International Journal of Systematic and Evolutionary Microbiology (2015), 65, 2345– 2350
DOI 10.1099/ijs.0.000261
Halorubrum gandharaense sp. nov., an alkaliphilic
haloarchaeon from commercial rock salt
Yusuke Kondo,1 Hiroaki Minegishi,1,2 Akinobu Echigo,1
Yasuhiro Shimane,2 Masahiro Kamekura,3 Takashi Itoh,4
Moriya Ohkuma,4 Naoko Takahashi-Ando,1 Yasumasa Fukushima,1
Yasuhiko Yoshida1 and Ron Usami1
Correspondence
1
Department of Biological Applied Chemistry, Graduate School of Engineering, Toyo University,
2100 Kujirai, Kawagoe-shi, Saitama 350-8585, Japan
2
Japan Agency for Marine-Earth Science and Technology, 2-15, Natsushima-cho, Yokosuka-shi,
Kanagawa 237-0061, Japan
3
Halophiles Research Institute, 677-1 Shimizu, Noda-shi, Chiba 278-0043, Japan
4
RIKEN BioResource Center, 3-1-1 Koyadai, Tukuba-shi, Ibaraki 305-0074, Japan
Yusuke Kondo
[email protected]
A Gram-stain-negative, non-motile, pleomorphic rod-shaped, orange–red-pigmented,
facultatively aerobic and haloalkaliphilic archaeon, strain MK13-1T, was isolated from
commercial rock salt imported from Pakistan. The NaCl, pH and temperature ranges for growth
of strain MK13-1T were 3.0–5.2 M NaCl, pH 8.0–11.0 and 15–50 8C, respectively. Optimal
growth occurred at 3.2–3.4 M NaCl, pH 9.0–9.5 and 45 8C. Addition of Mg2+ was not
required for growth. The major polar lipids of the isolate were C20C20 and C20C25 archaeol
derivatives of phosphatidylglycerol and phosphatidylglycerol phosphate methyl ester. Glycolipids
were not detected. The DNA G+C content was 64.1 mol%. The 16S rRNA gene sequence of
strain MK13-1T was most closely related to those of the species of the genus Halorubrum,
Halorubrum luteum CECT 7303T (95.9 % similarity), Halorubrum alkaliphilum JCM 12358T
(95.3 %), Halorubrum kocurii JCM 14978T (95.3 %) and Halorubrum lipolyticum JCM 13559T
(95.3 %). The rpoB9 gene sequence of strain MK13-1T had ,90 % sequence similarity to
those of other members of the genus Halorubrum. Based on the phylogenetic analysis and
phenotypic characterization, strain MK13-1T may represent a novel species of the genus
Halorubrum, for which the name Halorubrum gandharaense sp. nov. is proposed, with the type
strain MK13-1T (5JCM 17823T5CECT 7963T).
The genus Halorubrum (McGenity & Grant, 2001) is a
member of the family Halobacteriaceae. Species of this
genus are aerobic chemoorganotrophs and are widely
distributed in hypersaline environments (Kamekura,
1998; Ochsenreiter et al., 2002; Oren, 2002a, 2002b;
Papke et al., 2004). At present, the genus Halorubrum is
the largest within the family Halobacteriaceae, with 27
species, 23 neutrophilic and four alkaliphilic.
A distinguished feature of most of the neutrophilic species
is the presence of glycolipid S-DGD-3 in the cell membrane
(Kates, 1993). The four alkaliphilic species, able to grow at
pH 10.5, have been isolated from alkaline salt lakes;
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA
and RNA polymerase B9 gene sequences of strain MK13-1T are
AB563178 and AB820320, respectively.
Halorubrum vacuolatum was isolated from Lake Magadi,
Kenya (Mwatha & Grant, 1993; Kamekura et al., 1997),
Halorubrum tibetense from Lake Zabuye, Tibet, China (Fan
et al., 2004), Halorubrum alkaliphilum from a soda lake in
Xinjiang, China (Feng et al., 2005) and Halorubrum luteum
from Lake Chagannor, Inner Mongolia, China (Hu et al.,
2008). These species are able to grow at pH ranges of
pH 7.5–10.5, 8.0–10.5 or 8.5–10.5, with optimum pH 9.0–
10.0, and they possess no detectable amount of glycolipids
(Hu et al., 2008). In this study, we searched for haloalkaliphiles able to grow at pH higher than pH 11.0, and isolated
a novel alkaliphilic haloarchaeon strain, MK13-1T, that
belongs to the genus Halorubrum. Based on the phylogenetic
and phenotypic features, we would like to propose a novel
species of the genus Halorubrum.
