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Applied and Environmental Microbiology, November 2005, p. 6954-6962, Vol. 71, No. 11
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.11.6954-6962.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Putative Transposases Conserved in Exiguobacterium Isolates from Ancient Siberian Permafrost and from Contemporary Surface Habitats

NASA Astrobiology Institute and Department of Food Science, North Carolina State University, Raleigh, North Carolina 27695

Received 15 February 2005/ Accepted 13 July 2005

	ABSTRACT

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Gram-positive bacteria of the genus Exiguobacterium have been repeatedly isolated from Siberian permafrost ranging in age from 20,000 to 2 to 3 million years and have been sporadically recovered from markedly diverse habitats, including microbial mats in Lake Fryxell (Antarctic), surface water, and food-processing environments. However, there is currently no information on genomic diversity of this microorganism or on the physiological strategies that have allowed its survival under prolonged freezing in the permafrost. Analysis of the genome sequence of the most ancient available Exiguobacterium isolate (Exiguobacterium sp. strain 255-15, from 2 to 3 million-year-old Siberian permafrost) revealed numerous putative transposase sequences, primarily of the IS200/IS605, IS30, and IS3 families, with four transposase families identified. Several of the transposase genes appeared to be part of insertion sequences. Southern blots with different transposase probes yielded high-resolution genomic fingerprints which differentiated the different permafrost isolates from each other and from the Exiguobacterium spp. type strains which have been derived from diverse surface habitats. Each of the Exiguobacterium sp. strain 255-15 transposases that were used as probes had highly conserved homologs in the genome of other Exiguobacterium strains, both from permafrost and from modern sites. These findings suggest that, prior to their entrapment in permafrost, Exiguobacterium isolates had acquired transposases and that conserved transposases are present in Exiguobacterium spp., which now can be isolated from various modern surface habitats.

	INTRODUCTION

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Permafrost is a unique soil structure that embodies constantly frozen buried soils and sediments and underlies an estimated 20 to 25% of the earth's land surface (14). Permafrost reaches a thickness of more than 700 to 1,000 m in North America and Eurasia and also occurs in the ice-free regions of Antarctica and Greenland and in alpine regions. The permafrost dates back to as long as 2 to 3 million years in northeastern Siberia and is probably even older in Antarctica. The mean annual temperature of permafrost is between –10 and –12°C in the Arctic and between –18 and –27°C in the Antarctic. Permafrost sediments are firmly cemented by ice with content ranging from 12 to 70%, depending on the presence of ice wedges. The chemical composition of permanently frozen ground differs in different areas and depths in accordance with their origin (for a review, see reference 9).

Extensive studies have demonstrated that bacterial cells within these special soils survive for thousands of years and in deeper strata for up to several millions of years, with the age of the embedded cells corresponding to the age of the surrounding permafrost soil structure (9, 16, 21, 25). These bacteria pose quite unique opportunities for exploring the genomic organization of ancient, preserved microorganisms and the genetic relatedness between the permafrost microorganisms and their modern relatives from surface habitats. From an evolutionary point of view, permafrost bacteria have been naturally preserved in the frozen sediments in the absence of gene flow from any outside biota and represent ancestors of several bacterial species that can be isolated from modern surface habitats.

During previous work, we isolated representatives of several different taxa of gram-positive and gram-negative bacteria from Siberian permafrost core samples of markedly differing ages (10,000 to 2 to 3 million years) (24). Bacteria of the genus Exiguobacterium were unusual in being isolated from permafrost cores representing all available age ranges, including cores dated to 20,000 years (two strains), 600,000 years (one strain), and 2 to 3 million years (one strain) (T. A. Vishnivetskaya, S. Kathariou, and J. M. Tiedje, unpublished).

Exiguobacterium spp. are asporogenous gram-positive bacteria in the order Firmicutes (low-GC gram-positives). On the basis of the small-subunit rRNA sequences, Exiguobacterium has been clustered together with the round-spore-forming bacilli of the genus Bacillus and with the asporogenous taxa Caryophanon, Kurthia, and Planococcus (6, 7). To date, several Exiguobacterium species isolated from contemporary natural and food-processing environments have been described, including E. aurantiacum (type strain DSM6208, from alkaline potato processing plant effluent), E. undae (type strain DSM14481, from garden pond water in Germany), E. antarcticum (type strain DSM14480, from a microbial mat in Lake Fryxell in the Antarctic), and E. acetylicum (type strain DSM20416, from creamery plant waste) (3, 6-8).

