Antonie van Leeuwenhoek (2014) 105:443450

DOI 10.1007/s10482-013-0093-0

ORIGINAL PAPER

Nocardia bhagyanesis sp. nov., a novel actinomycete isolated

from the rhizosphere of Callistemon citrinus (Curtis), India
Radha Vaddavalli Sneha Peddi
Srilekha Yadav Kothagauni
Venkateswar Rao Linga

Received: 1 September 2013 / Accepted: 7 December 2013 / Published online: 24 December 2013
Springer Science+Business Media Dordrecht 2013

Abstract A novel actinomycete strain, designated

VRC07T, was isolated from a Callistemon citrinus
rhizosphere sample collected from Hyderabad, India.
Its taxonomic status was determined by using polyphasic approach. It is a Gram-positive, aerobic, nonmotile, weakly acid-fast strain. Phylogenetic analysis
based on the 16S rRNA gene sequence revealed that
strain VRC07T is a member of the genus Nocardia.
The highest levels of 16S rRNA gene sequence
similarity was found between the strains Nocardia
niwae W9241T (99.6 %), Nocardia amikacinitolerans
W9988T (99.3 %) and Nocardia arthritidis IFM
10035T (98.9 %); similarity to other type strains of
the genus Nocardia was below 98.7 %. The organism
The Genbank accession number for the 16S rRNA gene
sequence of Nocardia bhagyanesis VRC07T is JX076851.

Electronic supplementary material The online version of

this article (doi:10.1007/s10482-013-0093-0) contains supplementary material, which is available to authorized users.
R. Vaddavalli
Department of Botany, Osmania University,
Hyderabad 500007, Andhra Pradesh, India
S. Peddi
Department of Pharmacy, Aditya Pharmaceutical College,
Kakinada 533 437, India
S. Y. Kothagauni  V. R. Linga (&)
Department of Microbiology, Osmania University,
Hyderabad 500007, Andhra Pradesh, India
e-mail: vrlinga@gmail.com

had chemical and morphological features consistent

with its classification in the genus Nocardia such as
meso-diaminopimelic acid as the diagnostic diamino
acid in the cell wall peptidoglycan. Arabinose and
galactose as the diagnostic sugars. Diagnostic polar
lipids were phosphatidylinositol, diphosphatidylglycerol, and phosphatidylglycerol. The predominant
menaquinone was MK-8(H4, x-cycl). The major fatty
acids were C16:0, C18:0, C18:1 w9c, C18:0 10-methyl TBSA
and sum in feature 3 (16:1 w7c/16:1 w6c). The G?C
content of the genomic DNA was 68.5 mol%. The
DNADNA relatedness data, together with phenotypic differences clearly distinguished the isolate from
its closest relatives. On the basis of these phenotypic
and genotypic data, the isolate represents a novel
species, for which the name Nocardia bhagyanesis sp.
nov., is proposed. The type strain is VRC07T (=KCTC
29209T = MTCC 11725T = ATCC BAA-2548).
Keywords Nocardia bhagyanesis sp. nov. 
Rhizosphere soil  Polyphasic approach  16S
rRNA gene

Introduction
The genus Nocardia belongs to the family Nocardiaceae (Goodfellow and Lechevalier 1989). Members
of this genus are found worldwide in the soil, some are
non-pathogenic and some causes nocardiosis (Goodfellow 1998). The genus encompasses aerobic, Gram-

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positive, acid-fast, non-motile organisms which form

extensively branched mycelia and substrate hyphae
that fragment into rod-shaped elements. Members of
this genus are characterized chemotaxonomically by
the presence of meso-diaminopimelic acid in the cell
wall, arabinose and galactose as the characteristic
sugars in the whole-cell hydrolysates with isobranched and antesio-branched-chain fatty acids and
MK-8(H4) as the predominant menaquinone. Phosphatidylinositol, diphosphatidylglycerol, and phosphatidylglycerol containing polar lipids as major
components and mycolic-acid-containing actinomycetes (Goodfellow and Lechevalier 1989). At the time
of manuscript preparation, the genus contains 85
species with validly published names (http://eztaxone.ezbiocloud.net; 15/10/2013) (Kim et al. 2012).
During research work entitled Discovery of novel
antimicrobial agents against Streptococcus pneumoniaA multidisciplinary approach, strain VRC07T
was isolated from rhizosphere of Callistemon citrinus
(Curtis) Skeels, from Hyderabad, India, with the
prospect that it might produce novel antimicrobial
agents. The present study was carried out to determine
the taxonomic status of this strain by using a polyphasic approach. Based on phenotypic and genotypic
evidence, it is proposed that the strain VRC07T represents a novel species of the genus Nocardia, for
which the name Nocardia bhagyanesis sp. nov., is
proposed.

