Bacillus encimensis sp. nov. isolated from marine sediment

Abstract

Abstract A Gram-stain-positive, rod-shaped, endospore-forming, aerobic bacterium designated as SGD-V-25T was isolated from Veraval sediment sample, India. Strain SGD-V-25T is capable of growing at 25-50°C (optimum 37°C), pH 6-12 (optimum pH 7.0) and with 0-5% NaCl (w/v). Taxonomic position of this strain was deduced using a polyphasic approach and the 16S rRNA gene sequence analysis showed that the isolate belongs to the division Firmicutes, forming the cluster with B. badius MTCC 1548T, with which it shares highest similarity of 99.1% with 13 nucleotides differences. Other type strains of Bacillus species showed less than 96.0% similarity. The cell wall contained meso-diaminopimelic acid as the diagnostic diamino acid. The polar lipid profile of strain SGD-V-25T showed the presence of diphosphatidylglycerol (DPG), phosphatidyl glycerol (PG), phsophoglycolipid (PGL), and two aminophospholipid. The predominant isoprenoid quinone was MK-7. The major cellular fatty acids were iso-C15:0, anteiso-C15:0, anteiso-C17:0, iso-C16:0, C16:1 ω11c and C16:0. The G+C content of strain SGD-V-25T was 37.6 mol%. On the basis of phenotypic characteristics, phylogenetic analysis and DNA-DNA hybridization, strain SGD-V-25T was clearly distinguished from closely related member of the Bacillus genus, for which the name Bacillus encimensis sp. nov., is proposed. The type strain is SGD-V-25T (=NCIM 5513T=DSM 28241T).

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Bacillus encimensis sp. nov. isolated from marine
sediment
Syed G. Dastager,
1
Rahul Mawlankar,
1
Poonam Mual,
2
Ashish Verma,
2
Srinivasan Krishnamurthi,
2
Neetha Joseph
3
and Yogesh S. Shouche
3
Correspondence
Syed G. Dastager
sg.dastager@ncl.res.in or
syedmicro@gmail.com
1
NCIM-Resource Center, CSIR-National Chemical Laboratory, Pune-411008, Maharashtra, India
2
Microbial Type Culture Collection and Gene Bank (MTCC), CSIR-Institute of Microbial Technology,
Sector-39A, Chandigarh-160036, India
3
Microbial Culture Collection (MCC), National Centre for Cell Science, Pune 411007, Maharashtra,
India
A Gram-stain-positive, rod-shaped, endospore-forming, aerobic bacterium designated SGD-V-25
T
was isolated from Veraval sediment sample, India. Strain SGD-V-25
T
was capable of growing at
25–50 6C (optimum 37 6C), pH 6–12 (optimum pH 7.0) and with 0–5 % (w/v) NaCl. The taxonomic
position of this strain was deduced using a polyphasic approach and the 16S rRNA gene sequence
analysis showed that the isolate belongs to the phylum Firmicutes, forming the cluster with Bacillus
badius MTCC 1548
T
, with which it shares highest similarity of 99.1% with 13 nt differences. Other
type strains of the genus Bacillus showed less than 96 % similarity. The cell wall contained meso-
diaminopimelic acid as the diagnostic diamino acid. The polar lipid profile of strain SGD-V-25
T
showed the presence of diphosphatidylglycerol, phosphatidylglycerol, phsophoglycolipid and two
aminophospholipids. The predominant isoprenoid quinone was MK-7. The major cellular fatty acids
were iso-C
15 : 0
,anteiso-C
15 : 0
,anteiso-C
17 : 0
,iso-C
16 : 0
,C
16 : 1
v11cand C
16 : 0.
The genomic DNA
G+C content of strain SGD-V-25
T
was 37.6 mol%. On the basis of phenotypic characteristics,
phylogenetic analysis and DNA–DNA hybridization, strain SGD-V-25
T
could be clearly distinguished
from closely related members of the genus Bacillus, and the name Bacillus encimensis sp. nov., is
proposed to accommodate this strain. The type strain is SGD-V-25
T
(5NCIM 5513
T
5DSM 28241
T
).
