International Journal of Systematic and Evolutionary Microbiology (2016), 66, 801–806
DOI 10.1099/ijsem.0.000794
Bacillus cavernae sp. nov. isolated from cave soil
Liling Feng, Dongmei Liu, Xuelian Sun, Gejiao Wang3 and Mingshun Li3
Correspondence
Gejiao Wang
National Key Laboratory of Agricultural Microbiology, College of Life Science and Technology,
Huazhong Agricultural University, Wuhan, 430070, PR China
[email protected]
A Gram-stain-positive, spore-forming, motile, strictly aerobic, rod-shaped bacterium, designated
strain L5T, was isolated from soil of Tenglong cave, China. 16S rRNA gene sequence analysis
showed that strain L5T was related most closely to Bacillus asahii MA001T (96.5 %)
(the highest 16S rRNA gene sequence similarity), Bacillus kribbensis BT080T (96.4 %) and
Bacillus deserti ZLD-8T (96.2 %). The DNA G+C content of strain L5T was 45.6 mol%.
The major menaquinone was MK-7. The major fatty acids were iso-C14 : 0, anteiso-C15 : 0 and
iso-C16 : 0, and the major polar lipids were diphosphatidylglycerol, phosphatidylglycerol and
phosphatidylethanolamine. The diagnostic diamino acid in the cell-wall peptidoglycan was
meso-diaminopimelic acid. In addition, strain L5T had different characteristics compared with
the other Bacillus strains such as pink colony colour, low growth temperature and low nutrient
requirement. The results indicate that strain L5T represents a novel species of the genus
Bacillus, for which the name Bacillus cavernae sp. nov. is proposed. The type strain is L5T
(5KCTC 33637T5CCTCC AB 2015055T).
The genus Bacillus belongs to the family Bacillaceae, order
Bacillales, phylum Firmicutes, and was first proposed by
Cohn (1872) with Bacillus subtilis as the type species.
Additional Bacillus species were then identified, and
some of them were reclassified within other genera, such
as Alicyclobacillus, Aneurinibacillus, Brevibacillus, Geobacillus,
Marinibacillus, Paenibacillus, Salibacillus, Ureibacillus and
Virgibacillus. At the time of writing, the genus Bacillus comprised more than 300 species (http://www.bacterio.cict.fr/b/
bacillus.html) isolated from different environments.
Recently described species include Bacillus fengqiuensis
(Zhao et al., 2014), Bacillus huizhouensis (Li et al., 2014),
Bacillus filamentosus (Sonalkar et al., 2015), Bacillus pervagus
and Bacillus andreesenii (Kosowski et al., 2014), isolated
from sandy loam soil under long-term fertilization, paddy
field soil, sediment and composting reactor, respectively.
Common characteristics of members of the genus Bacillus
are Gram-reaction-positive, spore-forming, rod-shaped,
containing menaquinone 7 (MK-7) as the major menaquinone, diphosphatidylglycerol, phosphatidylglycerol and
phosphatidylethanolamine as the major polar lipids, and
meso-diaminopimelic acid as the diagnostic cell-wall diamino acid. Most Bacillus strains have iso-C14 : 0, iso-C15 : 0,
anteiso-C15 : 0, iso-C16 : 0 or anteiso-C17 : 0 as major fatty
acids. The DNA G+C content range is 36–52 mol%
3These authors contributed equally to this paper.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene
sequence of strain L5T is KT186244.
Three supplementary figures are available with the online Supplementary
Material.
000794 G 2015 IUMS
(Claus & Berkeley, 1986; Holt et al., 1994; Rheims et al.,
1999; Yumoto et al., 2004; Lim et al., 2007; Zhang et al.,
2011; You et al., 2013; Sonalkar et al., 2015).
