International Journal of Systematic and Evolutionary Microbiology (2013), 63, 464–469
DOI 10.1099/ijs.0.037622-0
Amphibacillus indicireducens sp. nov., an
alkaliphile that reduces an indigo dye
Kikue Hirota,1 Kenichi Aino,1,2 Yoshinobu Nodasaka,3 Naoki Morita1,2
and Isao Yumoto1,2
Correspondence
Isao Yumoto
[email protected]
1
Bioproduction Research Institute, National Institute of Advanced Industrial Science and
Technology (AIST), Tsukisamu-Higashi, Toyohira-ku, Sapporo 062-8517, Japan
2
Laboratory of Environmental Microbiology, Graduate School of Agriculture, Hokkaido University,
Kita-ku, Sapporo 060-8589, Japan
3
Laboratory of Electron Microscopy, Graduate School of Dentistry, Hokkaido University, Kita-ku,
Sapporo 060-8586, Japan
Two indigo-reducing alkaliphilic strains, designated strain C40T and strain N214, were isolated
from a fermented Polygonum Indigo (Polygonum tinctorium Lour.) liquor sample aged for 10
months and obtained from Date City, Hokkaido, Japan. 16S rRNA gene sequence phylogeny
suggested that strains C40T and N214 were members of the genus Amphibacillus with the
closest relative being Amphibacillus xylanus JCM 7361T (97.5 % 16S rRNA gene sequence
similarity with strain C40T), which is the only strain having a 16S rRNA gene sequence similarity
higher than 97 % with strain C40T. Cells of strain C40T were Gram-stain-positive, facultatively
anaerobic, straight rods that were motile by means of peritrichous flagella. The strains grew
between 17 and 39 6C (optimum, 35 6C) and in the pH range of 9.0–12.0. No isoprenoid
quinone was detected and the DNA G+C content was 37.5–37.7 mol%. The whole-cell fatty
acid profile mainly consisted of iso-C15 : 0 and anteiso-C15 : 0. DNA–DNA hybridization of strain
C40T with Amphibacillus xylanus JCM 7361T revealed a DNA–DNA relatedness value of
10±3 %. Owing to the differences in phenotypic characteristics and phylogenetic analyses
based on 16S rRNA gene sequences, as well as DNA–DNA relatedness data from reported
species of the genus Amphibacillus, the isolates merit classification as a novel species in the
genus Amphibacillus, for which the name Amphibacillus indicireducens sp. nov. is proposed. The
type strain is C40T (5JCM 17250T5NCIMB 14686T). An additional strain of the species is
N214. An emended description of the genus Amphibacillus is provided.
The traditional method of indigo dyeing is based on the
reduction of indigo by the action of bacteria. Indigoreducing bacteria have been isolated by Takahara & Tanabe
(1960) and Padden et al. (1999) and have been identified as
‘Bacillus alkaliphilus’ and Clostridium isatidis, respectively. In
addition to these isolates, we have also recently isolated the
indigo-reducing bacteria Alkalibacterium psychrotolerans,
Alkalibacterium iburiense and Alkalibacterium indicireducens
from indigo fermentation liquors obtained from Date City,
Iburi, Hokkaido, Japan and Tokushima Prefecture, Shikoku,
Japan (Nakajima et al., 2005; Yumoto et al., 2004, 2008). It
Abbreviation: meso-DAP, meso-Diaminopimelic acid.
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene
sequences of strains C40T and N214 are AB665218 and AB704542,
respectively.
A supplementary figure and a supplementary table are available with the
online version of this paper.
464
has also been demonstrated that, other than the abovementioned species, several isolates of micro-organisms from
indigo fermentation liquor were able to reduce indigo (Aino
et al., 2010). Among the isolates, a novel indigo-reducing
bacterium ‘Oceanobacillus indicireducens’ was recently
described (Hirota et al., in press).