Although a comparative analysis of physiological characteristics and of the phylogenetic relationships among these four Exiguobacterium spp. type strains has been described (8), there is currently no information on genomic diversity in this organism or on molecular subtyping tools that can be used to facilitate ecological and other studies. Such information and tools would be invaluable, not only because of the organism's remarkable ability to survive under subzero conditions in the permafrost but also because Exiguobacterium is emerging as a bacterium with impressive diversity in geographic distribution and habitats from which it has been isolated: in addition to the diverse origins of the type strains listed above, Exiguobacterium has been recently isolated from a glacial ice core sample in Greenland (13), the stem of potato plants in Austria (15), as well as hot springs in Colorado, in Yellowstone National Park, and in Southern India (Ganeshpuri, northeast of Bombay) (10; R. F. Ramaley, unpublished data).

Recently, the determination of the genomic sequence of Exiguobacterium sp. strain 255-15, isolated from one of the most ancient permafrost sediments (dated to 2 to 3 million years) has been undertaken in the context of the Joint Genome Institute Microbial Sequencing program (http://spider.jgi -psf.org/JGI_microbial/html/). Our analysis of the available genome sequence data revealed the presence of a large number of putative transposases, primarily of the IS200/IS605, IS30, and IS3 families. The putative transposase genes from this ancient permafrost strain were found to have highly conserved homologs in the genome of several other Exiguobacterium spp., not only from permafrost but also from modern environments. Strain specificity of the hybridization patterns obtained with transposase probes suggests the potential of these genes for the molecular subtyping for this microorganism.

	MATERIALS AND METHODS

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Bacterial strains and growth conditions.
The strains investigated in the present study and their sources are listed in Table 1. Permafrost strains 7-3 and 5138 had been isolated in earlier studies using direct plating of soil suspensions on Trypticase soy agar (Difco). Strains 190-11 and 255-15 had been isolated during these studies after incubation of natural permafrost sediments for 2 or 12 weeks, respectively, at 4°C, and subsequent plating on Trypticase soy agar (16, 24). Four strains, namely, E. aurantiacum DSM6208, E. undae DSM14481, E. antarcticum DSM14480, and E. acetylicum DSM20416, were purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ GmbH; Braunschweig, Germany). Unless otherwise indicated, bacteria were grown in Trypticase soy broth with 0.7% yeast extract at 24°C overnight in standing cultures. For selected experiments, bacteria were also grown in the same medium at 4°C for 1 week. Bacterial cells were preserved at –70°C in Trypticase soy broth supplemented with 20% glycerol.

Extraction of genomic DNA.
Bacterial cells were harvested by centrifugation of 7 ml of an overnight culture and resuspended in 360 µl of lysis buffer (20 mM Tris-HCl [pH 8.0], 2 mM EDTA, 1.2% Triton X-100) with lysozyme (40 mg/ml). After incubation for 1 h at 37°C, 50 µl of proteinase K (20 mg/ml) (QIAGEN, Valencia, CA) and 400 µl of buffer AL supplied by QIAGEN (catalog no. 19075) were added, followed by incubation for 30 min at 70°C. Further steps for DNA isolation were as described in the DNeasy Tissue Handbook (QIAGEN) for gram-positive bacteria.

16S rRNA gene amplification and sequencing.
The 16S rRNA genes were amplified with Bacteria-specific primers 8F and 1492R, targeted to E. coli 16S rRNA positions 8 to 27 (5'-AGA GTT TGA TCC TGG CTC AG-3') and positions 1512 to 1492 (5'-ACG GTT ACC TTG TTA CGA CTT-3'), respectively. The thermal PCR profile was as follows: initial denaturation at 95°C for 5 min, followed by 30 cycles of denaturation at 95°C for 1 min, primer annealing at 54°C for 1 min, and elongation at 72°C for 1.5 min. The final elongation step was 7 min at 72°C.