Three supplementary figures are available with the online Supplementary
Material.
Salt samples (1.0 g each) commercially available in Japan, 368
in total, were dissolved in 4.0 ml sterile 5 % NaCl solution,
000261 G 2015 IUMS
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Fri, 16 Jun 2017 22:11:49
Printed in Great Britain
2345
Y. Kondo and others
and 50 ml each was spread on MH4 medium agar plates.
The medium was prepared as follows; NaCl (200.0 g),
glycerol (1.0 g), sodium pyruvate (1.0 g), trisodium citrate
dihydrate (1.0 g), yeast extract (Difco) (1.0 g), tryptone
(Difco) (1.0 g), K2HPO4 (0.6 g), (NH4)2SO4 (0.6 g),
MgSO4.7H2O (0.2 g), K2SO4 (5.0 g), FeCl2.4H2O (36.0 mg),
MnCl2.4H2O (0.36 mg) and trace element solution (1.0 ml)
were dissolved in approximately 700 ml distilled water. The
trace element solution contained (l21): ZnSO4.7H2O, 0.1 g;
MnCl2.4H2O, 0.03 g; H3BO3, 0.3 g; CoCl2.6H2O, 0.2 g;
CuCl2.2H2O, 0.01 g; NiCl2.6H2O, 0.02 g; Na2MoO4.2H2O,
0.03 g; pH adjusted to 3.6 with HCl. The pH of the medium
was adjusted to pH 7.0 with 20 % KOH, and the volume
filled to 900 ml, 20.0 g agar added, and the medium
autoclaved for 20 min. One hundred millilitres 0.5 M KOH
solution was autoclaved separately. After cooling to approximately 60 8C, the two solutions were mixed aseptically, and
poured into Petri dishes. The final pH was 11.5–12.0
(measured with a pH meter) just after mixing, and the pH
remained 11.0 for 1 week (measured with a pH-test paper
(MACHEREY-NAGEL), but decreased to pH 10 when incubated at 37 8C for 2 weeks in a plastic bag, probably due to
absorption of carbon dioxide in the air.
After incubation of agar plates at 37 8C for 3 weeks, red
colonies (three to five) developed from five salt samples.
The colonies were transferred to fresh agar plates, and pure
cultures were obtained by repeated transfers on the agar
plates. Partial sequences (474 bp) of 16S rRNA genes (see
below) of 18 strains isolated suggested 10 strains were
very closely related to Halorubrum saccharovorum JCM
8865T (more than 99 % sequence similarity) and five strains
to Natrialba magadii ATCC 43099T (more than 99 %).
Sequences of the remaining three strains obtained from
salt sample no. 13 were the same, with less than 96 % similarity to those of species of the genus Halorubrum, suggesting
they represent novel species in the genus Halorubrum. Preliminary DNA–DNA hybridizations suggested that strain
MK13-1T and two other strains (MK13-2 and MK13-3)
shared less than 70 % relatedness in DNA–DNA hybridizations. In this study, therefore, strain MK13-1T was chosen
for further experiments. The strain was isolated from
sample no.13, rock salt labelled ‘Gandhara salt-Black rock
salt’ imported from Pakistan and sold in Japan by FAR
EAST. The pH of 25 % solution of this salt was as high as
pH 9.55.
Colony morphology was observed on agar medium after
incubation for 2–3 weeks at 37 8C. Gram staining was performed according to the method of Dussault (1955). Cell
morphology and motility were examined using a phase
contrast microscope (Axiovert 135; Zeiss). The range of
salinity for growth was determined by using the growth
medium MH4 containing various concentrations of NaCl
(0–5.2 M) at intervals of 0.43 M (2.5 %) or 0.17 M
(1.0 %) between 2.6 and 3.4 M NaCl. The pH range
for growth was assayed from pH 7.0 to 11.5 at intervals
of 0.5 pH unit in liquid medium containing 50 mM
pH buffers (HEPES/NaOH, pH 7.0–8.0; Tricine/NaOH,
2346
pH 7.5–9.0; CHES/NaOH, pH 9.0–10.0; Glycine/NaOH,
pH 9.5–10.5; CAPS/NaOH, pH 10.5–11.0) or media adjusted to pH 11.0 and pH 11.5 with 30 mM and 40 mM KOH,
respectively. The temperature range for growth was determined at 4, 8, 15, 20, 30, 34, 37, 45, 50 and 60 8C in a
medium of pH 9.5 with 3.4 M NaCl. Strain MK13-1T was
capable of growing in the range of 3.0–5.2 M NaCl,
pH 8.0–11.0 and 15–50 8C. Optimal growth occurred
at 3.2–3.4 M NaCl, pH 9.0–9.5 and 45 8C. Addition of
Mg2+ was not required for growth.
