International Journal of Systematic and Evolutionary Microbiology (2015), 65, 412–417
DOI 10.1099/ijs.0.064428-0
Rhizobium yantingense sp. nov.,
a mineral-weathering bacterium
Wei Chen, Xia-Fang Sheng, Lin-Yan He and Zhi Huang
Correspondence
Xia-Fang Sheng
Key Laboratory of Agricultural Environmental Microbiology, Ministry of Agriculture, College of Life
Sciences, Nanjing Agricultural University, Nanjing 210095, PR China
[email protected]
A Gram-stain-negative, rod-shaped bacterial strain, H66T, was isolated from the surfaces of
weathered rock (purple siltstone) found in Yanting, Sichuan Province, PR China. Cells of strain
H66T were motile with peritrichous flagella. Phylogenetic analysis based on 16S rRNA gene
sequences indicated that strain H66T belongs to the genus Rhizobium. It is closely related to
Rhizobium huautlense SO2T (98.1 %), Rhizobium alkalisoli CCBAU 01393T (98.0 %) and
Rhizobium cellulosilyticum ALA10B2T (98.0 %). Analysis of the housekeeping genes, recA, glnII
and atpD, showed low levels of sequence similarity (,92.0 %) between strain H66T and other
recognized species of the genus Rhizobium. The predominant components of the cellular fatty
acids were summed feature 8 (C18 : 1v7c and/or C18 : 1v6c) and C16 : 0. The G+C content of
strain H66T was 60.3 mol%. Strain H66T is suggested to be a novel species of the genus
Rhizobium based on the low levels of DNA–DNA relatedness (ranging from 14.3 % to 40.0 %)
with type strains of species of the genus Rhizobium and on its unique phenotypic characteristics.
The namehttp://dx.doi.org/10.1601/nm.1279Rhizobium yantingense sp. nov. is proposed for this
novel species. The type strain is H66T (5CCTCC AB 2014007T5LMG 28229T).
The genus Rhizobium was originally described as comprising stem and/or root nodule associated bacteria (Frank,
1889). Rhizobia are soil bacteria that fix nitrogen after
becoming established inside the root nodules of legumes.
At the time of writing the genus Rhizobium comprised
over 71 recognized species (http://www.bacterio.cict.fr/qr/
rhizobium.html). It is well established that in addition to
being symbiotically associated with legumes, a rather large
population of non-symbiotic rhizobia are found in the
bulk soil, the rhizospheres of legumes and other plants
(Chaintreuil et al., 2000; Peng et al., 2008; Berge et al.,
2009; Zhang et al., 2011). Rhizobia have also been isolated
from infertile or polluted environments such as beach sand,
wastewater and oil-contaminated soil (Ramana et al., 2013;
Yan et al., 2010; Zhang et al., 2012). Apart from their
capacity for nitrogen fixation, species of rhizobia can also
promote the growth of different plants including leguminous and nonleguminous plants, and weather silicate
minerals (Tan et al., 2001; Yanni et al., 1997; Zhao et al.,
2013). In this study we describe strain H66T, and propose
that it is the type strain of a novel species of the genus
Rhizobium.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene
sequence of strain H66T is KC934840. The GenBank/EMBL/DDBJ
accession number for the recA, atpD and glnII sequences of strain H66T
are KM029983, KM029981 and KM029982, respectively.
Three figures and two tables are available with the online Supplementary
Material.
412
Strain H66T was isolated from the surfaces of weathered
rock (purple siltstone) collected from Yanting, Sichuan
Province, PR China (31u 169 N 105u 279E ). The medium
used for isolation contained (l21): 10.0 g sucrose, 1 g
(NH4)2SO4, 0.1 g NaCl, 0.5 g MgSO4 . 7H2O, 2 g K2HPO4,
0.5 g yeast extract, 0.1 g CaCO3, and 20 g agar, pH 7.2.