The 16S rRNA gene of Exiguobacterium strain 5138 was sequenced at the University of North Carolina at Chapel Hill (UNC-CH) Genome Analysis Facility on a 3730 DNA Analyzer (Applied Biosystems). Primers 8F and 1492R (see above), and additional internal primers targeted to E. coli positions 342 to 357 (5'-CTA CGG G(AG)(GC) GCA GCA G-3') and positions 1100 to 1115 (5'-AGG GTT GCG CTC GTT G-3') were used to obtain overlapping DNA fragments.

Transposase coding sequences and primer design.
Genome sequence data of Exiguobacterium strain 255-15 are available in a number of contigs (http://genome.ornl.gov/microbial/exig/) and have been deposited in the NCBI database (accession number NZ_AADW00000000, contigs NZ_AADW01000001 to AADW01000138). Transposase genes were found in genome of strain 255-15 by a keyword search through the orthologous groups of proteins (COGs) tab-delimited datafile (http://genome.ornl.gov/microbial/exig/10feb04/cogs_summary). Nucleotide and amino acid sequences were obtained with annotated database files and Artemis_v5 (Software Foundation, Inc., Boston, MA) (18). Primers were designed by using Primer3 software (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3.cgi/) (17) and were purchased from QIAGEN. Primer sequences are listed in Table 2.

For Southern blots, genomic DNA (1 µg) was digested with EcoRI (New England Biolabs, Beverly, MA) overnight at 37°C, resolved on an 0.8% agarose gel, transferred to a nylon membrane (Osmonics, Inc.), and hybridized overnight with digoxigenin-labeled probes at 42°C in standard buffer (Roche) with 50% formamide. Membrane was washed at 42°C twice for 10 min in 2x SSC (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate) buffer with 0.1% sodium dodecyl sulfate and then twice for 20 min in 0.5x SSC buffer with 0.1% sodium dodecyl sulfate. Hybridizing bands were detected by chemiluminescence using CSPD (Roche) according to the instructions of the manufacturer.

Phylogenetic analyses.
Sequences were initially aligned with the most similar sequences in the small subunit rRNA database using the algorithm in the Ribosomal Database Project (5). Additional 16S rRNA sequences from closely related bacteria were retrieved from GenBank after identification by BLAST (1). Multiple sequence alignment was done by using the program CLUSTAL X (23). Phylogenetic 16S rRNA analyses were performed by the maximum-likelihood (19) and neighbor-joining (20) methods. Bootstrap values were based on 100 trees generated by using the program MEGA3 (11).

Nucleotide sequence accession number.
The 16S rRNA gene sequence of Exiguobacterium sp. strain 5138 was deposited in NCBI database under accession number AY831656.

	RESULTS

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Phylogenetic analysis of Exiguobacterium spp. from permafrost.
Sequences of 16S rRNA genes from the eight Exiguobacterium isolates in our collection and other Exiguobacterium spp. formed two distinct clusters. One cluster consisted of isolates mostly from freshwaters or Siberian permafrost, whereas the members of the other cluster were primarily from slightly alkaline and marine environments (Fig. 1). All studied Exiguobacterium strains except strain E. aurantiacum DSM6208 were grouped together in the first cluster with sequence identity range of 95.6 to 99.7%. Permafrost isolates 7-3 and 255-15 were closely (99.7%) related to each other, and they both had 99.0% sequence identity to E. antarcticum DSM 14480, whereas permafrost strains 190-11 and 5138 had 98.3 to 99.6% 16S rRNA sequence identity with each other and with E. undae DSM 14481, E. oxidotolerans T-2-2, and Exiguobacterium sp. strain ARCTIC-P28 (Fig. 1). 16S rRNA gene sequence identity between E. aurantiacum DSM6208 and the other seven Exiguobacterium spp. isolates characterized in the present study ranged from 92.4 to 95.5%, with highest similarity to isolates 7-3 and 255-15.

View larger version (34K): [in this window] [in a new window]   FIG. 1. Phylogenetic relationships of Siberian permafrost isolates and selected relatives. Strains studied are shown in boldface. The sequence of Arthrobacter globiformis DSM 20124T was used as an outgroup. Bootstrap values greater than 50% are shown. Accession numbers of previously published sequences are indicated in parentheses. The "T" indicates type strains.

