International Journal of Systematic and Evolutionary Microbiology (2008), 58, 2169–2173
DOI 10.1099/ijs.0.65511-0
Halolactibacillus alkaliphilus sp. nov., a moderately
alkaliphilic and halophilic bacterium isolated from a
soda lake in Inner Mongolia, China, and emended
description of the genus Halolactibacillus
Shu-Juan Cao,1 Jian-Hang Qu,1 Jin-Shui Yang,1 Qing Sun2
and Hong-Li Yuan1
Correspondence
Hong-Li Yuan
[email protected]
1
College of Biological Sciences, Key Laboratory of Agro-Microbial Resource and Application,
Ministry of Agriculture, China Agricultural University, Beijing 100193, PR China
2
National Research Center for GeoAnalysis, Chinese Academy of Geological Sciences, Beijing
100037, PR China
A moderately alkaliphilic and halophilic bacterium was isolated from sediment of Xiarinaoer soda
lake located in the Inner Mongolia municipality. This bacterium, designated strain H-5T, was a
facultative anaerobe, Gram-positive, rod-shaped and non-motile. Strain H-5T grew in complex
medium with 0.5–30 % (w/v) NaCl and at pH 7.5–13. The cell wall peptidoglycan contained
meso-diaminopimelic acid. The major isoprenoid quinones found in this strain were MK-9H4 and
MK-9H2, and the major cellular fatty acids were C16 : 0 and anteiso-C13 : 0. The DNA G+C
content of strain H-5T was 38.3 mol%. Phylogenetic analysis based on 16S rRNA gene
sequences revealed that strain H-5T was located in the genus Halolactibacillus. The 16S rRNA
gene sequence similarities between strain H-5T and the type strains of the two recognized
species of the genus Halolactibacillus were 98.6 and 98.0 %. The DNA–DNA relatedness values
between strain H-5T and the two type strains were 19 and 5 %. Based on the phenotypic and
chemotaxonomic data, the phylogenetic analysis and genomic distinctiveness, strain H-5T is
considered to represent a novel species of the genus Halolactibacillus, for which the name
Halolactibacillus alkaliphilus is proposed. The type strain is H-5T (5CGMCC AS 1.6843T
5NBRC 103919T).
Soda lakes represent the most alkaline, naturally occurring
environments on earth, with pH values generally greater
than 10, and occasionally reaching 12. These lakes are
characterized by the presence of large amounts of Na2CO3
(usually as Na2CO3 . 10H2O or Na2CO3 . NaHCO3 . 2H2O)
and depletion of Mg2+ and Ca2+ because of the
insolubility of these cations as carbonate minerals under
alkaline conditions. Such lakes are also somewhat saline
due to the concomitant increase in Cl2 (Duckworth et al.,
1996).
In order to investigate alkaliphilic and halophilic bacteria,
we isolated some strains from the sediment of Xiarinaoer
soda lake, which is located in Inner Mongolia, China.
Isolate H-5T was considered to be a Halolactibacillus-like
strain based on the results of the 16S rRNA gene sequence
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene
sequence of strain H-5T is EF554593.
A maximum-parsimony phylogenetic tree based on 16S rRNA gene
sequences is available as a supplementary figure with the online version
of this paper.
65511 G 2008 IUMS
analysis. The genus Halolactibacillus, which contains two
species, Halolactibacillus halophilus and Halolactibacillus
miurensis, isolated from decaying marine algae and living
sponge, was formally proposed by Ishikawa et al. (2005).
The aim of
position of
bacterium,
taxonomic
alkaliphilus
strain.
this study was to describe the exact taxonomic
a novel moderately alkaliphilic and halophilic
designated strain H-5T, using a polyphasic
approach. A novel species, Halolactibacillus
sp. nov., is proposed to accommodate this
Strain H-5T was isolated according to the method of
Horikoshi & Grant (1998). The base complex medium
used for isolation and maintenance contained (per litre
distilled water): 5 g polypeptone, 5 g yeast extract, 10 g
glucose, 1 g K2HPO4, 0.2 g MgSO4, 10 g Na2CO3 and 10 g
NaCl. When required, the medium was solidified by the
addition of agar (18 g l21). The pH was adjusted to 10
using 30 % (w/v) Na2CO3 solution. Incubation was at
28 uC. The recommended media and conditions for growth
were used to culture the reference type strains, including
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2169
S.-J. Cao and others
H. halophilus DSM 17073T and H. miurensis DSM 17074T
(Ishikawa et al., 2005).
