ORIGINAL PAPER

Microlunatus cavernae sp. nov., a novel actinobacteriumisolated from Alu ancient cave, Yunnan, south-west China

Juan Cheng • Wei Chen • Bing Huo-Zhang •

Salam Nimaichand • En-Min Zhou •

Xin-Hua Lu • Hans-Peter Klenk • Wen-Jun Li

Received: 20 February 2013 / Accepted: 29 April 2013 / Published online: 8 May 2013

� Springer Science+Business Media Dordrecht 2013

Abstract A Gram-positive, coccoid, non-endo-

spore-forming actinobacterium, designated YIM

C01117T, was isolated from a soil sample collected

from Alu ancient cave, Yunnan province, south-west

China. Based on the 16S rRNA gene sequence analysis,

strain YIM C01117T was shown to belong to the genus

Microlunatus, with highest sequence similarity of

97.4 % to Microlunatus soli DSM 21800T. The whole

genomic DNA relatedness as shown by the DNA–

DNA hybridization study between YIM C01117T and

M. soli DSM 21800T had a low value (47 ± 2 %).

Strain YIM C01117T was determined to contain LL-

diaminopimelic acid with Gly, Glu and Ala amino

acids (A3c0 type) in the cell wall. Whole-cell hydrol-

ysates were found to contain glucose, galactose,

mannose and ribose. The major polar lipids were

determined to be phosphatidylglycerol and diphos-

phatidylglycerol. The predominant menaquinone sys-

tem present is MK-9(H4), while the major fatty acids

were identified to be anteiso-C15:0 (24.1 %), iso-C16:0

(22.3 %) and iso-C15:0 (11.4 %). The G?C content of

the genomic DNA was determined to be 65.9 mol%.

The chemotaxonomic and genotypic data support the

affiliation of the strain YIM C01117T to the genus

Microlunatus. The results of physiological and bio-

chemical tests allow strain YIM C01117T to be

Juan Cheng and Wei Chen contributed equally to this work.

Electronic supplementary material The online version ofthis article (doi:10.1007/s10482-013-9929-x) containssupplementary material, which is available to authorized users.

J. Cheng � B. Huo-Zhang � S. Nimaichand �E.-M. Zhou � W.-J. Li (&)

Key Laboratory of Microbial Diversity in Southwest

China, Ministry of Education, and Laboratory for

Conservation and Utilization of Bio-resources, Yunnan

Institute of Microbiology, Yunnan University, Kunming

650091, People’s Republic of China

e-mail: liact@hotmail.com; wjli@ynu.edu.cn

W. Chen

Hongyun Honghe Tobacco (Group) Co., Ltd, Kunming

650202, People’s Republic of China

S. Nimaichand

Microbial Biotechnology Research Laboratory,

Department of Biochemistry, Manipur University,

Canchipur, Imphal 795003, Manipur, India

X.-H. Lu

The National Engineering Center for Research of Microbial

Pharmaceuticals and New Drug Research and Development

Center, North China Pharmaceutical Group Corporation,

Shijiazhuang 050015, People’s Republic of China

H.-P. Klenk

Leibniz Institute—DSMZ, German Collection of

Microorganisms and Cell Cultures, Inhoffenstrasse 7B,

38124 Braunschweig, Germany

W.-J. Li

Key Laboratory of Biogeography and Bioresource in Arid

Land, Xinjiang Institute of Ecology and Geography,

Chinese Academy of Sciences, Urumqi 830011, People’s

Republic of China

123

Antonie van Leeuwenhoek (2013) 104:95–101

DOI 10.1007/s10482-013-9929-x

differentiated phenotypically from recognized Micro-

lunatus species. Strain YIM C01117T is therefore

considered to represent a novel species of the genus

Microlunatus, for which the name Microlunatus

cavernae sp. nov. is proposed. The type strain is YIM

C01117T (= DSM 26248T = JCM 18536T).

Keywords Microlunatus cavernae sp. nov.

16S rRNA gene � Polyphasic taxonomy

Alu ancient cave

Introduction

The genus Microlunatus was established by Nakamura

et al. (1995). At the time of writing, the genus contained

six validly named species isolated from different

habitats: Microlunatus phosphovorus (Nakamura et al.