The genus Bacillus in the family Bacillaceae, belonging
to the phylum Firmicutes, includes obligately aerobic or
facultatively anaerobic, endospore-forming bacilli and is
metabolically and ecologically a diverse group. In the genus
Bacillus, several moderately halophilic or halotolerant
endospore-forming species have been described (de la
Haba et al., 2011). Tidal sediments have been utilized as
excellent sources for isolating novel and useful micro-
organisms (Yi et al., 2003; Yoon et al., 2003a). Some novel
genera or species have recently been isolated from tidal
sediments in Korea (Yi et al., 2003; Yoon et al., 2003a, b,
2004a, b). These bacteria are frequently isolated from saline
and hypersaline environments, such as saline soils and
saline aquatic habitats (Arahal & Ventosa, 2002; Ma
´rquez
et al., 2011; Ventosa, 2006; Ventosa et al., 1998). During
the study of the diversity of prokaryotes in coastal sediment,
a bacterial strain, designated SGD-V-25
T
, was isolated from
a sediment sample. The taxonomic position of this strain
was determined using a polyphasic approach that included
phenotypic properties and phylogenetic analysis based on
16S rRNA gene sequences.
Strain SGD-V-25
T
was isolated from a marine sediment sample
taken from Veraval coast (GPS position 20u53951.17˝N
70u22953.00˝ E). After primary isolation and purification on
marine agar 2216 (MA; Difco) at 30 uC for 2 weeks the
purified strain was subcultured on the same medium and
stored as slants at 4 uC and as 20 % (v/v) glycerol
suspensions at 270 uC. Biomass for chemical and molecular
studies was obtained by cultivation in shake flasks (about
140 r.p.m.) using trypticase soy broth (Hi-media, Mumbai)
medium at 30 uC for 48 h. Gram staining was carried out
using the standard Gram reaction, a non-staining method
was used to determine the Gram reactions (Buck, 1982) and
cell motility was confirmed by the development of turbidity
throughout a tube containing semisolid medium (Leifson,
1960). Morphological and physiological characterizations
were performed for strain SGD-V-25
T
. Cells were grown on
MA (pH 7.0) at 30 uC for 48 h. Cell morphology and surface
ornamentation was observed by light microscopy and
scanning electron microscopy. For scanning electron micros-
copy examination, 1 ml samples were fixed overnight at
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene
sequence of SGD-V-25T is KF413433.
Two supplementary figures and one supplementary table are available
with the online Supplementary Material.
International Journal of Systematic and Evolutionary Microbiology (2015), 65, 1421–1425 DOI 10.1099/ijs.0.000114
000114 G2015 IUMS Printed in Great Britain 1421

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4uC by adding formaldehyde to a final concentration of
7 %. Nine millilitres PBS (130 mM NaCl, 10 mM sodium
phosphate, pH 7±0.2) was added to the samples, which
were then filtered through 0.2 mm 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. Oxidase reagent (bioMe
´rieux) was
used for testing oxidase activity and catalase activity was
determined by bubble formation in a 3 % (v/v) H
2
O
2
solution. Semisolid agar (motility test medium; MA) was
used to examine motility (Tittsler & Sandholzer, 1936). In
order to determine the temperature range for growth, cells
were grown in marine broth (MB) at 4, 8, 20, 25, 30, 37, 42
and 50 uC for 72 h. Growth at various NaCl concentrations
(0, 1, 3, 5, 7, 10, 12, and 15 %, w/v, NaCl) was determined at
30 uC in broth medium that contained all of the constituents
of MB, except NaCl, supplemented with appropriate
concentrations of NaCl. The pH range for growth was
determined from pH 4 to 12 (at intervals of 1.0 pH unit)
using the buffer system described by Xu et al., (2005) in MB.
The strain was characterized biochemically using the API
50CH and API ZYM systems (bioMe
´rieux) as recommended
by the manufacturer. Nutrient plates were used to examine
hydrolysis of starch and Tweens 20, 40, 60 and 80 (final
concentration of 1 %, v/v).
