International Journal of Systematic and Evolutionary Microbiology (2015), 65, 1193–1198
DOI 10.1099/ijs.0.000079
Romboutsia sedimentorum sp. nov., isolated from
an alkaline-saline lake sediment and emended
description of the genus Romboutsia
Yanwei Wang,1 Jinlong Song,2 Yi Zhai,3 Chi Zhang,4 Jacoline Gerritsen,5,6
Huimin Wang,1 Xiaorong Chen,1 Yanting Li,1 Bingqiang Zhao,1 Bin Zhao4
and Zhiyong Ruan1
Correspondence
Zhiyong Ruan
[email protected]
Bin Zhao
[email protected]
1
Key Laboratory of Microbial Resources (Ministry of Agriculture, PR China), Institute of Agricultural
Resources and Regional Planning, CAAS, Beijing 100081, PR China
2
State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of
Sciences, Beijing 100101, PR China
3
Agricultural Engineering Institute, Chongqing Academy of Agricultural Sciences,
Chongqing 401329, PR China
4
State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology,
Huazhong Agricultural University, Wuhan 430070, PR China
5
Laboratory of Microbiology, Wageningen University, Dreijenplein 10, 6703 HB Wageningen,
The Netherlands
6
Winclove Probiotics, Hulstweg 11, 1032 LB Amsterdam, The Netherlands
A Gram-stain-positive, spore-forming, obligately anaerobic bacterium, designated LAM201T, was
isolated from sediment samples from an alkaline-saline lake located in Daqing oilfield, Daqing City,
PR China. Cells of strain LAM201T were non-motile and straight or spiral rod-shapes. Strain
LAM201T was able to utilize glucose, fructose, maltose, trehalose and sorbitol as the sole carbon
source. Acetic acid, ethanol, iso-butanoic acid and iso-valeric acid were the main products of
glucose fermentation. The major fatty acids of LAM201T were C16 : 0 (26.7 %) and C18 : 0
(11.2 %). The main polar lipids were four unknown glycolipids and five unknown phospholipids.
The predominant cell-wall sugars were ribose and galactose. The cell-wall peptidoglycan of strain
LAM201T contained alanine, glycine, glutamic acid and aspartic acid. Sodium sulfite was used as
the electron acceptor. The G+C content of the genomic DNA was 32±0.8 mol%, as determined
by the Tm method. Analysis of the 16S rRNA gene sequence indicated that the isolate belonged
to the genus Romboutsia and was most closely related to Romboutsia lituseburensis DSM 797T
and Romboutsia ilealis CRIBT with 97.3 % and 97.2 % similarities, respectively. The DNA–DNA
hybridization values between strain LAM201T and the two reference strains were 37 % and 31 %,
respectively. On the basis of its phenotypic, phylogenetic and chemotaxonomic characteristics,
strain LAM201T is suggested to represent a novel species within the genus Romboutsia, for
which the name Romboutsia sedimentorum sp. nov. is proposed. The type strain is LAM201T
(5ACCC 00717T5JCM 19607T).
The genus Clostridium belongs to the family Clostridiaceae,
which is one of the largest genera of the prokaryotes, and
was first described by Prazmowski (1880) and has been
adopted on the Approved Lists of Bacterial Names (Skerman
et al., 1980). At the time of writing, the genus Clostridium
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene
sequence of strain LAM201T is KF443808.
Eight supplementary figures are available with the online Supplementary
Material.
000079 G 2015 IUMS
comprises more than 200 species with validly published
names (http://www.bacterio.net/clostridium.html). Recently,
there was a proposal to reclassify a subset of species within the
genus Clostridium to four new genera within the family
Peptostreptococcaceae based on phenotypic and genetic
considerations (Gerritsen et al., 2014). This reclassification
included the proposal of the novel genus Romboutsia to
accommodate Romboutsia lituseburensis, previously named
Clostridium lituseburense, and the type species of the genus,
Romboutsia ilealis. While studying the anaerobic alkane-degrading
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 15:48:45
Printed in Great Britain
1193
Y. Wang and others
bacterial diversity of sediment samples from an alkalinesaline lake located in Daqing oilfield, Daqing City, PR China,
a Romboutsia-like strain, designated LAM201T, was isolated.