The following phenotypic tests were performed using the
optimal growth conditions, according to the proposed minimal standards for the description of new taxa in the order
Halobacteriales (Oren et al., 1997). Most tests were done in
our laboratory on both strain MK13-1T and Hrr. luteum
CECT 7303T. Physiological characteristics missing in the literature, more than 50, for Hrr. alkaliphilum JCM 12358T,
Hrr. tibetense JCM 11889T, Hrr. vacuolatum JCM 9060T
and Hrr. saccharovorum JCM 8865T were also determined
in our laboratory. Tests for catalase and oxidase activities
and for the hydrolysis of starch, gelatin, skimmed milk,
Tween 80 and Tween 20 were performed as described by
Gonzalez et al. (1978). H2S formation was determined by
black sulfide precipitate in soft agar medium containing 0.5 % (w/v) sodium thiosulfite. Indole production
from tryptophan and the utilization of sugars and organic
acids were assessed as described by Oren et al. (1997).
Reduction of nitrate and nitrite were detected by using
the sulfanilic acid and a-naphthylamine reagents (Smibert
& Krieg, 1994). Anaerobic growth was tested with
L -arginine, KNO3 and DMSO in screw-topped sealed vials.
The utilization of single or complex carbon sources (0.5 %,
w/v) was assessed in a modified MH4 medium (glycerol,
sodium pyruvate, trisodium citrate dehydrate and yeast extract
deleted, and 1.0 g l21 tryptone replaced by 0.1 g l21 tryptone).
Detailed results of the physiological tests are given in the
species description. Antibiotic sensitivity tests were performed
by spreading cell suspensions on culture plates and then
placing discs impregnated with antibiotics on top (Becton
Dickinson). Strain MK13-1T was sensitive to bacitracin
(10 U), novobiocin (30 mg) and rifampicin (5 mg), and resistant to ampicillin (10 mg), aztreonam (30 mg), carbenicillin
(100 mg), cefoxitin (30 mg), cefalotin (30 mg), chloramphenicol (30 mg), clindamycin (2 mg), erythromycin (15 mg),
gentamicin (120 mg), kanamycin (30 mg), linezoid (30 mg),
meropenem (10 mg), nalidixic acid (30 mg), neomycin
(30 mg), piperacillin tazobactam (110 mg), penicillin
G (10 U), polymyxin B (300 U), streptomycin (300 mg),
sulfisoxazole (250 mg), tetracycline (30 mg) and vancomycin
(30 mg).
Total DNA was extracted by the method of Cline et al.
(1989). The 16S rRNA gene sequences were analysed
as described previously (Nagaoka et al., 2010, 2011). The
full-length 16S rRNA gene of strain MK13-1T was amplified by PCR with the forward primer H16S-For and reverse
primer 23S-Rev2 using Ex Taq polymerase (TaKaRa). PCR
products were purified by agarose-gel electrophoresis
Downloaded from www.microbiologyresearch.org by
International Journal of Systematic and Evolutionary Microbiology 65
IP: 88.99.165.207
On: Fri, 16 Jun 2017 22:11:49
Halorubrum gandharaense sp. nov.