Weathered rock samples were added to flasks containing
physiological salt solution (0.85 %, w/v, NaCl) and were
shaken at 200 r.p.m. for 40 min to allow bacteria to
detach from the rock particles. The suspensions were then
allowed to stand for about 15 min. Serial 10-fold dilutions
of sample suspensions (1023–1025) were plated onto agar
plates to determine total culturable bacteria. The plates
were incubated for 3 days at 28 uC. Strain H66T was picked
and was able to weather biotite and feldspar (two kinds of
silicate minerals). Mineral dissolution experiments showed
that the amounts of Si, Al and Fe released from minerals by
strain H66T were between 2.7 and 3.7-fold, 2.5 and 3.5-fold
and 9.6 and 17-fold greater than uninoculated controls,
respectively. The strain was routinely cultured on YMA
medium (containing mannitol 10 g, K2HPO4 0.25 g,
KH2PO4 0.25 g, MgSO4 . 7H2O 0.2 g, NaCl 0.1 g, yeast
extract powder 0.8 g, agar 15 g) (Vincent, 1970) at 30 uC
for additional taxonomic experiments. The strain was
maintained as a glycerol suspension (40 %, v/v) at –80 uC.
Colonies of strain H66T were circular, convex, white and
opaque with a colony diameter of 3 mm after growth on
YMA agar at 30 uC for 3 days. Cellular morphology and the
presence of flagella were examined using light microscopy
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Rhizobium yantingense sp. nov.
(CX21; Olympus) and transmission electron microscopy
(H-7650; Hitachi) as described by Bogan et al. (2003) and
Zhang et al. (2008). The cells of Strain H66T were Gramreaction-negative, aerobic and non-spore-forming. Cells were
rod-shaped with a width of 0.8 mm and length of 2.8 mm
(Fig. S1, available in the online Supplementary Material).
Growth in YMA liquid medium at different temperatures
(4, 15, 30, 40, and 50 uC) and at pH 5.0–11.0 (at intervals
of 1.0) was assayed after 5 days of incubation. The buffers
used to control the pH of the YMA medium (each at a
final concentration of 100 mM) were acetic acid/acetate
(for pH 5.0), KH2PO4/NaOH (for pH 6.0–8.0), NaHCO3/
Na2CO3 (for pH 9.0–10.0) and Na2HPO4/NaOH (for
pH 11.0). Salt tolerance was tested in YMA broth
supplemented with 0–5 % (w/v) NaCl (at intervals of
0.5 %, w/v) after 5 days of incubation at 30 uC. The test for
utilization of different carbon sources was carried out using
a previously described method (Gao et al., 1994). Enzyme
activities were determined by using API ZYM according to
the manufacturer’s instructions (bioMérieux). Susceptibility
to antibiotics was tested on YMA plates containing the
following concentrations of antibiotics: ampicillin, 50 mg ml21,
100 mg ml21, 300 mg ml21; erythromycin, 10 mg ml21,
50 mg ml21, 100 mg ml21; kanamycin, 5 mg ml21,
25 mg ml21, 50 mg ml21; bacitracin, 300 mg ml21; streptomycin, 5 mg ml21; neomycin, 50 mg ml21; tetracycline,
5 mg ml21 (Gao et al., 1994). Phenotypic characteristics
clearly distinguish strain H66T from closely related species of
the genus Rhizobium (Table 1). Results for carbon and
nitrogen sources are given in the species description. Strain
H66T could use starch as the sole carbon source and urea as
the sole nitrogen source, but reference strains of related
species of the genus Rhizobium could not utilise either.
Cellular fatty acids of strain H66T and the type strains of
related species were analysed by using colonies grown on
YMA agar for 48 h at 30 uC. The fatty acid methyl ester
mixtures were separated using the Sherlock Microbial
Identification System (MIS) (MIDI, Microbial ID), which
consisted of a GC (6890N; Agilent) fitted with a 5 %
phenyl-methyl silicone capillary column (0.2 mm625 m),
a flame-ionization detector, an automatic sampler (7683A;
Agilent) and a computer (Hewlett Pacard). The results
were compared with the database (TSBA6) of fatty acids in
the Sherlock Microbial Identification System (MIS). The
cellular fatty acid profiles of strain H66T and related
species of the genus Rhizobium are shown in Table S1. The
main fatty acids of strains H66T are summed feature 8
(comprising C18 : 1v7c and/or C18 : 1v6c) and C16 : 0. Strain
H66T had a similar cellular fatty acid profile to the reference
strains of species of the genus Rhizobium. However, strain
H66T could be differentiated by the presence of summed
feature 1 (C13 : 1 AT 12-13) and summed feature 5 (C17 : 1v8c
and C17 : 1v6c).