On the basis of its phenotypic, phylogenetic and chemotaxonomic characteristics, the new isolate is considered to
represent a novel species of the genus Romboutsia.
Liquid culture medium (consisting of 5.0 g peptone, 1.0 g
yeast extract, 0.5 g L-cysteine, 0.4 g K2HPO4 . 3H2O, 0.5 g
NaCl, 0.1 g MgCl2, 1 mg resazurin and 40 ml salt solution
as DSMZ medium 104, in water, added to a volume of 1 l,
pH 7.0) was used as enrichment medium. Serum bottles
(100 ml) containing 20 ml of liquid enrichment culture
medium were autoclaved for 30 min at 121 uC. Sediment
samples (10 %, w/v) were inoculated into serum bottles
under an O2-free N2 atmosphere and incubated at 30 uC
for 10 days. The Hungate technique (Hungate, 1969; Bryant,
1972) was adopted to isolate single strains from the enrichment samples using brain heart infusion (BHI) agar (BD/
Difco 241830). Strain LAM201T was purified at least twice
before being preserved in 25 % (v/v) glycerol at 280 uC for
further studies.
The strain grew well in BHI broth (BD/BBL 211059) at
35 uC; the OD600 increased to 0.5 after 24 h of growth. The
morphological characteristics of cells in the exponential
growth phase were examined after 48 h of growth at 35 uC
using a light microscope (Nikon 80i) and transmission
electron microscope (Hitachi 7500) (Ruan, et al., 2014).
The Gram-staining reaction was conducted according to
the method described by Smibert & Krieg (1994). Sporeforming ability was observed after heat treatment at 80 uC
for 10 min by light microscopy (Nikon 80i). The motility
of strain LAM201T was examined from the detection of
turbidity through Hungate anaerobic tubes containing
semi-solid tryptic soy broth (TSB) (BD/Difco 211825) after
48 h of growth at 35 uC. The optimal growth conditions
for strain LAM201T were determined using BHI broth. All
tests were conducted independently and in duplicate. The
strain was incubated in Hungate tubes at: temperatures of
10 uC, 15 uC, 20 uC, 25 uC, 30 uC, 35 uC, 40 uC, 45 uC and
50 uC; pH values of 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 and 10,
and NaCl concentrations of 0 %, 1 %, 2 %, 2.5 %, 3 %,
3.5 %, 4 %, 4.5 %, 5 %, 5.5 %, 6 %, 6.5 %, 7 %, 8 % and
10 % (w/v). The pH was adjusted to the desired value by
using sterile solutions of citric acid/Na2HPO4 (pH 4.0 to
5.0), MES (pH 5.5 to 6.0), PIPES (pH 6.5 to 7.0), Tricine
(pH 7.5 to 8.5), CAPSO (pH 9.0 to 9.5) or CAPS (pH 10.0
to 11.5), added to a final concentration of 30 mM (Ruan
et al., 2014). Standard physiological identifications and
hydrolysis of casein were carried out according to methods
modified by Cowan & Steel (1965) and Lányi (1987). The
ability to grow under aerobic, trace-O2 and anaerobic conditions at 35 uC for up to 7 days was examined. The basal
medium used for testing physiological characteristics consisted of the following components in water (l21): 2 g
peptone, 1 g yeast extract, 0.6 g CaCl2, 0.1 g NH4Cl, 0.2 g
MgCl2, 0.1 g KCl, 2 g NaCl, 2.5 g NaHCO3, 0.5 g Na2S,
7.2 g HEPES, 0.5 g L-cysteine, 1 ml trace element solution
1194
as DSMZ medium 141 and 1 mg resazurin. The medium
was adjusted to pH 7.0 and sterilized by autoclaving at
121 uC for 30 min. By adding 20 mM of the respective
sugar to the basal medium growth on the following carbon
sources was examined: glucose, fructose, xylose, maltose,
arabinose, lactose, rhamnose, sucrose, mannose, ribose,
galactose, cellobiose, trehalose, sorbitol and mannitol. An
increase in the OD600 of medium containing an additional
carbon source, as compared to the basal medium lacking
an additional carbon source (control medium), was considered to represent positive growth. Tests for the utilization
of various energy sources were performed using the API 20A
system (bioMérieux) according to the manufacturer’s instructions. Enzyme activity was examined using the API ZYM
system (bioMérieux). To analyse the reduction of electron
acceptors, sodium nitrite (5 mM), sodium nitrate (20 mM),
sodium thiosulfate (20 mM), sodium sulfite (5 mM) or
sodium sulfate (20 mM) were added from filter-sterilized
solutions to basal medium (lacking the reductant L-cysteine,
Na2S and casamino acids), respectively. Elemental sulfur
(1 %, w/v) and amorphous Fe (III) oxyhydroxide (0.2 %, w/v)
were added to the basal medium immediately before autoclaving (Ramamoorthy et al., 2006). Ethanol, acetic acid, isobutanoic acid, butanoic acid, iso-valeric acid and iso-butanoic
acid from glucose fermentation in the basal medium were
analysed and quantified as described by Steer et al. (2001).
