International Journal of Systematic and Evolutionary Microbiology (2013), 63, 114–118
DOI 10.1099/ijs.0.036087-0
Rhodococcus canchipurensis sp. nov., an
actinomycete isolated from a limestone deposit site
Salam Nimaichand,1,23 Suchitra Sanasam,23 Liu-Qiang Zheng,1
Wen-Yong Zhu,1 Ling-Ling Yang,1 Shu-Kun Tang,1 Debananda
S. Ningthoujam2 and Wen-Jun Li1,3
Correspondence
Wen-Jun Li
wjli@ynu.edu.cn
Debananda S. Ningthoujam
ningthoujam@gmail.com
1
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, PR China
2
Microbial Biotechnology Research Laboratory, Department of Biochemistry, Manipur University,
Canchipur, Imphal, 795003 Manipur, India
3
Key Laboratory of Biogeography and Bioresource in Arid Land, CAS, Xinjiang Institute of Ecology
and Geography, Chinese Academy of Sciences, Ürűmqi 830011, China
A novel actinobacterial strain, MBRL 353T, was isolated from a sample collected from a limestone
quarry at Hundung, Manipur, India. Comparison of 16S rRNA gene sequences of strain MBRL
353T and other members of the genus Rhodococcus showed sequence similarities ranging from
95.5 to 98.2 %, with strain MBRL 353T showing closest sequence similarity to Rhodococcus
triatomae IMMIB RIV-085T (98.2 %) and Rhodococcus equi DSM 20307T (97.2 %). DNA–DNA
hybridization results, however, revealed that DNA–DNA relatedness values between strain MBRL
353T and R. triatomae DSM 44892T (43.4 %) and R. equi DSM 20307T (33.4 %) were well below
the 70 % limit for species identification. Strain MBRL 353T contained meso-diaminopimelic acid
as the diagnostic diamino acid and galactose and arabinose in the cell wall. Mycolic acids were
present. The major fatty acids were C16 : 0 (45.7 %), C18 : 1v9c (18.2 %) and 10-methyl C18 : 0
(11.3 %). The only menaquinone detected was MK-8(H2), while the major polar lipids were
diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol
mannoside and one unknown phospholipid. The G+C content of the genomic DNA was
69.2 mol%. The phenotypic and genotypic data showed that strain MBRL 353T merits recognition
as a representative of a novel species of the genus Rhodococcus for which the name
Rhodococcus canchipurensis sp. nov. is proposed; the type strain is MBRL 353T (5KCTC
19851T5JCM 17578T).
The genus Rhodococcus was first proposed by Zopf (1891),
but later elucidated by Tsukamura (1974) with Rhodococcus
rhodochrous as the type species of the genus and emended
subsequently by Rainey et al. (1995). The genus is placed in
the family Nocardiaceae of the suborder Corynebacterineae
(Stackebrandt et al., 1997). A special feature of this genus is
the simple rod–coccus developmental cycle (Fuhrmann
et al., 1997). Biotechnologically, the genus has acquired
significant value because of its ability to degrade a diversity
of organic compounds, including some of the most
difficult compounds with regard to recalcitrance and
3These authors contributed equally to this work.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene
sequence of strain MBRL 353T is JN164649.
Four supplementary figures and one supplementary table are available
with the online version of this paper.
114
toxicity (Larkin et al., 2005). At the time of writing, there
are more than 36 species with validly published names
in the genus, including the most recently described species
Rhodococcus jialingiae, Rhodococcus artemisiae and Rhodococcus nanhaiensis (Wang et al., 2010; Zhao et al., 2012; Li
et al., 2012).
The Microbial Biotechnology Research Laboratory (MBRL)
has been exploring actinomycete diversity in various
habitats of Manipur and also investigating their biotechnological potential for applications such as industrial enzyme
production (Ningthoujam et al., 2009a), biodegradation
of xenobiotics, and antimicrobial (Ningthoujam et al.,
2009b; Sanasam & Ningthoujam, 2010) and biocontrol
(Ningthoujam et al., 2009c) agents. As a part of our ongoing
studies on actinomycete diversity in Manipur, strain MBRL
353T was isolated from a soil sample collected from a
limestone quarry at Hundung, Manipur, India (25.05u N
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Rhodococcus canchipurensis sp. nov.