Putative IS elements in the genome of Exiguobacterium sp. strain 255-15.
Protein BLAST-based analysis of the deduced polypeptides suggested that the putative transposases were members of insertion sequence (IS) families: IS200/IS605, IS3, IS30, and ISNCY (data not shown). Based on the suggested nomenclature (12), these IS elements were designated ISEsp1 (2 copies) and ISEsp2 (1 copy), both of the IS200/IS605 family; ISEsp3 (5 copies) of the IS3 family; and ISEsp4 (11 copies) of the IS30 family. ISEsp1, ISEsp2, and ISEsp3 contain two consecutive ORFs for two putative unrelated transposases, orfA and orfB, whereas ISEsp4 contains just one ORF, orfA (Fig. 2).

View larger version (29K): [in this window] [in a new window]   FIG. 2. Organization of IS elements residing in the genome of Exiguobacterium sp. strain 255-15. The ORFs are represented as cross-hatched (orfA) and open (orfB) boxes; the direction of the transcription is indicated. The black boxes at each end represent the left (IRL) and right (IRR) terminal inverted repeats. (A and B) ISEsp1 (A) and ISEsp2 (B), both are IS605 elements; (C) ISEsp3 from IS3 family; (D) ISEsp4 from IS30 family.

ISEsp3 contained ORFs for two putative unrelated transposases transcribed in the same direction and separated by an intergenic region of either 161 or 134 bp (Fig. 2C). ISEsp3 was flanked by 41-bp imperfect inverted terminal repeats containing nine mismatches. As is the case with the majority of the elements of the IS3 family (12), ISEsp3 terminated with 5'-TG- - - -CA-3', and terminal repeats contained an internal block of G/C residues. The predicted primary amino acid sequence of the orfA protein of ISEsp3 exhibited a relatively strong helix-turn-helix motif, whereas orfB harbored a DDE motif. The leucine zipper harbored by many members of the IS3 family at the end of orfA (12) was not detected in ISEsp3.

Another IS element, ISEsp4 (1,056 bp), was present in 12 copies in the genome of 255-15 and belonged to the IS30 family. ISEsp4 contained a single ORF of 939 bp, which spanned almost the entire length. ISEsp4 was flanked by 9-bp imperfect inverted terminal repeats containing two mismatches (Fig. 2D). The predicted amino acid sequence of the orfA protein of ISEsp4 exhibited a relatively strong helix-turn-helix motif and a conserved DDE motif.

Southern blots with group-specific transposase probes confirm the number of putative transposases of each group in the genome of Exiguobacterium sp. strain 255-15.
Southern blots were performed by using as probes PCR-amplified internal fragments of selected transposase coding sequences, chosen so as to include one sequence from each of the nine groups identified on the basis of DNA homology (Table 3). In the genome of Exiguobacterium sp. strain 255-15, none of the 47 putative transposase coding sequences harbored internal EcoRI sites, and the number of EcoRI fragments expected to hybridize with each probe would therefore correspond to the number of transposase genes within each homology group. Exceptions may be due to fragments of similar sizes, or fragments harboring more than one transposase from the same group. The Southern blot results with Exiguobacterium sp. strain 255-15 were indeed in close agreement with those expected on the basis of the number of homologous genes identified in each group, and only minor deviations were observed. In one case, the probe derived from ORF0823 (IS605, homology group I) resulted in six bands (Fig. 3A) when seven were expected. Analysis of the genomic data indeed revealed that two homologous ORFs were located on EcoRI fragments of similar size (data not shown). In the second case, the probe derived from ORF0411 (IS30, homology group VI) resulted in 13 bands (Fig. 3F), 1 more than predicted. Further screening of the genomic sequences, however, identified one truncated fragment (580 bp) with 100% identity to an internal fragment of ORF0411 that had not been listed in the draft annotation, thus accounting for the additional hybridizing fragment observed in the Southern blot with this probe. In a third case, the probe derived from ORF0174 (IS3, homology group VIII) resulted in five hybridizing bands (Fig. 3H), whereas six were expected, due to the fact that two of the transposases in this group (ORF2227 and ORF2228) were localized on a single EcoRI fragment.

