The pH was adjusted

to 7.2–7.5 using 1 N NaOH and/or 1 N HCl. In

addition, betaine (0.125% w/v) and sodium pyruvate

(0.125% w/v) were added to every medium to

facilitate the isolation of strains that are difficult to

culture (Yue et al. 2006). Nystatin, rifampicin, nali-

dixic acid, aztreonam and novobiocin were added to

the media in groups to diminish the growth of fungi

and non-actinobacteria (Table 1). 0.2–0.3 ml pre-

pared soil sample suspension was spread on the

isolation media and then incubated at 28�C for

3–4 weeks. The pure cultures were maintained on

yeast extract-malt extract agar (ISP medium 2;

Shirling and Gottlieb 1966) slants at 4�C and as

20% (v/v) glycerol suspensions at -80�C. Biomass

for molecular systematic and chemotaxonomic stud-

ies was obtained by cultivation in shake flasks (about

214 Antonie van Leeuwenhoek (2010) 98:213–223

123

150 rpm) of ISP 2 medium or Tryptic Soy Broth

(TSB; Difco) at 28�C for 5–7 days.

Morphological observations

ISP 2, ISP 3, ISP 4, ISP 5 (Shirling and Gottlieb

1966), Czapek solution agar (Waksman 1961), nutri-

ent agar (Difco), and tomato paste-oat-meal agar

(Waksman 1961) seeded with the isolates were used

for observation of the colony and cultural character-

istics. Cultures were incubated at 28�C and properties

were recorded after 4, 7, 14, 21, and 28 days. For cell

morphology, the coverslip technique was used on ISP

2 agar, as described by Kawato and Shinobu (1959).

Light microscopy with a model BH-2 microscope

(Olympus) was employed to observe the morpholog-

ical properties. Some gold-coated dehydrated speci-

mens of 7–14 day cultures were examined by

scanning electron microscopy (SEM) (Quanta; FEI).

Metabolic properties

Carbohydrate utilization tests were carried out using

API 50 CH test kits (bioMerieux) and Biolog GEN III

MicroPlates (Biolog Inc.) according to the manufac-

turer’s instructions, and enzyme activities were

determined using API ZYM test kits (bioMerieux),

as recommended by the manufacturer. Growth was

tested at 4, 10, 20, 28–37 (at intervals of 1�C), 40

and 45�C on ISP 2 agar medium incubated for

15–30 days. The ability of the strains to grow at

different NaCl concentrations (0, 1, 3, 5–20%, w/v)

(at intervals of 0.5%) was determined according

to Wang et al. (2001). PH tolerances (5.0–11.0)

(at intervals of 0.5 pH units) were examined with TSB

medium. Other physiological and biochemical tests

were performed according to the established methods of

Williams et al. (1983) and Kampfer et al. (1991).

Chemotaxonomic analyses

Gram stains were performed according to the modified

Hucker method (Hucker 1921) and confirmed by using

3% KOH (Buck 1982). Whole-cell sugar compositions

and diagnostic isomers of diaminopimelic acid were

analysed by TLC as described by Lechevalier and

Lechevalier (1980). Purified peptidoglycan preparations

were obtained by the method of Schleifer and Kandler

(1972). The detailed procedure for isolation of pure

peptidoglycan was summarized as follows. After

collection the cultures from the agar free medium, the

pellet was washed three times with distilled water.

Table 1 The isolation media and their compositions

Medium M1 M2 M3 M4 M5 M6 M7

Composition Starch 5, Histidine 0.1, Amorphous Xylan 5, Humic acid

1.0,

Colloidal chitin

2,

Sodium

propionate 2,

(g l-1) Glycerol 5, Raffinose 1.0, Cellulose 5, Casein 0.3, Na2HPO4 0.5, K2HPO4 0.7 g, Casein

hydrolysate

0.3,

Proline 1, Na2HPO4 0.5, KH2PO4 0.5, KNO3 0.2, KCl 1.7, KH2PO4 0.3, KNO3 0.1.