Strain L5T was isolated from soil of Tenglong cave
(308 339 510 N 1088 989 290 E, elevation 2000 m) in
Enshi city, Hubei province, China. The soil texture was
clay, with pH of 6.0. Five grams of soil was serially diluted
with 0.85 % (w/v) NaCl and cultured on chemical defined
medium (CDM) plates (solution A: MgSO4, NH4Cl,
Na2SO4, K2PO4 . 3H2O, CaCl2 . 2H2O, sodium lactate;
solution B: FeSO4 . 7H2O; solution C: NaHCO3 – 100 ml
of solution A, 2.5 ml of solution B and 10 ml of solution
C were mixed and made up to 1 litre with water, 15 g
agar, pH 6.0; Weeger et al., 1999). A bacterial colony was
selected based on its pink colour and named L5T. Later,
the isolate was routinely cultivated on R2A agar (Bacton)
and preserved in 25 % glycerol at 280 8C.
Analyses of morphological, physiological and biochemical
characteristics were performed based on the recommended
minimal standards for the description of new aerobic sporeforming taxa (Logan et al., 2009). Strain L5T and related
type strains were cultivated on R2A agar at 28 8C, pH 7.0,
unless otherwise mentioned. Cells were observed by scanning electron microscopy (JSM-6390; JEOL). Flagella were
observed by light microscopy (10006, oil; Nikon). Spores
were observed by phase-contrast microscopy (10006, oil;
Nikon) after cultivation for 1 week. The motility of the
cells was detected by semi-solid puncturing in 0.3 % R2A
agar for 1 week. Gram staining was determined using a bioMérieux Gram-stain kit in combination with the 3 % KOH
method (Ryu, 1938). Growth temperature was tested on
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801
L. Feng and others
R2A agar at 4, 13, 20, 28, 32, 37 and 42 8C for 1 week.
To determine the tolerance to NaCl, the strains were
grown in R2A liquid medium with different NaCl concentrations (0, 1, 2, 3, 5, 7, 9, 11, 15 and 20 %, w/v) for
1 week. Experiments testing the pH range for growth at
pH 4.0–11.0 (1 pH unit interval) were performed in R2A
liquid medium with different buffer systems (pH 4–5,
0.1 M citric acid/0.1 M sodium citrate; pH 6–8, 0.1 M
KH2PO4/0.1 M NaOH; pH 9–10, 0.1 M NaHCO3/0.1 M
Na2CO3; pH 11, 0.05 M Na2HPO4/0.1 M NaOH) for
1 week. Growth in R2A liquid medium was determined
based on OD600 values with a blank culture medium as
the control. Anaerobic growth was determined in an
anaerobic chamber with an O2-absorbing and CO2-generating agent (Anaero-Pack; Mitsubishi Gas Chemical) for
2 weeks.
Growth was tested on trypticase soy agar (TSA; Bacto), 1/10
TSA, R2A agar, MacConkey agar, Luria–Bertani (LB) agar
(all from Difco) and CDM agar (Weeger et al., 1999). Catalase activity was determined using 3 % (v/v) H2O2 (Smibert & Krieg, 1994). Oxidase activity was determined using
1 % (w/v) tetramethyl-b-phenylenediamine (Merck).
Hydrolysis of starch, gelatin, casein and Tweens 20, 40, 60
and 80 was tested according to Cowan & Steel (1965) and
Arden Jones et al. (1979). Nitrate and nitrite reduction,
production of H2S and indole, and methyl red and Voges–
Proskauer tests were performed according to Dong & Cai
(2001). The ability to utilize sole carbon sources, acid production and enzyme activities were tested using traditional
methods (Dong & Cai, 2001) in combination with API
20E, API 32GN and API 50CH strips (bioMérieux) according to the manufacturer’s instructions.