1995) from activated sludge, Microlunatus ginsengisoli

(Cui et al. 2007) and Microlunatus panaciterrae (An

et al. 2008) from soil of a ginseng field, Microlunatus

aurantiacus (Wang et al. 2008) from a rhizosphere soil,

Microlunatus soli (Kampfer et al. 2010a) from a spawn

used for growing edible mushrooms in the laboratory

and Microlunatus parietis (Kampfer et al. 2010b) from

an indoor wall. All these species are characterized

chemotaxonomically by the presence of LL-diamino-

pimelic acid (DAP) in the cell-wall, MK-9(H4) as the

predominant menaquinone and anteiso-C15:0, iso-C15:0

and iso-C16:0 as the major fatty acids.

This paper reports the taxonomic position of a novel

strain, YIM C01117T, using a polyphasic character-

ization approach. The results suggest that strain YIM

C01117T represents a novel species of the genus

Microlunatus, for which the name Microlunatus

cavernae sp. nov. is proposed here.

Materials and methods

Strain and culture conditions

Strain YIM C01117T was isolated from a soil sample

collected from Alu ancient cave (E103�45.0000,N24�33.6730), a tourism cave located in Yunnan

province, south-west China. The soil sample after

serial dilution was spread on R2A medium (BD;

Becton, Dickinson and Company) and incubated for

2 weeks at 28 �C. Colonies were selected and

repeatedly re-streaked on the same medium to obtain

pure cultures. Strain YIM C01117T was routinely

cultivated on R2A medium at 28 �C and also stored as

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

for chemical and molecular studies was obtained by

cultivation on R2A medium at 28 �C for about

1 week. The reference strains M. soli DSM 21800T,

M. parietis DSM 22083T and M. phosphovorus DSM

10555T were obtained from DSMZ and cultured under

the same conditions as strain YIM C01117T.

Phenotypic characteristics

Gram staining was carried out by using the standard

Gram reaction and was confirmed by using the KOH

lysis test method (Cerny 1978). Morphology was

examined by light microscopy (BH-2; Olympus) and

scanning electron microscopy (QUANTA200; FEI).

For scanning electron microscopy, cultured cells were

harvested by centrifugation, washed and suspended in

20 mM phosphate buffer (pH 7.0). The suspended

cells were fixed with 3 % glutaraldehyde. The cells

were dehydrated in ethanol. After critical point drying,

samples were sputter coated with gold and observed

with a scanning microscope.

Growth at different temperatures (4, 10, 15, 22, 28,

37, 39, 40, 45 and 50 �C) and NaCl tolerance

(0–12.0 % w/v) were tested on tryptose soy agar

plates by incubating the cultures for 14–21 days. The

pH range (4.0–10.0, at intervals of 1.0 pH unit) for

growth was tested in tryptose soy broth medium

(28 �C, 14–21 days) using the buffer system described

by Xu et al. (2005). Carbon source utilization was

determined by the methods described by Shirling and

Gottlieb (1966) and Locci (1989). Nitrogen source

utilization was determined according to Williams et al.

(1989). Catalase activity was detected by assessing the

production of bubbles on addition of a drop of 3 % (v/

v) H2O2. Oxidase activity was determined by the

oxidation of tetramethyl-p-phenylenediamine (Kov-

acs 1956). Hydrolysis of cellulose, gelatin, tyrosine,

starch, and Tweens 20, 40, 60 and 80, milk coagulation

and peptonization, utilization of urea, hydrogen sul-

fide production and nitrate reduction were performed

as described by Gonzalez et al. (1978). Determination

of phosphate accumulation by strain YIM C01117T

was performed as described by Nakamura et al.

(1995), with M. phosphovorus DSM 10555T as the

reference strain.

96 Antonie van Leeuwenhoek (2013) 104:95–101

123

Chemotaxonomy

The isomer of amino acids in purified cell walls and

sugars of whole cell hydrolysates were determined

according to the procedures described by Hasegawa

et al. (1983), Lechevalier and Lechevalier (1970) and

Tang et al. (2009a, b). Polar lipids were extracted as

described by Minnikin et al. (1979) and identified by

two-dimensional TLC (Collins and Jones 1980).

Menaquinones were extracted (Collins et al. 1977)

and analyzed using HPLC (Kroppenstedt 1982).

Cellular fatty acid analysis was performed by using

the Microbial Identification System (Sherlock Version

6.1; MIDI database: TSBA6; Sasser 1990). Biomass

for fatty acid analysis was obtained from cell grown on

tryptose soy agar (Difco) at 28 �C for 4 days.