(NH4)2SO4 1, KCl 1.7, KNO3 0.2, KH2PO4 0.5, MgSO4�7H2O

0.05,

MgSO4�7H2O

0.5,

NaCl 1, MgSO4�4H2O

0.05,

KCl 0.1, KCl 0.1, FeSO4�7H2O

0.01,

FeSO4 0.01 g,

CaCl2 2, FeSO4�7H2O

0.01,

MgSO4�7H2O

0.3,

MgSO4�7H2O

0.3,

CaCl2 1 ZnSO4 0.001,

K2HPO4 1, CaCO3 0.02 NaCl 0.1, NaCl 0.1, MnCl2 0.001

MgSO4�7H2O

1

FeCl3 0.01, FeCl3 0.01,

NaNO3 2.5 NaNO3 2.5

Antibiotics

(mg l-1)

Nystatin 50,

aztreonam

25

Novobiocin 50,

rifampicin 20,

nalidixic acid

50

Nystatin 50,

nalidixic acid

50,

aztreonam 20

Nystatin 50,

nalidixic

acid 50,

Nystatin 50,

aztreonam

50

Novobiocin 50,

rifampicin 20,

nalidixic acid

50

Nystatin 50,

nalidixic acid

50,

aztreonam 20

pH = 7.2–7.5

Antonie van Leeuwenhoek (2010) 98:213–223 215

123

About 3 g cells were used for cell wall preparation.

Firstly, cells were grinded with the help of the liquid

nitrogen, and then suspended in 10 ml solution (con-

taining 1 mg proteinase K and 2 mg SDS). This

suspension was incubated at 55�C on a shaker for about

1 h and was centrifuged, and the pellet was washed two

times with distilled water. Then the sediment was

re-suspended in 10 ml potassium hydrate solution

(containing 0.2 g potassium hydrate solution) and was

incubated at 37�C for 20 h. The suspension was then

centrifuged and the pellet was washed two times with

ethanol 95% and 3 times with distilled water. The pellet

was suspended in 10 ml trypsin–phosphate buffer

(2 mg trypsin in 0.1 M phosphate buffer, pH 7.9) and

was incubated at 37�C on a shaker for about 2 h and was

centrifuged, and the pellet washed two times with

diluted hydrochloric (0.2 mol HCl in 10 ml water) and

three times with distilled water, at last was washed once

with chloroform. The final pellet was dried and the cell

wall was harvested. About 2 mg the cell wall prepara-

tion was hydrolyzed with 4 N HCl at 100�C for 16 h to

determine the quantitative amino acid content, and

another 2 mg cell wall was hydrolyzed with 4 N HCl in

a boiling-water bath for 45–60 min to determine the

peptide compared with those of known peptidoglycan

types.

Amino acids and peptides in cell-wall hydrolysates

were analysed by two-dimensional ascending TLC on

Table 2 16S rRNA gene

information of the 30

isolates studied in detail

Strain number Accession number Genus Similarity with the closest

described strain in GenBank (%)

6014 EU438912 Streptosporangium 96.1

03-9939 EU438907 Actinokineospora 96.8

CPCC202692 FJ529705 Actinokineospora 96.6

06-2230 EU438909 Actinopolymorpha 99.5

CPCC202699 FJ529702 Amycolatopsis 96.9

CPCC202698 FJ529703 Amycolatopsis 97.0

CPCC202695 FJ529717 Agromyces 97.0

06-3147 EU438911 Kineosporia 99.3

CPCC100076 FJ529700 Microlunatus 95.4

6015 EU438903 Saccharopolyspora 99.6

CPCC201357 EU438906 Actinomadura 99.7

CPCC202697 FJ529710 Actinomadura 98.2

6192 FJ529711 Actinomadura 98.9

06-2658 EU438910 Glycomyces 99.2

CPCC202694 FJ529715 Pseudonocardia 98.7

CPCC202696 FJ529716 Pseudonocardia 98.8

CPCC202724 FJ529719 Actinopolyspora 99.1

06-2143 EU531459 Micromonospora 98.9

07-1838 EU531461 Micromonospora 99.6

07-01839 EU531462 Micromonospora 99.8

06-1231-1 EU438908 Micromonospora 99.8

CPCC202691 FJ529699 Nonomuraea 97.5

CPCC202725 FJ529707 Nocardia 98.7

CPCC202726 FJ529708 Nocardia 98.6

CPCC202701 FJ529709 Nocardia 99.3

CPCC202693 FJ529714 Saccharothrix 97.9

6016 EU438904 Dermacoccus 95.9

CPCC201356 EU438905 Geodermatophilus 97.7

CPCC100074 FJ529713 Citricoccus 98.1

CPCC100077 FJ529706 Promicromonospora 97.3

216 Antonie van Leeuwenhoek (2010) 98:213–223

123

cellulose plates using the solvent systems of Schleifer

and Kandler (1972). The N-terminal amino acid of the

interpeptide bridge was determined by dinitropheny-

lation, as described by Schleifer (1985). Molar

ratios of amino acids were determined by GC and

GC-mass spectrometry of N-heptafluorobutyryl amino

acid isobutyl esters (MacKenzie 1987). Analysis of

enantiomers of peptidoglycan amino acids was per-

formed by GC of N-pentafluoropropionyl amino acid

isopropyl esters (Frank et al. 1980) on an L-Chirasil–

Val column (Macherey–Nagel) as described by Groth

et al. (1997). Menaquinones were extracted by using

the method of Collins (1985) and analyzed by HPLC

(Groth et al. 1997). Polar lipids were extracted

according to the method of Minnikin et al. (1984)

