International Journal of Systematic and Evolutionary Microbiology (2016), 66, 1768–1772
DOI 10.1099/ijsem.0.000943
Niastella vici sp. nov., isolated from farmland soil
Lu Chen,1 Dan Wang,1 Sining Yang2 and Gejiao Wang1
Correspondence
Gejiao Wang
[email protected]
1
State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology,
Huazhong Agricultural University, Wuhan, Hubei 430070, PR China
2
No. 1 Middle School Affiliated to Central China Normal University, Wuhan, Hubei 430223, PR China
Strain DJ57T is a Gram-reaction-negative, filamentous-shaped, non-flagellated, aerobic
bacterium isolated from farmland soil in Hunan province of China. 16S rRNA gene sequence
analysis demonstrated that this isolate belonged to the genus Niastella, with 95.83 %
nucleotide identity to Niastella populi THYL-44T, while the similarities to other type strains of
species of the genus Niastella were less than 95.76 %. The major isoprenoid quinone was
menaquinone-7 and the major fatty acids (.5 %) were iso-C15 : 0, iso-C17 : 0 3-OH, iso-C15 : 1
G, anteiso-C15 : 0 and iso-C16 : 0. The DNA G+C content was 44 mol%. Polar lipids were
phosphatidylethanolamine, three unknown aminophospholipids, three unknown phospholipids
and six unknown lipids. The chemotaxonomic, phenotypic and genotypic data indicated that
strain DJ57T represents a novel species of the genus Niastella, for which the name Niastella vici
sp. nov. is proposed. The type strain is DJ57T (5KCTC 42474T5CCTCC AB 2015052T).
Genus Niastella belongs to the family Chitinophagaceae
within the phylum Bacteroidetes (http://www.bacterio.net/p/
paenibacillus.html). So far, this genus consists of the type
strains of four species, all isolated from soil: Niastella koreensis
GR20-10T (Weon et al., 2006), Niastella yeongjuensis GR2013T (Weon et al., 2006), Niastella populi THYL-44T (Zhang
et al., 2010) and Niastella gongjuensis 5GH22-11T (Kim
et al., 2015). The genus was first proposed by Weon et al.
(2006) and emended by Zhang et al. (2010). Members of the
genus Niastella are Gram-reaction-negative, strictly aerobic,
long, filamentous rods. The DNA G+C content range is
41.4–45.8 mol%. The major respiratory quinone is menaquinone-7 (MK-7) and the predominant cellular fatty acids
are iso-C15 : 0, iso-C15 : 1 G and iso-C17 : 0 3-OH (Weon
et al., 2006; Zhang et al., 2010; Kim et al., 2015). In this
study, strain DJ57T was isolated from farmland soil near a
lead and zinc mine (308 179 540 N 1138 289 160 E) in the
city of Yueyang, Hunan province, PR China. The pH of the
soil was pH 6.23 and the soil Pb and Zn concentrations
were 127.6 and 54.41 mg kg21 (dry weight soil), respectively.
For the isolation, serial dilutions of the sample were spread on
1/10 tryptic soy agar (TSA; pH 7.3; Difco) and incubated at
28 8C for 7 days. Strain DJ57T was obtained after several subcultivation cycles on 1/10 TSA. However, it was later noticed
that the isolate grew better on R2A agar (pH 7.3; Difco).
The GenBank/EMBL/DDBJ accession number for the 16S rRNA
gene sequence of strain DJ57T is KT388748.
Two supplementary figures are available with the online Supplementary
Material.
1768
The nearly complete 16S rRNA gene fragment was amplified by
PCR using universal primers 27F and 1492R as described
by Fan et al. (2008), and the PCR products were sequenced
by TsingKe Biological Technology (Beijing, China). The
sequence (1491 bp) was compared with the available sequences
in NCBI GenBank using the BLASTN program and its close relatives were extracted by the Ezbiocloud server (Kim et al., 2012).