Table 1. Differential phenotypic characteristics between strain L5T and the type strains of closely related species of the genus
Bacillus
Strains: 1, L5T; 2, B. asahii JCM 12112T (Yumoto et al., 2004); 3, B. kribbensis DSM 17871T (Lim et al., 2007); 4, B. deserti KCTC 13246T (Zhang et al.,
2011); 5, B. circulans ATCC 4513T (Pettersson et al., 2000); 6, B. huizhouensis GSS03T (Li et al., 2014); 7, B. psychrosaccharolyticus DSM 6T (Priest et al.,
1988; Zhang et al., 2012); 8, B. muralis LMG 20238T (Heyrman et al., 2005); 9, ‘B. frigoritolerans’ DSM 8801 (Delaporte & Sasson, 1967); 10, B. simplex
NBRC 15720T (Li et al., 2014; Heyrman et al., 2005); 11, B. subtilis NRRL NRS-744T (type species; Nakamura et al., 1999). Data for taxa 1–4 are from
this study except for the growth characters and DNA G+C contents. NG , No growth; ND , no data available; V , variable; +, positive; 2, negative;
W , weakly positive. P, pink; W, white; C-W, cream-white. Spore position: C, central or paracentral; T, terminal or subterminal.
Characteristic
Colony colour
Spore shape
Spore position
Nitrate reduction
Growth at/with:
4 8C
40 8C
NaCl (%, w/v)
pH
Anaerobic growth
Growth on LB agar
Hydrolysis of:
Starch
Gelatin
Tween 20
Tween 80
Acid production from:
D -Mannitol
D -Glucose
Starch
D -Ribose
Glycerol
D -Arabinose
D -Xylose
Maltose
D -Fructose
DNA G+C content (mol%)
1
2
3
4
5
6
P
Oval
C
2
W
Oval
C/T
+
W
Oval
W
Oval
C
+*
C-W
Oval
2
W
Oval
C
2
+
2
0–1
6.0–9.0
2
2
2
+
0–1
6.0–9.0
2
+
2
+
0–5
5.0–9.0
2
+
2
+
0–4
6.0–9.0
2
+
0–5
5.6–6.5
+
+
2
+
2
+
2*
2
2
+
+
2
2
2
+
+
+
+
+
+
+
+
2
2
2
2
2
2
2
45.6
2
2
+
2
2
2
2
2
2
39.4
+
+
+
+
+
+
2
+
2
+
2
+
2
40.1
NG
ND
2
W
+
W
+
+
43.3
2
7
8
9
10
11
W
ND
+
P
Oval
C
+
2
W
Oval
C/T
+
W
Oval
C
+
2
+
0–2
6.5–8.0
+
+
+
+
0–2
5.2–8.0
2
+
0–7
7.0–9.0
2
2
0–4
6.0–10.0
2
2
0–5
5.0–9.0
ND
W
ND
V
+
+
+
+
2
+
0–7
5.5–5.7
2
+
2
2
+
+
+
2
+
+
+
2
+
+
2
+
+
+
+
+
+
+
2
+
2
+
2
+
ND
ND
2
+
2
ND
+
+
2
ND
ND
ND
ND
ND
ND
ND
+
2
+
+
2
+
+
+
2
+
+
2
+
+
+
+
+
ND
ND
ND
ND
ND
+
35.7
+
40.2
+
+
40.8
2
39.4
ND
ND
+
+
+
2
W
ND
ND
ND
W
W
2
+
+
41.2
ND
ND
+
+
ND
ND
+
+
ND
ND
ND
*Data different from the previous studies (Yumoto et al., 2004; Lim et al., 2007; Zhang et al., 2011).
802
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Bacillus cavernae sp. nov.
Table 2. Comparison of enzyme activities of strain L5T and
type strains of the most closely related Bacillus species
Strains: 1, L5T; 2, B. asahii JCM 12112T (Yumoto et al., 2004); 3,
B. kribbensis DSM 17871T (Lim et al., 2007); 4, B. deserti KCTC
13246T (Zhang et al., 2011). All data are from this study. +, Positive;
2, negative.
Enzyme activity
1
2
3
4
Alkaline phosphatase
Acid phosphatase
Trypsin
Cystine arylamidase
Valine arylamidase
+
2
2
2
2
2
2
+
2
2
2
2
2
2
+
+
+
2
+
2
The different characteristics of strain L5T and related type
strains are given in Tables 1 and 2. Cell morphology is
shown in Fig. 1. The flagella and spores are shown in Fig.
S1 (available in the online Supplementary Material). Strain
L5T was a Gram-reaction-positive, rod-shaped, spore-forming bacterium that was motile by means of polar flagella.