and then sequenced with the following primer set: forward
primers H16S-1F, H16S-627F, H16S-1134F and H16S1276F, and reverse primers H16S-256R, H16S-744R,
H16S-915R, H16S-1161R and H16S-1468R, using a Big
Dye Sequencing kit version 3.1 (Applied Biosystems) by
an ABI 310 DNA sequencer (Applied Biosystems). NCBI
BLAST analysis indicated that the 16S rRNA gene of
strain MK13-1T (1472 bp) showed the highest similarities
to those of Hrr. luteum CECT 7303T (95.9 % similarity),
Hrr. alkaliphilum JCM 12358T (95.3 %), Halorubrum
kocurii JCM 14978T (95.3 %) and Halorubrum lipolyticum
JCM 13559T (95.3 %). Lower sequence similarities
(,95.3 %) were found with other species of the genus
Halorubrum. The related 16S rRNA gene sequences
retrieved from the DNA Data Bank of Japan (Miyazaki
et al., 2003; Pearson & Lipman, 1988) were aligned using
CLUSTAL X 2.0.12 (Larkin et al., 2007). A phylogenetic
tree was reconstructed by the neighbour-joining (NJ)
method (Saitou & Nei, 1987) and was evaluated by bootstrap sampling, expressed as percentages of 1000 replicates
(Felsenstein, 1985). Maximum likelihood (ML) analysis
was performed with RAxML 7.2.8 using the GTR+C
model (Stamatakis et al., 2005), and confidence values
were obtained by bootstrapping (1000 replicates) using
CONSENSE in PHYLIP (Felsenstein, 2002). The NJ tree (Fig.
1) and ML tree (Fig. S1, available in the online Supplementary Material) also supported the conclusion that MK13-1T
was most closely related to the alkaliphilic species of the
genus Halorubrum.
T
998 Halorubrum distributum JCM 9100 (AB663410)
878 Halorubrum terrestre JCM 10247T (AB663422)
0.01
Halorubrum litoreum JCM 1356T (AB663416)
Halorubrum coriense JCM 9275T (AB663409)
Halorubrum arcis JCM 13916T (AB663405)
809
Halorubrum ezzemoulense CECT 7066T (AB663412)
Halorubrum californiense JCM 14715T (AB663406)
Halorubrum chaoviator DSM 19316T (AB663407)
837
1000
Halorubrum xinjiangense JCM 12388T (AB663426)
Halorubrum trapanicum JCM 10477T (AB663424)
Halorubrum sodomense JCM 8880T (AB663420)
Halorubrum tebenquichense JCM 12290T (AB663421)
Halorubrum ejinorense JCM 14265T (AB663411)
Halorubrum halophilum B8T (KF848217)
Halorubrum lipolyticum JCM 13559T (AB663415)
Halorubrum saccharovorum JCM 8865T (AB663419)
922
Halorubrum lacusprofundi JCM 8891T (AB663414)
Halorubrum kocurii JCM 14978T (AB663413)
Halorubrum aidingense JCM 13560T (AB663402)
Halorubrum orientale JCM 7145T (AB663418)
Halorubrum rubrum JCM 18365T (AB935411)
1000
Halorubrum cibi JCM 15757T (AB663408)
701
Halorubrum aquaticum JCM 14031T (AB663404)
Halorubrum tibetense JCM 11889T (AB663423)
Halorubrum alkaliphilum JCM 12358T (AB663403)
Halorubrum vacuolatum JCM 9060T (AB663425)
Halorubrum luteum CECT 7303T (AB663417)
Halorubrum gandharaense MK13-1T (AB563178)
954
Halorubrum gomorrense JCM 9908T (AB663364)
Halorubrum vallismortis JCM 8877T (AB663358)
Halorubrum salinarum JCM 8978T (AB663362)
Natronobacterium gregoryi JCM 8860T (AB663467)
Haloferax volcanii JCM 8879T (AB663383)
Fig. 1. Phylogenetic tree derived from 16S rRNA gene sequences showing the position of strain MK13-1T among related
haloarchaea. The tree was reconstructed by the neighbour-joining method. Bootstrap values .70 % (1000 replicates) are
shown. Bar, 0.01 sequence divergence.
http://ijs.sgmjournals.org
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Fri, 16 Jun 2017 22:11:49
2347
Y. Kondo and others
Determination of the RNA polymerase subunit B9 gene
(rpoB9) sequence and its analysis were done according
to the methods of Minegishi et al. (2010) and Shimane
et al. (2011). The full-length rpoB9 gene was amplified by
PCR from the 39 terminal of rpoB9 to the 59 terminal of
rpoA9, with the forward primer HrpoB2 1420F and the
reverse primer HrpoA 153R using Ex Taq polymerase.
PCR products were purified by agarose-gel electrophoresis
and then sequenced with the following primer set: forward
primers HrpoB-117F, HrpoB-458F, HrpoB-721F, HrpoB922F, HrpoB-1148F and HrpoB-1213F, and reverse primers HrpoB-323R, HrpoB-671R, HrpoB-1166R and
HrpoB-1457R. All other comparative sequences were
obtained from GenBank. Sequence similarity values
between strain MK13-1T and related taxa were calculated
by GENETYX version 11 (Genetyx); phylogenetic analysis
and tree reconstruction were performed with CLUSTAL X
2.0.12 for sequence alignment, TreeView (Page, 1996).
