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Polar Science 3 (2009) 163e169

http://ees.elsevier.com/polar/

Spore-forming halophilic bacteria isolated from Arctic terrains:

Implications for long-range transportation of microorganisms
Kise Yukimura a, Ryosuke Nakai a, Shiro Kohshima b, Jun Uetake c, Hiroshi Kanda c,
Takeshi Naganuma a,*
a
Graduate School of Biosphere Science, Hiroshima University, 1-4-4 Kagamiyama, Higashi-Hiroshima 739-8528, Japan
b
Wildlife Research Center, Kyoto University, Tanaka-Sekiden, Sakyo, Kyoto 606-8203, Japan
c
Department of Biology, National Institute of Polar Research, Itabashi, Tokyo 173-8515, Japan
Received 25 March 2009; revised 6 July 2009; accepted 6 July 2009
Available online 10 July 2009

Abstract

Organisms living in the Arctic terrains such as Greenland have to deal with low temperature conditions. The mechanisms by
which bacteria resist to low temperature are largely unknown; however, a well-known survival strategy of the microorganisms
inhabiting the Arctic is spore forming. Moreover, halophilic bacteria are often resistant to various stresses. We have attempted
isolation of spore-forming halophilic bacteria from Arctic terrains. We isolated 10 strains of spore-forming halophilic bacteria from
the samples collected from a glacial moraine in Qaanaaq, Greenland in July 2007. Identification based on 16S rRNA gene sequence
similarities showed that the isolates were closely related to the Oceanobacillus, Ornithinibacillus, Virgibacillus, Gracilibacillus,
and Bacillus genera. In addition, the 16S rRNA sequences of some isolates were extremely similar to those of strains from the desert
sand in China (100% identity, near full length), the source of the so-called ‘‘yellow dust.’’ Previous research indicated that yellow
dust had been transported to Greenland by the wind. Our research implies the long-range transportation of these microorganisms to
locations such as the Arctic.
Ó 2009 Elsevier B.V. and NIPR. All rights reserved.

Keywords: Greenland; Desert dust; Spore-forming bacteria; Halophilic bacteria; Transportation of microorganisms

1. Introduction 2005; Amato et al., 2007). The temperature in Qaa-

The Arctic terrains presents a hostile environment
for living organisms on account of its low temperature,
allowing the survival of only a handful of organisms
such as lichens and bacteria which are capable of
resisting such an adverse environment (Mueller et al.,
2005; Kaštovská et al., 2005; Miteva and Brenchley,
* Corresponding author. Tel.: þ81 82 424 7986; fax: þ81 82 424
7916.
E-mail address: takn@hiroshima-u.ac.jp (T. Naganuma).
naaq, Greenland, stays below 0  C for most of the year
and plunges to near 30  C (Weatherbase http://www.
weatherbase.com). Although little is known as to how
these Arctic bacteria have adjusted to low temperature
stresses, a well-known strategy is spore formation
(Zhang et al., 2001, 2002; Christner et al., 2003;
Sheridan et al., 2003; Miteva and Brenchley, 2005;
Yung et al., 2007). Some bacteria belonging to gram-
positive bacterial genera, such as Bacillus or Clos-
tridium form dormant cells called spores, which are
tolerant to stresses such as desiccation, high/low