CLUSTAL X software (Thompson et al., 1997) was used to align
the sequences. Maximum-parsimony (MP; Fitch, 1971), maximum-likelihood (ML; Felsenstein, 1981) and neighbour-joining (NJ; Saitou & Nei, 1987) trees were reconstructed by MEGA
version 5.0 software (Tamura et al., 2011), and the Kimura twoparameter model was used (Kimura, 1980) to calculate the
evolutionary distances. Bootstrap analysis was used to estimate
tree topologies according to 1000 resamplings (Felsenstein,
1985). The adjacent strains of DJ57T in the ML tree were N.
populi THYL-44T (Zhang et al., 2010), N. koreensis GR20-10T
(Weon et al., 2006), N. gongjuensis 5GH22-11T (Kim et al.,
2015) and N. yeongjuensis GR20-13T (Weon et al., 2006), and
their 16S rRNA gene sequence identities were 95.83, 95.76,
95.64 and 95.16 %, respectively (Fig. 1). Using MP and NJ
methods, strain DJ57T also formed the same cluster with the
four reference type strains (Figs S1 and S2, available in the
online Supplementary Material).
For morphological, biophysical and biochemical analyses,
strains DJ57T, N. populi CCTCC AB 208238T, N. koreensis
DSM 17620T and N. yeongjuensis DSM 17621T were
grown on R2A agar or in R2A broth at 28 8C. A transmission electron microscope (model 7650; Hitachi)
was used to examine the cell morphology (Fig. 2) and
oil-immersion phase-contrast microscopy (AXIO; Zeiss)
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Niastella vici sp. nov.
0.01
99
99
95
50
59
98
61
100
Terrimonas ferruginea DSM 30193T (AUDS01000006)
Terrimonas aquatica RIB1-6T (FJ347757)
Terrimonas rubra M-8T (JF803808)
Terrimonas lutea DYT (AB192292)
Terrimonas pekingensis QHT (JF834159)
Flavisolibacter ginsengisoli Gsoil 643T (AB267477)
100
Flavisolibacter ginsengiterrae Gsoil 492T (AB267476)
Niastella populi THYL-44T (EU877262)
Niastella gongjuensis 5GH22-11T (KM0071021)
Niastella vici DJ57T (KT388748)
100
Niastella koreensis GR20-10T (CP003178)
Niastella yeongjuensis GR20-13T (DQ244076)
Parasegetibacter luojiensis RHYL-37T (EU877263)
Flavitalea populi HY-50RT (HM130561)
Flavitalea gansuensis JCN-23T (GU295962)
97
Fig. 1. Maximum-likelihood phylogenetic tree based on 16S rRNA gene sequences, showing the relationship of strain DJ57T
and related taxa. Bootstrap values (,50 %) are shown at nodes as percentages of 1000 replicates. Bar, 0.01 substitutions
per nucleotide position.
Fig. 2. Cell morphology of strain DJ57T observed by transmission
electron microscopy. Cells were collected after growth for 2 days
at 28 8C on R2A medium. Bar, 2 mm.