RAxML 7.2.8 with the GTR+C model and TreeView.
The rpoB9 gene sequence of strain MK13-1T (1830 bp)
showed highest similarity to Hrr. luteum CECT 7303T
(90.0 %, GenBank acc. no. AB820300). The rpoB9 gene
sequence of strain MK13-1T formed a branch with species
of the genus Halorubrum on the maximum likelihood tree
(Fig. S2), similar to the 16S rRNA gene sequence analysis,
with an rpoB9 gene sequence similarity value of ,90 %
to members of the genus Halorubrum. The result supported
the view that strain MK13-1T was most closely related to
genus Halorubrum.
The DNA G+C content of the total DNA was determined
by the HPLC method of Tamaoka & Komagata (1984). The
DNA G+C content of strain MK13-1T was 64.1 mol%,
which was slightly higher compared with closely related
alkaliphilic species (see Table 1).
The total lipids were extracted with chloroform/methanol
as previously described (Kamekura, 1993). TLC was performed using HPTLC silica gel 60 plates (20610 cm;
Merck) with the solvent system chloroform/methanol/
acetic acid/water (85:22.5:10:4, by vol.). Glycolipids were
detected as purple spots by spraying with 0.5 % (w/v)
a-naphthol in methanol/water (1:1, v/v) and then with
sulfuric acid/ethanol (1:1, v/v), followed by brief heating
at 160 8C. Polar lipids were detected as brown spots
after prolonged heating. TLC of the total lipids (Fig. S3)
suggested that strain MK13-1T possessed C20C20 and
C20C25 archaeol derivatives of phosphatidylglycerol (PG)
and phosphatidylglycerol phosphate methyl ester (PGPMe), as shown by the double spots of PG and PGP-Me.
A glycolipid spot was not detected as in the case of
the other alkaliphilic species of the genus Halorubrum
[Hrr. luteum CGSA15T (Hu et al., 2008), Hrr. alkaliphilum DZ-1T (Feng et al., 2005), Hrr. tibetense 8W8T Fan
et al., 2004) and Hrr. vacuolatum M24T (Mwatha &
Grant 1993)].
Charactersistics that differentiate strain MK13-1T from Hrr.
luteum CGSA15T (5CECT 7303T), Hrr. alkaliphilum DZ-1T
2348
(5JCM 12358T), Hrr. tibetense 8W8T (5JCM 11889T),
Hrr. vacuolatum M24T (5JCM 9060T) and the type strain
of the type species, Hrr. saccharovorum ATCC 29252T
(5JCM 8865T), are summarized in Table 1. Strain MK131T differs from the four alkaliphilic species in growing at
pH 11.0 and growing anaerobically with nitrate, and can be
differentiated by many other features. The full-length 16S
rRNA gene sequence showed similarities of less than
96.1 % with those (determined anew by our group) of alkaliphilic as well as neutrophilic species of the genus Halorubrum. On the basis of these data, we show that strain
MK13-1T may represent a novel species of the genus Halorubrum, for which we propose the name Halorubrum gandharaense sp. nov.
Description of Halorubrum gandharaense sp. nov
Halorubrum gandharaense (gan.dhar.en9se. N.L. neut. adj.
gandharense of or belonging to Gandhara, an ancient
kingdom in Pakistan).
Cells are non-motile and pleomorphic rod-shaped (approximately 0.5–1.0|1.0–5.0 mm). Stains Gram-negative. Colonies
on agar medium containing 3.4 M NaCl are 0.5–1.5 mm in
diameter, translucent, orange–red-pigmented, circular,
slightly raised and smooth. Growth occurs at 3.0–5.2 M
NaCl (optimum, 3.2–3.4 M), 15–50 uC (optimum, 45 uC)
and at pH 8.0–11.0 (optimum, pH 9.0–9.5). Mg2+ was not
required for growth. Cells lyse in water. H2S production
from sodium thiosulfite is negative. Indole production
from tryptophan is positive. Nitrate is reduced to nitrite.
