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ORIGINAL PAPER
Bacillus enclensis sp. nov., isolated from sediment sample
Syed G. Dastager • Rahul Mawlankar •
Shan-Kun Tang • Krishnamurthi Srinivasan •
V. Venkata Ramana • Yogesh S. Shouche
Received: 19 September 2013 / Accepted: 24 October 2013 / Published online: 31 October 2013
� Springer Science+Business Media Dordrecht 2013
Abstract A novel bacterial strain, designated SGD-
1123T was isolated from Chorao Island, in Goa
Province, India. The strain was found to be able to
grow at 15–42 �C, pH 5–12 and 0–12 % (w/v) NaCl.
The whole cell hydrolysates were found to contain
meso-diaminopimelic acid, galactose and arabinose.
The major fatty acids were identified as iso-C15:0 and
anteiso-C15:0, MK-7 was identified as the predominant
menaquinone and the predominant polar lipids were
identified as diphosphatidylglycerol, phosphatidyl-
glycerol, phosphatidylethanolamine and an unidenti-
fied aminolipid. The genomic DNA G?C content was
determined to be 44.6 mol%. Phylogenetic analysis
based on 16S rRNA gene sequences placed the isolate
within the genus Bacillus and further revealed that
strain SGD-1123T had highest sequence similarity
with Bacillus aquimaris, and forms a separate clade
with its closest relatives i.e. B. aquimaris, Bacillus
vietnamensis and Bacillus marisflavi, with which it
shares 94.5, 94.1 and 94.1 % similarity respectively.
The phylogenetic, chemotaxonomic and phenotypic
analyses indicated that strain SGD-1123T represents a
novel species within the genus Bacillus, for which the
name Bacillus enclensis is proposed. The type strain is
SGD-1123T (NCIM 5450T=CCTCC AB 2011125T).
Keywords Bacillus sp � Marine sediment �Polyphasic
Introduction
Members of the genus Bacillus and related genera are
ubiquitous in nature. However, Bacillus species iso-
lated from marine sediments have attracted lessElectronic supplementary material The online version ofthis article (doi:10.1007/s10482-013-0066-3) contains supple-mentary material, which is available to authorized users.
S. G. Dastager (&) � R. Mawlankar
NCIM-Resource Center, CSIR-National Chemical
Laboratory, Pune 411008, Maharashtra, India
e-mail: [email protected];
[email protected]; [email protected]
S.-K. Tang
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, Yunnan, People’s Republic of China
K. Srinivasan
Microbial Type Culture Collection and Gene Bank
(MTCC), CSIR-Institute of Microbial Technology,
Sector-39A, Chandigarh 160036, India
V. V. Ramana � Y. S. Shouche
Microbial Culture Collection (MCC), National Centre for
Cell Science, Pune 411007, Maharashtra, India
123
Antonie van Leeuwenhoek (2014) 105:199–206
DOI 10.1007/s10482-013-0066-3
interest compared to their terrestrial relatives. The
genus Bacillus is one of the well-known genera of the
gram-positive low-G?C Firmicutes phylum. It
encompasses rod-shaped bacteria capable of aerobi-
cally forming resistant endospores which contribute to
their ubiquity in the environment. They have been
isolated from terrestrial and freshwater habitats and
are widely distributed in the world’s oceans. The
systematics of the genus has been subjected to many
revisions and it has been based on phenotypical
approaches, mainly, morphological, physiological and
biochemical properties (Smith et al. 1952), spore
shape and sporangium swelling (Gordon et al. 1973),
fatty acid composition and enzyme patterns (Baptist
et al. 1978). Molecular characterisation including
DNA–DNA reassociation (Priest et al. 1981) and DNA
base composition (Fahmy et al. 1985) have been also
applied, which has led to a proliferation of the number
of species within the genus. A major reorganization of
Bacillus taxonomy occurred when Ash et al. (1991)
used 16S small-subunit ribosomal RNA sequences,
allowing the definition of 51 species scattered in 5
distinct phylogenetic groups. Basing on polyphasic
approaches, the other rRNA groups have been subse-
quently redefined as separate Bacillus-derived genera
such as Paenibacillus, Halobacillus, Aneurinibacillus,
Brevibacillus (Shida et al. 1996), Virgibacillus (Hey-
ndrickx et al. 1998), Gracilibacillus and Salibacillus
(Waino et al. 1999). The genus Bacillus currently
consists of more than 222 recognized species (http://
www.bacterio.net/b/bacillus.html) distributed widely
across many terrestrial and aquatic habitats (Ivanova
et al. 1999; Siefert et al. 2000), including marine
sediments (Miranda et al. 2008).
