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: syed_micro@rediffmail.com;

sg.dastager@ncl.res.in; syedmicro@gmail.com

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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