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Bacillus paralicheniformis sp. Nov., isolated from fermented soybean paste

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International Journal of Systematic and Evolutionary Microbiology (2015), 65, 3487–3492 DOI 10.1099/ijsem.0.000441

Bacillus paralicheniformis sp. nov., isolated from

fermented soybean paste
Christopher A. Dunlap,1 Soon-Wo Kwon,2 Alejandro P. Rooney1 and
Soo-Jin Kim1,2
1
Correspondence Crop Bioprotection Research Unit, National Center for Agricultural Utilization Research,
Soo-Jin Kim Agricultural Research Service, United States Department of Agriculture, Peoria, IL, USA
sinhye@korea.kr 2
Korean Agriculture Culture Collection (KACC), Agricultural Microbiology Division, National
Academy of Agricultural Science, Rural Development Administration, Wanju-gun, Jeollabuk-do,
Republic of Korea

An isolate of a Gram-stain-positive, facultatively anaerobic, motile, rod-shaped, endospore-

forming bacterium was recovered from soybean-based fermented paste. Phylogenetic analysis
of the 16S rRNA gene indicated that the strain was most closely related to Bacillus sonorensis
KCTC-13918T (99.5 % similarity) and Bacillus licheniformis DSM 13T (99.4 %). In phenotypic
characterization, the novel strain was found to grow at 15–60 8C and to tolerate up to 10 %
(w/v) NaCl. Furthermore, the strain grew in media with pH 6–11 (optimal growth at
pH 7.0–8.0). The predominant cellular fatty acids were anteiso-C15 : 0 (37.7 %) and iso-C15 : 0
(31.5 %). The predominant isoprenoid quinone was menaquinone 7 (MK-7). The cell-wall
peptidoglycan contained meso-diaminopimelic acid. A draft genome sequence of the strain was
completed and used for phylogenetic analysis. Phylogenomic analysis of all published genomes
of species in the B. licheniformis group revealed that strains belonging to B. licheniformis
clustered into two distinct groups, with group 1 consisting of B. licheniformis DSM 13T and 11
other strains and group 2 consisting of KJ-16T and four other strains. The DNA G+C content
of strain KJ-16T was 45.9 % (determined from the genome sequence). Strain KJ-16T and
another strain from group 2 were subsequently characterized using a polyphasic taxonomic
approach and compared with strains from group 1 and another closely related species of the
genus Bacillus. Based upon the consensus of phylogenetic and phenotypic analyses, we
conclude that this strain represents a novel species within the genus Bacillus, for which the
name Bacillus paralicheniformis sp. nov. is proposed, with type strain KJ-16T (5KACC
18426T5NRRL B-65293T).

Cheonggukjang is a fermented soybean product in tra-
ditional Korean cuisine. It is produced by boiling soybeans
and allowing them to ferment for 2–3 days in the presence
of a ‘natural’ environmental inoculum present in the air or
sometimes added through the addition of rice straw (Cho
et al., 2014). The bacterial diversity of the cheonggukjang
inoculum is known to be dominated by members of the
genus Bacillus (Nam et al., 2012), some of which have
been identified previously (e.g. Bacillus subtilis), whereas
others have yet to be formally described. The purpose of
Abbreviation: DDH, DNA–DNA hybridization.
The GenBank/EMBL/DDBJ accession number for the genome
sequence of strain KJ-16T is LBMN01.
Two supplementary figures and two supplementary tables are available
with the online Supplementary Material.
this study was to describe one such species, for which we
propose the name Bacillus paralicheniformis sp. nov.
Strain KJ-16T was isolated from a 3-day-old cheonggukjang
fermentation on R2A agar at 28 8C. Growth was tested at 4,
10, 15, 28, 37, 45, 55 and 60 8C. The pH range for growth
was determined from pH 3.0 to 12.0 in steps of 1.0 pH unit
in R2A broth buffered with 0.1 M citrate/phosphate buffer
(pH 3–7), Tris (pH 8, 9), NaCO3 (pH 10, 11) or phosphate
(pH 12) and adjusted with HCl or NaOH as needed (Breznak
& Costilow, 1994). NaCl tolerance was investigated by using
R2A broth supplemented with 0, 1, 2, 3, 4, 5, 7 and 10 %
(w/v) NaCl. Growth under anaerobic conditions was deter-
mined on anaerobic agar (Difco) at 30 8C using a GasPak
jar (,1 % O2; ¢13 % CO2) (Merck) for 7 days (De Vos
et al., 2009). Gram staining was performed using the Gram
stain kit (Remel), according to the manufacturer’s instruc-
tions. Carbon source utilization was tested using the OmniLog