In the present study a novel isolate, strain SGD-
1123T, was characterized as a member of the genus
Bacillus using a polyphasic taxonomic approach.
Materials and methods
Isolation and cultivation of bacterial strain
Strain SGD-1123T, isolated on marine agar (MA)
medium after about 2 weeks incubation at 30 �C,
originates from a sediment sample collected from an
intertidal region of mangroves at Chorao Island in
Goa, India (GPS coordinates 15�3203400N and
73�5501500E). The strain was maintained on MA slants
at 4 �C and as glycerol suspensions (20 %, v/v) at
-80 �C. Biomass for chemical and molecular-sys-
tematic studies was obtained following growth in
shake flasks (about 200 rpm) of Tryptic Soy broth,
supplemented with the vitamin mixtures of the HV
medium (Hayakawa and Nonomura 1987) at 30 �C for
2 weeks.
Bacillus aquimaris JCM 11545T and Bacillus
marisflavi JCM 11544T were obtained from Microbial
Type Culture Collection, CSIR-Institute of Microbial
Technology, Chandigarh, India and cultured under
comparable conditions as reference strains.
Phenotypic characterisation
Morphological features were determined on MA after
48 h at 30 �C. Strain SGD-1123T was observed with
light microscopy (BH2; Olympus). For scanning
electron microscopy examination, 1 ml samples were
fixed overnight at 4 �C by adding formaldehyde to a
final concentration of 7 %. Nine milli litre PBS
(130 mM NaCl, 10 mM sodium phosphate, pH
7 ± 2) was added to the samples, which were then
filtered through 0.2 lm Millipore filters and washed
with PBS. The filters were then serially dehydrated in
25, 50, 70 and 100 % ethanol solutions (three times for
10 min at each stage), critical-point dried, mounted on
scanning electron microscope stubs, sputter-coated
with gold and viewed on a FEI Qunta 200 3D dual
beam scanning electron microscope. Transmission
electron microscopy was used to observe the flagella.
Wet mount preparations were used: bacterial suspen-
sions were settled onto specimen grids, stained with
1 % phosphotungstinate and viewed with a transmis-
sion electron microscope (JEOL 1200 EX). For
biochemical and physiological properties, cultures
were incubated at 30 �C and properties were recorded
for up to 4 days with 24 h intervals. Growth under
anaerobic conditions was determined after incubation
in an anaerobic chamber with MA that had been
prepared anaerobically. Growth at various NaCl
concentrations was investigated on MA or in MA
Broth. Growth at various temperatures was measured
on MA at 4–45 �C, as was NaCl concentrations
(0–15 %) (at intervals of 1 %, w/v), and pH (pH
5.0–12.0) at an intervals of 0.5 pH units using the
following buffer systems: pH 4.0–5.0: 0.1 M citric
acid/0.1 M sodium citrate; pH 6.0–8.0: 0.1 M
200 Antonie van Leeuwenhoek (2014) 105:199–206
123
KH2PO4/0.1 M NaOH; pH 9.0–10.0: 0.1 M NaHCO3/
0.1 M Na2CO3.
The utilization of sugars and acid production from
carbohydrates as carbon sources was determined using
API 50CH kits (bioMerieux) according to the manu-
facturer’s instructions, with API 50CHB as inocula-
tion medium. Fingerprints of enzymic activities were
obtained using API ZYM test strips (bioMerieux)
according to the manufacturer’s instructions. Nitrogen
assimilation was assessed using MA broth. Catalase
activity was determined by production of bubbles after
the addition of a drop of 3 % H2O2. Oxidase activity
was determined using the API oxidase reagent.