To determine cell morphology, bacterial cultures grown on
plates of the base complex medium for about 16 h were
stained using crystal violet and then examined by
microscopy. Spore formation was observed microscopically
with cultures grown for 2 weeks at 28 uC on medium with
the following composition (121): 5 g polypeptone, 5 g
yeast extract, 1 g K2HPO4, 0.2 g MgSO4, trace amount of
MnCl2, 10 g Na2CO3, 20 g NaCl and 18 g agar. Staining of
the cells was performed using the method described by
Dong & Cai (2001). The Gram reaction was determined
using the KOH lysis method (Gregersen, 1978). Motility
was tested using base medium (pH 10) containing 0.15 %
agar; the occurrence of diffused growth along the stab line
was determined (Ishikawa et al., 2003). Colony morphology was examined after incubation for 24 h on solid base
medium. The range of NaCl concentrations for growth was
determined using liquid base media containing 0–30 %
NaCl (w/v). The pH range for growth was determined by
adjusting the pH of the liquid base medium to pH 6.0–14.0
(in increments of 0.5 units) using 30 % (w/v) Na2CO3
solution. The temperature range for growth was determined by incubation in liquid base medium for 1 day to 3
weeks at temperatures of 4–50 uC.
The catalase activity test was performed by placing a small
amount of a culture that had been incubated for 24 h on a
slide and adding a drop of 3 % H2O2 solution. The
presence of gas bubbles indicated a positive reaction.
Oxidase activity was examined by daubing cultures that
had been incubated for 24 h on filter paper soaked with
N,N-dimethyl-p-phenylenediamine solution; the appearance of a red colour within 10 s indicated a positive
reaction. Nitrate reduction and hydrolysis of casein and
gelatin were examined according to the method of Dong &
Cai (2001) with modifications: the pH value was adjusted
to 10, and analysis was performed on days 1–3 of the
incubation. Utilization of carbohydrates was performed
using liquid medium containing (per litre distilled water):
2.0 g (NH4)2SO4, 0.5 g NaH2PO4, 0.5 g K2HPO4, 0.2 g
MgSO4, 0.02 % (w/v) yeast extract and 0.5 % (w/v)
carbohydrate. The incubation temperature used was below
28 uC, and the results were determined spectrophotometrically by measuring the OD600 after 3 days (Ishikawa
et al., 2003). The presence of lactate acid in the
fermentation products was determined by TLC according
to the method of Dong & Cai (2001).
For the analysis of isoprenoid quinones, cells grown
aerobically were freeze-dried and the lipid fraction was
extracted as described by Collins (1985). The presence of
meso-diaminopimelic acid in the cell wall peptidoglycan
was determined using TLC (Hasegawa et al., 1983). For
quantitative analysis of the fatty acid composition, fatty
acid methyl ester mixtures were prepared and identified
following the manufacturer’s instructions for the Microbial
Identification System (MIDI).
2170
The 16S rRNA gene of strain H-5T was amplified using
PCR with individual bacterial colonies (Güssow &
Clackson, 1989). The primers used for PCR were 27F
(59-GAGAGTTTGATCCTGGCTCAG-39) and 1495R (59CTACGGCTACCTTGTTACGA-39) (Escherichia coli numbering system; Brosius et al., 1978). PCR products were
sequenced using DNA Sequencer 3730 with the software
provided by the manufacturer (Applied Biosystems). An
almost-complete 16S rRNA gene sequence of strain H-5T
was obtained (1452 bp) and was compared initially with
reference sequences in the GenBank database by using
BLAST (Altschul et al., 1997). Alignment of sequences was
carried out using CLUSTAL W version 1.8 (Thompson et al.,
1994). The percentage sequence similarities were calculated
using the method of Jukes & Cantor (1969) in the CLUSTAL
W program. Phylogenetic analysis of multiple sequence
alignments was performed using MEGA version 3.1 (Kumar
et al., 2004). Phylogenetic tree construction was carried out
by using the neighbour-joining and maximum-parsimony
methods.
Genomic DNA was prepared according to the method of
Marmur (1961). The DNA G+C content was determined
by using the thermal denaturation method with a BIO-20
UV spectrophotometer, according to De Ley et al. (1970).
The equation of De Ley et al. (1970) was used to calculate
the DNA G+C content and was corrected using genomic
DNA of Escherichia coli K-12 as a reference. DNA–DNA
hybridization experiments were carried out spectrophotometrically (De Ley et al., 1970).
Morphological and physiological characteristics and other
taxonomic features are given in the species description and
Table 1.
