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Polar Science 3 (2009) 163e169 http://ees.elsevier.com/polar/

Spore-forming halophilic 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, , 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- naaq, Greenland, stays below 0 C for most of the year The Arctic terrains presents a hostile environment and plunges to near 30 C (Weatherbase http://www. for living organisms on account of its low temperature, weatherbase.com). Although little is known as to how allowing the survival of only a handful of organisms these Arctic bacteria have adjusted to low temperature such as lichens and bacteria which are capable of stresses, a well-known strategy is spore formation resisting such an adverse environment (Mueller et al., (Zhang et al., 2001, 2002; Christner et al., 2003; 2005; Kasˇtovska´ et al., 2005; Miteva and Brenchley, Sheridan et al., 2003; Miteva and Brenchley, 2005; Yung et al., 2007). Some bacteria belonging to gram- * Corresponding author. Tel.: þ81 82 424 7986; fax: þ81 82 424 positive bacterial genera, such as Bacillus or Clos- 7916. tridium form dormant cells called spores, which are E-mail address: [email protected] (T. Naganuma). 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., heated it at least 10 min at 80 C(Gerhardt et al., 1981). 2000; Onyenwoke et al., 2004). When the environment Heat-resistant spores were able to survive, while non- becomes more favorable these spores can germinate spore-forming bacteria died from the heat. After the and give rise to vegetative cells. Spore-forming heat treatment, the spores were enrichment cultured at bacteria have been isolated from diverse extreme room temperature for at least 2 weeks. Salt medium environments around the world, e.g. deep-sea, high containing 12% NaCl was used for screening halophilic altitude atmosphere, deserts, and salt lakes (Bae et al., bacteria. The composition of the medium in 1 l of 2005; Shivaji et al., 2006; Hua et al., 2007, 2008). distilled water was as follows: Bacto peptone (1.0 g/l), There has even been a report of a spore-forming yeast extract (0.5 g/l), NaCl (120 g/l), MgSO4$7H2O bacterium that was viable after being dormant for 250 (0.86 g/l), and MnSO4$H2O (12 mg/l). The pH was million years (Vreeland et al., 2000). Halophilic adjusted to 7.2 with NaOH. After enrichment culture, bacteria have often been reported to have not only the suspension was spread on 1.5% agar medium of the salinity resistance but also other various resistances same composition as the liquid medium and continued such as high/low temperature, pressure, and dryness. the cultivation. Subsequently, single colonies grown on Halophiles synthesize relatively low-molecular organic the agar medium were extracted and planted in a new compounds such as ectoine and betaine in order to medium, and this process was repeated at least three adjust intracellular osmotic pressure (Galinski, 1993). times for purification. Aside from osmoregulatory action, these osmor- egulators are known to function as protectors of 2.3. 16S rRNA gene analysis intracellular enzyme structures, endowing the bacteria with tolerance to diversified stresses (Lippert and We extracted the genomic DNA of the isolated Galinski, 1992; Malin and Lapidot, 1996; da Costa strain to determine the base sequence of the 16S rRNA et al., 1998; Lamosa et al., 2000; Borges et al., 2002; gene. The genomic DNA extraction method employed Mendum and Smith, 2002). here was that of Aljanabi and Martinez (1997). The Postulating that the existence of Arctic bacteria in extracted DNA was used as a template to amplify the hostile environments is because of formation of spores near full length of the 16S rRNA gene by PCR using and production of compatible solutes, we have tried the 16S rRNA gene specific primer set for eubacteria, isolating spore-forming moderately halophilic bacteria 27F (50-AGA GTT TGA TCC TGG CTC AG-30) and from samples collected in Qaanaaq, Greenland, in July 1492R (50-GGT TAC CTT GTT ACG ACT T-30) 2007. (DeLong, 1992). The amplified region corresponded to In this study we contribute to the study of the the sequence between the 8th and 1,509th bases of the diversity of bacteria that occur in the Arctic and Escherichia coli K12 strain’s 16S rRNA (Borosius examine whether these bacteria are endemic to et al., 1978). The DNA polymerase used for the PCR Greenland, or are cosmopolitan, on the basis of a 16S reaction was TaKaRa Ex TaqÒ (TaKaRa Bio Inc., Otsu, rRNA gene sequence analysis. Japan). TaKaRa Thermal Cycler Personal (TaKaRa Bio Inc., Otsu, Japan) was employed as the PCR amplifier. 2. Materials and methods As for the PCR reaction, after 1 cycle of thermal denaturation at 96 C (3 min), a cycle comprising 2.1. Sampling thermal denaturation (96 C; 40 s), annealing (56 C; 60 s), and extension (72 C; 60 s) was repeated 30 We used Arctic samples taken directly from cycles ending with a final extension reaction (72 C; a moraine of the Qaanaaq glacier in Greenland, ca. 77 5 min). After confirming the PCR product by 1.5% 310 N and 69 190 W, in July 2007. Rock debris were agarose gel electrophoresis, we purified it with the collected into sterilized plastic bags using sterilized QIAquick PCR Purification Kit (QIAGEN Science, plastic gloves, and kept at ambient temperature in the Maryland, USA). The determination of the base dark. sequence was outsourced to Macrogen (http://www. macrogen.com). A homology search of the obtained 2.2. Isolation of spore-forming moderately halophilic gene sequence was then performed using BLAST bacteria (Karlin and Altschul, 1990) through the database of the National Centre for Biotechnology Information To screen only spore-forming bacteria, we put the (NCBI, http://www.ncbi.nlm.nih.gov). By utilizing the sample in a 50 ml tube with 20 ml liquid medium and CLUSTAL X multiple alignment programme version K. Yukimura et al. / Polar Science 3 (2009) 163e169 165