The genomic DNA of strain LAM201T was extracted and
purified using a TIANamp Bacter DNA kit (Tiangen Biotech),
according to the manufacturer’s instructions. The 16S rRNA
gene was amplified by PCR with the bacterial universal
primers, 27F (59-AGAGTTTGATCCTGGCTCAG-39) and
1492R (59-GGTTACCTTGTTACGACTT-39). Purified PCR
products of approximately 1.4 kb were sequenced by the
Majorbio Company (Beijing, China). The EzTaxon-e service
(Kim et al., 2012) and CLUSTAL W (Thompson et al., 1994)
were used to determine sequence similarities and for multiple
sequence alignments. Phylogenetic trees were reconstructed
using the neighbour-joining and maximum-parsimony
methods with the MEGA4 program package (Tamura et al.,
2007). For the neighbour-joining method evolutionary distances were calculated according to the algorithm of Kimura’s
two-parameter model (Kimura, 1980). The DNA G+C
content was determined by the thermal denaturation method
using a Beckman DU 800 spectrophotometer (Beckman
Coulter). Escherichia coli K12 was used as a reference strain.
DNA–DNA reassociation analysis was performed as described by Chang et al. (2008).
Chemotaxonomic analyses were executed on strain LAM201T
and R. lituseburensis DSM 797T. The strains were grown in
TSB medium (BD/Difco 211825). Cells were harvested in the
late exponential phase after growth at 35 uC for 48 h. The
polar lipids were extracted and separated on silica gel plates
(10610 cm, Merck 5554) and further analysed with the
method described by Minnikin et al. (1984) and Xu et al.
(2011). Molybdatophosphoric acid was used to reveal total
polar lipids. Aminolipids were determined using ninhydrin
reagent and phospholipids were identified with Zinzadze
Downloaded from www.microbiologyresearch.org by
International Journal of Systematic and Evolutionary Microbiology 65
IP: 88.99.165.207
On: Sun, 18 Jun 2017 15:48:45
Romboutsia sedimentorum sp. nov.
LAM201T were extracted with the method described by
Minnikin et al. (1984) and analysed by HPLC as described by
Tindall (1990). The cell-wall peptidoglycan was obtained and
purified after 48 h of incubation in TSB medium. Quantitative analysis of the peptidoglycan amino acids was performed by GC (MacKenzie, 1987) and the whole-cell sugars
in the hydrolysate were analysed by TLC, according to
Staneck & Roberts (1974).
Colonies of strain LAM201T grown on BHI agar in Hungate
tubes were round, convex, white and 0.5 mm to 1 mm in
diameter after anaerobic incubation for 36 h at 35 uC. Cells
were non-motile and spore-forming and 1.2 mm–2 mm in
width62.3 mm–10 mm in length (Figs 1 and S1, available in
the online Supplementary Material). Strain LAM201T was
obligately anaerobic and formed free round spores after heat
treatment at 80 uC for 10 min (Fig. S2). The pH and
temperature ranges for growth were pH 6.0–9.0 (optimum:
pH 7.0) and 10 uC–40 uC (optimum: 30 uC), respectively.