Total DNA from each strain was prepared as described by
Terefework et al. (2001), and then used as the template in
the amplification of the 16S rRNA gene, atpD (ATP
http://ijs.sgmjournals.org
Table 1. Differential phenotypic characteristics between strain
H66T and the type strains of species of the genus Rhizobium
Strains: 1, H66T; 2, R. huautlense SO2T; 3, R. alkalisoli CCBAU
01393T; 4, R. cellulosilyticum ALA10B2T ; 5, R. vignae CCBAU 05716T;
6, R. galegae ATCC 43677T. Characteristics of the reference strains
were analysed in this study and the results were the same as those
from Lindstrom (1989), Lu et al. (2009), Wang et al. (1998), Ren et al.
(2011) and Garcı́a-Fraile et al. (2007). +, Positive; 2, negative, S,
sensitive; R, resistant.
Characteristic
Used as sole carbon source:
D-Arabitol
Maltose
Turanose
Dulcitol
Gluconate
Malic acid
Melibiose
Sorbin
Starch
Used as sole nitrogen source:
L-Arginine
N-Acetyl-glucosamine
Urea
Resistance to (mg ml21):
Ampicillin (100)
Bacitracin (300)
Erythromycin (50)
Neomycin sulfate (50)
Growth in NaCl (3 %, w/v)
Growth at pH 10.0
DNA G+C content (mol%)
1
2
3
4
5
6
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
2
2
2
R
S
R
R
S
R
R
S
S
R
R
R
S
R
R
S
S
R
R
S
S
R
S
R
+
2
+
+
2 2
+
+
+
+
2 2
60.3 57.0 65.5 57.0 60.8 63.0
synthase subunit beta), recA (recombinase A protein) and
glnII (glutamine synthetase II) housekeeping genes.
Strain H66T was identified as a member of the genus
Rhizobium based on the pairwise analysis of the 1430 bp 16S
rRNA gene sequence using the EzTaxon server (Kim et al.,
2012). The sequences of the 16S rRNA gene of other species
of the genus Rhizobium were obtained from the GenBank
database, and multiple alignment analysis was performed
using the CLUSTAL W program, version 1.81 (Thompson et al.,
1994). Phylogenetic trees were reconstructed using the
neighbour-joining, maximum-likelihood and minimumevolution methods according to the procedures in the
program MEGA version 4 (Tamura et al., 2007). Evolutionary
distances were calculated using the Kimura two-parameter
model (Kimura, 1980). The confidence levels of the clusters
were determined by using bootstrap analyses (Felsenstein,
1985) based on 1000 resamplings. As no significant
topological differences were found between the phylogenetic
trees reconstructed using the two methods, only the
tree reconstructed by using the NJ method after distance
analysis of aligned sequences is shown (Fig. 1). The
maximum-likelihood and minimum-evolution trees are
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W. Chen and others
89 Rhizobium vignae CCBAU 05176T (GU128881)
Rhizobium galegae ATCC 43677T (D11343)
100
Rhizobium alkalisoli CCBAU 01393T (EU074168)
0.02
Rhizobium huautlense S02T (AF025852)
Rhizobium yantingense H66T (KC934840)
Rhizobium cellulosilyticum ALA10B2T (DQ855276)
96
Rhizobium loessense CCBAU 7190BT (AY034029)
75
Rhizobium tubonense CCBAU 85046T (EU256434)
Rhizobium soli DS-42T (EF363715)
T
100 Rhizobium alamii GBV016 (AM931436)
52
98
97
Arthrobacter viscosus LMG 16473T (AJ639832)
Rhizobium indigoferae CCBAU 71042T (AF364068)
Rhizobium gallicum R602spT (U86343)
89
Rhizobium yanglingense SH 22623T (AF003375)
84
Rhizobium mongolense USDA 1844T (U89817)
50
Candidatus Rhizobium massiliae 90AT (AF531767)
Rhizobium vitis NCPPB 3554T (D14502)
100
Rhizobium taibaishanense CCNWSX 0483T (HM776997)
Novosphingobium panipatense SM16T (EF424402)
Fig. 1. Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences showing the position of strain H66T and
recognized species of the genus Rhizobium. Bootstrap values (expressed as percentages of 1000 replications) .50 % are
shown at the nodes. Novosphingobium panipatense SM16T was used as an outgroup. Bar, 0.02 substitutions per nucleotide
position.
shown in Figs S2 and S3. The 16S rRNA gene sequence of
strain H66T was closely related to Rhizobium huautlense
S02T, Rhizobium. alkalisoli CCBAU 01393T, Rhizobium
cellulosilyticum ALA10B2T, Rhizobium vignae CCBAU
05716T and Rhizobium galegae ATCC 43677T with gene
sequence similarities of 98.1 %, 98. 0 %, 98.0 %, 97.8 % and
97.5 %, respectively.