1873-9652/$ - see front matter Ó 2009 Elsevier B.V. and NIPR. All rights reserved.
doi:10.1016/j.polar.2009.07.002
164 K. Yukimura et al. / Polar Science 3 (2009) 163e169
temperature, and ultraviolet rays (Nicholson et al.,
2000; Onyenwoke et al., 2004). When the environment
becomes more favorable these spores can germinate
and give rise to vegetative cells. Spore-forming
bacteria have been isolated from diverse extreme
environments around the world, e.g. deep-sea, high
altitude atmosphere, deserts, and salt lakes (Bae et al.,
2005; Shivaji et al., 2006; Hua et al., 2007, 2008).
There has even been a report of a spore-forming
bacterium that was viable after being dormant for 250
million years (Vreeland et al., 2000). Halophilic
bacteria have often been reported to have not only
salinity resistance but also other various resistances
such as high/low temperature, pressure, and dryness.
Halophiles synthesize relatively low-molecular organic
compounds such as ectoine and betaine in order to
adjust intracellular osmotic pressure (Galinski, 1993).
Aside from osmoregulatory action, these osmor-
egulators are known to function as protectors of
intracellular enzyme structures, endowing the bacteria
with tolerance to diversified stresses (Lippert and
Galinski, 1992; Malin and Lapidot, 1996; da Costa
et al., 1998; Lamosa et al., 2000; Borges et al., 2002;
Mendum and Smith, 2002).
Postulating that the existence of Arctic bacteria in
hostile environments is because of formation of spores
and production of compatible solutes, we have tried
isolating spore-forming moderately halophilic bacteria
from samples collected in Qaanaaq, Greenland, in July
2007.
In this study we contribute to the study of the
diversity of bacteria that occur in the Arctic and
examine whether these bacteria are endemic to
Greenland, or are cosmopolitan, on the basis of a 16S
rRNA gene sequence analysis.
2. Materials and methods
2.1. Sampling
We used Arctic samples taken directly from
a moraine of the Qaanaaq glacier in Greenland, ca. 77
310 N and 69 190 W, in July 2007. Rock debris were
collected into sterilized plastic bags using sterilized
plastic gloves, and kept at ambient temperature in the
dark.
2.2. Isolation of spore-forming moderately halophilic
bacteria
To screen only spore-forming bacteria, we put the
sample in a 50 ml tube with 20 ml liquid medium and
heated it at least 10 min at 80  C (Gerhardt et al., 1981).
Heat-resistant spores were able to survive, while non-
spore-forming bacteria died from the heat. After the
heat treatment, the spores were enrichment cultured at
room temperature for at least 2 weeks. Salt medium
containing 12% NaCl was used for screening halophilic
bacteria. The composition of the medium in 1 l of
distilled water was as follows: Bacto peptone (1.0 g/l),
yeast extract (0.5 g/l), NaCl (120 g/l), MgSO4$7H2O
(0.86 g/l), and MnSO4$H2O (12 mg/l). The pH was
adjusted to 7.2 with NaOH. After enrichment culture,
the suspension was spread on 1.5% agar medium of the
same composition as the liquid medium and continued
the cultivation. Subsequently, single colonies grown on
the agar medium were extracted and planted in a new
medium, and this process was repeated at least three
times for purification.
2.3. 16S rRNA gene analysis
We extracted the genomic DNA of the isolated
strain to determine the base sequence of the 16S rRNA
gene. The genomic DNA extraction method employed
here was that of Aljanabi and Martinez (1997). The
extracted DNA was used as a template to amplify the
near full length of the 16S rRNA gene by PCR using
the 16S rRNA gene specific primer set for eubacteria,
27F (50 -AGA GTT TGA TCC TGG CTC AG-30 ) and
1492R (50 -GGT TAC CTT GTT ACG ACT T-30 )
(DeLong, 1992). The amplified region corresponded to
the sequence between the 8th and 1,509th bases of the
Escherichia coli K12 strain’s 16S rRNA (Borosius
et al., 1978). The DNA polymerase used for the PCR
reaction was TaKaRa Ex TaqÒ (TaKaRa Bio Inc., Otsu,
Japan). TaKaRa Thermal Cycler Personal (TaKaRa Bio
Inc., Otsu, Japan) was employed as the PCR amplifier.
As for the PCR reaction, after 1 cycle of thermal
denaturation at 96  C (3 min), a cycle comprising
thermal denaturation (96  C; 40 s), annealing (56  C;
60 s), and extension (72  C; 60 s) was repeated 30
cycles ending with a final extension reaction (72  C;
5 min). After confirming the PCR product by 1.5%
agarose gel electrophoresis, we purified it with the
QIAquick PCR Purification Kit (QIAGEN Science,
Maryland, USA). The determination of the base
sequence was outsourced to Macrogen (http://www.
macrogen.com). A homology search of the obtained
gene sequence was then performed using BLAST
(Karlin and Altschul, 1990) through the database of
the National Centre for Biotechnology Information
(NCBI, http://www.ncbi.nlm.nih.gov). By utilizing the
CLUSTAL X multiple alignment programme version
 K. Yukimura et al. / Polar Science 3 (2009) 163e169