According to the similarity searches of the 16S rRNA gene
sequence conducted within GenBank, strain H-5T was most
closely related to members of the genus Halolactibacillus
(98.6–98.0 %). The sequence similarities with members of
other genera were less than 96 %. Pairwise analysis using
CLUSTAL W version 1.8 revealed that the novel isolate
exhibited high similarity values to H. miurensis DSM
17074T (98.6 %), H. halophilus DSM 17073T (98.0 %),
Paraliobacillus ryukyuensis DSM 15140T (95.9 %),
Gracilibacillus halotolerans DSM 11805T (94.4 %),
Gracilibacillus dipsosauri DSM 11125T (94.2 %),
Gracilibacillus boraciitolerans DSM 17256T (93.9 %),
Bacillus oleronius ATCC 700005T (93.0 %), Bacillus oleronius DSM 9356T (92.9 %) and Bacillus acidicola DSM
14754T (92.6 %). The phylogenetic analysis revealed that
strain H-5T was a member of a branch of the genus
Halolactibacillus. The neighbour-joining tree is shown in
Fig. 1; a maximum-parsimony tree is available as Supplementary Fig. S1 in IJSEM Online.
The DNA G+C content of strain H-5T was 38.3 mol%.
The levels of DNA–DNA relatedness between the novel
isolate and the type strains of the recognized species of the
genus Halolactibacillus were 19 and 5 %.
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Halolactibacillus alkaliphilus sp. nov.
Table 1. Characteristics that differentiate strain H-5T from recognized species of the genus Halolactibacillus
Strains: 1, H. halophilus DSM 17073T (data from Ishikawa et al., 2005); 2, H. miurensis DSM 17074T (Ishikawa et al., 2005); 3, strain H-5T
(H. alkaliphilus sp. nov.). +, Positive; 2, negative; W, weak.
Characteristic
1
2
3
Anaerobic growth
Glucose requirement in aerobic
cultivation
NaCl (%):
Range
Optimum
pH:
Range
Optimum
Major isoprenoid quinones
DNA G+C content (mol%)
Major cellular fatty acids
Source of isolation
+
+
+
+
2
0–24
2–3
0–25.5
2–3
0.5–30
2.5
6.5–9.5
9–10
None
40.2
anteiso-C13 : 0, C16 : 0
Decaying marine algae, living
sponge
6.0–10
9.5
None
38.5
anteiso-C13 : 0, C16 : 0
Decaying marine algae, living
sponge
7.5–13
12
MK-9H4, MK-9H2
38.3
C16 : 0, anteiso-C13 : 0
Sediment of soda lake
In conclusion, the similarity of the 16S rRNA gene
sequence of strain H-5T to those of the type strains of
recognized Halolactibacillus species was high. Phylogenetic
analysis revealed that strain H-5T fell within the branch of
the genus Halolactibacillus. Although there were some
differences in cell motility, fatty acid composition (Table 2)
and major cellular isoprenoid quinones, and some minor
differences that included the range of pH values and NaCl
concentrations for growth, glucose requirement under
aerobic growth conditions and the ability to ferment some
carbohydrates, between strain H-5T and recognized species
of the genus Halolactibacillus, strain H-5T was similar to
the recognized Halolactibacillus species in morphological
characteristics and most of the physiological characteristics.
Thus it is evident that the newly isolated strain represents a
member of the genus Halolactibacillus. DNA–DNA
W
hybridization is considered to be a key marker for the
identification of a novel species (Wayne et al., 1987). In our
study, the levels of DNA–DNA relatedness obtained were
low enough to identify strain H-5T as representing a novel
species of the genus Halolactibacillus.
Based on the aforementioned data, we conclude that strain
H-5T should be classified as representing a novel
Halolactibacillus species, for which the name Halolactibacillus alkaliphilus sp. nov. is proposed.
Emended description of the genus
Halolactibacillus Ishikawa et al. 2005
Halolactibacillus [Ha.lo.lac9ti.ba.cil9lus. Gr. n. hals salt; L. n.
lac lactis, milk; L. masc. n. bacillus stick, a small rod; N.L.
masc. n. Halolactibacillus salt (loving) lactic acid rodlet.
Fig. 1. Neighbour-joining tree showing the phylogenetic position of strain H-5T, recognized Halolactibacillus species and other
reference strains based on 16S rRNA gene sequences. Percentage bootstrap values, based on 1000 replications, greater than
50 % are shown at branch points. Bar, 0.005 substitutions per nucleotide position. A maximum-parsimony tree is available as
Supplementary Fig. S1 in IJSEM Online.
http://ijs.sgmjournals.org
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2171
S.-J. Cao and others
Table 2. Cellular fatty acid profiles (%) of strain H-5T and
recognized species of the genus Halolactibacillus
Strains: 1, strain H-5T (H. alkaliphilus sp. nov.); 2, H. halophilus DSM
17073T (data from Ishikawa et al., 2005); 3, H. miurensis DSM 17074T
(Ishikawa et al., 2005). Strain H-5T was grown on base agar medium
(pH 10), prepared following the manufacturer’s instructions for the
Microbial Identification System (MIDI). 2, Not detected.