and were then identified by separating with two-

dimensional TLC and spraying the chromatogram with

appropriate detection reagents (Minnikin et al. (1984).

The resultant fatty acids were prepared and analyzed

following the instructions of the standard Sherlock

MIDI (Microbial Identification) system (Sasser 1990;

Kampfer and Kroppenstedt 1996).

Molecular analyses

Extraction of genomic DNA and amplification of 23S

rRNA and 16S rRNA genes were done as described by

Xu et al. (2003). The products of amplification of the

23S rRNA gene sequence of some of the isolates,

comprising 380 bp, indicated that these strains were

high-G ? C-DNA Gram-positive bacteria, i.e., actino-

bacteria (Stackebrandt et al. 1997; Yu et al. 2001). The

G ? C content of the genomic DNA was determined

using the thermal denaturation method (Marmur and

Doty 1962) with Streptomyces griseus ATCC 23345T

as a control. Multiple alignments with sequences of

validly described members of related taxa and calcu-

lation of levels of sequence similarity were carried out

using the EzTaxon server (Chun et al. 2007). Phylo-

genetic trees and distance matrices were reconstructed

using the neighbour-joining method (Saitou and Nei

1987) from Knuc values (Kimura 1980, 1983) using

MEGA version 4.0 (Tamura et al. 2007). The topology

of the phylogenetic tree was evaluated by the bootstrap

resampling method of Felsenstein (1985) with 1,000

replicates. Taxon-specific 16S rRNA signature nucle-

otides analysis was carried out using the CLUSTAL W

tool in MEGA version 4.0 (Tamura et al. 2007).

Results

Isolation and primary identification results

A total of 1800 mycelium-forming actinobacterial

strains and 130 non-mycelium forming strains were

purified from the isolation plates. After Gram

staining (confirmed by the KOH method), 97 isolates

were identified as Gram positive bacteria among the

130 non-mycelium forming isolates. Amplification

of 23S rRNA genes of the strains was carried out on

the 97 Gram positive bacteria. As a result, the

amplification of the 23S rRNA from the 97 Gram

positive bacteria showed that 63 strains produced the

expected 380 bp-fragment, which indicated that

these 63 strains were actinobacteria (Stackebrandt

et al. 1997; Yu et al. 2001). From an examination of

the colonies and cells of the Gram positive bacteria,

35 of them looked like Rhodococcus (most colony

color was pink or orange, and amino acids in cell-

wall hydrolysates contained meso-DAP as isomers)

strains and 24 of them looked like Micrococcaceae

(most colony color was pale yellow or orange–

yellow, while amino acids in cell-wall hydroly-

sates did not contain meso-DAP or LL-DAP as

diaminopimelic acid isomers) strains. The remaining

four unclassified bacterial strains, namely 6016,

CPCC 201356, CPCC 10074 and CPCC10077 were

selected to be subjected to a taxonomic study.

Among the 1800 actinomycetes, 817 isolates

formed extensively branched substrate mycelia that

carried abundant aerial hyphae that differentiated into

chains of spores on ISP 2 and ISP 4 plates. Another

634 isolates gave orange or black colonies on ISP 2

and ISP 4 plates. The isomer of diaminopimelic acid

in the cell hydrolysates indicated that the 817 strains

mainly contained LL-diaminopimelic acid and the

634 strains contained meso-diaminopimelic acid.

Combining the data on the isomers of diaminopimelic

acid with the cultural characteristics, the 817 strains

were assigned to the genus Streptomyces and the 634

isolates were assigned to the genus Micromonospora

(Williams et al. 1989). The colonies and cultural

characteristics of the remaining 349 actinomycetes

were further observed on ISP 3, ISP 5, Czapek

solution agar, nutrient agar (Difco), and tomato paste-

oat-meal agar. Twenty-six mycelium-forming strains

were chosen for further 16S rRNA gene sequencing.