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C. A. Dunlap and others

Data Collection system (Biolog). Strains selected for character-
ization were cultured overnight on Biolog universal growth
plates and prepared according to manufacturer’s instructions
for the GEN III MicroPlate test panel and for phenotype
array plates PM1 and PM2a (Biolog). Catalase and oxidase
activities were examined using 3 % (v/v) hydrogen peroxide
solution and 1 % (w/v) tetramethyl p-phenylenediamine dihy-
drochloride (Difco), respectively. Urease activity was
measured using Stuart’s urease broth (Stuart et al., 1945).
Other biochemical tests were carried out on API 20NE, API
ID 32GN and API ZYM strips (bioMérieux) according to the
manufacturer’s instructions. API 20NE and API ID 32GN
tests were performed and results were recorded after incu-
bation of 7 days at 28 8C, and API ZYM test results were
checked after 4 h at 37 8C.
Polar lipids were determined by two-dimensional silica gel
TLC using the method of Tindall (1990). Determination of
cell-wall meso-diaminopimelic acid was performed according
to the method of Staneck & Roberts (1974) but modified to
use 200 mg (wet wt) bacterial cells (incubated at 28 8C for
2 days on R2A plates). Total cellular fatty acid content was
measured using the MIDI protocol (Microbial Identification
Inc.) and analysed on an Agilent 7890 gas chromatograph.
In addition to KJ-16T, strain ATCC 12759 was included as a
reference strain of B. paralicheniformis for phenotype testing.
In addition to B. licheniformis ATCC 14580T, strain KJ-10
was included as a reference strain of B. licheniformis. Strain
KJ-10 was isolated under the same conditions as KJ-16T and
identified through 16S rRNA gene sequencing. Strains were
grown for 24 h at 28 8C on TSA and prepared using the stan-
dard MIDI protocol for extraction and production of fatty
acid methyl esters.
The genome of strain KJ-16T was sequenced on an Ion Torrent
Personal Genome Machine using an Ion PGM 400 Hi-Q
sequencing kit (Life Technologies) following the manufac-
turer’s suggested protocols. The resulting reads were qual-
ity-trimmed to the Q20 confidence level. The draft genome
was assembled using CLCbio Genomics Workbench 7.1
(Qiagen) using default parameters. Genome comparisons
and alignments for phylogenetic trees were made using
BIGSdb software (Jolley & Maiden, 2010). Bacillus aerius (Shi-
vaji et al., 2006) could not be included in the comparisons,
because its type strain is no longer available from a culture col-
lection or from the original authors (Dunlap, 2015; Lai et al.,
2014). The phylogenetic tree was reconstructed using MEGA
6.06 software (Tamura et al., 2013). Neighbour-joining trees
were reconstructed using the Tamura–Nei model (Tamura
& Nei, 1993) with a gamma correction (alpha value50.5);
this model was chosen on the basis of the likelihood test
implemented in MEGA 6.06. A dataset of 799 genes identified
as the core genome of type strains of the B. subtilis species
complex was found using BIGSdb software (Jolley &
Maiden, 2010). Digital DNA–DNA hybridizations (DDH)
were determined online (http://ggdc.dsmz.de/distcalc2.php)
using formula 2 of the Genome-to-Genome Distance Calcu-
lation (GGDC ) version 2.0 as described by Meier-Kolthoff
et al. (2013). Formula 2 of the GGDC is the only function
appropriate to analyse draft genomes, which is the case for
most of the genomes in this study (Meier-Kolthoff et al.,
2013). The genomes were screened for phages using PHAST
software (Zhou et al., 2011).
Cells of strain KJ-16T were Gram-stain-positive, faculta-
tively anaerobic, motile, endospore-forming rods, 0.63–
0.71 mm in diameter and 1.90–3.94 mm long when cultured
on R2A agar. Central or subterminal ellipsoidal endospores
were observed with swollen sporangia (Fig. S1, available in
the online Supplementary Material). Colonies formed on
R2A agar were creamy white, mucoid, translucent and
raised, and were 3–4 mm in diameter after 2 days of incu-
bation at 37 8C on R2A agar. The strain was able to grow
on nutrient agar, LB agar, R2A agar and TSA, but not
on MacConkey agar. The strain grew at pH 6–11 and at
15–60 8C. It tolerated 0–10 % (w/v) NaCl. The optimum
temperature and pH were 37 8C and pH 7.0.

Table 1. Fatty acid profiles of strains of the B. licheniformis group

Values are percentages of total fatty acids; only fatty acids making up more than 0.4 % of total are shown. Data were obtained in this study.