These features are typical characteristics of the genus Bacillus. However, compared with other Bacillus strains, such
as Bacillus asahii JCM 12112T (Yumoto et al., 2004), Bacillus
kribbensis DSM 17871T (Lim et al., 2007) and Bacillus deserti
KCTC 13246T (Zhang et al., 2011), strain L5T has some distinctive characteristics. Colonies of strain L5T were pink,
whereas those of the other strains were white. Strain L5T
was unable to grow on nutrient-rich media such as LB
agar and TSA. In addition, the growth temperature of
strain L5T was lower than the other Bacillus strains, possibly
related to the cave environment from where it was found.
The nearly full-length 16S rRNA gene sequence of strain
L5T was amplified using 16S rRNA gene primers 27F and
Fig. 1. Scanning electron micrograph of cells of strain L5T after
growth for 2 days. Bar, 1 mm.
http://ijs.microbiologyresearch.org
1492R as described by Fan et al. (2008). The PCR product
was cloned into pGEM-T easy vector (Promega) for
sequencing. The nearly complete 16S rRNA gene sequence
of strain L5T (1472 bp) was compared with sequences
available in the EzTaxon-e server (Kim et al., 2012) and
NCBI GenBank database. Multiple alignments of sequences
were carried out using CLUSTAL X (Thompson et al., 1997)
within MEGA 6.0 (Tamura et al., 2013). Phylogenetic trees
were reconstructed using the maximum-likelihood
(Guindon et al., 2010), neighbour-joining (Saitou & Nei,
1987) and maximum-parsimony (Fitch, 1971) algorithms
in the MEGA 6.0 software. Distance calculations and bootstrap analysis were performed according to Kimura’s
two-parameter method (Kimura, 1980) and 1000 replications (Felsenstein, 1985), respectively.
Based on 16S rRNA gene sequencing analysis (Ash et al.,
1991), strain L5T was related most closely to members of
the genus Bacillus and clustered with B. asahii MA001T
(96.5 %) (the highest 16S rRNA gene sequence similarity),
B. kribbensis BT080T, Bacillus circulans ATCC 4513T and
B. deserti ZLD-8T. In the maximum-likelihood tree (Fig. 2),
strain L5T was located within a group containing
B. kribbensis BT080T and B. deserti ZLD-8T, adjacent to
B. asahii MA001T, while B. circulans ATCC 4513T was located
in another cluster. The neighbour-joining and maximumparsimony trees showed similar phylogenetic positions (Figs
S2 and S3). B. asahii JCM 12112T, B. kribbensis DSM
17871T and B. deserti KCTC 13246T were then used as reference strains for further analyses (see below).
For fatty acid analysis, strain L5T and the three most closely
related type strains were cultivated on R2A agar and collected in the exponential phase based on the quadrant
streak pattern described in the MIS operating manual
(2008). Whole-cell fatty acids were analysed by GC (6890;
Hewlett Packard) according to the standard protocol of
the Sherlock Microbial Identification System (MIDI Sherlock version 6.1, MIDI database TSA40 4.10) (Kroppenstedt, 1985; Sasser, 1990). The major whole-cell fatty
acids of strain L5T were iso-C14 : 0 (35.9 %), anteisoC15 : 0 (24.9 %) and iso-C16 : 0 (17.8 %), indicating that
strain L5T belonged to the genus Bacillus, but there were
differences in content compared with B. asahii JCM
12112T, B. kribbensis DSM 17871T, B. deserti KCTC
13246T, B. circulans ATCC 4513T and B. subtilis NRRL
NRS-744T (Table 3).