16S rRNA gene sequence analysis showed that these

Antonie van Leeuwenhoek (2010) 98:213–223 217

123

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218 Antonie van Leeuwenhoek (2010) 98:213–223

123

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Antonie van Leeuwenhoek (2010) 98:213–223 219

123

26 strains belonged to 16 different genera (Table 2).

Together with the four non-mycelium forming

actinobacteria, 30 isolates (Table 2) were selected

for detailed taxonomic characterization.

Taxonomic results

The chemotaxonomic properties, including cell-wall

peptidoglycan diamino acid, predominant menaqui-

nones, genomic DNA G ? C contents, phospholipids

and fatty acid profiles for the selected 30 test strains

are listed in Table 3. Combined with the results of

phylogenetic analyses and physiological tests (data

not shown), ten novel taxa are proposed in this study.

The 16S rRNA signature nucleotide pattern anal-

ysis indicated that strain 6014 possessed the unique

signature nucleotides 1116 (G), 1137 (C), 1436–1465

(C–U) instead of 1116 (G), 1137 (U), 1436–1465

(G–C) of the family Streptosporangiaceae of the sub-

order Streptosporangineae (this study and Stackebrandt

et al. 1997), and strain 03-9939 possessed unique 16S

rRNA signature nucleotides compared to other genera

of the family Actinosynnemataceae, namely, 603–635

(C–G), 617–623 (U–C), 619 (U) (this study and

Labeda and Kroppenstedt 2007). In the phylogenetic

tree, strain 6014 (Fig. 1) and 03-9939 (Fig. 2) formed

a distinct lineage, respectively. Therefore, these two

strains may represent two new genera of the above

two families. The detailed taxonomic study results

are going to be published in another paper. According

to the morphological, physiological and biochemical,

chemotaxonomic and phylogenetic data, strain

06-2230 could be identified as a new member of the

genus Actinopolymorpha (Supplementary Fig. S1);

strain CPCC202699 represents a new species of

the genus Amycolatopsis (Supplementary Fig. S2);

strain CPCC100076 represents a new species of the

genus Microlunatus (Supplementary Fig. S3); strain

CPCC202695 represents a new species of the genus

Agromyces (Supplementary Fig. S4); strain 6016

represents a new species of the genus Demetria

(Supplementary Fig. S5); strain CPCC201356 repre-

sents a new species of the genus Geodermatophilus

(Supplementary Fig. S6); strain CPCC100074 repre-

sents a new species of the genus Citricoccus (Sup-

plementary Fig. S7); strain CPCC100077 represents a

new species of the genus Promicromonospora (Sup-

plementary Fig. S8). It will be necessary to carry out

further experiments (including DNA–DNA hybrid-

ization analysis) to determine whether the remaining

20 strains belong to recognized species.

Conclusion

In this study, gellan gum was used as a setting agent

instead of agar, and betaine and sodium pyruvate were

added to the isolation media. This study showed that a

Planotetraspora mira IFO 15435T (D85496)Acrocarpospora pleiomorpha R-31T ( AB006174) Acrocarpospora pleiomorpha IFO 16266T (AB025318)

Acrocarpospora corrugata DSM 43316T (X89941)

Thermopolyspora flexuosa strain DSM 43186T (AY039253) Planobispora longispora IFO 13918T (D85494)

NonomuraeaPlanomonospora parontospora IFO 13880T (D85495)

Streptosporangium

Streptosporangium violaceochromogenes DSM 43849T (X89951)

