International Journal of Systematic and Evolutionary Microbiology (2011), 61, 1149–1152
DOI 10.1099/ijs.0.023119-0
Halostagnicola alkaliphila sp. nov., an alkaliphilic
haloarchaeon from commercial rock salt
Shuhei Nagaoka,1 Hiroaki Minegishi,2 Akinobu Echigo,2
Yasuhiro Shimane,3 Masahiro Kamekura4 and Ron Usami2,3
Correspondence
Shuhei Nagaoka
[email protected]
1
Department of Biological Applied Chemistry, Graduate School of Engineering, Toyo University,
2100 Kujirai, Kawagoe-shi, Saitama 350-8585, Japan
2
Bio-Nano Electronics Research Centre, Toyo University, 2100 Kujirai, Kawagoe-shi, Saitama 3508585, Japan
3
Graduate School of Interdisciplinary New Science, Toyo University, 2100 Kujirai, Kawagoe-shi,
Saitama 350-8585, Japan
4
Halophiles Research Institute, 677-1 Shimizu, Noda-shi, Chiba 278-0043, Japan
A Gram-negative, pleomorphic, aerobic, haloalkaliphilic archaeon, strain 167-74T, was isolated
from commercial rock salt imported into Japan from China. Phylogenetic analysis based on 16S
rRNA gene sequence similarities showed that strain 167-74T is closely related to Halostagnicola
larsenii XH-48T (98.3 %) and Halostagnicola kamekurae 194-10T (97.2 %). The major polar lipids
of the isolate were C20C20 and C20C25 derivatives of phosphatidylglycerol and
phosphatidylglycerol phosphate methyl ester. A glycolipid was not detected, in contrast to the two
existing, neutrophilic species of the genus Halostagnicola. The DNA G+C content of strain 16774T was 60.7 mol%. and it gave DNA–DNA reassociation values of 19.5 and 18.8 %,
respectively, with Hst. larsenii JCM 13463T and Hst. kamekurae 194-10T. Therefore, strain 16774T represents a novel species, for which the name Halostagnicola alkaliphila sp. nov. is
proposed, with the type strain 167-74T (5JCM 16592T 5CECT 7631T).
The genus Halostagnicola belonging to the family
Halobacteriaceae was first described by Castillo et al.
(2006) and, at the time of writing, the genus comprised
two species, Halostagnicola larsenii (Castillo et al., 2006)
and Halostagnicola kamekurae (Nagaoka et al., 2010). Cells
of the type strains of both species are pleomorphic,
neutrophilic and strictly aerobic. In this report, we describe
an alkaliphilic haloarchaeon, strain 167-74T, isolated
from commercial rock salt, that belongs to the genus
Halostagnicola.
Strain 167-74T was isolated from a sample of rock salt
produced in Hubei Province, China. The rock salt is
labelled ‘BEST ancient rock salt’ and is sold in Japan by
Hanamasa Co., Ltd. The salt sample (1.0 g) was dissolved
in 5 ml sterile 5 % NaCl solution and spread on JCM no.
167 medium agar plates. JCM no. 167 medium contains
(l21) 200 g NaCl, 5.0 g Na2CO3, 0.24 g MgSO4 . 7H2O,
1.0 g KCl, 1.0 g NH4Cl, 5.0 g yeast extract (Difco), 5.0 g
Abbreviations: PG, phosphatidylglycerol; PGP-Me, phosphatidylglycerol
phosphate methyl ester.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene
sequence of strain 167-74T is AB533255.
Two supplementary figures are available with the online version of this
paper.
023119 G 2011 IUMS
Casamino acids (Difco), 1.0 g sodium glutamate, 1.0 g
KH2PO4, 0.17 g CaSO4 . 2H2O and 1.0 ml trace metal
solution. The trace metal solution contained (l21) 0.1 g
ZnSO4 . 7H2O, 0.03 g MnCl2 . 4H2O, 0.3 g H3BO3, 0.2 g
CoCl2 . 6H2O, 0.01 g CuCl2 . 2H2O, 0.02 g NiCl2 . 6H2O
and 0.03 g Na2MoO4 . 2H2O (adjusted to pH 3.6 with
HCl). All components, except Na2CO3, were dissolved in
distilled water and made to 900 ml. The medium was
adjusted to pH 6.5 with 40 % KOH, and autoclaved.