Molecular analysis and DNA–DNA hybridizations

Extraction of genomic DNA and PCR amplification

of the 16S rRNA gene were performed from strain

YIM C01117T as described previously (Li et al.

2007). The sequence obtained was compared

with available 16S rRNA gene sequences of

cultured species from the EzTaxon-e server

(http://eztaxon-e.ezbiocloud.net/; Kim et al. 2012).

Multiple alignments with sequences of the most

closely related taxa and calculations of levels of

sequence similarity were carried out using CLUS-

TAL_X program (Thompson et al. 1997). Phylo-

genetic analyses were performed by using three

tree-making algorithms i.e. the neighbour-joining

(Saitou and Nei 1987), maximum-likelihood (Fel-

senstein 1981) and maximum-Parsimony (Fitch

1971) methods using the software package MEGA

version 5.0 (Tamura et al. 2011). Kimura’s two

parameter model was used to calculate evolutionary

distance matrices of the neighbour-joining method

(Kimura 1980). Bootstrap analysis was performed

with 1,000 replications (Felsenstein 1985). Aeris-

cardovia aeriphila T6T (AY174107) was used as an

outgroup.

The G?C content of the genomic DNA for strain

YIM C01117T was determined by using the HPLC

method described by Mesbah et al. (1989). DNA–

DNA relatedness was studied using the fluorometric

micro-well method (Ezaki et al. 1989; Christensen

et al. 2000; He et al. 2005). The hybridizations were

performed with eight replications.

Results and discussion

Phenotypic characteristics

Strain YIM C01117T was observed to be a Gram-

positive, coccus-shaped nonmotile, nonendospore-

forming actinobacterium. The cells measured

0.6–1.0 lm in diameter and occurred either singly

or in pairs. Small irregular clusters of cells were

also found (see Supplementary Fig. S1). Growth of

strain YIM C01117T was observed at 10–37 �C,

0–9.0 % NaCl (w/v) and pH 5.0–10.0, with optimal

growth at 22–28 �C, 0–1.0 % NaCl (w/v) and pH

7.0–8.0. The strain was determined to be positive

for catalase test and negative for oxidase, gelatin-

ase, amylase and urease activities. The strain

showed negative results for milk coagulation, milk

peptonization, nitrate reduction, hydrogen sulfide

production, and hydrolysis of tyrosine, Tweens 20,

40, 60, 80 and cellulose. The differential charac-

teristics of strain YIM C01117T and the type strains

in the genus Microlunatus are shown in Table 1

while the detailed characteristics of the strain are

given in the species description. Strain YIM

C01117T was observed to accumulate phosphate

only weakly in comparison with M. phosphovorus

DSM 10555T. As shown in Fig. 1, cells of YIM

C01117T exhibited weak phosphate-accumulating

activity after incubation for 5 h in the absence of

any carbon substrate in the medium; after 13 h, the

phosphate-accumulating activity of YIM C01117T

became more stable.

Chemotaxonomic characteristics

The peptidoglycan type of strain YIM C01117T was

determined to be A3c0 based on LL-diaminopimelic

acid with Gly, Glu and Ala amino acids in its purified

cell wall (Fig. S2). The major whole-cell sugars were

identified to be glucose, galactose, mannose and

ribose. The polar lipids were found to consist of

phosphatidylglycerol and diphosphatidylglycerol with

a few unknown/uncharacterized lipids: one glycolipid,

two phospholipids and one polar lipid (see Supple-

mentary Fig. S3). The menaquinone system of strain

YIM C01117T was determined to mainly comprise of

MK-9(H4) with a small amount of MK-8(H6). The

major fatty acids ([10 %) of strain YIM C01117T

Antonie van Leeuwenhoek (2013) 104:95–101 97

123

were identified as the saturated branched-chain fatty

acids anteiso-C15:0 (24.1 %), iso-C16:0 (22.3 %) and

iso-C15:0 (11.4 %) (Table 2). The major fatty acids

profile of strain YIM C01117T were consistent with

those of three type strains, but there were also some

differences with the latter. The G?C content of

genomic DNA was determined to be 65.9 mol%,

which is consistent with the range for the genus

Microlunatus. Thus, the chemotaxonomic data for

strain YIM C01117T are consistent with its assignment

to the genus Microlunatus.