6014 Streptomyces megasporus DSM 41476T (Z68100)

100

93

64

95

60

100

7688

64

80

74

79

0.01

Microbispora

HerbisporaMicrotetraspora

Sphaerisporangium

Nonomuraea

Streptosporangium

Fig. 1 Neighbour-joining tree showing the phylogenetic rela-

tionships between strain 6014 (1497 bp) and the type strains of

related genera within the suborder Streptosporangineae based on

16S rRNA gene sequences. Numbers at the branch nodes are

bootstrap values (percentages of 1000 replicates). Bar, 1 nt

substitution per 100 nt

220 Antonie van Leeuwenhoek (2010) 98:213–223

123

greater number of rare actinomycete colonies were

recovered on gellan gum media containing betaine and

sodium pyruvate than on agar-containing media with-

out betaine and sodium pyruvate, which showed that

this was a great improvement for rare actinomycete

isolation. Furthermore, the parallel experiments suggest

that the isolation procedure is a critically important step

in studies of actinobacteria. In this study, rare actino-

bacteria appeared in all media we used, while media

M3, M4, M5 and M6 were more useful than other media

for isolation of rare genera of actinobacteria. Here, we

presumed that the main reason was that non-

monosaccharide was used as the carbon source. As

we know, if monosaccharide is used as the main carbon

and energy source in isolation media, O2- will be

accumulated rapidly when the predominant microbes in

soil samples assimilate the monosaccharide, while O2-

is not good for microbe growth, especially for the non-

predominant and poor growth microbes. Therefore, it is

necessary to improve traditional isolation methods to

explore the untapped actinobacteria. In summary, this

study has demonstrated that there is a rich diversity of

actinobacteria and some undiscovered actinobacterium

species in the soils of Qinghai–Tibet plateau, and it

Lentzea violacea IMSNU 50388T (AJ242633)

Lentzea albidocapillata DSM 44073T (X84321)

Lentzea flaviverrucosa JCM 11373T (AF183957)

Lentzea californiensis NRLL B-16137T (AF174435)

Lentzea albida NRRL B-24073T (AB006176)

Lentzea waywayandensis NRRL B-16159T (AF114813)

Lentzea kentuckyensis NRRL B-24416T (DQ291145)

Lechevalieria fradiae AS 4.3506T (AY114175)

Lechevalieria xinjiangensis strain R24T (DQ898283)

Lechevalieria flava NRRL B-16131T (AF114808)

Lechevalieria aerocolonigenes NRRL B-3298T (AF114804)

Actinosynnema mirum DSM 43827T (X84447)

Actinosynnema pretiosum subsp. pretiosum NRRL B-16060T (AF114800)

Saccharothrix mutabilis subsp. mutabilis DSM 43853T (X76966)

Saccharothrix mutabilis subsp. capreolus DSM 40225T (X76965)

Saccharothrix espanaensis NRRL 15764T (AF114807)

Saccharothrix australiensis NRRL 11239T(AF114803)

Saccharothrix algeriensis NRRL B-24137T (AY054972)

Saccharothrix coeruleofusca NRRL B-16115T (AF114805)

Saccharothrix syringae NRRL B-16468T (AF114812)

Saccharothrix xinjiangensis AS 4.1731T (AY135693)

Saccharothrix texasensis NRRL B-16134T (AF114814)

Umezawaea tangerina MK27-91F2T (AB020031)

Saccharothrix longispora NRRL B-16116T (AF114809)

CPCC 202692 03-9939

Actinokineospora riparia DSM 44259T (AF114802)

Actinokineospora inagensis DSM 44258T (AF114799)

Actinokineospora enzanensis DSM 44649T (AB058395

Actinokineospora diospyrosa B-24047T (AF114797)

Actinokineospora auranticolor IFO16518T (AB058396)

Actinokineospora terrae AB058394T (IFO15668)

Actinokineospora globicatena NRRL B-24048T (AF114798)

Pseudonocardia thermophila IMSNU 20112T (AJ252830)

100

99

99

68 86

93

59

54

59

94

93

89

61

72

98 60

99

64

75

79

85

79

71 65

78

0.01

Fig. 2 Neighbour-joining tree showing the phylogenetic rela-

tionships between strain 03-9939 (1442 bp), CPCC 202692

(1456 bp) and the type strains of related genera within the family

Actinosynnemataceae based on 16S rRNA gene sequences.

Numbers at the branch nodes are bootstrap values (percentages

of 1000 replicates). Bar, 1 nt substitution per 100 nt

Antonie van Leeuwenhoek (2010) 98:213–223 221

123

suggests that these strains might represent a valuable

source of new taxa for further microbial development

and utilization. Additionally, this study indicates that

the biogeographic distribution of actinobacteria in

Qinghai–Tibet plateau is an interesting topic for further

study.

Acknowledgments The authors are grateful to the two

anonymous reviewers and Miss Cao Yan-Ru from Yunnan

University for their critical helping to improve the quality of

this manuscript. This research was supported by National

Facilities and Information Infrastructure for Science and

Technology (2005DKA21203), National Basic Research

Program of China (2010CB833800), the National Natural

Science Foundation of China (nos. 30970008 and 30970038),

Key Projects of International Cooperation (2007DFB31620),

State-level Public Welfare Scientific Research Institutes for

Basic R&D special fund (no. IMBF200915) and Yunnan

Provincial Natural Science Foundation (Nos. 2009AC017 and

2009DA002).

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