B. paralicheniformis sp. nov. B. licheniformis B. sonorensis

Fatty acid KJ-16T ATCC 12759 ATCC 14580T KJ-10 NRRL B-23154T

iso-C14 : 0 0.6 0.7 0.9 1.0 0.8

C14 : 0 0.6 0.7 0.6 0.6 0.7
iso-C15 : 0 31.5 30.6 29.2 33.0 36.2
anteiso-C15 : 0 37.7 38.6 39.7 37.4 38.9
iso-C16 : 0 2.7 3.2 3.6 3.9 2.4
C16 : 1v11c 0.6 0.7 0.6 1.0 0.6
C16 : 0 5.3 4.8 3.5 3.1 3.3
iso-C17 : 1v10c 0.4 0.5 0.5 0.8 0.4
iso-C17 : 0 8.9 8.6 8.2 7.5 7.1
anteiso-C17 : 0 11.3 11.4 12.5 11.1 8.9

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 Bacillus paralicheniformis sp. nov.

The predominant respiratory quinone was menaquinone
with seven isoprene units (MK-7). The cell-wall peptidogly-
can contained meso-diaminopimelic acid. The major
polar lipids were diphosphatidylglycerol, phosphatidyl-
glycerol, phosphatidylethanolamine, a glycolipid, three
unknown aminolipids and two unknown lipids (Fig. S2).
The glycolipid appears to be b-gentiobiosyldiacylglycerol,
based on similar chromatographic properties seen in other
strains of the B. subtilis group (Fischer et al., 1978; Kämpfer
et al., 2006). The total cellular fatty acid profile showed a
large amount of branched fatty acids; the major components
(.5.0 %) were anteiso-C15 : 0 (37.7 %), iso-C15 : 0 (31.5 %),
anteiso-C17 : 0 (11.3 %), iso-C17 : 0 (8.9 %) and C16 : 0
(5.3 %) (Table 1). The profiles of strains of the close relatives
Bacillus licheniformis and B. sonorensis were consistent with
previously reported profiles (Palmisano et al., 2001; Sumpa-
vapol et al., 2010). The novel strain could not be
distinguished from B. licheniformis or B. sonorensis on the
basis of the fatty acid profile.
A phylogenomic analysis of KJ-16T and all published
genomes of species in the B. licheniformis group revealed
that strains belonging to B. licheniformis clustered
into two distinct groups, with group 1 consisting of
B. licheniformis DSM 13T and 11 other strains, and group
2 consisting of KJ-16T and four other strains (Fig. 1).

100 CGMCC 3963 (AMWQ01)

3F-3 (JFYM01)
100
CG-B52 (AVEZ01)
5NAP23 (JYBQ01)
0.05 100 DSM 13T (CP000002) Group 1-
B. licheniformis
GB2 (JYGX01)

100 F1-1 (AZSL01)

100 5-2-D (AJLW01)
100 10-1-A (AJLV01)

F2-1 (AZSM01)

100 WX-02 (AHIF01)

T
100 KJ-16 (LBMN01)
Group 2-
ATCC 9945A (CP005965)
B. paralicheniformis
100 100 ATCC 12759 (JMPZ01)
100 BL-09 (CP010524)
100 G-1 (AZSK01)

B. sonorensis KCTC 13918T (AYTN01)

81 B. siamensis KCTC 13613T (AJVF01)

100 B. methylotrophicus KACC 13105T (JTKJ01)
B. amyloliquefaciens DSM 7T (FN597644)
100 B. atrophaeus 1942 (CP002207)
B. mojavensis KCTC 3706T (AFSI01)
100
B. tequilensis KCTC 13622T (AYTO01)
100
B. vallismortis DV1-F-3T (AFSH01)
100
87 B. subtilis subsp. inaquosorum KCTC 13429T (AMXN01)
100 B. subtilis subsp. spizizenii TU-B-10T (CP002905)
B. altitudinis 41KF2bT (ASJC01)

100 B. safenesis F0-36bT (ASJD01)

100 B. pumilus SAFR-032T (cp000813)

Fig. 1. Phylogenetic tree reconstructed from the core genomes of type strains of species from the B. subtilis group (799
genes). Bootstrap values .50 %, based on 1500 pseudoreplicates, are indicated on branch points. Bar, 0.05 nucleotide
substitutions per site.
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C. A. Dunlap and others