Materials and methods

Isolation and maintenance of organism
Strain VRC07T was isolated from rhizosphere of C.
citrinus (Curtis) Skeels, from botanical garden of
Osmania University, Hyderabad, India (GPS coordinates for the sampling site is 17230 6.737400 N
78290 11.97900 E). Soil samples were collected in
sterile tubes and brought to the laboratory of Osmania
University, India. Samples were dried in laminar flow
under aseptic conditions. By using serial dilution
technique, samples were made up to 10-5 dilutions,
0.1 ml suspension from each dilution was spread on
yeast extract malt extract agar plates (ISP 2) (Shirling
and Gottlieb 1966). The plates were observed intermittently during incubation. The pinpoint colony
which was white in color and having clear zone of

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Antonie van Leeuwenhoek (2014) 105:443450

inhibition was selected and maintained at 4 C on ISP

2 medium, and glycerol suspensions (20 % v/v) at
-20 C. Strain VRC07T was deposited in the Korean
Collection for Type Cultures (KCTC) as strain KCTC
29209T and Microbial Type Culture Collection
(MTCC) as strain MTCC 11725T.
Phenotypic characteristics
Cultural characteristics were determined after incubation for 23 weeks at 30 C on media from the
International Streptomyces project (ISP) (Shirling and
Gottlieb 1966), starch casein, 5 % skim milk, potato
dextrose (PDA), Czapeks and nutrient agars (NA)
(Waksman 1967). The colors of substrate and aerial
mycelia and any soluble pigments produced were
determined according to the ISCC-NBS centroid color
chart (Kelly 1964). The morphological characteristics
of strain VRC07T, including spore-chain morphology
and spore surface ornamentation, were examined
under light microscopy (Olympus microscope BH-2)
and scanning electron microscopy (Hitachi; S-3700N)
using 14 days culture grown on ISP 2 agar (growth at
30 C). Specimens were prepared according to Williams and Davies (1967). A range of physiological
tests such as growth at different temperatures 4, 10, 15,
20, 28, 35, 40 and 45 C, pH tolerance 4.010.0 and
tolerance of NaCl concentrations 3, 5, and 7 % (w/v)
were examined according to Gordon et al. (1974).
Biochemical characteristics such as, acid production
from carbohydrates, the assimilation of sole carbon
and sole nitrogen sources, H2S production and sensitivity of the strain to different antibiotics were
determined by using the previous methods (Gordon
and Mihm 1962; Tsukamura 1966).
Chemosystematics
Biomass for chemosystematics and molecular studies
was obtained from trypticase soy broth (TSB) (HiMedia) which were shaken on a rotary shaker at
150 rpm for 7 days at 30 C. Cells were harvested by
centrifugation, washed twice with distilled water and
re-centrifuged. The resultant preparation was freezedried until further use. Cell wall amino acids and
whole cell sugars were determined by TLC as
described previously (Staneck and Roberts 1974).
Cellular fatty acids were determined by the method of
Sasser (1990), using MIDI Sherlock version 6.0, MIDI

Antonie van Leeuwenhoek (2014) 105:443450

database RTSBA6. Phospholipids were extracted and

analyzed using the procedure of Komagata and Suzuki
(1987). Mycolic acids were tested by the acid methanolysis method of Minnikin et al. (1979, 1980).
Menaquinones were extracted and examined by HPLC
as described previously by Collins et al. (1977). The
G?C content of the DNA was determined by the
method of Mesbah et al. (1989).

445

method (Chung et al. 1999) and a simple fluorimetric method for estimation of DNADNA relatedness
was based on thermal denaturation temperatures
(Gonzalez and Saiz-Jimenez 2005). DNADNA
relatedness was calculated as the mean of triplicate
measurements.