2.0 (Larkin et al., 2007), we aligned the obtained 16S
rRNA gene sequences of the isolates with the
sequences of the type strains of the corresponding
taxa that the isolates were affiliated with by BLAST.
Then, a phylogenetic tree was created using the NJ
method (Saitou and Nei, 1987) with MEGA 4.0
(Tamura et al., 2007). We registered the determined
gene sequences in GenBank/EMBL/DDBJ (accession
numbers, AB489106eAB489115) (Fig. 1).
3. Results and discussion
3.1. Spore-forming moderately halophilic bacteria
isolated from the Greenland sample
Qaanaaq is the northernmost city in the world
located on the northwest coast of Greenland, an
autonomous region of Denmark. From the samples
taken in Qaanaaq, Greenland in 2007, we obtained
a total of 10 spore-forming moderately halophilic
bacteria strains (Oceanobacillus: 5 strains; Bacillus: 2
strains; Ornithinibacillus: 1 strain; Gracilibacillus: 1
strain; Virgibacillus: 1 strain). All these genera belong
to the Bacillaceae family and are known to form
spores. Although it is not yet known whether these
bacteria actively grow in the Arctic, this study has
corroborated that at the least, they have the ability to
germinate and give rise to vegetative cells. The strains
obtained were either halophilic or halotolerant bacteria
capable of multiplication in 12% NaCl medium. Many
of these bacteria adjust to external osmotic pressure by
accumulation of low-molecular organic compounds
such as ectoine and betaine (Galinski, 1993). There are
also many reports that some compatible solutes
contribute not only to osmoregulation, but also to
strengthening the tolerance to low/high temperature,
dryness, and pressure (Lamosa et al., 2000; Mendum
and Smith, 2002; Borges et al., 2002; Malin and
Lapidot, 1996; Galinski, 1993; Lippert and Galinski,
1992; da Costa et al., 1998).
It is possible that they are adjusting to low
temperature stresses in the Arctic by means of these
solutes.
3.2. Long-range transport of microorganisms
As shown in Table 1, 16S rRNA gene sequences of
the isolated strains exhibited high similarities at sub-
species levels (99.44e100%) with the sequences
registered in the database. Top-hit sequences were
obtained from strains isolated in various places
worldwide or from environmental DNA.
165
The 16S rRNA gene sequence of the GL1-2 strain
was 100% identical to that of the Bacillus lichen-
iformis YS2-3 strain isolated from Dunhuang, the
oasis located between the Gobi and Taklamakan
deserts in China in 2006. This Gobi YS2-3 strain has
also been reported to completely match the DstI-4
strain isolated from yellow dust collected in Hir-
oshima, Japan during a yellow dust event in 2006 (Hua
et al., 2007). This yellow-dust-born strain DstI-4 and
the Gobi YS2-3 strain have shown high similarity not
only in the genotypes (16S rRNA and other genes) but
also in phenotypes, implying a high probability that
microorganisms in the Gobi deserts are transported to
Japan with the yellow dust (Hua et al., 2007). These
results, combined with those of this study, have
demonstrated the obtained strains with completely
identical 16S rRNA genes from three locations: the
Gobi/Taklamakan deserts, the source of the yellow
dust; Japan, the destination of the yellow dust; and,
Greenland.
Furthermore, the 16S rRNA gene of the GL3-1
strain, closely related to Oceanobacillus picturae, was
completely identical to a strain (100% identity, near
full length) isolated from Dunhuang in 2007 (Yuki-
mura, unpublished). This species has been identified as
one of the yellow-dust-borne halophilic bacteria iso-
lated from non-saline soil in Japan by Echigo et al.
(2005).
Apart from the yellow dust, a phenomenon in
which the sands of the Gobi/Taklamakan deserts and
Loess Plateau were transported to Japan, it is well
known that millions of tons of deserts dust are trans-
ported around the world on air currents every year
(Duce et al., 1980; Parrington et al., 1983; Betzer
et al., 1988; Uematsu et al., 2002; Jickells et al.,
2005). Moreover, mineral particles of the Gobi/
Taklamakan deserts, one of the sources of the yellow
dust, have been observed in Greenland (Biscaye et al.,
1997; Bory et al., 2003). The results of this study
suggested the possibility that not only mineral parti-
cles but also microorganisms are carried from inland
China to Greenland. Although there have been
a number of studies in which bacteria possibly
deriving from African or Asian dusts were isolated
from the atmosphere (Yongyi et al., 1993; Bauer et al.,
2002; Yeo and Kim, 2002; Griffin and Kellogg, 2004;
Echigo et al., 2005; Prospero et al., 2005; Griffin et al.,
2006; Griffin, 2007; Brodie et al., 2007), almost all of
them have concentrated on tracing either the destina-
tions or the sources of the dusts. Comparison of
bacteria isolated from both dusts source areas and end-
point sites has rarely been done (Hua et al., 2007).
166 K. Yukimura et al. / Polar Science 3 (2009) 163e169