Hydrolysis of urea was determined on peptone-
glucose agar (L-1): peptone 1 g, glucose 1 g, NaCl
5 g, KH2PO4 2 g, containing 2 % (w/v) urea and
0.001 % (w/v) phenol red. Hydrolysis of starch was
determined on peptone-beef extract agar containing
0.2 % (w/v) soluble starch by flooding of the plates
with iodine solution. Hydrolysis of casein was tested
on casein agar by observation of clear zones around
the colonies. The incubation period for hydrolysis of
urea, starch and casein was 24–48 h day at 30 �C.
Gelatin hydrolysis was determined by incubation for
one week at 30 �C on peptone–gelatin medium (L-1)
(peptone 5 g, gelatin 120 g). Milk coagulation and
peptonization was determined using 20 % (w/v)
skimmed milk as medium incubation for 48–72 h at
30 �C.
Determination of 16S rRNA gene sequence,
phylogenetic analysis and genomic relatedness
Extraction of genomic DNA, PCR amplification and
sequencing of the 16S rRNA gene from strain SGD-
1123T was performed as described by Li et al. (2007).
The resulting 16S rRNA gene sequence was compared
with available 16S rRNA gene sequences from
GenBank using the BLAST program to determine an
approximate phylogenetic affiliation. Multiple align-
ments with sequences of the most closely related
bacteria and calculations of levels of sequence simi-
larity were carried out using CLUSTAL_X (Thomp-
son et al. 1997). Phylogenetic analyses were
performed using four tree-making algorithms: the
neighbour-joining (Saitou and Nei 1987), maximum-
likelihood (Felsenstein 1981) and maximum-parsi-
mony (Fitch 1971) methods. A phylogenetic tree was
constructed using the neighbour-joining method of
Saitou and Nei (1987) from Knuc values (Kimura
1980) using MEGA version 5.0 (Tamura et al. 2011).
The topology of the phylogenetic tree was evaluated
by the bootstrap resampling method of Felsenstein
(1985) with 1,000 replicates.
Chemotaxonomic characterisation
Sugar analysis of the purified cell walls followed
procedures described by Staneck and Roberts (1974).
Polar lipids were extracted, examined by two-dimen-
sional TLC and identified using previously described
procedures (Minnikin et al. 1984) with spraying the
molybdenum-Blue and drying in an incubator at
100 �C. Reaction sites were outlined with a soft pencil
before executing a second spraying treatment with
ninhydrin and developing at 120–160 �C in an incu-
bator. Menaquinones were isolated according to
Minnikin et al. (1984) and separated by reversed
phase HPLC (Kroppenstedt 1982). For fatty acids
analysis, cells of strain SGD-1123T were cultured on
tryptic soy agar (TSA, Difco) at 30 �C for 48 h.
Cellular fatty acids analysis was performed as
described by Sasser (1990) according to the MIDI
protocols by gas chromatography with flame ioniza-
tion detector (GC–FID) and identified using the
Microbial Identification Software (MIDI Sherlock
aerobe method and TSBA library version Aerobic
Bacteria Library TSBA6/RTSBA6 v6.10; Newark,
DE, USA). For the determination of G?C composi-
tion, genomic DNA was prepared according to the
method of Marmur and Doty (1962). Genomic DNA
was hydrolysed and the resultant nucleotides were
analysed by reversed phase HPLC (Tamaoka and
Komagata 1984).
DNA finger printing by arbitrary primed PCR
(AP-PCR)
To study the genetic relatedness of SGD-1123T with
the two closest type strains, B. aquimaris JCM 11545T
and B. marisflavi JCM 11544T, the strains were
analyzed by AP-PCR. In this method, arbitrarily
selected primers are annealed to template DNA
under low stringency conditions for the initial cycles
of DNA amplifications, which allows interactions
between the primers and target DNA in regions
containing base mismatches. The AP-PCR fingerprint-
ing were performed by using the M13F primer [(-20):
Antonie van Leeuwenhoek (2014) 105:199–206 201
123
GTAAAACGACGGCCAGT] and the following PCR
program: two cycles of 94 �C for 5 min, 40 �C for
5 min, and 72 �C for 5 min; followed by 40 high-
stringency cycles of 94 �C for 1 min, 60 �C for 1 min
and 72 �C for 2 min. Amplified DNA product were
resolved by electrophoresis n agarose 2 % w/v gels.