Results and discussion

Molecular analysis
Morphological and biochemical characteristics
Determination of 16S rRNA gene sequence
Genomic DNA was extracted as described previously
by Marmur (1961). The 16S rRNA was amplified
using the bacterial universal primers and sequencing
was performed under contract by Macrogen Inc.
(Geumchen-gu, Seoul 158-781, Korea) using a
3730XL DNA analyzer (Applied Biosystems). The
16S rRNA sequence has been deposited in the
Genbank data library and the assigned accession
number is JX076851.
Phylogenetic analysis and DNADNA hybridization
studies
The 16S rRNA gene sequence was aligned with
closely related sequences belonging to the genus
Nocardia, by using CLUSTAL W (Larkin et al.
2007). Pairwise evolutionary distances were calculated using the DNADIST program with the Kimura
two parameter model as developed by (Kimura
1980). The identification of phylogenetic neighbours
and the calculation of pairwise 16S rRNA gene
sequence identities were achieved by using the
Eztaxon-e database (Kim et al. 2012). The phylogenetic relationship between the isolate and closely
related strains was investigated using the neighborjoining (Saitou and Nei 1987), maximum-parsimony
(Kluge and Farris 1969) and maximum-likelihood
(Felsenstein 1981) algorithms. Phylogenetic trees
were generated using molecular evolutionary genetics analysis (MEGA) software version 5 (Tamura
et al. 2011). The stability of the clades in the trees
was appraised using a bootstrap value with 1,000
repeats (Felsenstein 1985). DNADNA hybridization was performed by using dot-blot hybridization

Strain VRC07T exhibited good growth on ISP 1, ISP 2,

ISP 3, ISP 6, ISP 9, 5 % skim milk agar and nutrient
agar with white color aerial and substrate mycelium.
Good growth was observed on ISP 4 agar with white
color aerial mycelium white to orange color substrate
mycelium and produced a light yellow color pigment.
Sparse growth was observed on ISP 5, Czapeks and
starch casein agars with white color aerial and
substrate mycelium. No growth on PDA. Extensively
branched substrate mycelium was well developed. The
spores were non-motile, smooth surfaced and rod like
elements (Fig. 1). Strain VRC07T growth occurred in
the presence of 05 % (w/v) NaCl (optimum 3 %), at
pH 410 (optimum 7) and at 440 C (optimum
30 C). The morphological and physiological characteristics described above are consistent with those of
the genus Nocardia. It utilizes the majority of sugars
tested for its growth. Cells are resistant to ofloxacin
(1 lg l-1), vancomycin (1 lg l-1), cephalothin
(1 lg l-1), clindamycin (1 lg l-1), erythromycin
(1 lg l-1), kanamycin (25 lg l-1), amphicillin
(10 lg l-1), but susceptible to pencillin G
(1 lg l-1), co-trimoxazole (1 lg l-1) and gentamycin
(1 lg l-1). The detailed physiological and biochemical properties are given in the species description and
Table 1. Strain VRC07T can be distinguished from its
closest relatives by many phenotypic characteristics,
including differences in utilization of sole carbon and
nitrogen sources, degradation activity and different
cultural characteristics on tested media. Strain
VRC07T exhibited positive for chitin and starch
degradation. Grow on L-histidine and L-serine as sole
nitrogen sources, on D-galactose and D-raffinose as
sole carbon sources, but negative for growth at 45 C,
tolerates up to 5 % NaCl (w/v).

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Antonie van Leeuwenhoek (2014) 105:443450

Fig. 1 Scanning electron micrograph of strain VRC07T grown on ISP 2 medium for 14 days at 30 C. Bar 10 lm

Phylogenetic analysis based on 16S rRNA gene

sequence comparison and DNADNA relatedness
An almost complete 16S rRNA gene sequence
(1,465 bp) was determined strain VRC07T. Blast
sequence analysis of the 16S rRNA gene sequence
showed that the strain was affiliated to the genus
Nocardia. The closest phylogenetic relatives were
Nocardia niwae W9241T (99.6 %) (Benjamin et al.
2011), Nocardia amikacinitolerans W9988T (99.3 %)
(Ezeoke et al. 2013) and Nocardia arthritidis IFM
10035T (98.9 %) (Akiko et al. 2004), lower sequence
similarities (\98.8 %) were found with the type
strains of all other members of the genus Nocardia.
It is evident from the phylogenetic tree (Fig. 2) that
strain VRC07T formed a monophyletic clade with
Nocardia niwae DSM 45340T and Nocardia amikacinitolerans W9988T and this phylogenetic relationship was supported by a bootstrap value of 65 %.
The phylogenetic relationship was also found and
supported in trees constructed with other maximumparsimony and maximum-likelihood tree-making
algorithms (supplementary data). It has been
observed that some Nocardia species have high
16S rRNA gene sequence similarities, but have