The strain grew in medium without NaCl and tolerated
up to 5 % (w/v) NaCl. Strain LAM201T was able to utilize
glucose, fructose, maltose, trehalose and sorbitol as the sole
carbon source. Xylose, arabinose, lactose, rhamnose, sucrose, mannose, ribose, galactose, cellobiose and mannitol
could not be utilized by strain LAM201T. In the API ZYM
system, strain LAM201T gave positive reactions for alkaline
phosphatase, cystine arylamidase and a-chymotrypsin;
weakly positive reactions for a-galactosidase, a-glucosidase
and a-mannosidase, and negative reactions for esterase (C4),
Fig. 1. Transmission electron micrograph of cells of strain
LAM201T. Bar, 0.5 mm.
reagent. The results were analysed as described by Fang et al.
(2012). Analysis of major fatty acids was performed on strain
LAM201T and R. lituseburensis DSM 797T. The fatty acids
were extracted from fresh cells of the two strains, which had
been incubated in TSB medium for 48 h at 35 uC. Identification and quantification of the major acids were performed
using the Sherlock Microbial Identification System with the
standard MIS Library Generation Software (Version 6.0 and
Date 4, Microbial ID). The isoprenoid quinones of strain
Table 1. Differential phenotypic, physiological and genotypic characteristics of strain LAM201T and related strains
Strains: 1, LAM201T; 2, R. lituseburensis DSM 797T; 3, R. ilealis CRIBT. Data for strain LAM201T were all obtained from this study. +, Positive; 2,
negative; W, moderately positive; ND, not determined; GL, glycolipid; PL, phospholipid; L, lipid.
Characteristic
Cell width (mm)
Cell length (mm)
Motility
Gelatin hydrolysis
Utilization of:
Arabinose
Fructose
Galactose
Maltose
Ribose
Sorbitol
Sucrose
Trehalose
Polar lipids
DNA G+C content (mol%)
16S rRNA gene sequence identity
DNA–DNA reassociation
1
2
3D
1.2–2.0
2.3–10.0
2
–
1.4–1.7*
3.1–6.5*
+*
+*
1.0–2.0
1.0–5.3
–
–
–
+
–
+
–
+
–
+
4 GL, 5 PL
32dD
100 %
–D
+D
–D
+D
+D
–D
+D
–D
8GL, 5PL*
30*d
97.3 %
37 %
W
ND
–
W
–
–
–
+
–
6 GL, 4 PL, L
28.1§
97.2 %
31 %
*Data from this study.
DData from Gerritsen et al. (2014).
dDNA G+C content was determined by the Tm method.
§DNA G+C content was determined by HPLC.
http://ijs.sgmjournals.org
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 15:48:45
1195
Y. Wang and others
esterase lipase (C8), lipase (C14), leucine arylamidase, valine
arylamidase, trypsin, acid phosphatase, naphthol-ASBI-phosphohydrolase, b-galactosidase, b-glucuronidase,
b-glucosidase, N-acetyl-b-glucosaminidase and a-fucosidase. In the API 20A system, positive reactions to maltose,
trehalose, sorbitol and glucose were observed. Reactions to
lactose, salicin, xylose, arabinose, glycerol, cellobiose,
mannose, melezitose, raffinose, rhamnose, urea, tryptophan,
gelatin and aesculin were negative. Strain LAM201T could
not hydrolyse casein, while R. lituseburensis DSM 797T
could. A comparison between the physiological characteristics of strain LAM201T and its related reference strains is
shown in Table 1. Sodium sulfite was used as an electron
acceptor. The main end products of glucose fermentation in
the basal medium were acetic acid (65.5 %), ethanol
(16.8 %), iso-butanoic acid (6 %) and iso-valeric acid (4 %).
The 16S rRNA gene sequence (1339 nt) was obtained from
strain LAM201T. Phylogenetic analysis based on 16S rRNA
gene sequences indicated that strain LAM201T is a member
of the genus Romboutsia and is most closely related to R.
lituseburensis DSM 797T and R. ilealis CRIBT with sequence
similarities of 97.3 % and 97.2 %, respectively (Fig. 2). The
DNA–DNA hybridization values between strain LAM201T
and the two reference strains were 37 % and 31 %, respectively. The topologies of the phylogenetic trees reconstructed
by using the maximum-parsimony method support strain
100
0.01
52
LAM201T forming a stable clade with related species (Fig.