This was consistent with other recent genotyping studies of
B. licheniformis, which have shown that it is split into at
least two distinct groups (Dhakal et al., 2013, 2014; Madslien
et al., 2012). All the available genome data for group 1 and
group 2 strains were compared using in silico DDH estimates
(Table 2). The DDH varied from 91.4 to 99.4 % for intragroup
comparisons and 57.3 to 59.0 % for intergroup comparisons,
well below the threshold of 70 % for species delineation
(Wayne et al., 1987).
The genomes of group 1 strains were generally smaller
(4.26¡0.11 vs 4.40¡0.03 Mbp) and more G+C-rich
(46.02¡0.24 vs 45.88¡0.04 %) relative to the genomes
of group 2 strains. In addition, comparisons of the core
genomes of group 1 and group 2 strains revealed many
further differences. The core genomes contained 92 and
179 unique genes for group 1 and group 2, respectively
(Tables S1 and S2). Of interest is a 14-gene cluster
unique to group 1 strains that encodes lichenicidin. Liche-
nicidin is a lantibiotic that has broad antibacterial activity
against Gram-positive organisms (Dischinger et al.,
2009). A recent study of isolates of B. licheniformis from
powdered infant formula found that the group could be
partitioned into lichenicidin producers and non-producers
that had greater antibacterial activity (Alvarez-Ordóñez
et al., 2014). Group 2 strains contained some low-similarity
orthologues to some of the lichenicidin genes found in
group 1, specifically LanT, LanM and a LanA-like protein
with protein sequence similarities of 48, 33 and 43 %,
respectively. These genes appear to encode a novel lantibio-
tic in group 2 strains. Group 1 strains also share a 12.5 kb
intact phage (highest similarity to Enterococcus phage
phiFL3A, GenBank accession no. NC_013648; Yasmin
et al., 2010).
Group 2 has many distinguishing gene clusters. It contains
three antimicrobial clusters that encode plipastatin
(Tsuge et al., 2007), bacitractin (Konz et al., 1997) or the
bacitractin-like peptide subpeptin (Wu et al., 2006), and an
unknown lantipeptide (BaLi_c01740-02380). Interestingly,
group 2 has many unique carbohydrate-metabolizing enzymes:
type II chitinase (BaLi_c8870), xylosidase (BaLi_c29190),
glucanase (BaLi_c31440) and arabinofuranohydrolase
(BaLi_c36440). Group 2 also contains a nine-gene urease clus-
ter (BaLi_c20330-20410) and an eight-gene nitrate reductase
cluster (BaLi_c28700-28780). Group 2 strain G-1 was pre-
viously reported to have urease activity, while other group 1
strains did not (Dhakal et al., 2014).
Results of the Biolog tests showed that both strains from
group 2 (KJ-16T and ATCC 12759) could metabolize dex-
trin, maltose, trehalose, cellobiose, sucrose, methyl b-D -
glucoside, D -salicin, a-D -glucose, D -mannose, D -fructose,
D -mannitol, glycerol, L -alanine, D -gluconic acid and L -
malic acid. Results of API 20NE tests showed that the
group 2 strains tested were positive for nitrate reduction,
arginine dihydrolase, urease, aesculin hydrolysis, gelatin
hydrolysis and b-galactosidase activity. In API assays,
group 2 strains could assimilate D -glucose, L -arabinose,
D -mannose, D -mannitol, N-acetylglucosamine, maltose,
potassium gluconate, malic acid, trisodium citrate, L -rham-
nose, D -ribose, inositol, sucrose, sodium acetate, lactic acid,
L -alanine, glycogen, melibiose, D -salicin, D -sorbitol and L -
proline. The group 2 strains produced acid from glucose
and did not utilize citrate. They were positive for hydrolysis
of starch, casein and gelatin. The cell-wall peptidoglycan
contained meso-diaminopimelic acid. A summary of dis-
tinguishing phenotypic properties is provided in Table 3.
Group 2 strains could be distinguished from group 1
strains and B. sonorensis on the basis of urease activity
using Stuart’s urease broth. Group 2 strains could be dis-
tinguished from group 1 strains on the basis of not assim-
ilating stachyose, glycyl L -glutamic acid and L -

Table 2. Genome-to-genome distance comparisons of strains of group 1 (B. licheniformis) and group 2 (B. paralicheniformis
sp. nov.)

Regression-based DDH values are indicated. Values .70 % are highlighted in bold.

Strain 1 2 3 4 5 6 7 8 9 10 11

Group 1
1. ATCC 14580T –
2. F1-1 98.0 –
3. WX-02 96.6 97.2 –
4. 10-1-A 97.8 98.4 96.4 –
5. 5-2-D 97.7 99.4 97.3 98.4 –
6. CGMCC 3963 97.1 97.2 96.5 96.0 96.9 –
7. CG-B52 97.5 96.6 96.5 96.3 97.2 95.7 –
Group 2
8. 9945a 58.3 58.3 58.8 58.1 58.2 58.0 57.8 –
9. G-1 57.9 58.1 58.6 57.6 58.1 57.6 57.5 92.0 –
10. KJ-16T 58.5 58.6 59.0 58.2 58.7 58.2 58.2 95.3 93.4 –
11. ATCC 12759 57.7 57.8 58.4 57.7 57.8 57.4 57.3 91.7 93.5 91.4 –
BL-09 58.0 57.9 58.7 57.9 57.9 57.5 57.5 92.7 94.9 92.4 95.3

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 Bacillus paralicheniformis sp. nov.