Housekeeping genes (atpD, glnI, glnII, recA, dnaK etc.)
throughout the genome are being used increasingly to
investigate the phylogeny of bacteria. We determined
partial atpD, recA and glnII sequences for strain H66T using
the methods reported previously (Martens et al., 2007, 2008).
Distances were calculated and clustering was performed
with the neighbour-joining algorithm as described above.
Bootstrap analysis was performed using 1000 replications. In
this study PCR amplifications and sequencing of partial atpD
(510 bp) and recA (530 bp) genes were performed according
to Gaunt et al. (2001) and a 660 bp intragenic fragment of
glnII was amplified according to Turner & Young (2000).
Sequence similarities of atpD, recA and glnII genes between
strain H66T and reference strains are given in Table S2.
Neighbour-joining trees reconstructed based on these
sequences are shown in Fig. 2. The atpD gene sequence of
strain H66T showed highest similarities (92 %) to those of
R. alkalisoli CCBAU 01393T, R. cellulosilyticum ALA10B2T
and R. galegae ATCC 43677T. The recA and glnII gene
sequences of strain H66T showed less than 89 % similarities
with those of the reference strains. The low similarities found
between the housekeeping gene sequences of strain H66T and
those of known sequences held in the GenBank database
414
indicate that strain H66T represents a separate species of the
genus Rhizobium.
The DNA G+C content of strain H66T was determined by
the thermal denaturation method (Marmur & Doty, 1962)
using Escherichia coli K-12 as a reference. DNA–DNA
hybridizations among strain H66T and type strains of
the genus Rhizobium were determined using a UV/VIS
spectrophotometer (UV1201; Rayleigh) as described by De
Ley et al. (1970). The genomic DNA G+C content of
strain H66T was 60.3 mol%, which was in the range previously determined for members of the genus Rhizobium
(57–66 %; Jordan, 1984). DNA–DNA hybridization studies
showed relatively low relatedness values of strain H66T
with R. huautlense S02T (14.3 %), R. alkalisoli CCBAU
01393T (26.4 %), R. cellulosilyticum ALA10B2T (27.3 %),
R. vignae CCBAU 05716T (40.0 %) and R. galegae ATCC
43677T (40.0 %). All of the values were significantly lower
than 70 %, the threshold value recommended for the
assignment of genomic species (Wayne et al., 1987). These
results indicate that strain H66T represents a novel species
of the genus Rhizobium.
Nodulation and nitrogen fixation abilities are important
characteristics for Rhizobium species and the host range is
an important feature for the description of novel rhizobial
species (Graham et al., 1991). In this study, the nifH and
nodC genes of strain H66T were analysed according to the
methods of Ueda et al. (1995) and Laguerre et al. (2001).
However, the nifH and nodC genes could not be amplified
from strain H66T.
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Rhizobium yantingense sp. nov.
T
72 Rhizobium yanglingense CCBAU 01590 (AY907359)
T
72 Rhizobium gallicum R602sp (AY907357)
35 Rhizobium mongolense USDA 1844T (AY907358)
(a)
0.2
Rhizobium leguminosarum USDA 2370T (AJ294376)
26
Rhizobium cellulosilyticum ALA10B2T (AM286427)
23
Rhizobium lusitanum p1-7T (DQ431674)
Rhizobium leucaneae USDA 9039T (AJ294372)
Rhizobium yantingense H66T (KM029983)
66
99
Rhizobium galegae ATCC 43677T (AM182127)
Rhizobium huautlense S02T (AY688601)
Rhizobium alkalisoli CCBAU 01393T (EU672490)
Rhizobium vignae CCBAU 05176T (GU128888)
(b)
42
0.1
73 Rhizobium yanglingense SH 22623T (AY907359)
77 Rhizobium gallicum R602spT (AY907357)
Rhizobium mongolense USDA 1844T (AY907358)
100
Rhizobium leguminosarum USDA 2370T (AJ294376)
Rhizobium leucaneae USDA 9039T (AJ294372)
Rhizobium lusitanum p1-7T (DQ431674)
44
Rhizobium rhizogenes NCPPB 2991T (AJ294374)
39
Rhizobium alkalisoli CCBAU 01393T (EU672461)
76
Rhizobium vignae CCBAU 05176T (GU128888)
Rhizobium galegae ATCC 43677T (AM418779)
57
52
Rhizobium huautlense S02T (AY688589)
93
Rhizobium yantingense H66T (KM029981)
Rhizobium cellulosilyticum ALA10B2T (AM286426)
(c)
0.01
99
Rhizobium galegae ATCC 43677T (AF169587)
Rhizobium leguminosarum USDA 2370T (AF169586)
48
21
Rhizobium vignae CCBAU 05176T (GU128895)
Rhizobium etli HBR19T (JN580697)
47
Rhizobium huautlense CCBAU 65679 (EU618011)
Rhizobium alkalisoli CCBAU 01393T (EU672475)
64
100 Rhizobium yanglingense SH 22623T (AY929462)
Rhizobium gallicum bv. gallicum USDA 3736T (AY929427)
Rhizobium lusitanum p1-7T (EF639841)
25
Rhizobium leucaneae USDA 9039T (AJ294372)
85
88
Rhizobium rhizogenes LMG152 (AY929470)
Rhizobium yantingense H66T (KM029982)
Fig. 2. Neighbour-joining phylogenetic trees based on partial recA (a), atpD (b), and glnII (c) gene sequences showing the
position of strain H66T and recognized species of the genus Rhizobium. Bootstrap values (expressed as percentages of 1000
replications) are shown at each node. Bars, 0.2, 0.1 or 0.01 substitutions per nucleotide position.