123

DNADNA relatedness values below the 70 % cutoff point. Although, the strain VRC07T showed
relatively high 16S rRNA gene sequence similarities
between the nearest relatives. The levels of DNA
DNA relatedness with these three strains were
47.4 2.7, 43.3 1.9 and 38 2.3 %, respectively. These values were below the threshold value
of 70 % recommended by Wayne et al. (1987) for
assignment of strains to the same species, suggesting
strongly that the strain VRC07T represents a novel
species of the genus Nocardia.
Chemotaxonomic characteristics
The results of chemical analysis indicated that strain
VRC07T has chemotaxonomic markers characteristic
of the genus Nocardia. The strain contains mesodiaminopimelic acid as the wall diamino acid. Arabinose and galactose were detected as the major
components of sugars in whole-cell hydrolysates.
The predominant menaquinone was MK-8(H4, x-cycl).
The polar lipids were phosphatidylinositol, diphosphatidylglycerol, and phosphatidylglycerol. In the
TLC analysis revealed that the strain contained monoand di-unsaturated mycolic acids with 52, 54 and 56

Antonie van Leeuwenhoek (2014) 105:443450

Table 1 Different
characteristics of strain
VRC07T and closely related
Nocardia type strains

Characteristics

447

Aerial mycelium

White

White

White

Pale orange

Substrate mycelium

White

Orange-coral

Light-orange

Orange

Soluble pigment

None

None

None

None

NaCl tolerance(%,w/v)

05

07

09

05

Temperature range(C)

440

1045

2545

2545

Adenine
Casein

?
?

Chitin

Elastine

Starch

Tyrosine

Xanthine

Hypoxanthine

Reduction of nitrate

L-Arabinose

D-Galactose

D-Lactose

D-Maltose

Colour of

Degradation of

Growth on sole carbon source

D-Raffinose

L-Rhamnose
D-Sucrose

?
?

?
?

?
?

?
-

D-Xylose

L-Aspargine

L-Histidine

L-Lysine

L-Serine

L-Valine

Growth on sole nitrogen source

Strains 1 VRC07T; 2 N.
niwae W9241T; 3 N.
amikacinitolerans W9988T
and 4 N. arthritidis IFM
10035T. All the data are
from this study
? Positive, - negative

L-Glutamic

acid

carbon atoms; this chain length was in the range of the

mycolic acids expected for species of the genus
Nocardia C50C62 (Klatte et al. 1994; Baba et al.
1997). The major cellular fatty acids were C16:0
(31.1 %), C18:0 (8.7 %), C18:1 w9c (4.8 %), C18:0
10-methyl TBSA (13.5 %) and sum in feature 3
(21.1 %) (16:1 w7c/16:1 w6c) (Table 2). This fatty
acid profile was similar to the closest phylogenetic
Nocardia species, although there were differences in
the amount of some components. The DNA G?C
content value of 68.5 mol %, was within the range
reported for the genus Nocardia (Goodfellow and
Maldonado 2006).

Taxonomic conclusion
The results of morphological and chemotaxonomic
investigations and phylogentic analysis supported the
affiliation of strain VRC07T to the genus Nocardia.
However, strain VRC07T could be distinguished from
its closest relatives by many phenotypic characteristics, such as differences in the degradation of chitin,
starch, casein and production of soluble pigment only
on ISP 4 medium, assimilation of sole carbon and
nitrogen sources and the acid production, in tolerance
to NaCl, in the temperature ranges for growth and in
the composition of fatty acids. In conclusion, strain

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Antonie van Leeuwenhoek (2014) 105:443450

54

Nocardia mikamii W8061 (EU484388)

T

Nocardia aobensis IFM 0372 (AB126876)

67

Nocardia veterana DSM 44445 (AY191253)

98

Nocardia africana SD769 (AF277198)

T

Nocardia elegans IMMIB N-402 (AJ854057)

T

Nocardia amamiensis TT 00-78 (AB275164)

T

92

Nocardia pneumoniae IFM 0784 (AB108780)

65

Nocardia bhagyanesis KCTC 29209 (JX076851)

Nocardia niwae W9241 (FJ765056)

T

Nocardia amikacinitolerans W9988 (GU985442)

T

Nocardia beijingensis AS4.1521 (AF154129)