Nitrite is reduced and forms gas. Anaerobic growth with
DMSO does not occur, but occurs with L -arginine and
nitrate. Oxidase and catalase activity is positive. Starch, gelatin
and skimmed milk are not hydrolysed but Tween 80 and
Tween 20 are hydrolysed. The following single carbon sources
are utilized for growth: cellobiose, D -glucose, glycerol, maltose, D -mannose, sucrose, trehalose, sodium citrate, sodium
fumarate, sodium a-ketoglutarate, sodium D,L -lactate,
sodium L -malate, sodium pyruvate, sodium succinate, L -arginine chloride, sodium L -aspartate and sodium L -glutamate.
No growth occurs on L -arabinose, D -fructose, D -galactose, lactose, D -mannitol, raffinose, a-L -rhamnose, ribitol, ribose,
D -sorbitol, L -sorbose, D -xylose, sodium acetate, sodium
propionate, L -alanine, glycine or L -lysine chloride. The
following complex carbon sources are utilized for growth:
Bacto yeast extract, Bacto Casamino acids, Bacto tryptone,
Bacto neopeptone and peptone (Oxoid). No growth occurs
on Bacto malt extract or Bacto peptone. Polar lipids
are C20C20 and C20C25 archaeol derivatives of phosphatidylglycerol and phosphatidylglycerol phosphate methyl
ester, but phosphatidylglycerol sulfate and glycolipids are
not detected.
The type strain is strain MK13-1T (5JCM 17823T5CECT
7963T), isolated from commercial rock salt imported
from Pakistan. The DNA G+C content of the type strain
is 64.1 mol% (HPLC).
Downloaded from www.microbiologyresearch.org by
International Journal of Systematic and Evolutionary Microbiology 65
IP: 88.99.165.207
On: Fri, 16 Jun 2017 22:11:49
Halorubrum gandharaense sp. nov.
Table 1. Distinguishing characteristics of strain MK13-1T, four alkaliphilic species (Hrr. luteum, Hrr. alkaliphilum, Hrr. tibetense
and Hrr. vacuolatum) and the type species, Hrr. saccharovorum, of the genus Halorubrum
Strains: 1, MK13-1T; 2, Hrr. luteum CECT 7303T; 3, Hrr. alkaliphilum DZ-1T; 4, Hrr. tibetense 8W8T; 5, Hrr. vacuolatum M24T; 6, and Hrr.
saccharovorum ATCC 29252T. Data for strains 2–6 are from Hu et al. (2008), Feng et al. (2005), Fan et al. (2004), Mwatha & Grant (1993), Tomlinson & Hochstein (1976) and Pesenti et al. (2008) except where indicated otherwise. +, Positive; 2, negative; ¡, weak reaction.
Characteristic
Cell size (mm)
Cell shape
Motility
Colony colour
Growth conditions
NaCl range (M)
NaCl optimum (M)
Temperature range (8C)
Temperature optimum (8C)
pH range
pH optimum
Mg2+ requirement
Tween 80 hydrolysis
Indole production
Nitrate reduction
H2S formation
Anaerobic growth in nitrate
Assimilation as sole carbon
and energy source
D -Fructose
D -Galactose
Lactose
Maltose
D -Mannitol
D -Mannose
Ribose
D -Sorbitol
Sucrose
D -Xylose
Sodium acetate
Sodium citrate
Sodium fumarate
Sodium L -malate
Sodium pyruvate
Sodium succinate
L -Alanine
L -Arginine chloride
Sodium L -aspartate
Sodium L -glutamate
Glycine
L -Lysine chloride
Sensitivity to antibiotics
Bacitracin (10 U)
Erythromycin (15 mg)
Novobiocin (30 mg)
Rifampicin (5 mg)
PGS present
S-DGD-3 present
DNA G+C content (mol%)
1
2
3
0.5–1.061.0–5.0 0.5–0.960.6–1.5 0.8–1.061.8–2.0
Pleomorphic
Pleomorphic
Short rods
rods
rods
2
+
+
Orange–red
Orange
Red
4
0.5–161.5–2.5
Irregular rods
2
Red
5
6
0.5–0.761.5–3.0 0.6–1.262.5–5
Pleomorphic
Rods
short rods
2
+
Bright pink
Orange–red
3.0–5.2
3.2–3.4
15–50
45
8.0–11.0
9.0–9.5
2
+
+
+
2
+
2.5–5.2
4.0–4.3
17–41
33–37
7.5–10.5
9.5–10.0
2*
2*
+*
+*
+*
2*
1.8–5.2
3.9–4.3
20–44
38
8.0–10.5
9.0–10.0
2
2
+
+
+
2
1.7–5.2
3.0–3.4
22–45
37–40
8.0–10.5
9.0–9.5
2
+
2
+
2
2
2.5–5.2
3.5
20–50
35–40
8.5–10.5
9.5
2
+
2
+
2*
2
1.5–5.2
3.5–4.5
30–56
50
(9.5*
7.0–7.5
0.005 M
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*
+
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*
2*
+
2
+
+
2
2
64.1
2*
+*
+*
2*
2
2
60.2
2
+*
2*
2
2
2
62.1
2
2
+
2
2
2
63.3
+*
2
+
+*
2
2
62.7
+*
+
+*
+*
+
+
71.2
*Determined in this study.