Extraction of genomic DNA, PCR amplification and
sequencing of the 16S rRNA gene from strain SGD-V-25
T
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 affili-
ation. Multiple alignments with sequences of the most closely
related species of the genus Bacillus and calculations of levels
of sequence similarity were carried out using CLUSTAL_X
(Thompson et al., 1997). Phylogenetic trees were recon-
structed using the neighbour-joining (Saitou & Nei, 1987),
maximum-parsimony (Fitch, 1971) and maximum-like-
lihood (Felsenstein, 1981) algorithms with Kimura’s two-
parameter calculation model (Kimura, 1980) implemented
in the program MEGA, version 6.0 (Tamura et al., 2013). The
resultant tree topologies were evaluated by bootstrap analysis
based on 1000 replicates (Felsenstein, 1985). Phylogenetic
analysis (Fig. 1) showed that strain SGD-V-25
T
grouped with
members of the genus Bacillus,Bacillus badius MTCC 1458
T
(99.1 % similarity) with 13 nt variation. Very similar tree
topologies were obtained using the other algorithms (Fig. 1).
For the genomic DNA G+C content determination, DNA
was prepared according to the method of Marmur and Doty
(1962). DNA was hydrolysed and the resultant nucleotides
were analysed by reversed phase HPLC (Mesbah et al.,
1989). For DNA–DNA hybridization experiments, genomic
DNA was extracted and purified according to the method of
Marmur (1961). Hybridization was carried out based on the
principles and equations described by De Ley et al. (1970)
under the consideration of modifications carried out by
Huss et al. (1983), and the optimized fluorimetric procedure
of Loveland-Curtze et al., (2011) was evaluated by using a
Step One Plus Real-Time PCR system (Applied Biosystems)
fitted with 96-well thermal cycling blocks. DNA suspended
in the 26SSC was used for the analysis in three
independent samples. The reassociation was carried out at
an optimum renaturation temperature of 75 uC (Gillis et al.,
1970; Marmur & Doty, 1962). The DNA–DNA relatedness
value determined between strain SGD-V-25
T
and B. badius
MTCC 1458
T
, which is the only strain that shared greater
than 99 % similarity, was 54.5 % (3.0±2.0) (readings are the
mean of triplicates; Table S1, available in the online
Supplementary Material), 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). This suggests that strain SGD-V-25
T
should be considered a different genomic species of the
genus Bacillus. The genomic DNA G+C content of strain
SGD-V-25
T
was 37.6 mol%, which was similar to those of
related reference species of the genus Bacillus.
Whole-cell sugars were analysed according to the procedures
developed by Hasegawa et al. (1983). Polar lipids were
extracted, examined by using two-dimensional TLC and
identified using standard procedures (Minnikin et al., 1984).
Polar lipids were separated by using two-dimensional TLC
(silica-gel plate 60; Merck). The first direction was developed
in chloroform/methanol/water (65 : 25 : 4, by vol.) and the
second was developed in chloroform/methanol/acetic acid/
water (80 : 12 : 15 : 4, by vol.). Total lipid material and
specific functional groups were detected by using Dittmer
and Lester reagent (phosphate), ninhydrin (free amino
groups), Dragendorff reagent (quaternary nitrogen) and
anisaldehyde-sulfuric acid (glycolipids). Menaquinones
were isolated according to Minnikin et al., (1984) and were
separated by using HPLC (Kroppenstedt, 1982). For analysis
of fatty acids, strain SGD-V-25
T
was cultured on trypticase
soy agar (Difco) at 28 uC for 72 h. Preparation and analysis
of fatty acid methyl esters were performed as described by
Sasser (1990) by using the Microbial Identification System
(MIDI) and the Microbial Identification software package
(Sherlock version 6.1; MIDI database, TSBA6).
The colonies of strain SGD-V-25
T
were aerobic, Gram-
stain positive, motile rods with cell size of 0.4–0.761.1–
2.0 mm (Fig. S1), and showed terminal round spores. Strain
SGD-V-25
T
grew well on MA and nutrient agar (NA).