T

Nocardia arthritidis IFM 10035 (AB108781)

T

Nocardia araoensis IFM 0575 (AB108779)

T

Nocardia thailandica IFM 10145 (AB126874)

T

Nocardia asiatica IFM 0245 (AB092566)

T

Nocardia puris DSM 44599 (AJ508748)

T

Nocardia farcinica ATCC 3318 (Z36936)

T

Nocardia higoensis IFM 10084 (AB108778)

59
88

Nocardia shimofusensis IFM 10311 (AB108775)

T

Nocardia blacklockiae ATCC 700035 (EU099360)

T

Nocardia altamirensis OFN S17 (EU006090)

T

Nocardia brasiliensis ATCC 19296 (Z36935)

T

83

Nocardia iowensis UI 122540 (DQ925490)

57

Nocardia tenerifensis GW39-1573 (AJ556157)

Nocardia abscessus IMMIB D-1592T (AF218292)
T

Nocardia paucivorans D-1632 (AF179865)

53

Nocardia cyriacigeorgica DSM 44484 (AF430027)

T

Nocardia takedensis MS1-3 (AB158277)

99

Nocardia exalbida IFM 0803 (AB187522)

T

Nocardia gamkensis CZH20 (DQ235272)

T

Nocardia xishanensis AS 4.1860 (AY333115)

T

Nocardia lijiangensis YIM 33378 (AY779043)

93
100

Nocardia polyresistens YIM 33361 (AY626158)

T

Mycobacterium sphagni DSM 44076 (FR733719)

Fig. 2 Neighbour joining (NJ) phylogenetic tree derived from

aligned 16S rRNA gene sequences, showing the position of
strain VRC07T among members of the genus Nocardia. The tree
is based on a comparison of *1,465 bp. Numbers on branch

nodes are percentage bootstrap support values (1,000 replicates). Mycobacterium sphagni DSM 44076T (FR733719) taken
as an outgroup. Only values [50 % are given. Bar 0.01
nucleotide substitutions per position

VRC07T is considered to represent a novel species of

the genus Nocardia, for which the name Nocardia
bhagyanesis sp. nov. is proposed.

description about the novel species. An aerobic,

Gram-positive, non motile, weakly acid-fast actinomycete that forms extensively branched substrate
mycelium, with white color aerial mycelium. Diffusible pigments are not produced. White spore layer on
broth surface. Colony size is small, 24 mm, powdery,
opaque, elevation is convex to irregular and colony
margins are entire. Grows well on ISP 1, ISP 2, ISP 3,
ISP 6, ISP 9, 5 % skim milk agar and nutrient agar
with white color aerial and substrate mycelium. Good
growth on ISP 4 with white to orange color substrate
mycelium produces light yellow color pigment, sparse

Description of Nocardia bhagyanesis sp. nov.,

Nocardia bhagyanesis sp. nov. (bha.gya.ne.sis. N.L.
fem. N. bhagyanesis of Bhagyanarayana, named in the
honour of Prof. Bhagyanarayana Gaddam, the
renowned Indian mycologist).
The following taxonomical, physiological and
biochemical characteristics gives more detailed

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Antonie van Leeuwenhoek (2014) 105:443450

449

Table 2 Fatty acid profiles of (%) stain VRC07T and closely

related Nocardia type strains
Fatty acid

C10:0

0.52

C11:0

1.09

C12:0

Anteiso-C13:0

Anteiso-C15:0

0.68

C14:0

1.97

C15:0

0.56

1.92

1.10

1.31

2.7

1.0

C16:0

31.13

34.26

32.14

C17:0

1.97

0.92

1.00

0.63

C18:0

8.77

9.79

14.67

13.32

Iso-C17:1 w10c

0.51

1.9

Iso-C15:1 G

Iso-C15:0

Iso-C17:1 w10c

0.54

Iso-C20:0

0.81

C13:1 at 1213

1.62

1.78

2.45

30.62

1.32

0.57

0.65

1.02

1.45
2.53

Cis-9-C16:1

Cis-8-C17:1

1.55

Cis-9-C18:1

12.45

C15:1 w8c

0.63

0.56

C15:1 w5c

0.58

0.69

C17:1 w8c

1.24

1.11

C18:3 w6c (6,9.12)