was used to observe gliding motility. A Gram staining kit
(Baso Diangostigs) was used to determine the Gram stain
reaction. Motility tests were performed using R2A with
0.15 % agar. Anaerobic growth was tested on R2A in an
anaerobic chamber (Mitsubishi Gas Chemical) incubated
at 28 8C for 7 days. The temperature range for growth
was tested at 4, 16, 20, 28, 32, 37 and 42 8C on R2A for
7 days. Tolerance to salt was tested in R2A broth containing 0–3 % (w/v) NaCl (0.5 % intervals). Tolerance to pH
was tested in R2A broth at pH 3–10 (1 pH unit intervals)
after 7 days of incubation at 28 8C. The following buffer
systems were used to prepare R2A broth at pH 3–10:
pH 3.0–7.0,
0.1 M
citric
acid/0.2 M
Na2HPO4;
pH 8.0–9.0, 0.2 M Tris/0.2 M HCl; pH 10.0, 0.05 M
NaHCO3/0.1 M NaOH. Growth was also tested on R2A
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agar, 1/10 TSA, TSA, MacConkey agar, nutrient agar
(NA) and Luria–Bertani (LB) agar (all from Difco) after
7 days of incubation at 28 8C. Nitrate reduction, methyl
red test, H2S production and the Voges–Proskauer reaction
were tested as described by Dong & Cai (2001). Hydrolysis
of gelatin, casein, tyrosine, starch, chitin, agar, DNA,
cellulose and Tweens 20, 40, 60 and 80 was tested according
to the methods of Cowan & Steel (1965). Oxidase activity
was tested by oxidation of tetramethyl-p-phenylenediamine. The production of bubbles after exposing cell
biomass to 3 % H2O2 (Merck) was used to confirm catalase
activity. The presence of flexirubin pigment was tested
according to the methods of Bowman (2000) and Bernardet et al. (2002). Congo red absorption was tested by
directly flooding colonies on agar plates with 0.01 % aqueous Congo red dye (Bernardet et al., 2002). Other activities were determined with the API 20NE, ZYM and 50CH
systems according to the manufacturer’s instructions
(bioMérieux). The results of API ZYM tests were observed
after incubation at 28 8C for 6 h, while the other API tests
were examined after incubation at 28 8C for 2 days.
Compared with the other strains representing the genus
Niastella, strain DJ57T showed several different characteristics such as activities for esterase (C4) and trypsin
(Table 1).
For fatty acid analysis, the novel strain was cultivated in R2A at
28 8C with 150 r.p.m. shaking and the cell biomass was
collected in the mid-exponential phase. The whole-cell fatty
acids were extracted and analysed by using the Sherlock
Microbial Identification System (Sherlock version 4.5; database TSBA40 4.10; MIDI) (Kroppenstedt, 1985; Sasser,
1990). The major fatty acids of strain DJ57T were iso-C15 : 0
(19.5 %), iso-C15 : 1 G (15.8 %), iso-C17 : 0 3-OH (9.4 %)
anteiso-C15 : 0 (7.2 %) and iso-C16 : 0 (5.9 %) (Table 2).
These fatty acid types were similar to those of the
other strains representing members of the genus Niastella
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1769
L. Chen and others
Table 1. Different characteristics among strain DJ57T and
other type strains of species of the genus Niastella
Table 2. Fatty acid contents (percentages) of strain DJ57T
and the type strains of species of the genus Niastella
Strains: 1, DJ57T; 2, N. koreensis DSM 17620T; 3, N. yeongjuensis DSM
17621T; 4, N. populi CCTCC AB 208238T; 5, N. gongjuensis 5GH2211T. Data for taxa 1, 2, 3 and 4 are from this study except the DNA
G+C contents of taxa 2, 3 and 4. Data for taxa 5 are from Kim
et al. (2015). +, Positive; 2, negative; ND , no data.
Strains: 1, DJ57T; 2, N. koreensis DSM 17620T; 3, N. yeongjuensis DSM
17621T; 4, N. populi CCTCC AB 208238T; 5, N. gongjuensis 5GH2211T (Kim et al., 2015). Data for taxa 1, 2, 3 and 4 are from this
study. Data for taxa 5 are from Kim et al. (2015). –, Not detected
or ,1 %. The major fatty acids are indicated in bold type.
Characteristic
Colony colour
1
2
White Light
yellow
Oxidase activity
2
2
Gliding motility
+
+
Hydrolysis of chitin 2
+
Nitrate reduction
+
2
Gelatin hydrolysis
+
2
Esterase (C4)
+
2
Trypsin
+
2
a-Chymotrypsin
+
2
a-Galactosidase
+
+
a-Glucosidase
+
+
a-Mannosidase
2
2
a-Fucosidase
+
2
Assimilation of:
N-Acetyl-b2
2
glucosamine
L -Arabinose
+
2
D -Glucose
+
2
L -Histidine
2
2
Maltose
+
2
D -Mannose
+
2
Melibiose
2
2
L -Rhamnose
+
2
Salicin
2
2
L -Serine
2
2
Sucrose
+
2
DNA G+C content 44.0
45.8
(mol%)
3
4
5
Milky Yellow Yellow
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
44.3
+
+
+
+
+
+
+
+
+
+
45.2
+
+
2
+
+
2
2
2
2
2
41.4
ND
ND
+
+
2
2
2
2
2
2
2
*Reported as weakly positive according to Weon et al. (2006) and
Zhang et al. (2010).