Strain SGD-V-25
T
formed opaque, cream, circular colonies
with entire margins after incubation on NA (pH 7.0) at
37 uC for 48 h. Strain SGD-V-25
T
was catalase-positive,
but oxidase-negative. It grew at temperatures between 25
and 50 uC (optimum, 37 uC), at pH 6.0–12.0 (optimum,
pH 7.0) and in the presence of 0–5 % (w/v) NaCl. NaCl
was not required for growth. Strain SGD-V-25
T
hydrolysed
starch, but not casein, CM-cellulose, urea, or Tweens 20,
40, 60 or 80. A detailed species description is presented
below. A phenotypic comparison of strain SGD-V-25
T
and
S. G. Dastager and others
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related species of the genus Bacillus is presented in Table 1
and in the species description. It is evident from Table 1
that there were phenotypic differences between strains
SGD-V-25
T
and B. badius MTCC 1458
T
. Strain SGD-V-25
T
showed positive reactions for catalase and amylase (starch)
but was negative for oxidase and nitrate was reduced.
The nearly complete 16S rRNA gene sequence of strain
SGD-V-25
T
(1522 bp) was determined and compared with
Bacillus drentensis LMG 21831T (AJ542506)
Bacillus circulans ATCC 4513T (AJ542508)
Bacillus bataviensis LMG 21833T (FN995266)
Bacillus isabeliae CVS-8T (AY724690)
Bacillus canaveralius KSC SF8bT (DQ870688)
Bacillus lentus IAM 12466T (D16272)
Bacillus sporothermodurans M215T (U49079)
Bacillus shackletonii LMG 18435T (AJ250318)
Bacillus niacini IFO 15566T (AM503357)
Domibacillus robiginosus WS 4628T (HE577175)
Bacillus encimensis SGD-V-25T (KF413433)
Bacillus badius ATCC 14574T (X77790)
Bacillus thermophiles SgZ-9T (JX274437)
Paenibacillus polymyxa IAM 13419T (D16276)
100*
100*
51
99*
90*
79*
91*
69
91*
0.01 Fig. 1. Neighbour-joining tree based on nearly
complete 16S rRNA gene sequences, show-
ing the phylogenetic position of strain SGD-V-
25
T
and type strains of related species. The
sequence of Paenibacillus polymyxa IAM
13419
T
(D16276) was used as an out-group.
Only bootstrap values .50 % (expressed as
percentages of 1000 replications) are shown
at branch points. Asterisks indicate that the
corresponding nodes (groupings) are also
recovered in maximum-parsimony and max-
imum-likelihood trees. Bar, 0.01 substitutions
per nucleotide position.
Table 1. Characteristics that serve to differentiate the novel strain SGD-V-25
T
from B. badius MTCC 1458
T
All data from present study otherwise indicated. Both strains have subterminal or terminal spore positions and both strains grow at 50 uC. Both
strains produced acid from gentiobiose. Both strains were positive for lysine and ornithine decarboxylase, citrate, maltose, fructose, glucose,
raffinose, trehalose, melibiose, sucrose, DL-arabinose, sodium-gluconate, glycerol, inositol, sorbitol, mannitol, adonitol, arabitol, erythritol,
rhamnose, xylitol and naphthol-AS-BI-phosphohydrolase. Negative for b-galactosidase, phenyl deamination, methyl red, indole production, lipase
(C14), cystine arylamidase, trypsin, a-chromotrypsin, a-galactosidase, b-galactosidase, a-glucosidase, b-glucosidase, N-acetyl-b-glucosaminidase,
a-mannosidase, a-fucosidase and oxidase activity. +, Positive; 2, negative; W, weakly positive.