Table 3. Distinguishing phenotypic properties in the B. licheniformis group

B. paralicheniformis B. licheniformis B. sonorensis

Property KJ-16T ATCC 12759 ATCC 14580T KJ-10 NRRL B-23154T

Urease (Stuart’s urease medium) + + 2 2 2

Biolog Phenotype array
Succinic acid + + + + 2
D -Glucuronic acid + + + + 2
Tween 20 2 2 + + 2
Tween 40 2 2 + + 2
Tween 80 2 2 + + 2
Stachyose 2 2 + + 2
Glycyl L -glutamic acid 2 2 + + 2
L -Pyroglutamic acid 2 2 + + 2
Biolog Gen III plates
D -Galactose 2 2 2 2 +
myo-Inositol 2 2 2 2 +
L -Alanine + + 2 2 +
D -Galacturonic acid 2 2 2 2 +
Mucic acid 2 2 2 2 +
Nalidixic acid 2 2 2 2 +

pyroglutamic acid and assimilating L -alanine. Group 2
strains could be distinguished from B. sonorensis on the
basis of not assimilating D -galactose, myo-inositol, D -galac-
turonic acid and mucic acid and being susceptible to nali-
dixic acid.
Based upon the consensus of phylogenomic and pheno-
typic analyses, we conclude that strains belonging to
B. licheniformis group 2 represent a novel species within
the genus Bacillus, for which the name Bacillus paralicheni-
formis sp. nov. is proposed.
Description of Bacillus paralicheniformis sp. nov.
Bacillus paralicheniformis [Gr. prep. para beside, alongside,
near, like; N.L. masc. adj. licheniformis lichen-shaped,
a bacterial specific epithet; N.L. masc. adj. paralicheniformis
similar to Bacillus licheniformis ].
Cells are Gram-stain-positive, facultatively anaerobic,
motile, endospore-forming rods, 0.63–0.71 mm in diameter
and 1.90–3.94 mm long when cultured on R2A agar.
Central or subterminal ellipsoidal endospores are observed
with swollen sporangia. Colonies formed on R2A agar are
creamy white, mucoid, translucent and raised, 3–4 mm in
diameter after 2 days of incubation at 37 uC. Produces cat-
alase but not oxidase. Positive results for nitrate reduction,
arginine dihydrolase, urease, aesculin hydrolysis, gelatin
hydrolysis, casein hydrolysis, starch hydrolysis and
b-galactosidase activity. Acid is produced from glucose.
Does not utilize citric acid. Can be distinguished from its
closest relatives on the basis of utilizing L -alanine, succinic
acid and D -glucuronic acid and not utilizing D -galactose,
L -pyroglutamic acid, D -galacturonic acid, mucic acid or
D -sorbitol. Produces bacitracin, fengycin and a lantipep-
tide. Does not produce lichenicidin. The predominant
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menaquinone is MK-7. The cell-wall peptidoglycan
contains meso-diaminopimelic acid. The major fatty acids
are anteiso-C15 : 0, iso-C15 : 0, anteiso-C17 : 0 and iso-C17 : 0.
The major polar lipids are diphosphatidylglycerol,
phosphatidylglycerol, phosphatidylethanolamine, a glyco-
lipid, three unknown aminolipids and two unknown lipids.
The type strain is KJ-16T (5KACC 18426T5NRRL
B-65293T), isolated from cheonggukjang, a Korean fermen-
ted soybean food product. The DNA G+C content of the
type strain is 45.9 % (determined from the genome
sequence).
Acknowledgements
This study was supported by the Research Program for Agricultural
Science & Technology Development, National Academy of Agricul-
tural Science, Rural Development Administration, Republic of
Korea (project no. PJ011248). The authors would like to thank
Heather Walker for expert technical assistance. Any opinions, find-
ings, conclusions, or recommendations expressed in this publication
are those of the author(s) and do not necessarily reflect the view of
the US Department of Agriculture. The mention of firm names or
trade products does not imply that they are endorsed or rec-
ommended by the USDA over other firms or similar products not
mentioned. USDA is an equal opportunity provider and employer.
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