Table 1 Characteristics that serve to differentiate the novel strain SGD-1123T from recognized Bacillus species
Character Bacillus
SGD-1123TB. aquimaris
JCM 11545TB. vietnamensis
JCM 11124TB. marisflavi
JCM 11544T
Gram staining ? ? (or V) ? V
Flagellum type Single polar Single polar Peritrichous Peritrichous
Spore position Central Sub terminal or central Central Central
Colony colour Pale orange yellow Pale yellow Pale yellow Pale orange-yellow
Growth at 45 �C - ? – –
Optimal pH for growth 7.0–7.5 6.0–8.0 6.5–10.0 6.0–7.0
Growth at pH
4.5 - ? - -
9.0 ? ? ? -
Growth in NaCl at (%, w/v)
0 ? ? ? W
17 - - - ?
Hydrolysis of
Aesculin - ? ? -
Starch ? - ? ?
Acid production from
Aesculin – ? ? –
Arbutin ? ? – –
D-Cellobiose – ? – –
D-Galactose ? W – –
Gentiobiose ? ? – –
Glycerol ? ? ? –
Glycogen ? – ? ?
5-Ketogluconate ? – – ?
D-Mannitol ? ? ? –
D-Mannose ? ? – –
Melibiose – ? – –
Methyl a-D-mannoside ? ? – –
D-Raffinose – W – –
Salicin ? ? – –
Starch ? – ? ?
D-Xylose – ? – –
Major fatty acids iso-C15:0,
anteiso-C15:0
Anteiso-C15:0,
iso-C15:0
Anteiso-C15:0, iso-C15:0
and anteiso-C17:0
iso-C15:0,
anteiso-C15:0
G?C content (mol%) 44.6 49.0 43.0–44.0 38.0
Source of Isolation Marine sediment Tidal flat Fish sauce Tidal flat
SGD-1123T (data from this study); Data for B. aquimaris from Yoon et al. (2003), B. vietnamensis from Noguchi et al. (2004) and B.
marisflavi from Yoon et al. (2003)
? positive, - negative, W weakly positive, V variable
202 Antonie van Leeuwenhoek (2014) 105:199–206
123
Results and discussion
Colonies of strain SGD-1123T were observed to be
pale orange yellow on nutrient agar, tryptone soy agar,
and no aerial mycelia observed. No diffusible pigment
was observed. Strain SGD-1123T was found to have
morphological characteristics typical of the genus
Bacillus, including endospore formation. Cells were
observed to be Gram-positive short rods,
0.3–0.4 9 1.3–4.0 lm, and motile with a single
flagellum (Online Supplementary Fig. 1A & B).
Strain SGD-1123T was found to grow at 15–42 �C
temperature, pH 5.0–12.0 and with 0–12 % (w/v)
NaCl, and optimum growth was observed at 30 �C,
pH 7.0–7.5 and 0–5 % (w/v) NaCl concentration. No
growth observed below 15 �C, pH 5.0 and above
12 % (w/v) NaCl. The detailed physiological and
biochemical characteristics of strain SGD-1123T are
given in the species description and Table 1.