Characteristic SGD-V-25
T
B. badius MTCC 1458
T
Sporangium type Spherical Ellipsoidal, swollen
Colony colour Light yellowish cream Cream
Optimal pH for growth 7.0–7.5 6.0–8.0
Growth at pH:
6.0 +2
12.0 +2
Maximum NaCl tolerance at 5% (w/v) +2
Hydrolysis of:
Aesculin +2
Starch +2
Urea +W
Nitrate reduction +2
H
2
S production 2+
Utilized as sole carbon source
Galactose, lactose, salicin, dulicitol, CM-cellobiose, melezitose,
sorbose, methyl a-D-glucoside and methyl a-D-mannoside
+2
Glucose 2+
Acid production from:
L-Arabinose, glucose, inositol, D-mannitol, melibiose, sodium
gluconate, sorbitol, sorbose, sucrose
2+
Glycerol, salicin +2
Production of (API ZYM test):
Alkaline phosphatase, esterase (C4), esterase lipase (C8) +2
Leucine arylamidase, valine arylamidase W2
Acid phosphatase 2+
DNA G+C content (mol%) 37.6 43.8
Source of isolation Marine sediment Tidal flat
Bacillus encimensis sp. nov.
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the corresponding sequences of other bacterial strains in
the GenBank database. Phylogenetic analysis based on 16S
rRNA gene sequences revealed that strain SGD-V-25
T
should be assigned to the genus Bacillus. The phylogenetic
tree (based on 16S rRNA gene sequence data from strain
SGD-V-25
T
and corresponding sequences from the type
strains of the genus Bacillus) was reconstructed according
to the neighbour-joining algorithm (Fig. 1). Comparative
analysis of 16S rRNA gene sequences and phylogenetic
relationships showed that strain SGD-V-25
T
lies in a
subclade in the tree with B. badius MTCC 1458
T
(supported
by a bootstrap value of 100 %, Fig. 1), with which it shares a
16S rRNA gene sequence similarity of 99.1 %. The affiliation
of strain SGD-V-25
T
and it closest neighbour, B. badius
MTCC 1458
T
, was also supported by the maximum-
parsimony and maximum-likelihood algorithms with high
bootstrap values (PHYLIP version 3.6), thus suggesting that
strain SGD-V-25
T
should be considered as a novel member
of the genus Bacillus. The genomic DNA G+C content of
strain SGD-V-25
T
was 37.6 mol%, which was similar to
those of related reference species of the genus Bacillus.
Chemotaxonomically, strain SGD-V-25
T
contained meso-
diaminopimelic acid as the diagnostic diamino acid and an
A1ctype of peptidoglycan. Ribose and glucose were major
cell-wall sugars, while galactose was present in minor
quantities. The polar lipid profile contained diphosphatidyl-
glycerol, phosphatidylglycerol, phsophoglycolipid and two
aminophospholipids (Fig. S2). MK-7 (92.3 %) was the major
menaquinone and MK-6 and MK-8 were minor menaqui-
nones detected. The fatty acids (¢5 %) found for strain SGD-
V-25
T
were iso-C
15 : 0
(32.1 %), anteiso-C
15 : 0
(16.0 %),
anteiso-C
17 : 0
(8.9 %), iso-C
16 : 0
(6.4 %), C
16 : 1
v11c(5.5 %),
C
16 : 0
(4.9 %) and C
16 : 1
v6c/v7c(5.6 %). The fatty acid
methyl ester profiles of strain SGD-V-25
T
and the closest type
strain, B. badius MTCC 1458
T
, showed high levels of similarity
in major fattyacids, some per cent variation was also detected
(Table 2). The chemotaxonomic features observed support
the assignment of strain SGD-V-25
T
to the genus Bacillus.
The phenotypic and chemotypic properties of strain SGD-
V-25
T
, the 16S rRNA gene sequence comparison results
and DNA–DNA hybridization results support the proposal
of a novel species in the genus Bacillus. The phenotypic,
genotypic and phylogenetic data distinguish strain SGD-V-
25
T
from other members of the genus Bacillus with validly
published names. Therefore, we propose that this isolate
represents a novel species within the genus, for which the
name Bacillus encimensis sp. nov., is proposed.
Description of Bacillus encimensis sp. nov.
Bacillus encimensis (en.cim9en.sis. N.L. masc. adj. encimen-
sis arbitrary name formed from NCIM, the acronym for the
NCIM Resource Center, CSIR-National Chemical labor-
atory, India, where taxonomic studies on this species were
performed).