Sodium carbonate was dissolved in 100 ml distilled water
and autoclaved separately. After autoclaving, the two
solutions were mixed aseptically. The final pH was 9.0.
After incubation of agar plates at 37 uC for 2–4 weeks,
various coloured colonies developed. Four colonies were
transferred to fresh agar plates, and pure cultures were
obtained by plating serial dilutions and repeated transfers
on agar plates. Partial 16S rRNA gene sequences of the four
isolates were almost the same, and the most alkaliphilic
strain, 167-74T, was chosen as a representative for further
experiments.
Hst. larsenii JCM 13463T, obtained from the Japan
Collection of Microorganisms, and Hst. kamekurae 19410T, isolated in our previous study (Nagaoka et al., 2010),
were used as reference strains.
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S. Nagaoka and others
Colony morphology was observed on agar medium after
incubation for 2–3 weeks at 37 uC. Gram-staining was
performed according to Dussault (1955). Cell morphology
and motility were examined using phase-contrast microscopy (Axiovert 135; Zeiss). Total DNA was extracted by
the method of Cline et al. (1989). The 16S rRNA gene was
analysed as described previously (Nagaoka et al., 2010). 16S
rRNA gene sequences retrieved from the DDBJ (Miyazaki
et al., 2003; Pearson & Lipman, 1988) were aligned using
CLUSTAL_X version 2.0.12 (Larkin et al., 2007). A phylogenetic tree was reconstructed by the neighbour-joining
method (Saitou & Nei, 1987) and evaluated by bootstrap sampling, from 1000 replicates (Felsenstein, 1985).
Maximum-likelihood analysis was performed with RAxML
7.0.4 using the GTR+C model (Stamatakis et al., 2005),
and confidence values for the maximum-likelihood tree
were obtained by bootstrapping (1000 replicates) using
CONSENSE in PHYLIP (Felsenstein, 2002). The 16S rRNA gene
sequence of strain 167-74T was most similar to those of
Hst. larsenii JCM 13463T (98.5 %) and Hst. kamekurae 19410T (97.2 %). Lower similarities (,94.6 %) were found
with sequences from other species of the family
Halobacteriaceae with validly published names. The
neighbour-joining (Fig. 1) and maximum-likelihood
(Supplementary Fig. S1, available in IJSEM Online) trees
also supported the conclusion that strain 167-74T was most
closely related to members of the genus Halostagnicola.
Total lipids were extracted with chloroform/methanol as
described previously (Kamekura, 1993). TLC of polar lipids
(Supplementary Fig. S2) suggested that strain 167-74T and
the type strains of the other two species of the genus
Halostagnicola contained C20C20 and C20C25 archaeol
derivatives of phosphatidylglycerol (PG) and phosphatidylglycerol phosphate methyl ester (PGP-Me), as shown by
the double spot for PGP-Me. Phospholipids were the same
as for the two type strains of the genus Halostagnicola, but
no glycolipid spot was detected in strain 167-74T.
Results of physiological characterization are given in the
species description, with methods mentioned in the
proposed minimal standards for the descriptions of new
taxa in the order Halobacteriales (Oren et al., 1997), as
described previously (Castillo et al., 2006; Nagaoka et al.,
2010). The range and optimal NaCl concentration for
growth were determined by using the growth medium
containing various concentrations of NaCl (0–30 %, w/v, at
intervals of 5 %, w/v). The pH range for growth was
assayed from pH 7.0 to 11.0 at intervals of 0.5 pH units in
liquid medium with various buffers (glycyl glycine/NaOH,
pH 7.0–8.0; glycine/NaOH, pH 8.5–11.0), each at 25 mM.