Phylogenetic analysis and DNA–DNA relatedness

Sequence analysis of the almost complete 16S rRNA

gene sequence of strain YIM C01117T (1,480 bp;

GenBank accession no. KC693588) using the EzTax-

on-e server showed highest similarity to sequences of

strains belonging to the genus Microlunatus: M. soli

DSM 21800T (97.4 %), M. parietis DSM 22083T

(96.6 %), M. panaciterrae Gsoil 954T (95.1 %), M.

phosphovorus DSM 10555T (94.6 %), M. ginsengisoli

Gsoil 633T (94.6 %) and M. aurantiacus YIM 45721T

Table 1 Physiological characteristics of strain YIM C01117T and the type strains of the genus Microlunatus

Characteristics 1 2 3 4 5 6 7

Temperature range for growth (�C) 20–37 20–30 20–30

NaCl range (%) 0–9.0 0–9.0 0–6.0 0–7.0 7.0–7.5 5.5–8.5 5.0–9.0

pH range 5.0–10.0 6.0–10.0 5.0–10.0 5.0–9.0 0 0–5.0 0–5.0

Oxidase – ? – ? – – ?

Nitrate reduction – ? ? ? ? – –

Hydrolysis of cellulose – – – – – ? –

Gelatin – – – – ND ? ?

H2S – – – – – – –

Starch – – – – ND – ?

Tween 20 – – – – ? ND ND

Tween 60 – – – ? – ND ND

Tyrosine – – – ? ND ND ND

Urea – – – – ? – –

Utilization of sole carbon source sources

L-arabinose – ? ? – ? ? ?

Cellobiose ? ? – ? ? ? –

D-fructose – – ? ? ? ? –

Galactose ? – ? ? ? ? –

Raffinose ? ? ? ? ? ? –

L-rhamnose ? – ? ? – ? ?

Xylitol ? ? – ? ? ? –

D-xylose ? ? – ? ? ? –

Utilization of sole nitrogen source

L-histidine ? ? – ? – ? –

L-lysine ? – ? ? – – –

L-glycine ? ? ? ? ND – ND

L-methionine ? ? ? ? ND – ND

Strains: 1 YIM C01117T; 2 M. soli DSM 21800T; 3 M. parietis DSM 22083T; 4 M. phosphovorus DSM 10555T; 5 M. aurantiacusYIM 45721T (Wang et al. 2008); 6 M. ginsengisoli DSM 17942 T (Cui et al. 2007); 7 M. panaciterrae Gsoil 954 T (An et al. 2008).

All the data for strains YIM C01117T, M. soli DSM 21800T, M. parietis DSM 22083T and M. phosphovorus DSM 10555T are from

the present study

? Positive, - negative, ND not determined

98 Antonie van Leeuwenhoek (2013) 104:95–101

123

(94.4 %). The phylogenetic tree based on the neigh-

bour-joining (NJ) method showed the formation of

clade with M. soli DSM 21800T with a bootstrap

support of 62 % (Fig. 2), which indicates the affiliation

of the strain YIM C01117T to the genus Microlunatus.

Based on the sequence analysis from EzTaxon-e and the

NJ tree, the strain M. soli DSM 21800T was the closely

related phylogenetic neighbor, and thus it was selected

for DNA–DNA hybridization studies with strain YIM

C01117T. The determined DNA–DNA relatedness

value of 47 ± 2 %, which is well below the 70 %

cut-off point for the recognition of genomic species

(Wayne et al. 1987). Additionally, the differences in the

phenotypic characters of YIM C01117T from the

reference type strain M. soli DSM 21800T (including

utilization of galactose and L-lysine as sole C/N sources

but not arabinose, along with negative results in the

oxidase and nitrate reduction tests) support the place-

ment of strain YIM C01117T as representative of a

novel species within the genus Microlunatus, for which

the name M. cavernae sp. nov. is proposed.

Description of Microlunatus cavernae sp. nov.

Microlunatus cavernae (ca.ver’na.e. L. gen. n. caver-

nae, of a cave; M. L. gen. fem. n. cavernae of a cave,

referring to the habitat of the organism) Gram-positive,

coccus-shaped nonmotile, non-endospore-forming.