View larger version (60K): [in this window] [in a new window]   FIG. 3. Southern hybridization analysis of genomic DNAs from Exiguobacterium strains. EcoRI-digested genomic DNA was resolved on 0.8% agarose gel, transferred to nylon membrane, and hybridized with the corresponding probe as described in Materials and Methods. Unless otherwise indicated, bacteria were grown at 24°C. The ORF designation and size of probe are indicated on the right-hand side. Lane 1, DNA molecular weight marker II digoxigenin-labeled, 0.12 to 23.1 kbp (Roche). Lanes 2 to 15 are Exiguobacterium strains, as follows: 2, strain 7-3; 3, strain 7-3 grown at 4°C; 4, strain 7-3 after eight cycles of freeze-thawing and subsequent growth at 24°C; 5, strain 5138; 6, strain 5138 grown at 4°C; 7, strain 190-11; 8, strain 190-11 grown at 4°C; 9, strain 255-15; 10, strain 255-15 grown at 4°C; 11, E. aurantiacum DSM 6208; 12, E. aurantiacum DSM 6208 grown at 4°C; 13, E. antarcticum DSM 14480; 14, E. undae DSM 14481; 15, E. acetylicum DSM 20416.

The type strains included in the present study represented four different species, isolated from different environments (Table 1). Interestingly, several of the transposase genes in the genome of Exiguobacterium sp. strain 255-15 appeared to have closely related homologs in the genomes of one or more of the type strains. The ORF0823-based probe, which hybridized with all four permafrost isolates, hybridized strongly with the genomes of the type strains as well, except for E. aurantiacum DSM 6208 (Fig. 3A and Table 4). E. antarcticum DSM 14480 was noteworthy among the type strains in hybridizing with all but one of the nine probes, yielding multiple hybridization bands with several of the probes. A strong hybridization signal was obtained with the genome of this strain even with the probe derived from ORF1723 (cassette chromosome recombinase), which had not hybridized with any of the isolates from permafrost (other than Exiguobacterium sp. strain 255-15) (Fig. 3I and Table 4).

The data summarized in Table 4 indicate that Exiguobacterium spp. from permafrost sediments, microbial mat from Lake Fryxell in the Antarctic, or surface water had variable but typically multiple (15-19) transposase hybridization bands with transposase probes derived from Exiguobacterium sp. strain 255-15 (Table 4). Food processing plant-derived strains E. aurantiacum DSM 6208 and E. acetylicum DSM 20416 appeared to have a significantly smaller copy number of sequences homologous to these probes, yielding a total of only six and five hybridizing bands, respectively.

Transposase-derived probes as molecular subtyping tools for Exiguobacterium.
Each of the eight Exiguobacterium spp. isolates could be distinguished from each other not only on the basis of its reactivity with the panel of the nine probes but also by its hybridization pattern (number and size of hybridizing fragments) with specific probes. The probes derived from ORF0823, ORF1938, or ORF0411 appeared most suitable for such differentiation, since they yielded multiple, strongly hybridizing fragments with several isolates (Fig. 3A, B, and F and Table 4).

The hybridization patterns following growth at 24 and 4°C or repeated freeze-thaw cycles were stable in all strains (Fig. 3 and data not shown). Selected strains were also examined after prolonged (4 months) storage at –20°C, and no changes in hybridization patterns were detected (data not shown).

Tandem transposases in the genomes of Exiguobacterium spp. correspond to bipartite elements IS3 and IS200/IS605.
Probes derived from ORF0173 (orfA of IS3 family) and its immediately adjacent ORF0174 (orfB of IS3 family) hybridized with the same EcoRI fragment in the genome of Exiguobacterium sp. strain 255-15, as well as in several other genomes (permafrost isolate Exiguobacterium sp. strain 5138, as well as E. antarcticum DSM 14480, E. undae DSM 14481, and E. acetylicum DSM 20416). In the case of E. antarcticum DSM 14480 the same five hybridizing bands were obtained with both probes, suggesting five copies of the element, each with an orfA and orfB transposase on the same EcoRI fragment (Fig. 3G and H and Table 4). Isolates that harbored orfA also harbored orfB, with the exception of Exiguobacterium sp. strain 190-11, derived from permafrost, which hybridized with ORF0174 (orfB) but not ORF0173 (orfA) (Fig. 3G and H and Table 4).