S3). The genomic DNA G+C content of strain LAM201T
was 32±0.8 mol%, as determined by the Tm method; this
value is higher than those of R. lituseburensis DSM 797T and
R. ilealis CRIBT.
The major fatty acids of strain LAM201T were C16 : 0 (26.7 %)
and C18 : 0 (11.2 %). The C16 : 0 content of strain LAM201T
was lower than those of R. lituseburensis DSM 797T (29.2 %)
and R. ilealis CRIBT (31.3 %), while the C18 : 0 content was
higher than those of R. lituseburensis DSM 797T (8.5 %) and
R. ilealis CRIBT (6.6 %). The detailed fatty acid compositions of strain LAM201T, R. lituseburensis DSM 797T and R.
ilealis CRIBT are shown in Table 2. The main polar lipids of
strain LAM201T were four unknown glycolipids and five
unknown phospholipids (Figs S4, S5 and S6). The main
polar lipids of strain LAM201T and R. lituseburensis DSM
797T were similar, while differences existed in their relative
contents and other minor compositions (Figs. S4 and S7).
Respiratory quinones were not found in strain LAM201T,
while its predominant cell-wall sugars were ribose and
galactose. The peptidoglycan of strain LAM201T contained
alanine, glycine, glutamic acid and aspartic acid (Fig. S8).
On the basis of this phenotypic, phylogenetic and chemotaxonomic characterization, strain LAM201T is considered
to represent a novel species within the genus Romboutsia, for
which the name Romboutsia sedimentorum sp. nov. is proposed.
Terrisporobacter mayombei DSM 6539T (FR733682)
Terrisporobacter glycolicus DSM 1288T (X76750)
25
Intestinibacter bartlettii DSM 16795T (ABEZ02000021)
Asaccharospora irregularis DSM 2635T (X73447)
70
Romboutsia lituseburensis DSM 797T (AB075770)
Romboutsia sedimentorum LAM201T (KF443808)
97
53
71
Romboutsia ilealis CRIBT (JN381505)
Clostridium bifermentans ATCC 638T (AB075771)
Clostridium sordellii ATCC 9714T (X73447)
99
33
Eubacterium tenue DSM 20695T (FR749984)
96
60
Clostridium ghonii NCIMB 10636T (X73451)
Peptostreptococcus anaerobius ATCC 27337T (L04168)
Clostridium difficile ATCC 9689T (AB075770)
82
100
Clostridium hiranonis TO-931T (AB023970)
Tepidibacter mesophilus B1T (GQ231514)
99
Peptostreptococcus canis CCUG 57081T (HE687281)
Clostridium mangenotii DSM 1289T (FR733662)
Sporacetigenium mesophilum ZLJ115T (AY682207)
69
100
Peptostreptococcus russellii CCUG 58235T (AY167952)
Fig. 2. Neighbour-joining phylogenetic tree based on a comparison of the 16S rRNA gene sequences of strain LAM201T and
its closest relatives. GenBank accession numbers are given in parentheses. Bootstrap values (percentages) based on 1000
replications are shown at nodes. Bar, 0.01 nucleotide substitutions per nucleotide position.
1196
Downloaded from www.microbiologyresearch.org by
International Journal of Systematic and Evolutionary Microbiology 65
IP: 88.99.165.207
On: Sun, 18 Jun 2017 15:48:45
Romboutsia sedimentorum sp. nov.
Table 2. Fatty acid compositions of strain LAM201T and
related strains
Strains: 1: LAM201T (data from this study); 2, R. lituseburensis DSM
797T (this study); 3, R. ilealis CRIBT (Gerritsen et al., 2014). Data are
presented as percentages of the total fatty acid contents. Major
components of each strain are indicated in bold type. TR, Trace
amounts (less than 1.5 %). ND, not detected.
Fatty acid
C12 : 0
C14 : 0
C15 : 0
C16 : 0
C17 : 0
C18 : 0
C 16 : 1v9c
C 16 : 1v11c
C 17 : 1v9c
C 18 : 1v9c
Summed Features*
3
8
1
2
3§
2.4
5.4
4.2
26.7
8.0
11.2
4.5
1.7
6.9
6.5
29.2
11.0
8.5
1.6
3.8
6.2
31.3
9.9
6.6
6.5
TR
5.9
5.0
5.2
ND
4.8
7.4
3.7
9.9
3.1
11.1
TR
TR
ND
3.4
ND
*Summed Feature 3 includes C16 : 1v7c and/or C16 : 1v6c. Summed
Feature 8 includes C18 : 1v7c and/or C18 : 1v6c.