http://ijs.sgmjournals.org
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Fri, 16 Jun 2017 22:11:49
2349
Y. Kondo and others
References
Mwatha, W. E. & Grant, W. D. (1993). Natronobacterium vacuolata
Cline, S. W., Schalkwyk, L. C. & Doolittle, W. F. (1989).
Transformation of the archaebacterium Halobacterium volcanii with
genomic DNA. J Bacteriol 171, 4987–4991.
sp. nov., a haloalkaliphilic archaeon isolated from Lake Magadi,
Kenya. Int J Syst Bacteriol 43, 401–404.
Nagaoka, S., Minegishi, H., Echigo, A. & Usami, R. (2010).
Dussault, H. P. (1955). An improved technique for staining red
Halostagnicola kamekurae sp. nov., an extremely halophilic archaeon
from solar salt. Int J Syst Evol Microbiol 60, 2828–2831.
Fan, H., Xue, Y., Ma, Y., Ventosa, A. & Grant, W. D. (2004).
Nagaoka, S., Minegishi, H., Echigo, A., Shimane, Y., Kamekura, M. &
Usami, R. (2011). Halostagnicola alkaliphila sp. nov., an alkaliphilic
halophilic bacteria. J Bacteriol 70, 484–485.
Halorubrum tibetense sp. nov., a novel haloalkaliphilic archaeon
from Lake Zabuye in Tibet, China. Int J Syst Evol Microbiol 54,
1213–1216.
haloarchaeon from commercial rock salt. Int J Syst Evol Microbiol
61, 1149–1152.
Ochsenreiter, T., Pfeifer, F. & Schleper, C. (2002). Diversity of
Felsenstein, J. (1985). Confidence limits on phylogenies: an approach
using the bootstrap. Evolution 39, 783–791.
Archaea in hypersaline environments characterized by molecularphylogenetic and cultivation studies. Extremophiles 6, 267–274.
Felsenstein, J. (2002). PHYLIP (phylogeny inference package), version
3.6a. Distributed by the author. Department of Genome Sciences,
University of Washington, Seattle, USA.
Oren, A. (2002a). Diversity of halophilic microorganisms: environments, phylogeny, physiology, and applications. J Ind Microbiol
Biotechnol 28, 56–63.
Feng, J., Zhou, P., Zhou, Y. G., Liu, S. J. & Warren-Rhodes, K. (2005).
Oren, A. (2002b). Molecular ecology of extremely halophilic Archaea
Halorubrum alkaliphilum sp. nov., a novel haloalkaliphile isolated
from a soda lake in Xinjiang, China. Int J Syst Evol Microbiol 55,
149–152.
Gonzalez, C., Gutierrez, C. & Ramirez, C. (1978). Halobacterium
vallismortis sp. nov. An amylolytic and carbohydrate-metabolizing,
extremely halophilic bacterium. Can J Microbiol 24, 710–715.
Hu, L., Pan, H., Xue, Y., Ventosa, A., Cowan, D. A., Jones, B. E., Grant,
W. D. & Ma, Y. (2008). Halorubrum luteum sp. nov., isolated from
Lake Chagannor, Inner Mongolia, China. Int J Syst Evol Microbiol
58, 1705–1708.
Kamekura, M. (1993). Lipids of extreme halophiles. In The Biology
of Halophilic Bacteria, pp. 135–161. Edited by R. H. Vreeland &
L. I. Hochstein. Boca Raton, FL: CRC Press.
and Bacteria. FEMS Microbiol Ecol 39, 1–7.
Oren, A., Ventosa, A. & Grant, W. D. (1997). Proposed minimal
standards for description of new taxa in the order Halobacteriales.