94.33u E) using starch casein nitrate agar (SCNA; Küster &
Williams, 1964) as the selective medium. Soil samples were
air dried for a week, crushed and sieved. Sieved soil (1 g) was
treated with 0.1 g CaCO3 for a week to prevent the growth of
fast-growing bacteria and was then suspended in 99 ml
quarter-strength Ringer’s salt solution (pH 7.0) and kept in
an orbital shaker at 150 r.p.m. for 30 min. The suspension
was centrifuged, 0.1 ml of the supernatant was decimally
diluted and 0.1 ml aliquots were spread on SCNA (pH 7.2)
and incubated at 28 uC for 3 weeks. The isolates were
subcultured in yeast extract-malt extract agar (ISP 2)
(Shirling & Gottlieb, 1966) to obtain pure cultures. Strain
MRBL 353T, one of the Hundung isolates, was preserved in
glycerol (20 %, v/v) at 280 uC.
For observing its morphological characteristics, strain
MBRL 353T was cultivated aerobically at 28 uC on ISP 2
medium. Cell morphology was observed by using light
microscopy (Axioscope A1; Zeiss) and scanning electron
microscopy (JSM-6360; Jeol). Growth on various ISP
media, tryptic soy agar (Difco), SCNA, Czapek’s agar and
nutrient agar were observed. The colony colour was
determined using the ISCC-NBS colour chart (Kelly, 1964).
Strain MBRL 353T formed smooth, circular, convex,
opaque and pink colonies after 7 days of incubation at
28 uC. The cells were Gram-positive, non-spore-forming
and non-motile. Observation by light and scanning electron
microscopy revealed the typical rod–coccus developmental
cycle for the genus Rhodococcus (Wang et al., 2008). The
strain showed an elementary rod structure during the early
growth phase (26 h), which started to disintegrate in shorter
rods at 47–56 h, and finally formed cocci at around 109 h
(Fig. S1, available in IJSEM Online). The strain grew well in
most of the media tested, but showed poor growth on ISP 3,
ISP 4 and ISP 5 media.
Utilization of sole carbon and nitrogen sources was
determined as described by Shirling & Gottlieb (1966).
Tests for decomposition of adenine, casein, tyrosine, urea
and xanthine were performed following the methods of
Gordon et al. (1974). Hydrolysis of starch, gelatin and
Tweens 20, 40, 60 and 80 was determined as described by
Smibert & Krieg (1994), and the esterase enzyme method,
which observes the formation of fuzzy halos on solid
medium containing 0.5 % NaCl, 0.1 % peptone, 0.01 %
CaCl2 . H2O and 0.9 % agar, was used. Nitrate reduction was
monitored as described by Lányı́ (1987). Antibiotic
susceptibility test was performed as described by Groth
et al. (2004) using antibiotic discs (HiMedia). Growth at 5,
15, 28, 37, 42, 50 and 60 uC, at pH 4, 5, 6, 7, 8, 9 and 10, and
with 0, 2, 5, 7 and 10 % NaCl was determined on TSA as
described by Goodfellow (1986). Catalase activity was
observed by assessing bubble production in 3 % (v/v)
H2O2 and oxidase activity was determined by using 1 % (w/
v) solution of tetramethyl-p-phenylenediamine (Kovacs,
1956). Other biochemical tests including Voges–Proskauer,
methyl red and the production of H2S and indole were
performed as described by Goodfellow (1986).