Cells are Gram-stain-positive, aerobic, motile rods, 0.4–
0.761.1–2.0 mm in size. Grows well on MA and NA. Forms
opaque, cream coloured, circular colonies with entire margins
after incubation on NA (pH 7.0) at 37 uC for 48–72 h.
Catalase-positive, but oxidase-negative. It grows between 25
and 50 uC(optimumat37uC) and in the presence of 0–5 %
(w/v) NaCl. NaCl is not required for growth. Hydrolyses
starch, casein and urea but not CM-cellulose, or Tweens 20,
40, 60 or 80. Nitrate is reduced. Negative for b-galactosidase,
H
2
S production, phenyl deamination, methyl red, Voges–
Proskauer test, indole production, malonate and glucose.
Positive for utilization of DL-arabinose, adonitol, arabitol,
glucose, dulicitol, erythritol, fructose, galactose, glycerol,
inositol, lactose, mannitol, maltose, melibiose, raffinose,
rhamnose, salicin, sucrose, sorbitol and xylitol, ornithine
decarboxylase and lysine decarboxylase. Acid is produced
from glycerol, gentiobiose and salicin. Acid is not produced
from other carbon sources tested. In the API ZYM substrate
utilization kit, positive for alkaline phosphatase, esterase (C4),
esterase lipase (C8) and naphthol-AS-BI-phosphohydrolase;
weakly positive for leucine arylamidase and valine arylami-
dase. Negative for the following substrates: lipase (C14),
cystine arylamidase, trypsin, a-chromotrypsin, a-galactosi-
dase, b-galactosidase, a-glucosidase, b-glucosidase, N-acetyl-
b-glucosaminidase, a-mannosidase and a-fucosidase. The
major fatty acids detected are iso-C
15 : 0
,anteiso-C
15 : 0
,
anteiso-C
17 : 0
,iso-C
16 : 0,
C
16 : 1
v11cand C
16 : 1
v7c/v6c.
The predominant quinones are MK-7 as the major and MK-
6 and MK-8 as the minor menaquinone. The polar lipid
profile contains diphosphatidylglycerol, phosphatidyl gly-
cerol, phsophoglycolipid and two aminophospholipids.
Table 2. Fatty acid composition of SGD-V-25
T
compared with
B. badius MTCC-1458
T
All data was from the present study.
Fatty acid Profile (%)
SGD-V-25
T
B. badius MTCC 1458
T
iso-C
14 : 0
1.9 2.0
C
14 : 0
3.1 2.5
iso-C
15 : 0
32.1 42.6
anteiso-C
15 : 0
16.0 7.0
C
16 : 1
v7calcohol 3.6 2.6
iso-C
16 : 0
6.4 8.6
C
16 : 1
v11c5.5 5.9
C
16 : 0
4.9 9.0
iso-C
17 : 1
v10c3.8 4.3
anteiso-C
17 : 1
A 0.9 –
iso-C
17 : 0
2.2 4.1
anteiso-C
17 : 0
8.9 4.6
C
17 : 0
– 0.4
C
18 : 1
v9c– 0.2
C
18 : 0
– 0.5
Summed feature 3* 5.6 2.8
Summed feature 4* 4.5 2.1
*Summed feature 3 contained C
16 : 1
v7c/v6cand summed feature 4
contained iso-C
17 : 1
I/anteiso-C
17 : 1
B.
S. G. Dastager and others
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meso-Diaminopimelic acid (type A1c) is present in the cell-
wall peptidoglycan and the major whole-cell sugars are
glucose and ribose.
The type strain, SGD-V-25
T
(5NCIM 5513
T
5DSM 28241
T
),
isolated from a marine sediment sample in Veraval coast,
Gujarat Province of India. The genomic DNA G+Ccontent
of the type strain is 37.6 mol%.
Acknowledgements
Author S. G. D. thanks the Council for Scientific and Industrial
Research (CSIR), New Delhi for financial supported from the grants
under 12th five year plan. S. G. D acknowledges the financial support
from the CSIR-National Chemical Laboratory, Pune, India (grant no.
MLP-027426).
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