Int J Syst Bacteriol 47, 233–238.
Page, R. D. M. (1996). TreeView: an application to display
phylogenetic trees on personal computers. Comput Appl Biosci 12,
357–358.
Papke, R. T., Koenig, J. E., Rodrı́guez-Valera, F. & Doolittle, W. F.
(2004). Frequent recombination in a saltern population of
Halorubrum. Science 306, 1928–1929.
Pearson, W. R. & Lipman, D. J. (1988). Improved tools for biological
sequence comparison. Proc Natl Acad Sci U S A 85, 2444–2448.
Kamekura, M. (1998). Diversity of extremely halophilic bacteria.
Pesenti, P. T., Sikaroodi, M., Gillevet, P. M., Sánchez-Porro, C.,
Ventosa, A. & Litchfield, C. D. (2008). Halorubrum californiense
Kamekura, M., Dyall-Smith, M. L., Upasani, V., Ventosa, A. & Kates,
M. (1997). Diversity of alkaliphilic halobacteria: proposals for transfer
sp. nov., an extreme archaeal halophile isolated from a crystallizer
pond at a solar salt plant in California, USA. Int J Syst Evol
Microbiol 58, 2710–2715.
Extremophiles 2, 289–295.
of Natronobacterium vacuolatum, Natronobacterium magadii, and
Natronobacterium pharaonis to Halorubrum, Natrialba, and
Natronomonas gen. nov., respectively, as Halorubrum vacuolatum
comb. nov., Natrialba magadii comb. nov., and Natronomonas
pharaonis comb. nov., respectively. Int J Syst Bacteriol 47, 853–857.
Kates, M. (1993). Biology of halophilic bacteria. Part II. Membrane
lipids of extreme halophiles: biosynthesis, function and evolutionary
significance. Experientia 49, 1027–1036.
Larkin, M. A., Blackshields, G., Brown, N. P., Chenna, R., McGettigan,
P. A., McWilliam, H., Valentin, F., Wallace, I. M., Wilm, A. & other
authors (2007). Clustal W and Clustal X version 2.0. Bioinformatics
23, 2947–2948.
McGenity, T. J. & Grant, W. D. (2001). Genus VII. Halorubrum.
Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new
method for reconstructing phylogenetic trees. Mol Biol Evol 4,
406–425.
Shimane, Y., Hatada, Y., Minegishi, H., Echigo, A., Nagaoka, S.,
Miyazaki, M., Ohta, Y., Maruyama, T., Usami, R. & other authors
(2011). Salarchaeum japonicum gen. nov., sp. nov., an aerobic,
extremely halophilic member of the Archaea isolated from
commercial salt. Int J Syst Evol Microbiol 61, 2266–2270.
Smibert, R. M. & Krieg, N. R. (1994). Phenotypic characterization.
In 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.
In Bergey’s Manual of Systematic Bacteriology, pp. 320–324. Edited
by D. R. Boone, R. W. Castenholz & G. M. Garrity. 2nd edn,
vol. 1, New York: Springer.
Stamatakis, A., Ludwig, T. & Meier, H. (2005). RAxML-III: a fast
Minegishi, H., Kamekura, M., Itoh, T., Echigo, A., Usami, R. &
Hashimoto, T. (2010). Further refinement of the phylogeny of the
Tamaoka, J. & Komagata, K. (1984). Determination of DNA base
Halobacteriaceae based on the full-length RNA polymerase subunit
B9 (rpoB9) gene. Int J Syst Evol Microbiol 60, 2398–2408.
Miyazaki, S., Sugawara, H., Gojobori, T. & Tateno, Y. (2003). DNA
Data Bank of Japan (DDBJ) in XML. Nucleic Acids Res 31, 13–16.
2350
program for maximum likelihood-based inference
phylogenetic trees. Bioinformatics 21, 456–463.
of
large
composition by reverse-phase high-performance liquid chromatography. FEMS Microbiol Lett 25, 125–128.
Tomlinson, G. A. & Hochstein, L. I. (1976). Halobacterium
saccharovorum sp. nov., a carbohydrate-metabolizing, extremely
halophilic bacterium. Can J Microbiol 22, 587–591.
Downloaded from www.microbiologyresearch.org by
International Journal of Systematic and Evolutionary Microbiology 65
IP: 88.99.165.207
On: Fri, 16 Jun 2017 22:11:49