The genus Amphibacillus was first described by Niimura
et al. (1990) to accommodate a facultatively anaerobic,
obligately alkaliphilic, spore-forming bacterium, Amphibacillus xylanus isolated from an alkaline compost of manure
with grass and rice straw. Since the first description of the
genus Amphibacillus, additional species have been described
as follows: Amphibacillus fermentum and Amphibacillus
tropicus from the sediment of a soda lake in Kenya
(Zhilina et al., 2001), Amphibacillus sediminis from the
sediment of Lake Hamana in Japan (An et al., 2007) and
Amphibacillus jilinensis from the sediment of a soda lake in
Jilin province, China (Wu et al., 2010). Members of the
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Amphibacillus indicireducens sp. nov.
genus Amphibacillus are moderately alkaliphilic and lack
isoprenoid quinones and oxidase activity (Arai et al., 2009).
A fermented Polygonum Indigo (Polygonum tinctorium
Lour.) liquor sample aged for 10 months was used for the
screening of indigo-reducing bacteria (Aino et al., 2010).
An aliquot of the obtained sample was inoculated on a
reinforced clostridial agar (RCA; Sigma) plate containing
100 mM NaHCO3/Na2CO3 buffer, pH 10.0 (alkali RCA)
and aerobically incubated at 30 uC for 10 days. The
NaHCO3/Na2CO3 buffer for media used in this study was
sterilized separately by autoclaving. Twenty-eight strains
were isolated which were inoculated onto a PYA (peptone/
yeast extract/alkaline)-indigo carmine agar plate containing
8 g peptone (Kyokuto), 3 g yeast extract (Merck), 1 g
K2HPO4, 2 g indigo carmine and 15 g agar in 1 l NaHCO3/
Na2CO3 buffer (100 mM in deionized water, pH 10.0),
followed by incubation at 30 uC for 2 days. After checking
for the microbial reduction of indigo carmine, the culture
was transferred to 100 ml of fresh 0.01 % indigo-containing PYA broth to further check the indigo-reducing ability
of the isolates. The resulting strains were transferred to
an alkali RCA plate and incubated at 30 uC. Three strains
were identified as positive for indigo reduction during the
screening process. Among the three strains, two isolates, C40T
and N214, are characterized in this study. Amphibacillus
xylanus JCM 7361T, Amphibacillus sediminis IAM 15428T
and Amphibacillus jilinensis JCM 16149T were used as the
reference strains. Cells for chemotaxonomic analysis (fatty
acid composition, DNA G+C content, isoprenoid quinone
and cell wall) were harvested in the late exponential phase
after cultivation with reciprocal shaking (90 strokes min21)
at 27 uC in Clostridial Nutrient Medium (CNM; Fluka
BioChemika) containing 100 mM NaHCO3/Na2CO3 buffer,
pH 10.0 (alkali CNM).
For phenotypic characterization, alkali RCA or alkali CNM
was used as the basal medium. The culture was incubated
at 35 uC for 2 weeks and experiments were performed three
times to confirm the reproducibility of results. API ZYME
strips (bioMérieux) were used according to the manufacturer’s instructions. Growth experiments at pH 7–12 were
performed using PYA medium containing 100 mM
NaH2PO4/Na2HPO4 buffer for pH 7–8, 100 mM NaHCO3/
Na2CO3 buffer for pH 9–10, Na2HPO4/NaOH buffer for
pH 11 or KCl/NaOH buffer for pH 12. Growth under
anaerobic conditions was determined by checking the
growth on the alkali RCA plate in an argon gas exchange
jar. Other physiological and biochemical characteristics were
examined according to the methods described in Cowan and
Steel’s Manual (Barrow & Feltham, 1993) and by Yumoto et
al. (1998). Acid production from carbohydrate was tested by
Alkali-OF medium (pH 10) containing of 2 g peptone
(Kyokuto), 2 g yeast extract (Kyokuto), 0.3 g K2HPO3,
0.03 g thymol blue, 3.0 g agar, 0.29 g NaHCO3, 0.7 g
NaCO3 and 1 % carbohydrate. Anaerobiosis in the inoculated test tubes was achieved by overlaying with sterile
paraffin oil. Hydrolysis of xylan and cellulose were
determined using alkali-RCA containing 1 % of each
http://ijs.sgmjournals.org
substrate and judged by the method of Teather & Wood
(1982). For the observation of negatively stained cells by
transmission electron microscopy (TEM), the cells were
grown on alkali RCA. TEM preparation and observations
were performed as described previously (Yumoto et al.,
2001). The growth of the isolate at pH 10 under aerobic
conditions in the temperature range of 10–40 uC was
determined using a temperature gradient incubator (BioPhotorecorder, type TN-2612; Advantec) and monitoring
optical density at 650 nm (OD650). Strains C40T and N214
can be differentiated from previously reported phylogenetic
neighbour species belonging to the genus Amphibacillus on
the basis of on the 16S rRNA gene sequence and
characteristics listed in Table 1.