Strain SGD-1123T was found to contain meso-
diaminopimelic acid as the diagnostic amino-acid in
the cell-wall peptidoglycan and ribose, glucose, gal-
actose as cell-wall sugars. The predominant mena-
quinone was identified as unsaturated menaquinone
with seven isoprene units (MK-7). The major polar
lipids detected in strain SGD-1123T were diphosphat-
idylglycerol, phosphatidylglycerol, phosphatidyletha-
nolamine and an unidentified aminolipid
(Supplementary Fig. 2). The genomic DNA G?C
content of strain SGD-1123T was determined to be
44.6 mol%. The major fatty acids detected ([5 %)
were iso-C15:0 (39.0 %), anteiso-C15:0 (30.7 %), C16:0
Brevibacterium frigoritolerans DSM 8801T (AM747813)
Bacillus muralis LMG 20238T (AJ628748)
Bacillus simplex NBRC 15720T (AB363738)
Bacillus purgationiresistens DS22T (FR666703)
Bacillus horneckiae DSM 23495T (FR749913)
Bacillus kochii WCC 4582T (FN995265)
Bacillus gottheilii WCC 4585T (FN995266)
Bacillus infantis SMC 4352-1T (AY904032)
Bacillus firmus NCIMB 9366T (X60616)
Bacillus oceanienclensis H2T (GQ292772)
Bacillus acidicola 105-2T (AF547209)
Bacillus shackletonii LMG 18435T (AJ250318)
Bacillus ginsengihumi Gsoil 114T (AB245378)
Bacillus licheniformis ATCC 14580T (AE017333)
Bacillus aerius 24KT (AJ831843)
Bacillus atrophaeus JCM 9070T (AB021181)
Bacillus subtilis subsp. spizizenii NRRL B-23049T (CP002905)
Bacillus seohaeanensis BH724T (AY667495)
Bacillus coahuilensis M4-4T (ABFU01000135)
Bacillus marisflavi TF-11T (AF483624)
Bacillus aquimaris TF-12T (AF483625)
Bacillus vietnamensis 15-1T (AB099708)
Anaerobacillus alkalilacustris Z-0521T (DQ675454)
100*
98*
100*
90*
100*
88*
84*
76*
90*
86*
97*
70
0.005
Bacillus enclensis SGD-1123T (KF265350)
Fig. 1 Neighbour-joining tree showing the phylogenetic
positions of strains SGD-1123T and representatives of related
taxa based on 16S rRNA sequences. The topology of the entire
tree was conserved in all trees using different algorithms.
Asterisks indicate the branches found in phylogenetic consensus
trees generated with the maximum-parsimony and maximum-
likelihood methods. Numbers at nodes are levels of bootstrap
support from 1,000 resample datasets; values greater than 70 %
are shown at branch-points. Bar 0.005 nucleotide substitutions
per position
Antonie van Leeuwenhoek (2014) 105:199–206 203
123
(8.0 %), iso-C17:0 (6.49 %) and anteiso-C17:0 (5.7 %).
These are the key characteristics of the members of the
genus Bacillus (Claus and Berkeley 1986).
The almost complete 16S rRNA gene sequence
(1,435 bp) of strain SGD-1123T was obtained (Gen-
Bank/EMBL/DDBJ accession number KF265350).
Strain SGD-1123T shares highest sequence similarity
with B. aquimaris JCM 11545T, Bacillus vietnamensis
JCM 11124T and B. marisflavi JCM 11544T with 94.5,
94.1 and 94.1 %, and nucleotide differences of 77, 77
and 83 nucleotides respectively. A phylogenetic tree,
based on 16S rRNA gene sequence data from strain
SGD-1123T and corresponding sequences from the
type strains of members of the genus Bacillus was
constructed using the neighbour-joining algorithm
(Fig. 1). The comparative analysis of 16S rRNA gene
sequences and phylogenetic relationships showed that
strain SGD-1123T lies in a subclade in the tree with B.
aquimaris JCM 11545T and B. marisflavi JCM 11544T
(supported by a bootstrap value of 76 %, Fig. 1), with
which it shares a highest 16S rRNA gene sequence
similarity. The affiliation of strain SGD-1123T and it
closest neighbours was also supported by the maxi-
mum parsimony and maximum-likelihood algorithms
with above 70 % bootstrap values. DNA–DNA
hybridizations (DDH) play a key role in microbial
species discrimination in cases when 16S rRNA gene
sequence similarities are 97 % or higher. Depending
on the investigated taxonomic group, the threshold
value has been increased to between 98.2 and 99.0 %
and it appears reasonable (Stackebrandt and Goebel
1994; Stackebrandt and Ebers 2006; Jan et al. 2013).