2.0 (Larkin et al., 2007), we aligned the obtained 16S The 16S rRNA gene sequence of the GL1-2 strain rRNA gene sequences of the isolates with the was 100% identical to that of the Bacillus lichen- sequences of the type strains of the corresponding iformis YS2-3 strain isolated from Dunhuang, the taxa that the isolates were affiliated with by BLAST. oasis located between the Gobi and Taklamakan Then, a phylogenetic tree was created using the NJ deserts in China in 2006. This Gobi YS2-3 strain has method (Saitou and Nei, 1987) with MEGA 4.0 also been reported to completely match the DstI-4 (Tamura et al., 2007). We registered the determined strain isolated from yellow dust collected in Hir- gene sequences in GenBank/EMBL/DDBJ (accession oshima, Japan during a yellow dust event in 2006 (Hua numbers, AB489106eAB489115)(Fig. 1). et al., 2007). This yellow-dust-born strain DstI-4 and the Gobi YS2-3 strain have shown high similarity not 3. Results and discussion only in the genotypes (16S rRNA and other genes) but also in phenotypes, implying a high probability that 3.1. Spore-forming moderately halophilic bacteria microorganisms in the Gobi deserts are transported to isolated from the Greenland sample Japan with the yellow dust (Hua et al., 2007). These results, combined with those of this study, have Qaanaaq is the northernmost city in the world demonstrated the obtained strains with completely located on the northwest coast of Greenland, an identical 16S rRNA genes from three locations: the autonomous region of Denmark. From the samples Gobi/Taklamakan deserts, the source of the yellow taken in Qaanaaq, Greenland in 2007, we obtained dust; Japan, the destination of the yellow dust; and, a total of 10 spore-forming moderately halophilic Greenland. bacteria strains (Oceanobacillus: 5 strains; Bacillus:2 Furthermore, the 16S rRNA gene of the GL3-1 strains; Ornithinibacillus: 1 strain; Gracilibacillus:1 strain, closely related to Oceanobacillus picturae, was strain; Virgibacillus: 1 strain). All these genera belong completely identical to a strain (100% identity, near to the family and are known to form full length) isolated from Dunhuang in 2007 (Yuki- spores. Although it is not yet known whether these mura, unpublished). This species has been identified as bacteria actively grow in the Arctic, this study has one of the yellow-dust-borne halophilic bacteria iso- corroborated that at the least, they have the ability to lated from non-saline soil in Japan by Echigo et al. germinate and give rise to vegetative cells. The strains (2005). obtained were either halophilic or halotolerant bacteria Apart from the yellow dust, a phenomenon in capable of multiplication in 12% NaCl medium. Many which the sands of the Gobi/Taklamakan deserts and of these bacteria adjust to external osmotic pressure by Loess Plateau were transported to Japan, it is well accumulation of low-molecular organic compounds known that millions of tons of deserts dust are trans- such as ectoine and betaine (Galinski, 1993). There are ported around the world on air currents every year also many reports that some compatible solutes (Duce et al., 1980; Parrington et al., 1983; Betzer contribute not only to osmoregulation, but also to et al., 1988; Uematsu et al., 2002; Jickells et al., strengthening the tolerance to low/high temperature, 2005). Moreover, mineral particles of the Gobi/ dryness, and pressure (Lamosa et al., 2000; Mendum Taklamakan deserts, one of the sources of the yellow and Smith, 2002; Borges et al., 2002; Malin and dust, have been observed in Greenland (Biscaye et al., Lapidot, 1996; Galinski, 1993; Lippert and Galinski, 1997; Bory et al., 2003). The results of this study 1992; da Costa et al., 1998). suggested the possibility that not only mineral parti- It is possible that they are adjusting to low cles but also microorganisms are carried from inland temperature stresses in the Arctic by means of these China to Greenland. Although there have been solutes. a number of studies in which bacteria possibly deriving from African or Asian dusts were isolated 3.2. Long-range transport of microorganisms from the atmosphere (Yongyi et al., 1993; Bauer et al., 2002; Yeo and Kim, 2002; Griffin and Kellogg, 2004; As shown in Table 1, 16S rRNA gene sequences of Echigo et al., 2005; Prospero et al., 2005; Griffin et al., the isolated strains exhibited high similarities at sub- 2006; Griffin, 2007; Brodie et al., 2007), almost all of species levels (99.44e100%) with the sequences them have concentrated on tracing either the destina- registered in the database. Top-hit sequences were tions or the sources of the dusts. Comparison of obtained from strains isolated in various places bacteria isolated from both dusts source areas and end- worldwide or from environmental DNA. 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 aAliment length. b Reference taken from sequence databases, NCBI.

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