Bacterial DNA was prepared according to the method of
Marmur (1961). The DNA base composition was determined by the method of Tamaoka & Komagata (1984). The
DNA G+C contents of strains C40T and N214 were 37.5
and 37.7 mol%, respectively. Analysis of whole-cell fatty
acids and isoprenoid quinones were performed as
described previously (Yumoto et al., 2002). The presence
of meso-diaminopimelic acid (meso-DAP) in the cell wall
was analysed in strains C40T and N214 by thin-layer
chromatography (TLC) (Staneck & Roberts, 1974). Gas
chromatography (GC) analysis revealed the cellular fatty
acid composition of strains C40T and N214 was similar to
those of other species of the genus Amphibacillus in that
anteiso-C15 : 0 and iso-C15 : 0 are the predominant fatty acids
(Table S1, available in IJSEM Online; data are means of two
datasets obtained from two independent cultures). There
were differences in the ratio of the amount of anteiso-C15 : 0
to that of iso-C15 : 0 and in the absence of C20 : 0 (except
for A. xylanus) compared with other species of the genus
Amphibacillus. Isoprenoid quinone could not be detected
by TLC and HPLC analyses from the concentrated
extracted fractions in strains C40T and N214. The cell
walls of strains C40T and N214 contained meso-DAP.
The 16S rRNA gene was amplified by PCR using primers
9F (59-GAGTTTGATCCTGGCTCAG-39) and 1541R (59AAGGAGGTGATCCAGCC-39). The resulting PCR product was purified with a QIAquick PCR purification kit
(Qiagen) and sequenced directly by the dideoxynucleotide
chain-termination method using a DNA sequencer (ABI
PRISM 3100; Applied Biosystems) with a BigDye
Termination RR mix version 3.1 (Applied Biosystems)
according to the manufacturer’s instructions. The sequences
were aligned and the consensus sequence was determined
using the program CLUSTAL W (Thompson et al., 1994).
Phylogenetic trees were constructed by the neighbourjoining (Saitou & Nei, 1987), maximum-parsimony
(Felsenstein, 1981), minimum-evolution (Fitch, 1971) and
maximum-likelihood (Guindon & Gascuel, 2003) methods
using MEGA 5 software (Tamura et al., 2011). For neighbourjoining analysis, the distance between sequences (Kunc value)
was calculated using Kimura’s two-parameter model
(Kimura, 1980). The similarity between sequences was
calculated using the GENETYX program (Genetec). The 16S
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465
K. Hirota and others
Table 1. Differential characteristics of strains C40T and N214 and closely related members of the genus Amphibacillus
Strains: 1, C40T; 2, N214; 3, A. xylanus JCM 7361T; 4, A. sediminis IAM 15428T; 5, A. jilinensis JCM 16149T. All data except DNA G+C content and
data of growth temperature are from this study resulting from API ZYME, hydrolyses of macromolecules and sensitivity for antibiotics (aerobic
condition). Data for A. xylanus JCM 7361T, A. sediminis IAM 15428T and A. jilinensis JCM 16149T are cited from Niimura et al. (1990), An et al.