Emended description of the genus Romboutsia
Gerritsen 2014
The description of the genus is as given by Gerritsen et al.
(2014) with the following modifications. The DNA G+C
content is 27–32 mol%.
Description of Romboutsia sedimentorum
sp. nov.
Romboutsia sedimentorum (se.di.men.to9rum. L. gen. pl. n.
sedimentorum of sediments, pertaining to the isolation
source).
Gram-stain-positive, non-motile, spore-forming, obligately
anaerobic and straight or spiral rod-shaped. Cells are
1.2 mm–2 mm in width62.3 mm–10 mm in length. The pH
and temperature ranges for growth are pH 6.0–9.0
(optimum: pH 7.0) and 10 uC–40 uC (optimum: 30 uC),
respectively. Grows in medium without NaCl and can
tolerate up to 5 % (w/v) NaCl. Able to utilize glucose,
fructose, maltose, trehalose and sorbitol as the sole carbon
source. Gives positive reactions to alkaline phosphatase,
cystine arylamidase and a-chymotrypsin in the API ZYM
system. The end products of glucose fermentation in basal
medium are acetic acid, ethanol, iso-butanoic acid and isovaleric acid. The major fatty acids are C16 : 0 and C18 : 0. The
predominant cell-wall sugars are ribose and galactose.
The main polar lipids are four unknown glycolipids and
five unknown phospholipids. No respiratory quinone is
detected.
http://ijs.sgmjournals.org
The type strain is LAM201T (5ACCC 00717T5JCM
19607T), which was isolated from sediment samples of an
alkaline-saline lake located in the Daqing oilfield, Daqing
City, PR China. The genomic DNA G+C content of the
type strain is 32±0.8 mol%, as determined by the Tm
method.
Acknowledgements
Many thanks to Professor Min Wu from Zhejiang University for his
technical assistance. This work was supported by National Key
Technology R&D Program of China (no. 2013BAD05B04F02 and
2011BAD11B05), National Natural Science Foundation of China
(NSFC, no. 31270159), Foundation of the Key Laboratory of
Development and Application of Rural Renewable Energy (MOA,
China) (no. 2013002), and Science Foundation of Modern Farming
Group (no. MF20100518).
References
Bryant, M. P. (1972). Commentary on the Hungate technique
for culture of anaerobic bacteria. Am J Clin Nutr 25, 1324–
1328.
Chang, Y.-H., Jung, M. Y., Park, I.-S. & Oh, H.-M. (2008).
Sporolactobacillus vineae sp. nov., a spore-forming lactic acid
bacterium isolated from vineyard soil. Int J Syst Evol Microbiol 58,
2316–2320.
Cowan, S. T. & Steel, K. J. (1965). Manual for the Identification of
Medical Bacteria. London: Cambridge University Press.
Fang, M.-X., Zhang, W.-W., Zhang, Y.-Z., Tan, H.-Q., Zhang, X.-Q., Wu,
M. & Zhu, X.-F. (2012). Brassicibacter mesophilus gen. nov., sp. nov., a
strictly anaerobic bacterium isolated from food industry wastewater.
Int J Syst Evol Microbiol 62, 3018–3023.
Gerritsen, J., Fuentes, S., Grievink, W., van Niftrik, L. A., Tindall, B. J.,
Timmerman, H. M., Rijkers, G. T. & Smidt, H. (2014).
Characterization of Romboutsia ilealis gen. nov., sp. nov., isolated
from the gastro-intestinal tract of a rat, and proposal for the
reclassification of five closely related members of the genus
Clostridium into the genera Romboutsia gen. nov., Intestinibacter
gen. nov., Terrisporobacter gen. nov. and Asaccharospora gen. nov. Int
J Syst Evol Microbiol 64, 1600–1616.
Hungate, R. E. (1969). A roll tube method for cultivation of strict
anaerobes. Methods Microbiol 3B, 117–132.