The temperature for growth was determined at 4, 10, 15,
20, 25, 30, 35, 37, 40, 45, 50, 55 and 60 uC in a medium at
pH 8.0 with optimal NaCl concentration. Strain 167-74T
was capable of growth in the presence of 20–30 % (w/v)
NaCl, at pH 8.0–10.0 and at 20–55 uC. Optimal growth of
strain 167-74T occurred in 25 % (w/v) NaCl, at pH 9.0 and
at 37 uC. Phenotypic testing of the strain used the above
optimal growth conditions.
Tests for catalase and oxidase activities and for hydrolysis
of starch, gelatin, casein and Tween 80 were performed as
described by Gonzalez et al. (1978). Reduction of nitrate
was detected by using sulfanilic acid/a-naphthylamine
reagent (Smibert & Krieg, 1994). H2S formation was
determined by formation of a black sulfide precipitate in
medium containing 0.5 % (w/v) sodium subsulfite. Indole
production from tryptophan and the utilization of sugars
and organic acids were assessed as described by Oren et al.
(1997). Antibiotic sensitivity tests were performed by
spreading cell suspensions on culture plates and then
placing discs impregnated with antibiotics on top (Becton
Dickinson). Utilization of single or complex carbon
sources was assessed in a modified JCM no. 167 medium
(Casamino acids and sodium glutamate omitted; tested
under optimal growth conditions) with 0.5 g yeast extract
l21, supplemented with 1.0 % (w/v) test sugar or organic
acid.
The G+C content of the total DNA of strain 167-74T,
determined by the HPLC method (Tamaoka & Komagata,
1984), was 60.7 mol%. DNA–DNA hybridization between
strain 167-74T, Hst. larsenii JCM 13463T and Hst.
kamekurae 194-10T was assessed by using the fluorometric
method of Ezaki et al. (1989). The relatedness of strain 16774T to Hst. larsenii JCM 13463T and Hst. kamekurae 19410T was 19.5 and 18.8 %, respectively. These values are well
below the threshold value of 70 % DNA–DNA relatedness
generally accepted for the definition of a novel species
(Wayne et al., 1987; Stackebrandt & Ebers, 2006).
Fig. 1. Phylogenetic tree derived from 16S rRNA gene sequences
showing the position of strain 167-74T among related haloarchaea. The tree was reconstructed by the neighbour-joining method.
Bootstrap values .600 (from 1000 replicates) are shown. Bar,
1 % sequence divergence.
1150
Detailed results are included in the species description and
differences between strain 167-74T, Hst. larsenii JCM
13463T and Hst. kamekurae 194-10T are highlighted in
Table 1. On the basis of the data presented, we conclude
that strain 167-74T represents a novel species of the genus
Halostagnicola, for which we propose the name Halostagnicola alkaliphila sp. nov.
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Halostagnicola alkaliphila sp. nov.
Table 1. Characteristics that distinguish strain 167-74T from
Hst. larsenii JCM 13463T and Hst. kamekurae 194-10T
Strains: 1, 167-74T; 2, Hst. larsenii JCM 13463T; 3, Hst. kamekurae
194-10T. Data are from Castillo et al. (2006), Nagaoka et al. (2010)
and this study. All strains were positive for reduction of nitrate to
nitrite and assimilation of D-galactose, D-glucose, glycerol, maltose, Dmannose, propionate and trehalose. All strains were negative for
anaerobic growth in arginine and DMSO, hydrolysis of gelatin and
skimmed milk and assimilation of D-sorbitol, succinate and L-malate.
All strains were sensitive to bacitracin (10 U), novobiocin (30 mg),
anisomycin (50 mg) and pravastatin (50 mg) and resistant to
ampicillin (10 mg), chloramphenicol (30 mg), gentamicin (120 mg),
kanamycin (30 mg), neomycin (30 mg), penicillin G (10 U), streptomycin (300 mg), tetracycline (30 mg) and vancomycin (30 mg).
