Spore-forming halophilic bacteria isolated from Arctic terrains - Core

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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 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; Kasˇtovska´ et al., 2005; Miteva and Brenchley, * Corresponding author. Tel.: þ81 82 424 7986; fax: þ81 82 424 7916. E-mail address: [email protected] (T. Naganuma).

2005; Amato et al., 2007). The temperature in Qaanaaq, 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 grampositive bacterial genera, such as Bacillus or Clostridium form dormant cells called spores, which are tolerant to stresses such as desiccation, high/low

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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 osmoregulators 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 nonspore-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


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 subspecies 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.

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The 16S rRNA gene sequence of the GL1-2 strain was 100% identical to that of the Bacillus licheniformis 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 Hiroshima, 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 (Yukimura, unpublished). This species has been identified as one of the yellow-dust-borne halophilic bacteria isolated 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 transported 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 particles 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 destinations or the sources of the dusts. Comparison of bacteria isolated from both dusts source areas and endpoint sites has rarely been done (Hua et al., 2007).

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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.


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Table 1 Samples and bacterial isolates obtained in this study. Isolate (accession no.)

GL1-1 (AB489106) GL1-2 (AB489107) GL3-1 (AB489109) GL4-1 (AB489108) GL4-2 (AB489110) GL5-1 (AB489111) GL5-2 (AB489112) GL5-3 (AB489113) GL6-1 (AB489114) GL6-2 (AB489115)

Closest sequence (accession no.) Specie/strain/clone

Similarity (%)

Originb

Uncultured Bacillus sp. clone ACf125 (AM489496) Bacillus licheniformis YS2-3 (AB305274) Bacillus (Oceanobacillus) sp. MB-1 (AF326359) Virgibacillus pantothenticus M1-4 (AB039331) Gracilibacillus sp. JM-Gb (AB189324) Bacillus (Oceanobacillus) sp. MB-1 (AF326359) Bacillus licheniformis ATCC 14580 (CP000002) Oceanobacillus picturae strain JL85 (EF512732) Bacillus (Oceanobacillus) sp. MB-1 (AF326359) Bacillus (Oceanobacillus) sp. MB-1 (AF326359)

99.44

Italy

100

Gobi Desert, China

99.93

Mission Bay sediment, San Diego, USA Mixed-culture system, Japan

99.79 99.93

99.85

Joumon Cave, Gifu, Japan Mission Bay sediment, San Diego, USA No data

99.93

No data

99.79

Mission Bay sediment, San Diego, USA Mission Bay sediment, San Diego, USA

99.93

99.86

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 transportation. 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 targeted in this study possess such capabilities. Our data support the transport via the atmosphere of microorganisms, including stress-tolerant spore-forming halophiles, that eventually can become globally disseminated.

forming moderately halophilic bacteria of the Bacillaceae family. Among the strains isolated in Greenland, there were two strains of which the 16S rRNA sequences were identical to those of two strains isolated from a location in China which is the source of yellow dust (Dunhuang between the Gobi and Taklamakan 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 longrange transportation of microorganisms all the way to Greenland, in addition to Japan. Dust-borne microorganisms may be transported by adhering to dusts mineral particles and/or in a free-drifting state separated from the particles. The challenge is to elucidate the actual circumstances of the airborne transportation of microorganisms by proceeding with further analyses.

4. General overview

We are very much obliged to Dr. Martin Bay Hebsgaard, University of Copenhagen, for his technical assistance. This study was partly supported by the Grants-in-Aid for Scientific Research (No. 18255005) from Japan Society for the Promotion of Science.

The data have revealed the existence of bacteria tolerant to various stresses, such as salinity and temperature in the Arctic. They belong to spore-

Acknowledgment

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