http://ijs.sgmjournals.org
The isolate could degrade adenine, but not guanine, tyrosine
or xanthine. The strain could hydrolyse starch and Tweens
20, 40, 60 and 80, but not casein, gelatin or urea. The strain
was positive for catalase and Voges–Proskauer tests, but
negative for oxidase, methyl red, citrate utilization, indole
production, nitrate reduction and H2S production. The
strain was resistant to (mg per disc, unless otherwise
indicated) amikacin (30), ampicillin (10), amphotericin
(100 U per disc), chloramphenicol (30), erythromycin (15),
fluconazole (25), gentamicin (10), kanamycin (30), meticillin (5), nalidixic acid (30), neomycin (30), novobiocin
(30), nystatin (100 U per disc), penicillin (10), streptomycin
(10), trimethoprim (50) and vancomycin (30). However,
strain MBRL 353T showed sensitivity to ampicillin/sulbactam (10), amoxicillin (10), carbamicillin (10), ciprofloxacin
(10), gentamicin (30), nitrofurantoin (300), rifampicin (5),
tetracycline (30) and tobramycin (10). The detailed
phenotypic properties of strain MBRL 353T are listed in
Table 1 and other phenotypic characteristics are mentioned
in the species description.
The amino acid content of the cell wall was determined
according to Staneck & Roberts (1974) and the sugars of
the whole-cell hydrolysate were analysed as described by
Tang et al. (2009). For other chemotaxonomic analyses,
strain MBRL 353T was grown in tryptic soy broth shake
flasks for 1 week at 28 uC. Cell biomass was harvested by
centrifugation, washed with distilled water and lyophilized.
Mycolic acids were extracted and analysed according to the
protocol of Minnikin et al. (1980). Polar lipids were
extracted and analysed by two-dimensional TLC as
described by Minnikin et al. (1984). The extraction of
Table 1. Phenotypic characteristics that differentiate strain
MBRL 353T from its closest phylogenetic neighbours
Strains: 1, Rhodococcus canchipurensis sp. nov. MBRL 353T; 2, R. equi
DSM 20307T; 3, R. kunmingensis YIM 45607T; 4, R. triatomae DSM
44892T. All data were taken from this study.
Characteristic
Utilization as sole C source
Dulcitol
D-Galactose
D-Mannose
D-Xylose
Utilization as sole N source
L-Arginine
L-Phenylalanine
Potassium nitrate
L-Serine
L-Valine
Hydrolysis of:
Starch
Urea
pH for growth
NaCl for growth (%)
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1
2
3
4
2
2
+
+
+
+
+
+
2
2
+
+
2
2
2
2
+
+
+
+
+
+
+
+
+
+
+
+
+
2
+
2
2
2
2
2
+
2
6–10
0–5
2
+
6–8
0–2
2
+
6–8
0–5
2
2
6–10
0–5
115
S. Nimaichand and others
menaquinones was done as described by Collins et al.
(1977) and menaquinones were analysed by HPLC
(Tamaoka et al., 1983). The cellular fatty acids were
extracted, methylated and analysed by using the Sherlock
Microbial Identification System (MIDI) according to the
method of Sasser (1990) and the manufacturer’s instructions. The fatty acid methyl esters were then analysed by
GC (7890A GC; Agilent Technologies) by using the
Microbial Identification software package (Sherlock version 6.1; MIDI database TSBA6).
Strain MBRL 353T had meso-diaminopimelic acid as the
diagnostic diamino acid and galactose and arabinose in
the cell-wall hydrolysate (cell-wall chemotype IV sensu
Lechevalier & Lechevalier, 1970). The non-diagnostic sugars
mannose, ribose, rhamnose and glucose were also detected in
the whole-cell hydrolysate. Mycolic acids were present (Fig.