12.45

0.56
1.56

0.54

1.13

C18:1 w9c

4.87

0.60

14.07

C18:1 w7c11-methyl

1.67

1.14

0.98

0.52

0.78

1.11

8.61

10.81

11.53

C17:0 10-methyl
C18:0 10-methyl

C18:0 10-methyl, TBSA

13.55

2.67

Iso-C19:1I

C16:1 3-OH

C17:1 3-OH

2.36

C16:1 2-OH

1.12

C18:0 2-OH

1.00

1.62

0.87

21.07

6.23

Sum in feature 6a

1.11

Sum in feature 7a

0.72

Sum in feature 8a

3.93

Sum in feature 9a

0.62

Sum in feature 1a
Sum in feature 3

1.23
1.16

1.18

8.36

16.36

1.57

1.35

0.60

1.76

0.58

3.60

0.61

Strains 1 VRC07T, 2 N. niwae W9241T, 3 N. amikacinitolerans

W9988T and 4 N. arthritidis IFM 10035T. All the data are from this
study. Values are percentages of total fatty acids; fatty acids amounting to \0.50 % in all species are not shown; not detected. Sum in
features 1, 3, 6, 7, 8, 9 contains 15:1 iso H and/or 13:0 3OH, 16:1 w7c
and/or 16:1 w6c, 19:1 w11c and/or 19:1 w9c, 19:1w7c and/or 19:1 w6c,
18:1 w7c and/or 18:1 w6c, 16:0 10-methyl and/or 17:1 iso w9c,
respectively
a

Sum in features are groups of two or three fatty acids that cannot be
separated by GC with the MIDI system

growth on ISP 5, Czapeks and starch casein agars. No

growth on potato dextrose agar. Growth occurs at
440 C (optimum 30 C) and pH 410 (optimum pH
7). The NaCl tolerance range is 5 % (w/v). Catalase,
oxidase, urease, lysine, ornithine and methyl red tests
are positive. Acid produced from inositol, sorbitol and
mannitol, but not from arabinose, xylose, adonitol,
rhamnose, glucose and lactose. Nitrate is reduced to
nitrite and H2S gas is not produced. Negative for
phenylalanine deamination. Negative for Voges Proskauers tests. Uses urease, citrate, malonate, inulin,
sodium gluconate, glycerol, salicin, dulcitol, arbitol,
erythritol, L-arabinose, dextrin, fructose, D-galactose,
mannose, maltose, starch, raffinose, D-lactose and
alpha-methyl-D-gluconate as sole carbon sources for
growth, but not indole, cellobiose, melibiose, saccharose and trehalose. Uses L-arginine, L-asparagine, Lhistidine, L-methionine, L-proline and L-tyrosine. Decarboxylations of sodium salts are positive for acetate,
oxalate, succinate and tartarate, negative for propionate and benzoate. Positive results for gelatin liquefaction, milk peptonization. Tweens 20, 80 and
aesculin are hydrolysed. Able to degrade chitin,
casein, starch, urea, xanthine and hypoxanthine.
Unable to degrade adenine, elastin, tyrosine. Growth
is not seen in the presence of inhibitory compounds
such as lysozyme (0.005 %, w/v), potassium tellurite
(0.005 %, w/v), phenol (1.5 %, w/v) and sodium azide
(0.001 %, w/v). The diagnostic diamino acid of the
peptidoglycan is meso-diaminopimelic acid. Whole-cell
sugars are arabinose and galactose. MK-8(H4, x-cycl) as
the predominant menaquinone, diagnostic polar lipids
are phosphatidylinositol, diphosphatidylglycerol and
phosphatidylglycerol. The major cellular fatty acids
are C16:0, C18:0, C18:1 w9c, C18:0 10-methyl TBSA and sum
in feature 3 (16:1 w7c/16:1 w6c). The G?C content of
the genomic DNA of the type strain is 68.5 mol %.
The type strain, VRC07T (=KCTC 29209T = MTCC
11725T = ATCC BAA-2548) was isolated from rhizosphere soil of C. citrinus (Curtis) Skeels, Hyderabad,
India.
Acknowledgments We are grateful to Dr. Ch. Mohan Rao,
Director (Center for Cellular and Molecular Biology,
Hyderabad) for providing facilities to do key experiments,
Dr. R.B.N. Prasad, Head, Lipid Science & Technology (Indian
Institute of Chemical Technology, Hyderabad) for providing
facilities for chemosystematics studies. The authors thank
Professor Jean Paul Marie Euzeby for his help with the
nomenclature.

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