Kim, O. S., Cho, Y. J., Lee, K., Yoon, S. H., Kim, M., Na, H., Park, S. C.,
Jeon, Y. S., Lee, J. H. & other authors (2012). Introducing EzTaxon-
e: a prokaryotic 16S rRNA gene sequence database with phylotypes
that represent uncultured species. Int J Syst Evol Microbiol 62, 716–
721.
Kimura, M. (1980). A simple method for estimating evolutionary
rates of base substitutions through comparative studies of nucleotide
sequences. J Mol Evol 16, 111–120.
Lányi, B. (1987). Classical and rapid identification methods for
medically important bacteria. Methods Microbiol 19, 1–67.
MacKenzie, S. L. (1987). Gas chromatographic analysis of amino
acids as the N-heptafluorobutyryl isobutyl esters. J Assoc Off Anal
Chem 70, 151–160.
Minnikin, D. E., O’Donnell, A. G., Goodfellow, M., Alderson, G.,
Athalye, M., Schaal, A. & Parlett, J. H. (1984). An integrated
procedure for the extraction of bacterial isoprenoid quinones and
polar lipids. J Microbiol Methods 2, 233–241.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 15:48:45
1197
Y. Wang and others
Prazmowski, A. (1880). Untersuchung über die Entwickelungsgeschichte
Staneck, J. L. & Roberts, G. D. (1974). Simplified approach to
und Fermentwirking einiger Bacterien-Arten. Inaugural Dissertation.
Leipzig: Hugo Voigt, pp. 1–58.
identification of aerobic actinomycetes by thin-layer chromatography.
Appl Microbiol 28, 226–231.
Ramamoorthy, S., Sass, H., Langner, H., Schumann, P.,
Kroppenstedt, R. M., Spring, S., Overmann, J. & Rosenzweig, R. F.
(2006). Desulfosporosinus lacus sp. nov., a sulfate-reducing bacterium
Steer, T., Collins, M. D., Gibson, G. R., Hippe, H. & Lawson, P. A.
(2001). Clostridium hathewayi sp. nov., from human faeces. Syst Appl
isolated from pristine freshwater lake sediments. Int J Syst Evol
Microbiol 56, 2729–2736.
Tamura, K., Dudley, J., Nei, M. & Kumar, S. (2007). MEGA4: molecular
Ruan, Z., Wang, Y., Song, J., Jiang, S., Wang, H., Li, Y., Zhao, B., Jiang,
R. & Zhao, B. (2014). Kurthia huakuii sp. nov., isolated from biogas
Microbiol 24, 353–357.
evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol
Evol 24, 1596–1599.
Skerman, V. B. D., V. McGowan & P. H. A. Sneath (ed.). (1980).
CLUSTAL W:
improving the sensitivity of progressive multiple sequence alignment
through sequence weighting, position-specific gap penalties and
weight matrix choice. Nucleic Acids Res 22, 4673–4680.
Approved lists of bacterial names. Int J Syst Bacteriol 30, 225–
420.
Tindall, B. J. (1990). Lipid Composition of Halobacterium lacusprofundi. FEMS Microbiol Lett 66, 199–202.
Smibert, R. M. & Krieg, N. R. (1994). Phenotypic characterization. In
Xu, X.-W., Huo, Y.-Y., Wang, C.-S., Oren, A., Cui, H.-L., Vedler,
E. & Wu, M. (2011). Pelagibacterium halotolerans gen. nov., sp. nov. and
slurry, and emended description of the genus Kurthia. Int J Syst Evol
Microbiol 64, 518–521.
Methods for General and Molecular Bacteriology, pp. 607–654. Edited
by P. Gerhardt, R. G. E. Murray, W. A. Wood & N. R. Krieg.
Washington, DC: American Society for Microbiology.
1198
Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994).
Pelagibacterium luteolum sp. nov., novel members of the
family Hyphomicrobiaceae. Int J Syst Evol Microbiol 61, 1817–1822.
Downloaded from www.microbiologyresearch.org by
International Journal of Systematic and Evolutionary Microbiology 65
IP: 88.99.165.207
On: Sun, 18 Jun 2017 15:48:45