S2). The major polar lipids detected were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol,
phosphatidylinositol mannoside and one unknown phospholipid (Fig. S3; phospholipid type II sensu Lechevalier et al.,
1977). MK-8(H2) was the only menaquinone detected. The
79
Genomic DNA isolation and PCR amplification of the 16S
rRNA gene was done as described by Li et al. (2007). The
almost-complete 16S rRNA gene sequence (1522 bp) of the
strain was submitted to the EzTaxon server (Kim et al.,
2012) and aligned with the 16S rRNA gene sequences of
other Rhodococcus species using CLUSTAL X version 2.1
(Larkin et al., 2007). Phylogenetic analysis was done using
the software package MEGA version 5.0 (Tamura et al.,
2011). Distances (using distance options according to
Kimura’s two-parameter model; Kimura, 1983) were
calculated and clustering was performed with the neighbour-joining method (Saitou & Nei, 1987). To determine
the support of each clade, bootstrap analysis was
performed with 1000 resamplings (Felsenstein, 1985). The
validity of the neighbour-joining tree was compared with
the maximum-parsimony tree (Kluge & Farris, 1969)
drawn using MEGA 5.0 (Fig. S4). The G+C content of the
genomic DNA was determined by the HPLC method
Rhodococcus wratislaviensis NCIMB 13082T (Z37138)
70
95
cellular fatty acid composition is shown in Table S1. The
observed chemotaxonomic features support the assignment
of strain MBRL 353T to the genus Rhodococcus.
Rhodococcus imtechensis RKJ300T (AY525785)
Rhodococcus opacus DSM 43205T (X80630)
Rhodococcus percolatus MBS1T (X92114)
Rhodococcus koreensis DNP505T (AF124343)
61 58
0.01
Rhodococcus jostii IFO 16295T (AB046357)
Rhodococcus marinonascens DSM 43752T (X80617)
63
Rhodococcus tukisamuensis Mb8T (AB067734)
89
Rhodococcus maanshanensis M712T (AF416566)
Rhodococcus globerulus DSM 43954T (X80619)
98
Rhodococcus erythropolis DSM 43066T (X79289)
65
62
99
89
Rhodococcus qingshengii djl-6T (DQ090961)
Rhodococcus jialingiae djl-6-2T (DQ185597)
Rhodococcus baikonurensis GTC 1041T (AB071951)
Rhodococcus equi DSM 20307T (X80614)
92
Rhodococcus kunmingensis YIM 45607T (DQ997045)
Rhodococcus canchipurensis MBRL 353 T (JN164649)
56
52
Rhodococcus triatomae IMMIB RIV-085T (AJ854055)
78
100
Rhodococcus corynebacterioides DSM 20151T (AF430066)
Rhodococcus kroppenstedtii K07-23T (AY726605)
92
100
Rhodococcus cercidiphylli YIM 65003T (EU325542)
Rhodococcus fascians DSM 20669T (X79186)
Rhodococcus yunnanensis YIM 70056T (AY602219)
50
Rhodococcus kyotonensis DS472T (AB269261)
Corynebacterium diphtheriae NCTC 11397T (X84248)
Fig. 1. Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences showing the relationships between strain
MBRL 353T and type strains of species of the genus Rhodococcus. Corynebacterium diphtheriae NCTC 11397T was used as
an outgroup. Bootstrap values (.50 %) based on 1000 resamplings are shown at branch nodes. Bar, 1 % sequence
divergence.
116
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Rhodococcus canchipurensis sp. nov.
according to Mesbah et al. (1989). DNA–DNA relatedness
was studied in triplicate by the thermal renaturation
method (De Ley et al., 1970) using a Lambda 35 UV/Vis
spectrophotometer (Perkin Elmer) equipped with a PTP
6+6 Peltier temperature programmer (Perkin Elmer) and
a BG-chiller E15 (Baygene Biotech).
The G+C content of the genomic DNA was 69.2 mol%.
EzTaxon analysis and the phylogenetic tree showed that
strain MBRL 353T formed a distinct lineage most closely
related to R. triatomae IMMIB RIV-085T and R. equi DSM
20307T (Fig. 1) (16S rRNA gene sequence similarities of
98.2 and 97.2 %, respectively). However DNA–DNA
hybridization experiments showed that strain MBRL 353T
displayed low DNA–DNA reassociation values with R.
triatomae DSM 44892T (43.4±6.2 %) and R. equi DSM
20307T (33.4±2.3 %) (mean±standard deviation), thereby
indicating that the whole-genome DNA–DNA relatedness
values with the isolate’s closest phylogenetic neighbours are
well below the delineating 70 % cut-off point for species
identification (Wayne et al., 1987).