Fig. 1. Neighbor-joining tree showing the relationships of isolates from Greenland with other selected bacteria. GL strains (accession numbers,
AB489106eAB489115) isolated from Greenland in this study. GDS (AB491178eAB491187), DH (AB491188eAB491191) and YS
(AB305271eAB305276) strains isolated from the Gobi desert. DstI-4 strain (AB305269) isolated from yellow dust in Japan. Bootstrap values of
1000 replications greater than 50% are shown at the nodes. Names in bold indicate strains identified in this research. 16S rRNA gene sequence
accession numbers are in parentheses. Scale bar: 0.02 nucleotide substitution per site.
 K. Yukimura et al. / Polar Science 3 (2009) 163e169 167

Table 1
Samples and bacterial isolates obtained in this study.
Isolate (accession no.) Closest sequence (accession no.)
Specie/strain/clone Similarity (%) Originb
GL1-1 (AB489106) Uncultured Bacillus sp. clone 99.44 Italy
ACf125 (AM489496)
GL1-2 (AB489107) Bacillus licheniformis 100 Gobi Desert, China
YS2-3 (AB305274)
GL3-1 (AB489109) Bacillus (Oceanobacillus) sp. 99.93 Mission Bay sediment,
MB-1 (AF326359) San Diego, USA
GL4-1 (AB489108) Virgibacillus pantothenticus 99.79 Mixed-culture system, Japan
M1-4 (AB039331)
GL4-2 (AB489110) Gracilibacillus sp. JM-Gb 99.93 Joumon Cave,
(AB189324) Gifu, Japan
GL5-1 (AB489111) Bacillus (Oceanobacillus) sp. 99.93 Mission Bay sediment,
MB-1 (AF326359) San Diego, USA
GL5-2 (AB489112) Bacillus licheniformis 99.85 No data
ATCC 14580 (CP000002)
GL5-3 (AB489113) Oceanobacillus picturae strain 99.93 No data
JL85 (EF512732)
GL6-1 (AB489114) Bacillus (Oceanobacillus) sp. 99.79 Mission Bay sediment,
MB-1 (AF326359) San Diego, USA
GL6-2 (AB489115) Bacillus (Oceanobacillus) sp. 99.86 Mission Bay sediment,
MB-1 (AF326359) San Diego, USA
a
Aliment length.
b
Reference taken from sequence databases, NCBI.

However, some studies reported that spore-forming
bacteria were frequently isolated from the dusts
samples collected using a balloon in Dunhuang
(Kobayashi et al., 2007; Maki et al., 2008).
Some of the bacteria strains isolated from
Greenland had a completely identical 16S rRNA gene
with that of the strains isolated from the Gobi Desert,
China, or from yellow dust which had flown over to
Japan. This suggested that these bacteria were not
endemic to Greenland, but were cosmopolitan species
which may have been disseminated globally through
some unspecified presses such as atmospheric trans-
portation. Only the species capable of withstanding the
stress during the dissemination and the environment of
the destination could possibly become cosmopolitan.
The spore-forming moderately halophilic bacteria tar-
geted in this study possess such capabilities. Our data
support the transport via the atmosphere of microor-
ganisms, including stress-tolerant spore-forming halo-
philes, that eventually can become globally
disseminated.
forming moderately halophilic bacteria of the Bacil-
laceae family. Among the strains isolated in Greenland,
there were two strains of which the 16S rRNA
sequences were identical to those of two strains iso-
lated from a location in China which is the source of
yellow dust (Dunhuang between the Gobi and Takla-
makan deserts). These strains were closely affiliated
with B. licheniformis and O. picturae.
Although sequences of genes other than 16S rRNA
genes are to be compared, this finding raises the
possibility that yellow dust events are capable of long-
range transportation of microorganisms all the way to
Greenland, in addition to Japan. Dust-borne microor-
ganisms may be transported by adhering to dusts
mineral particles and/or in a free-drifting state sepa-
rated from the particles. The challenge is to elucidate
the actual circumstances of the airborne transportation
of microorganisms by proceeding with further
analyses.
Acknowledgment

4. General overview We are very much obliged to Dr. Martin Bay

Hebsgaard, University of Copenhagen, for his tech-
The data have revealed the existence of bacteria nical assistance. This study was partly supported by the
tolerant to various stresses, such as salinity and Grants-in-Aid for Scientific Research (No. 18255005)
temperature in the Arctic. They belong to spore- from Japan Society for the Promotion of Science.
168 K. Yukimura et al. / Polar Science 3 (2009) 163e169
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