The nearest neighbours of strain SGD-1123T are noted
so share less than 97 % 16S rRNA gene sequence
identity and so DNA–DNA hybridization was not
carried out in the present study. Further, AP-PCR
amplicon fingerprint profiles showed marked differ-
ences in the banding patterns between strains SGD-
1123T, B. aquimaris JCM 11545T and B. marisflavi
JCM 11544T (Supplementary Fig. 3), consistent with
the assignment of these strains to separate species.
Conclusion
The phenotypic and chemotypic properties of strain
SGD-1123T, and the 16S rRNA gene sequence compar-
ison results, support a proposal to classify the novel
isolate as a member of the genus Bacillus. The
phenotypic, genotypic and phylogenetic data distinguish
strain SGD-1123T from other validly named members of
the genus Bacillus. Therefore we propose that this isolate
represents a novel species within the genus, for which the
name Bacillus enclensis sp. nov., is proposed.
Description of Bacillus enclensis sp. nov.
Bacillus enclensis (e.ncl.en’ sis. N.L. masc. adj. encl-
ensis arbitrary name formed from NCL, the acronym for
the National Chemical laboratory, India, where taxo-
nomic studies on this species were performed).
Cells are aerobic rods, 0.3–0.4 9 1.3–4.0 lm.
Gram-positive. Motile by means of a single polar
flagellum. Central ellipsoidal endospores are
observed in swollen sporangia. Colonies are smooth,
circular to slightly irregular, slightly raised, pale
orange yellow in colour and 2–3 mm in diameter
after 72 h at 30 �C on marine and nutrient agar.
Optimal growth temperature is 30 �C. Growth occurs
at 15 and 42 �C, but not at below 15 or above 45 �C.
Optimal pH for growth was 7.0–7.5. Growth is
observed at pH 5.0–12.0, but not at pH 4.0. Growth
occurs in the presence of 0–12 % (w/v) NaCl.
Growth does not occur under anaerobic conditions
on marine agar. Catalase-positive. Starch, oxidase-
and urease tests were negative. Aesculin and casein
are hydrolysed. Hypoxanthine, Tween 80, tyrosine
and xanthine are not hydrolysed. Acid is produced
from D-cellobiose, D-fructose, D-galactose, D-glucose,
maltose, D-mannitol, D-mannose, melibiose, D-raffi-
nose, D-ribose, sucrose and D-trehalose. Results using
the API 50CHB system show that acid is produced
from aesculin, arbutin, erythritol, glycerol, methyl a-
D-mannoside, amygdalin, D-arabitol, inulin, N-ace-
tylglucosamine, D-lyxose, D-turanose, and salicin, but
not from D-arabinose, dulcitol, D-fucose, L-fucose,
gentiobiose, gluconate, glycogen, 2-ketogluconate,
5-ketogluconate, methyl a-D-glucopyranoside,
methyl b-D-xylose, sorbose, D-tagatose, xylitol and
L-xylose. The cell-wall peptidoglycan contains meso-
diaminopimelic acid. The predominant menaquinone
is MK-7. The predominant polar lipids are diphos-
phatidylglycerol, phosphatidylglycerol, phosphati-
dylethanolamine and an unidentified aminolipid.
The major fatty acids are iso-C15:0, and anteiso-
C15:0. The G?C content of the type strain is
44.6 mol%.
204 Antonie van Leeuwenhoek (2014) 105:199–206
123
The type strain, SGD-1123T (=NCIM 5450T=
CCTCC AB 2011125T), was isolated from sediment
sample of Chorao Island of Goa Province, India. The
GenBank/EMBL/DDBJ accession number for the 16S
rRNA gene sequence of strain SGD-1123T is
KF265350.
Acknowledgments SGD acknowledges the financial supports
received under the Start up Grant Nos. MLP-027426 from the
CSIR-National Chemical Laboratory, Pune, India.
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