Fatty acid
C10 : 0
iso-C11 : 0
anteiso-C11 : 0
iso-C12 : 0
C12 : 0
iso-C13 : 0
anteiso-C13 : 0
iso-C14 : 0
C14 : 0
iso-C15 : 0
anteiso-C15 : 0
C15 : 0
iso-C16 : 0
C16 : 0
C16 : 1
C16 : 1v9
iso-C17 : 0
anteiso-C17 : 0
C17 : 0
C18 : 0
C18 : 1v9
C18 : 2
1
2
3
0.31
0.49
1.01
1.40
2.35
5.51
18.05
0.47
6.45
1.77
3.81
1.16
0.32
52.36
2
2
1.11
0.58
0.50
2.35
2
2
2
2
2
2
2.5
6.5
19.1
2
4.1
3.5
6.2
1.4
0.9
43.1
2
2
1.5
2
2
4.6
2.7
1.1
2
2
2
2
2.1
5.7
18.8
0.5
4.0
3.7
7.6
1.6
0.8
37.2
1.3
0.7
3.5
2.5
0.9
5.5
2.2
1.2
The description is the same as that given by Ishikawa et al.
(2005). In addition, cells are non-motile or motile. Growth
occurs under conditions with or without sugars. Optimum
pH for growth is 8.0–12, with a range of 6.0–13.
Table 3. Profiles of sugar fermentation of strain H-5T and
recognized species of the genus Halolactibacillus
Strains: 1, H. halophilus DSM 17073T (data from Ishikawa et al.,
2005); 2, H. miurensis DSM 17074T (Ishikawa et al., 2005); 3, strain
H-5T (H. alkaliphilus sp. nov.). +, Positive; –, negative; W, weakly
positive. All strains ferment D-glucose, D-galactose, D-mannose,
D-lactose, D-ribose, maltose, sucrose, raffinose, D-salicin, trehalose,
D-glucoside, starch and sodium gluconate, but not adonitol, inositol,
D-arabinose, D-rhamnose or D-sorbitol.
Carbon compound
1
2
3
L-Arabinose
2
+
W
D-Xylose
2
+
+
Melezitose
Glycerol
Inulin
2
+
2
+
+
+
+
2172
W
+
Predominant menaquinones include MK-9H4 and MK9H2. The G+C content of the DNA is 38.3–40.7 mol%.
Description of Halolactibacillus alkaliphilus
sp. nov.
Halolactibacillus alkaliphilus (al.ka.li.phi9lus. N.L. n. alkali
alkali; Gr. adj. philos loving; N.L. masc. adj. alkaliphilus
alkali loving).
Cells are 0.5–0.963.5–4.7 mm, Gram-positive, non-sporulating straight rods, occurring singly, in pairs or in short
chains, and non-motile. Colonies grown on the base agar
medium are round, convex, entire, yellow and semitransparent, with diameters of 1–2 mm after incubation for
1 day at 28 uC. NaCl concentration range for growth is
0.5–30 % (w/v) (optimum, 2.5 %). pH range for growth is
7.5–13 (optimum, pH 12). Moderately alkaliphilic and
halophilic. Temperature range for growth is 15–45 uC
(optimum, 28 uC). Catalase- and oxidase-negative.
Negative for nitrate reduction. Hydrolyses starch and
gelatin, but not casein. The following carbohydrates are
fermented: D-ribose, D-glucose, D-galactose, D-mannose,
D-lactose, maltose, sucrose, raffinose, D-salicin, trehalose, D-glucoside, glycerol, starch, sodium gluconate,
L-arabinose (weak), D-xylose, melezitose and inulin.
Fructose, D-mannitol, D-rhamnose, D-sorbitol, inositol
and adonitol are not fermented (Table 3). Lactate acid
is a fermentation product from glucose. Cell-wall
peptidoglycan contains meso-diaminopimelic acid.
Major cellular fatty acids are C16 : 0 (52.36 %) and
anteiso-C13 : 0 (18.05 %). Predominant menaquinones
are MK-9H4 and MK-9H2. The DNA G+C content of
the type strain is 38.3 mol%.
The type strain, strain H-5T (5CGMCC AS
1.6843T5NBRC 103919T), was isolated from sediment of
Xiarinaoer soda lake in Inner Mongolia, China.
Acknowledgements
This work was supported by a project from the Chinese National
Natural Science Foundation (no. 30670071 and no. 40572101) and
the Hi-tech Research and Development Program (863 Program) of
China (No. 2006AA10A213).
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