A nodulation test was carried out as described by Zhang
et al. (2012). Six legume plant species were selected:
Leucaena leukocephala, Phaseolus vulgaris, Pisum sativum,
Medicago sativa, Astragalus smicus and Vicia faba. Strain
H66T could not nodulate any of the plants tested.
during long-term evolution; they may be lost or horizontally transferred across species boundaries (Kuykendall et al.,
2005). This may explain why strain H66T could not form
nodules with legume plants and why the nifH and nodC
genes could not be amplified from strain H66T.
Many well-defined nitrogen fixation genes are clustered on
large plasmids or MEGA plasmids, which are not very stable
Phylogenetic analysis, enzyme activities and differences
in other physiological and biochemical characteristics
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W. Chen and others
(Table 1) together with the fatty acid profile (Table S1)
clearly distinguish strain H66T from closely related species
of the genus Rhizobium. Thus, on the basis of the data
presented, we suggest that strain H66T represents a novel
species of the genus Rhizobium, for which the name
Rhizobium yantingense H66T sp. nov. is proposed.
natural endophytes of the African wild rice Oryza breviligulata. Appl
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Description of Rhizobium yantingense sp. nov.
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Cells are Gram-stain-negative, aerobic, non-motile, nonspore-forming and rod-shaped (0.6–0.8 mm61.2–2.8 mm).
Colonies are circular, convex, white and opaque with a
colony diameter of 3 mm after growth on YMA agar at
28 uC for 3 days. Growth occurs at 10–40 uC with an
optimum temperature of 28 uC. Growth occurs at pH 7.0–
10.0 (optimum, pH 7.0) and with 0–3 % (w/v) NaCl.
Positive for alkaline phosphatase, esterase, leucine arylamidase, trypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase and a-glucosidase activities. Negative for
b-galactosidase and b-glucosidase activities. Oxidase- and
urease-positive, but catalase-negative. Nitrate is not reduced
to either nitrite or nitrogen gas. Utilizes D-arabinose, Larabinose, D-manntitol, D-xylose, D-galactose, D-fructose, Dglucose, D-mannose, aesculin ferric citrate, maltose and
malic acid as single carbon sources. Utilizes urea, N-acetylglucosamine, L-aspartic acid, -L-glutamic acid, D-glutamic
acid, L-alanine, DL-threonine, hypoxanthine, L-cystine, Lisoleucine, L-lysine, L-phenylalanine, L-threonine, L-methionine and L-valine as sole nitrogen sources. The major fatty
acids (.10 % of total) are summed feature 8 (comprising
C18 : 1v7c and/or C18 : 1v6c) and C16 : 0.
The type strain is H66T (5CCTCC AB 2014007T5LMG
28229T), which was isolated from the surfaces of weathered
rock (purple siltstone) in Yanting (Sichuan, PR China). It
can weather silicate minerals and release Si, Al, and Fe from
silicate minerals. The genomic DNA G+C content of the
type strain is 60.3 mol%.
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This work was supported by National Natural Science Foundation of
China (project no. 41071173, 41473075).
Lu, Y. L., Chen, W. F., Li Han, L., Wang, E. T. & Chen, W. X. (2009).
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