(Weon et al., 2006; Zhang et al., 2010; Kim et al., 2015), but
iso-C16 : 0 was only found in strain DJ57T (Table 2).
Biomass for molecular systematic and chemotaxonomic studies
was obtained from R2A broth in shaken flasks at 28 8C for
2 days. The DNA of strain DJ57T was purified on hydroxyapatite according to the procedure of Cashion et al. (1977), and the
DNA G+C content was determined by HPLC according to the
method of Mesbah et al. (1989). The DNA G+C content of the
strain DJ57T was 44.0 mol%. Respiratory quinones of DJ57T
were extracted and identified by HPLC as described by Xie &
Yokota (2003). The respiratory quinone of DJ57T was MK-7.
Polar lipids of strain DJ57T and N. populi CCTCC AB
208238T were analysed by two-dimensional TLC according to
the protocols of Tindall (1990) and Ventosa et al. (1993).
1770
Fatty acid
1
Saturated fatty acids
C14 : 0
–
C15 : 0
3.0
2.2
C16 : 0
Unsaturated fatty acids
2.4
C17 : 1v6c
Branched-chain fatty acids
7.2
anteiso-C15 : 0
2.1
anteiso-C15 : 1 A
iso-C14 : 0
2.6
–
iso-C14 : 0 3-OH
19.5
iso-C15 : 0
15.8
iso-C15 : 1 G
iso-C15 : 0 3-OH
1.0
5.9
iso-C16 : 0
4.9
iso-C16 : 0 G
3.2
iso-C16 : 0 3-OH
iso-C17 : 0 3-OH
9.4
Hydroxy fatty acids
3.1
C15 : 0 2-OH
C16 : 0 2-OH
1.0
1.3
C16 : 0 3-OH
1.4
C17 : 0 2-OH
1.3
C17 : 0 3-OH
Summed feature 3
3.6
2
3
4
5
–
–
1.7
–
2.1
1.9
1.1
1.1
2.1
–
–
2.6
–
–
–
–
5.4
1.2
–
1.0
19.6
20.1
1.5
–
–
–
15.5
2.7
–
–
–
25.2
17.6
2.7
–
–
–
15.9
3.7
–
–
–
30.2
17.5
1.6
–
–
–
11.2
5.3
2.3
–
–
34.3
18.2
1.5
–
–
1.4
24.8
1.9
–
1.0
2.0
1.7
10.7
–
–
2.2
1.3
1.0
10.7
1.4
1.6
3.4
–
–
11.0
–
–
1.4
2.1
–
3.1
*Summed feature 3 comprises C16 : 1v7c and/or iso-C15 : 0 2-OH,
which could not be separated by the MIDI system.
Strain DJ57T and N. populi CCTCC AB 208238T both contained
phosphatidylethanolamine and several unknown aminophospholipids, phospholipids and lipids, which were very similar
to those of N. gongjuensis 5GH22-11T (Kim et al., 2015). Besides,
N. populi CCTCC AB 208238T contained a glycolipid (Fig. 3).
All the data suggested that strain DJ57T was affiliated to the
genus Niastella but had distinguishing characteristics when
compared with the other type strains.
On the basis of phylogenetic inference and distinctive
phenotypic features, it is concluded that strain DJ57T represents a novel species of the genus Niastella, which is
named Niastella vici sp. nov.
Description of Niastella vici sp. nov.
Niastella vici (vi9ci. L. gen. n. vici of a village, where the
type strain was isolated).
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Niastella vici sp. nov.