(2007) and Wu et al. (2010), respectively. All strains were negative for hydrolysis of gelatin, casein, DNA and Tweens 40, 60 and 80; and lipase
(C14), cystine arylamidase, trypsin, acid phosphatase, N-acetyl-b-glucosaminidase, a-mannosidase and a-fucosidase activities. All strains were
positive for hydrolysis of starch and xylan; p-Nitrophenyl-b-D-galactopyranosidase, esterase/lipase (C8), naphthol-AS-BI-phoshohydrolase and aglucosidase activities; and sensitivity to azithromycin (15 mg), erythromycin (15 mg), kanamycin (30 mg) and nalidixic acid (30 mg). +, Positive; 2,
negative; W, weakly positive.
Characteristic
Hydrolysis of:
Cellulose
Tween 20
Enzyme activities (API ZYME)
Alkaline phosphatase
Esterase (C4)
Leucine arylamidase
Valine arylamidase
Chymotrypsin
a-Galactosidase
2-Naphthol-b-D-Galactosidase
b-Glucuronidase
b-Glucosidase
Sensitivity to:
Amikacin (30 mg)
Gentamicin (10 mg)
Polymyxin B (300 U)
Streptomycin (10 mg)
Tenebrimycin (10 mg)
Neomycin (30 mg)
Novobiocin (30 mg)
Nystatin (100 mg)
Temperature for growth (uC)
Range
Optimum
pH for growth
Range
Optimum
DNA G+C content (mol%)
1
2
3
4
5
+
2
+
2
+
+
+
+
2
+
2
+
+
2
2
+
+
+
+
2
+
+
2
2
+
+
+
+
W
+
+
+
+
2
+
2
2
2
2
+
2
2
2
2
+
2
2
2
2
2
2
+
+
+
+
2
+
2
2
+
+
+
+
2
+
2
2
+
+
2
+
+
+
+
+
2
2
+
2
+
2
2
2
+
2
2
+
2
+
2
2
17–39
35
17–39
35
25–45
40
17–55
27
15–45
32
9.0–12.0
10.0
37.5
9.0–12.0
10.0
37.7
8.0–10.0
9.0
36.0
7.0–9.0
8.5
42.3
7.5–10.5
9.0
37.7
rRNA gene sequences of strain C40T (1537 bp) and N214
(1540 bp) were obtained and compared with those of
previously reported strains to determine sequence similarities, and phylogenetic trees of the two strains together with
their closely related neighbours were constructed. The
maximum-likelihood and maximum-parsimony phylogenetic trees (Figs 1 and S1c, respectively) show that the node
involving strains C40T, N214 and A. xylanus JCM 7361 is
most closely related to the independent node of A. sediminis
IAM 15428T. However, in the neighbour-joining and
minimum-evolution phylogenetic trees, the node involving
strains C40T, N214 and A. xylanus JCM 7361T is most closely
related to the node consisting four species of the genus
Amphibacillus including A. sediminis Shu-P-Ggiii25-2T (Fig.
S1a, b). The neighbour-joining and minimum-evolution
trees were very similar to each other. 16S rRNA gene
466
+
+
+
W
sequence similarity between strains C40T and N214 was
100 %. Strain C40T showed the highest 16S rRNA gene
sequence similarity with A. xylanus JCM 7361T (97.5 %). 16S
rRNA gene sequence similarities of strain C40T with other
members of the genus Amphibacillus were in the range 93.7–
95.4 %: A. sediminis Shu-P-Ggiii25-2T (95.4 %), Amphibacillus cookii JW/BP-GSL-QDT (94.6 %), A. jilinensis JCM
16149T (94.4 %), A. fermentum Z-7984T (94.2 %) and A.
tropicus Z-7792T (93.7 %).