The menaquinone was extracted from lyophilized cells and
then analysed by the HPLC method as described by Xie &
Yokota (2003). Polar lipids were detected and identified by
two-dimensional TLC (Collins & Jones, 1980). The DNA
G+C content was determined by HPLC according to the
method of Tamaoka & Komagata (1984). The diagnostic
diamino acid in the cell-wall peptidoglycan was identified
by the Deutsche Sammlung von Mikroorganismen und
Zellkulturen (DSMZ) according to a published protocol
(Schumann, 2011). HPLC analyses revealed that the DNA
G+C content of strain L5T was 45.6 mol% and the
major menaquinone was MK-7. The polar lipids were
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803
L. Feng and others
0.01
*99
57
Bacillus jeotgali YKJ-10T (AF221061)
Bacillus boroniphilus JCM 21738T (BAUW01000204)
Bacillus subterraneus DSM 13966T (FR733689)
Bacillus thioparans BAS-1T (DQ371431)
Bacillus foraminis CV53T (A7717382)
*77
Bacillus novalis LMG 21837T (AJ542512)
Bacillus pocheonensis Gsoil 420T (AB245377)
Bacillus niacini IFO 15566T (AB021194)
Bacillus canaveralius KSC SF8bT (DQ870688)
Bacillus benzoevorans DSM 5391T (D783 11)
Bacillus circulans ATCC 4513T (AY724690-3)
57
99
51
75
*
Bacillus nealsonii DSM 15077T (EU656111)
Bacillus horneckiae MTCC 9535T (FR74 9913)
Bacillus purgationiresistens DS22T (FR666703)
Bacillus kochii WCC 4582T (FN995265)
Bacillus gottheilii WCC 4585T (FN995266)
Bacillus infantis SMC 4352-1T (AY904032)
66
Bacillus oceanisediminis H2T (GQ292772)
Bacillus firmus NCI MB 9366T (X60616)
61
*99
57
Bacillus kribbensis BT080T (DQ280367-2)
Bacillus deserti ZLD-8T (GQ465 041-4)
Bacillus cavernae L5T (KT186244)
Bacillus asahii MA001T (AB109209-1)
*97
Bacillus huizhouensis GSSO3T (KJ4 64756)
*88
Bacillus psychrosaccharolyticus ATCC 23296T (AB021195)
*94
Bacillus simplex NBRC 15720T (AB363738)
Bacillus muralis LMG 20238T (AJ628748)
*9962 Brevibacterium filgoritolercms DSM 8801T (AM747813)
Bacillus shackletonii LMG 18435T (AJ250318)
Virgibacillus pantothenticus IAM 11061T (D16275)
Pullulanibacillus naganoensis ATCC 53909T (AB021193)
89
Fig. 2. Maximum-likelihood phylogenetic tree based on partial 16S rRNA gene sequences (1472 nt) showing the position of
strain L5T. Bootstrap values (.50 %) based on 1000 replications are shown at branch nodes. Nodes that were also recovered
in trees reconstructed with both the maximum-likelihood and the maximum-parsimony methods are indicated by asterisks. Bar,
0.01 substitutions per nucleotide position.
diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, aminophospholipid and several unidentified phospholipids (Fig. 3). The diagnostic diamino acid
in the cell-wall peptidoglycan was meso-diaminopimelic
acid.
In summary, strain L5T has characteristics similar to those of
the type species B. subtilis NRRL NRS-744T and the other
members of the genus Bacillus such as being Gram-reaction-positive, spore-forming, motile, strictly aerobic and
rod-shaped. In addition, the respiratory quinone, fatty
acids, polar lipids and diagnostic diamino acid (meso-diaminopimelic acid) indicate that strain L5T is a member of the
genus Bacillus. However, based on its 16S rRNA gene
sequence, strain L5T shares less than 97 % similarity with
other Bacillus members. Moreover, strain L5T has differential characteristics such as colony colour, lower growth
temperature and low nutrient requirement. Taking these
data together, strain L5T is thus considered to represent a
novel species of the genus Bacillus, for which the name
Bacillus cavernae sp. nov. is proposed.
804
Description of Bacillus cavernae sp. nov.
Bacillus cavernae (ca.ver9nae. L. gen. n. cavernae of a cavern).