(a)
(b)
L
L
L
L
PL
PL
PL
PE
L
AL
L
L
PL
PE
AL
AL
L
L
AL
AL
AL
AL
L
L
L
PL
GL
AL
Fig. 3. Two-dimensional thin-layer chromatograms of polar lipids of strain DJ57T (a) and N. populi CCTCC AB 208238T
(b) detected by molybdophosphoric acid after separation by two-dimensional TLC. L, unidentified lipid; AL, unknown
aminolipid; PL, unknown phospholipid; PE, phosphatidylethanolamine; GL, unknown glycolipid.
Cells are filamentous rods, approximately 0.2–0.5 mm wide
and 6–15 mm long (Fig. 2), Gram-reaction-negative, strictly
aerobic, devoid of flagella and exhibit gliding motility. Colonies on R2A agar are smooth, convex, regular and white.
Growth occurs at 20–42 uC (optimal at 28 uC), at pH 5.0–
8.0 (optimal at pH 6.5) and in the absence of NaCl.
Grows on R2A, NA and 1/10 TSA, but not on LB, TSA or
MacConkey agar. Flexirubin-type pigments are not present
and Congo red is not absorbed. Casein and gelatin are
hydrolysed, but starch, Tween 80, Tween 60, Tween 40,
Tween 20, urea, DNA, chitin, tyrosine, agar and CM-cellulose are not. Oxidase- and catalase-negative. Negative for
H2S and indole production. Positive for nitrate reduction
and aesculin hydrolysis and negative for glucose fermentation, arginine dihydrolase and urease. Positive for alkaline
phosphatase, esterase (C4), leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, a-chymotrypsin, acid
phosphatase, naphthol-AS-BI-phosphohydrolase, a-galactosidase, b-galactosidase, a-glucosidase, b-glucosidase, Nacetyl-b-glucosaminidase and a-fucosidase and negative for
esterase lipase (C8), lipase (C14), b-glucuronidase and amannosidase. Positive for acid production from aesculin,
turanose, lyxose, tagatose, D -fucose, L -fucose, D -arabinitol,
gluconate and 5-keto-D -gluconate after 24 h and from D arabinose, galactose, glucose, aesculin, melibiose, sucrose,
trehalose, turanose, lyxose, tagatose, D -fucose, L -fucose, D arabinitol, L -arabinitol, gluconate and 5-keto-D -glunconate
after 48 h. D -Glucose, L -arabinose, D -mannose, L -rhamnose,
maltose and sucrose are assimilated. N-Acetyl- b-glucosamine, L -serine, salicin, melibiose, L -histidine D -mannitol,
potassium glucosamine, capric acid, adipic acid, malic
acid, trisodium citrate, phenylacetic acid, D -ribose, inositol,
itaconic acid, suberic acid, sodium malonate, sodium
acetate, lactic acid, L -alanine, potassium 5-ketogluconate,
glycogen, 3-hydroxybenzoic acid, L -fucose, D -sorbitol,
propionic acid, valeric acid, potassium 2-ketogluconate,
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3-hydroxybutyric acid, 4-hydroxybenzoic acid and L -proline
are not assimilated. The predominant isoprenoid quinone is
MK-7. The major fatty acids are iso-C15 : 0, iso-C17 : 0 3-OH
and iso-C15 : 1 G, anteiso-C15 : 0 and iso-C16 : 0. The polar
lipids are phosphatidylethanolamine, three unknown aminophospholipids, three unknown phospholipids and six
unknown lipids.
The type strain, DJ57T (5KCTC 42474T5CCTCC AB
2015052T), was isolated from farmland soil near a lead and
zinc mine in Yueyang city, Hunan province, PR China.
The DNA G+C content of the type strain is 44.0 mol%.
Acknowledgements
The present study was supported by the National Natural Science
Foundation of China (31470226). We are grateful to Dr Jean
Euzéby (École Nationale Vétérinaire, Toulouse, France) for the
etymology of the novel species name.
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