On the basis of the results of 16S rRNA gene sequence
analysis, A. xylanus JCM 7361T and A. sediminis IAM 15428T
were considered as reference strains for DNA–DNA hybridization. All strains were cultivated by the same method. The
hybridization temperature was 41.0 uC. The level of DNA–
DNA relatedness was determined fluorometrically by the
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Amphibacillus indicireducens sp. nov.
T
100 Amphibacillus indicireducens C40 (AB665218)
0.01
100
Amphibacillus indicireducens N214 (AB704542)
60
Amphibacillus x ylanus JCM 7361T (D82065)
35
Amphibacillus sediminis Shu-P-Ggiii25-2T (AB243866)
Amphibacillus jilinensis Y1T (FJ169626)
53
99
Amphibacillus cookii JW/BP-GSL-QDT (HM057160)
62
Amphibacillus tropicus Z-7792T (AF418602)
Amphibacillus fermentum Z-7984T (AF418603)
53
Halolactibacillus halophilus M2-2T (AB196783)
33
100
Halolactibacillus miurensis M23-1T (AB196784)
Gracilibacillus orientalis XH-63T (AM040716)
Gracilibacillus halotolerans NN T (AF036922)
58
68
Gracilibacillus dipsosauri DD1T (X82436)
Halobacillus halophilus NCIMB 2269T (X62174)
100
Halobacillus trueperi DSM 10404T (AJ310149)
Bacillus subtilis DSM 10T (AJ276351)
Fig. 1. Maximum-likelihood phylogenetic tree derived from 16S rRNA gene sequence data of strains C40T and N214, and
closely related members of the family Bacillaceae. Bootstrap values based on 1000 replicates are shown at branch nodes. Bar,
0.01 changes per nucleotide position.
method of Ezaki et al. (1989) using photobiotin-labelled DNA
probes and black microplates. The DNA–DNA relatedness
value between strains C40T and N214 was 100 %. DNA–DNA
hybridization results indicated that strain C40T was different
from A. xylanus JCM 7361T (10±3 %, n54) and A. sediminis
IAM 15428T (9±1 %, n53).
In addition to the phylogenetic position based on the 16S
rRNA gene sequence, the characteristic of growth at
pH 12.0 is specific to the novel isolates when comparing
all recognized species of the genus Amphibacillus, as is not
requiring NaCl for growth (except for A. sediminis and A.
jilinensis). Further comparison of phenotypic characteristics under the same experimental conditions was
performed using phylogenetically closest neighbours based
on the 16S rRNA gene sequence: A. xylanus JCM 7361T, A.
sediminis IAM 15428T and A. jilinensis JCM 16149T (Table
1). Strains C40T and N214 can be differentiated from A.
xylanus JCM 7361T, A. sediminis IAM 15428T and A.
jilinensis JCM 16149T on the basis of several phenotypic
and chemotaxonomic characteristics. For example, strains
C40T and N214 can be differentiated from A. xylanus JCM
7361T on the basis of their inability to hydrolyse Tween 20,
the negative result in tests for valine arylamidase activity,
sensitivity to polymyxin B and resistance to tenebrimycin,
novobiocin and nystatin, as well as growth characteristics
(temperature and pH). Strains C40T and N214 can be
differentiated from A. sediminis IAM 15428T and A. jilinensis
JCM 16149T on the basis of positive results for bglucuronidase and b-glucosidase activities (API ZYME), and
growth characteristics (temperature and pH). Strains C40T
and N214 can also be differentiated from other phylogenetically related members of the genus Amphibacillus on the basis
of fatty acid composition, namely, the ratio of the amount of
http://ijs.sgmjournals.org
anteiso-C15 : 0 to that of iso-C15 : 0 and the absence of C20 : 0
(except for A. xylanus) (Table S1).