Cells are Gram-stain-positive, rod-shaped (0.3–0.8 mm|1.0–
6.0 mm), round-ended or occasionally slightly tapered, occur
in chains and sometimes singly or in pairs and are motile by
means of polar flagella. Oval spores are formed centrally or
paracentrally in swollen sporangia. Colonies on R2A agar
medium are pink, smooth, convex, non-transparent and circular. Strictly aerobic. The temperature range for growth is
4–32 uC (optimum, 28 uC) and the pH range is 6.0–9.0
(optimum, pH 7.0). Growth occurs with 0–1 % (w/v) NaCl
(optimum, 0 %). Catalase is positive but oxidase is negative.
Enzyme activities are observed for alkaline phosphatase,
esterase (C4), esterase lipase (C8) and naphthol-AS-BIphosphohydrolase, but no activities are observed for leucine
arylamidase, acid phosphatase, trypsin, cystine arylamidase,
valine arylamidase, a-glucosidase, b-galactosidase, arginine
dihydrolase, lipase (C14), valine arylamidase, a-chymotrypsin, a-galactosidase, b-glucuronidase, b-glucosidase,
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Bacillus cavernae sp. nov.
Table 3. Cellular fatty acid compositions of strain L5T and the
type strains of closely related Bacillus species
Strains: 1, L5T; 2, B. asahii JCM 12112T; 3, B. kribbensis DSM 17871T; 4,
B. deserti KCTC 13246T; 5, B. circulans ATCC 4513T (Venkateswaran
et al., 2003); 6, B. subtilis NRRL NRS-744T (type species; Rooney
et al., 2009). Data for taxa 1–4 are from this study. Values are percentages of total fatty acids; –, trace amount (,1 %) or not detected.
Fatty acid
Saturated
C14 : 0
C15 : 0
C16 : 0
iso-C14 : 0
iso-C15 : 0
iso-C16 : 0
anteiso-C15 : 0
anteiso-C17 : 0
Others
C16 : 1v7c alcohol
C16 : 1v11c
1
2
3
4
5
6
2.2
–
3.5
35.9
1.6
17.8
24.9
1.9
2.4
–
3.9
23.6
23.1
1. 8
15.5
–
–
–
3.2
5.4
4.2
2.8
70.0
8.0
–
1.4
1.7
11.3
4.6
2.1
56.3
2.9
2.9
1.0
2.7
4.0
13.9
4.4
58.4
4.8
–
–
1.9
–
19.4
2.5
38.2
12.1
7.4
13.1
–
–
5.3
4.8
–
2.6
–
1.6
2.7
1. 7
b-fucosidase, N-acetyl-b-glucosaminidase or a-mannosidase.
Acid is produced from D -glucose, D -mannitol and aesculin
but not D -ribose, glycerol, D -arabinose, D -xylose, D -fructose
or maltose. Assimilates D -arabinose, D -glucose, D -mannose,
L -lactate, maltose, D -fructose, lactose, D -ribose, D -sorbitol,
valerate, glycogen, cellobiose, sodium glutamate, sodium
caprylate, sodium butyrate, L -serine, L -phenylalanine, L -sarcosine and L -threonine. Gelatin and Tweens 40, 60 and 80 are
hydrolysed, but not starch, casein or Tween 20. Negative reactions for citrate utilization, H2S production, nitrate reduction,
indole production, Voges–Proskauer test and methyl red
test. Unable to grow on LB agar, MacConkey agar or TSA.
Significantly, produces pink pigment and is able to grow at
4 uC, in contrast to closely related Bacillus strains. The major
menaquinone is MK-7. The major fatty acids are iso-C14 : 0,
anteiso-C15 : 0 and iso-C16 : 0. The major polar lipids are
diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. The diagnostic diamino acid in the cellwall peptidoglycan is meso-diaminopimelic acid.
The type strain is L5T (5KCTC 33637T5CCTCC AB
2015055T), isolated from soil of Tenglong cave, Hubei,
China. The DNA G+C content of the type strain is
45.6 mol% (HPLC).
Acknowledgements
This work was supported by the National Natural Science Foundation
of China (31170106) and the Natural Science Foundation of Hubei
Province (2014CFB931). The cell-wall peptidoglycan was analysed
by the DSMZ.
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