Characteristic
1
Cell width (mm)
0.8–1.0
2.0–2.5
Cell length (mm)
Pigmentation
White/pink
Motility
+
NaCl range (%, w/v)
20–30
NaCl optimum (%, w/v)
25
pH range
8.5–10.0
pH optimum
9.0
Temperature range (uC)
20–55
Temperature optimum (uC)
37
Enzyme activities
Catalase
2
Oxidase
+
b-Galactosidase
2
Indole production
2
Anaerobic growth with nitrate
+
Hydrolysis of starch
2
Hydrolysis of Tween 80
2
Assimilation of:
L-Arabinose
2
Cellobiose
2
D-Fructose
+
Lactose
2
D-Mannitol
2
Ribitol
2
Ribose
2
Raffinose
2
Sucrose
+
D-Xylose
+
Starch
2
Acetate
2
Pyruvate
+
DL-Lactate
+
Fumarate
+
Glutamate
2
Citrate
2
Antibiotic sensitivity
Erythromycin (15 mg)
+
Nalidixic acid (30 mg)
2
Rifampicin (5 mg)
+
DNA G+C content (mol%)
60.7
2
3
0.5–1.0
0.8–1.0
1.0–3.0
2.0–2.5
Pink White/pink
2
+
15–30
10–30
20
15
6.0–9.0
6.0–9.0
7.0–8.0
6.5–7.0
25–50
20–50
37
30
+
+
+
2
2
+
2
2
2
2
+
2
2
+
+
+
+
+
+
+
+
2
+
+
+
+
+*
+*
+
+
+
+
2
2
+
+
+
2
+
2
2
2
+
+*
+*
2
+
2
2
+
2
61.0
2
+
+
59.8
*Determined in this study; not reported by Castillo et al. (2006) or
Nagaoka et al. (2010).
Description of Halostagnicola alkaliphila sp. nov.
Halostagnicola alkaliphila [al.ka.li.phi9la. N.L. n. alkali (from
Arabic al-qalyi the ashes of saltwort) alkali; N.L. adj. philus a -um (from Gr. adj. philos -ê -on) friend to, loving; N.L.
fem. adj. alkaliphila loving alkaline conditions].
Cells are motile, pleomorphic and rod-shaped (approx.
0.8–1.062.0–2.5 mm). Stains Gram-negative. Colonies on
agar medium are white/pink and circular, 2–3 mm in
diameter. Growth occurs at 20–30 % (w/v) NaCl
(optimum, 25 %, w/v), 20–55 uC (optimum, 37 uC) and
pH 8.0–10.0 (optimum, pH 8.5–9.0). Cells lyse in water.
H2S is not produced from sodium sulfite. Indole is not
produced from tryptophan. Nitrate is reduced to nitrite.
Nitrite is not reduced and no dinitrogen gas is formed.
Anaerobic growth does not occur with DMSO and
arginine, but does occur with nitrate. Tests for oxidase
activity are positive, but catalase activity is absent. Starch,
Tween 80, casein and gelatin are not hydrolysed. Tests
positive for urease activity, but negative for b-galactosidase
activity. The following substrates are utilized for growth:
D-fructose, D-galactose, D-glucose, glycerol, maltose, Dmannose, sucrose, trehalose, D-xylose, pyruvate, propionate, DL-lactate and fumarate. No growth occurs on Larabinose, cellobiose, citrate, lactose, D-mannitol, ribitol,
ribose, raffinose, D-sorbitol, starch, acetate, succinate,
L-malate or L-glutamate. Sensitive to anisomycin
(50 mg), bacitracin (10 U), erythromycin (15 mg), novobiocin (30 mg), pravastatin (50 mg) and rifampicin
(5 mg). Resistant to ampicillin (10 mg), chloramphenicol
(30 mg), gentamicin (120 mg), kanamycin (30 mg),
nalidixic acid (30 mg), neomycin (30 mg), penicillin G
(10 U), streptomycin (300 mg), tetracycline (30 mg) and
vancomycin (30 mg). Polar lipids are C20C20 and C20C25
archaeol derivatives of PG and PGP-Me. The G+C
content of the type strain is 60.7 mol% (HPLC).
The type strain, strain 167-74T (5JCM 16592T 5CECT
7631T), was isolated from commercial rock salt imported
into Japan from Hubei Province, China.
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