On the basis of the above results, the isolates represent a
novel species of the genus Amphibacillus, for which the
name Amphibacillus indicireducens sp. nov. is proposed; the
type strain is C40T. An additional strain of the species is
N214.
Emended description of the genus Amphibacillus
Niimura et al. 1990
Amphibacillus (Am.phi.ba.cil9lus. Gr. pref. amphi both
sides or double; L. dim. n. bacillus a small rod; N. L. masc.
n. Amphibacillus rod capable of both aerobic and anaerobic
growth).
The description is based on those given by Niimura et al.
(1990) and An et al. (2007) with the following additions.
Cells are straight or slightly curved and motile by means of
peritrichous flagella, or are non-motile rods (0.3–1.060.9–
4.0 mm). Growth occurs at pH 7–12.
Description of Amphibacillus indicireducens
sp. nov.
Amphibacillus indicireducens (in.di.ci.re.du9cens. L. n.
indicum indigo; L. part. adj. reducens bringing or leading
back, used to mean ‘reducing’; N.L. part. adj. indicireducens
indigo-reducing).
Cells are Gram-stain-positive, peritrichously flagellated
straight rods (0.3–0.561.0–3.0 mm) and facultatively
anaerobic. Spherical endospores are formed in the terminal
position. Colonies are circular, convex and white on alkali
RCA. Catalase- and oxidase-negative. Temperature range
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K. Hirota and others
for growth is 17–39 uC with optimum growth at 35 uC
(pH 10.0). The NaCl range for growth is 0–7 % with
optimum growth at 0–1 % (pH 10.0, 35 uC). The pH range
for growth is pH 9.0–12.0 with optimum growth at
pH 10.0 (30 uC). Hydrolyses starch, xylan and cellulose,
but not casein, gelatin, DNA, or Tweens 20, 40, 60 and 80.
Positive result in API ZYME tests for esterase (C4),
esterase/lipase (C8), leucine arylamidase, naphthol-AS-BIphosphohydrolase, a-galactosidase, b-galactosidase, b-glucuronidase, a-glucosidase and b-glucosidase, but negative
result for alkaline phosphatase, lipase (C14), valine
arylamidase, cysteine arylamidase, trypsin, chymotrypsin,
acid phosphatase, N-acetyl-b-glucosaminidase, a-mannosidase and a-fucosidase. Ferments (no gas) L-arabinose, Darabinose, D-xylose, D-glucose, D-fructose, D-mannose, Drhamnose, D-galactose, sucrose, trehalose, cellobiose raffinose and melibiose, but not xylitol, sorbitol, mannitol or
myo-inositol. Weakly ferments lactose. Sensitive to amikacin (30 mg), gentamicin (10 mg), polymyxin B (300 U),
streptomycin (10 mg) and neomycin (30 mg), but resistant
to tenebrimycn (10 mg), novobiocin (30 mg) and nystatin
(100 mg).The cellular fatty acids are iso-C13 : 0, anteisoC13 : 0, iso-C14 : 0, C14 : 0, iso-C15 : 0, anteiso-C15 : 0, C15 : 0, isoC16 : 0, C16 : 0, iso-C17 : 0, anteiso-C17 : 0, C18 : 0, C18 : 1v9c,
C18 : 2v6c and C20 : 0. The cell-wall peptidoglycan was based
on meso-DAP. No isoprenoid quinone was detected and
the DNA G+C content was in the range 37.5–37.7 mol%.
T
T
T
The type strain, C40 (5JCM 17250 5NCIMB 14686 ),
was isolated from a Polygonum Indigo (Polygonum
tinctorium Lour.) fermentation liquor from Date City,
Hokkaido, Japan. The DNA G+C content of the type
strain is 37.5 mol%. An additional strain of the species is
strain N214.
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Acknowledgements
We would like to thank Mr Noboru Ichijyou for providing the indigo
fermentation liquor aged for 10 months.
composition by reversed-phase high-performance liquid chromatography. FEMS Microbiol Lett 25, 125–128.
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