Cells measure 0.6–1.0 lm in diameter and occur either

singly or in pairs. Irregular clusters of cells are also

observed. Growth occurs at 10–37 �C, pH 5.0–10.0 and

in the presence of up to 9.0 % NaCl (w/v). Utilizes

cellobiose, galactose, maltose, D-mannose, raffinose,

rhamnose, sodium malate, xylitol and D-xylose as sole

carbon sources, but not L-arabinose and D-fructose.

Utilizes L-alanine, L-glycine, L-histidine, L-lysine, L-

methionine, L-phenylalanine and L-valine as sole nitro-

gen sources. Positive in the catalase test and negative in

oxidase, milk coagulation, milk peptonization, nitrate

reduction and hydrogen sulfide production tests. Does

not hydrolyze cellulose, gelatin, starch, tyrosine,

Tweens 20, 40, 60, 80 and urea. The peptidoglycan

type is A3c0 based on LL-diaminopimelic acid. The

0

50

100

150

200

250

0 5 10 15 20 25 30

Time (h)

Pho

spha

te c

once

ntra

tion

(mg

P-1

)

Fig. 1 Phosphate uptake profiles of strain YIM C01117T and

M. phosphovorus DSM 10555T under aerobic conditions. Both

strains were grown on R2A medium (removed KH2PO4 and

MgSO4�7H2O) and harvested in the late exponential phase. The

amount of phosphate was determined by the molybdenum blue

method. Filled square YIM C01117T, filled diamond M.phosphovorus DSM 10555T (reference strain)

Table 2 Fatty acid profiles ([1 %) of strains YIM C01117T,

M. soli DSM 21800T, M. parietis DSM 22083T and M. phos-phovorus DSM 10555T

Fatty acids YIM

C01117TDSM

21800TDSM

22083TDSM

10555T

C16:0 1.1 – – 1.5

C14:1x5c 2.4 1.4 2.7 –

C17:1x9c – 2.2 – –

C17:1x6c – 11.5 – –

Iso-C14:0 2.4 2.2 2.5 16.1

Iso-C15:0 11.4 7.5 30.5 13.7

Iso-C15:1 3.7 – – –

Iso-C16:0 22.3 21.2 6.0 22.7

Iso-C16:1 9.5 – – –

Iso-C17:0 3.1 – – 2.5

Anteiso-C15:0 24.1 18.0 44.3 32.9

Anteiso-C15:1 1.2 – – –

Anteiso-C17:0 8.9 2.6 1.4 1.1

C8:0 3OH 1.0 – 1.2 –

Iso-C14:0 3OH – 4.1 3.1 2.0

C14:0 2OH – 1.8 1.2 –

Summed feature 3a – – – 1.5

Summed feature 4a – – 3.0 –

Summed feature 8a – 20.6 – –

Summed feature 3 consisted of C16:1x6c and/or C16:1x7c

Summed feature 4 consisted of anteiso-C17:1 and/or iso-C17:1

Summed feature 8 consisted of C18:1x7c.and/or C18:1x6c

- Negative or absenta Summed features are groups of two or three fatty acids that

cannot be separated by GLC with the MIDI system

Antonie van Leeuwenhoek (2013) 104:95–101 99

123

major whole cell sugars are glucose, galactose, man-

nose and ribose. The polar lipids consist of phosphat-

idylglycerol, diphosphatidylglycerol, an unknown

glycolipid, two unknown phospholipids and an unchar-

acterized polar lipid. The menaquinone system com-

prises of MK-9(H4) and MK-8(H6). The fatty acid

profile includes anteiso-C15:0, iso-C16:0 and iso-C15:0 as

major components. The G?C content of the genomic

DNA of the type strain is 65.9 mol %.

The GenBank accession number for the 16S rRNA

gene sequence of strain YIM C01117T is KC693588.

The type strain YIM C01117T (= DSM 26248T =

JCM 18536T) was isolated from a soil sample

collected at Alu Ancient cave (E103�45.0000,N24�33.6730), Yunnan province, south-west China.

Acknowledgments The authors are grateful to Prof. Jean P.

Euzeby for construction of the Latin name of the new taxon.

This work was funded jointly by National New Drug Research

and Development Project (No. 2010ZX09401-403) and project

of China tobacco Yunnan industrial Co. Lid. (Nos. 2012JC07

and 2012FL02). W-J Li was also supported by ‘Hundred Talents

Program’ of the Chinese Academy of Sciences.

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7163*

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