The genotypic and phenotypic features described above
suggest that strain MBRL 353T could be clearly distinguished
from its closest phylogenetic relatives. Besides low DNA–
DNA relatedness to its closest phylogenetic neighbours, the
strain was also distinguished by several phenotypic properties: for example, dulcitol was utilized by R. equi DSM
20307T but not by strain MBRL 353T; mannose and xylose
were utilized by strain MBRL 353T but not by R. triatomae
DSM 44892T; and, starch hydrolysis was positive for strain
MBRL 353T but negative for the three reference strains
(Table 1). Therefore, strain MBRL 353T represents a novel
species of the genus Rhodococcus, for which the name
Rhodococcus canchipurensis sp. nov. is proposed.
Description of Rhodococcus canchipurensis
sp. nov.
Rhodococcus canchipurensis (can.chi.pur.en9sis. N.L. masc.
adj. canchipurensis of or belonging to Canchipur, Manipur
University, India, where the MBRL research group which
isolated the type strain is located).
Gram-positive, aerobic, non-spore-forming and nonmotile. Cells are mostly rods in the early growth phase
but occur as cocci in the stationary phase. Growth occurs at
15–37 uC and pH 6–10, with optimum growth at 28 uC
and pH 7. Growth occurs in the presence of up to 5 %
NaCl. Utilizes fructose, D-mannose, sodium malate, Lsorbose, succinic acid and D-xylose as sole carbon sources
and L-arginine, glycine, L-histidine, L-hydroxyproline, DLmethionine, L-phenylalanine, potassium nitrate, L-serine
and L-valine as sole nitrogen sources, but does not utilize
cellobiose, dulcitol, D-galactose, myo-inositol, lactose,
maltose, mannitol, raffinose, rhamnose, trehalose, xylitol,
L-alanine, cysteine, L-ornithine, proline or threonine.
Hydrolyses starch and Tweens 20, 40, 60 and 80, but not
casein or urea. Decomposes adenine, but not guanine,
tyrosine or xanthine. Positive for catalase and Voges–Proskauer
http://ijs.sgmjournals.org
test, but negative for oxidase, methyl red test, citrate
utilization, indole production, nitrate reduction and H2S
production. Contains meso-diaminopimelic acid, arabinose
and galactose in the cell wall. Mycolic acids are present. MK8(H2) is the only menaquinone, while the polar lipids consist
of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol mannoside and one
unknown phospholipid. The fatty acid composition (.1 %)
is C16 : 0, C18 : 1v9c, 10-methyl C18 : 0 (tuberculostearic acid),
summed feature 3 (containing C16 : 1v7c and/or C16 : 1v6c),
C18 : 0, C14 : 0, C17 : 0, C17 : 1v8c, iso-C19 : 0 and summed feature
9 (containing C19 : 1v11c and/or C19 : 1v9c).
The type strain, MBRL 353T (5KCTC 19851T5JCM 17578T),
was isolated from a limestone quarry at Hundung, Manipur,
India. The genomic DNA G+C content of the type strain is
69.2 mol%.
Acknowledgements
The authors are grateful to Professor Hans-Peter Klenk (DSMZ) for
kindly providing the reference type strains and to Foo Cheau Yee and
Ming Hong for their skilful technical assistance. S. N. wishes to thank
the University Grants Commission (UGC), Government of India, for
offering him the Rajiv Gandhi National Fellowship. S. S. would like to
thank Dr S. Mayilraj, IMTECH, Chandigarh, India for hosting her in
his laboratory as a DST-NE Visiting Student where part of the
characterization work on strain MBRL 353T was done. The authors
wish to thank SAIF, NEHU, Shillong for providing the SEM facility.
W.-J.L. was also supported by ‘Hundred Talents Program’ of the
Chinese Academy of Sciences.
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