Identical hybridizing bands were also obtained with probes derived from ORF1017 (orfA of IS200 family) and its immediately adjacent ORF1018 (orfB of IS605 family) in Exiguobacterium spp. 255-15 and 190-11, as well as E. aurantiacum DSM 6208 and E. antarcticum DSM 14480, with a single band hybridizing in each case (Fig. 3C and D). Each IS200 element was immediately adjacent to transposase IS605 orfB in the genome of strain 255-15 (Table 3), and the Southern blot data suggest that the tandem gene arrangement of IS200/IS605 transposases also exists in Exiguobacterium sp. strain 190-11, E. aurantiacum DSM 6208 and E. antarcticum DSM 14480. There was only one possible exception, E. acetylicum DSM 20416, which hybridized with ORF1018 (orfB), but not with the ORF1017 (orfA)-derived probe (Fig. 3C and D and Table 4). It is not clear whether the lack of hybridization signals reflected the absence of the second ORF sequences or the presence of a divergent ORF that lacked sufficient homology with the probes.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

 
Exiguobacterium spp. have been isolated from or molecularly detected in an impressive diversity of habitats, including Arctic permafrost, mats of Lake Fryxell in the Antarctic, surface water, several types of food processing plants, and a range of saline or alkaline environments. In the present study, we have shown that putative transposase genes identified in the genome of Exiguobacterium sp. strain 255-15, isolated from ancient (2 to 3 million years old) Siberian permafrost, had highly conserved homologs in the genomes of other Exiguobacterium spp. not only from permafrost but also from other, diverse habitats.

Transposase probes (such as ORF0823) hybridized strongly with most screened Exiguobacterium spp. genomes, and each of the nine probes had homologs in one or more of the other Exiguobacterium spp. isolates, including representatives of four different species. The findings suggest that the transposases (and associated IS elements) in the genome of the ancient permafrost isolates entered the Exiguobacterium spp. gene pool prior to the time that selected lineages became entrapped in the permafrost, and that modern Exiguobacterium spp. lineages, in spite of their extensive geographical range and their ecologically markedly diverse habitats, still harbor transposase genes highly conserved with those of strain 255-15. This may indicate that Exiguobacterium isolates from diverse sources comprise a common gene pool permissive of IS element transfer. Alternatively, currently unidentified selective pressures have driven conservation and maintenance of the transposase genes (and associated IS elements) in these isolates.

Of the different isolates investigated here, including those from permafrost, it was E. antarcticum DSM 14480 that was found to have the greatest similarity to the ancient permafrost isolate (Exiguobacterium sp. strain 255-15) in terms of the reactivity pattern with the nine transposase probes. Exiguobacterium sp. strain 255-15 was also phylogenetically closer to E. antarcticum DSM 14480 than to any of the other type strains, suggesting that their shared transposase repertoire preceded their geographic separation and, in the case of the permafrost isolate, the entrapment into the frozen sediment. Further studies are needed to characterize the genomic differentiation between these two isolates and to identify possible transposases and IS elements that may be harbored by E. antarcticum DSM 14480 but not by Exiguobacterium sp. strain 255-15. In addition, the actual nucleotide sequences of the homologous transposases in E. antarcticum DSM 14480 (and other Exiguobacterium strains) will need to be determined in order to more accurately evaluate their evolutionary relationship with their counterparts in the genome of the ancient permafrost isolate. Such studies are currently under way in our laboratory.

Multiple, strongly hybridizing bands suggested multiple copies of the corresponding transposase gene in several of the genomes (especially IS605 orfB ORF0823 and IS30 ORF0411 in the permafrost isolates, E. antarcticum DSM 14480 and E. undae DSM 14481). Multiple copies with high genetic similarity to each other would be expected to result from transposition events within the genome, suggesting that the corresponding IS elements are active in these organisms. Hybridization data did not yield evidence for activity under selected laboratory conditions and signals that may induce activity of the transposases remain to be identified. Functional transposases hold promise as tools for transposon mutagenesis of Exiguobacterium spp. Although a mercury resistance transposon has been described in Exiguobacterium (2), genetic tools for these bacteria have not been reported.

In conclusion, we describe the detection of highly conserved putative transposases in the genomes of Exiguobacterium spp. from ancient permafrost and from widely diverse environments. These sequences hold potential as molecular subtyping tools for tracing the place of origin or the genetic ancestry of these hitherto poorly characterized organisms that exhibit impressive diversity in ecological habitats and geographic distribution.

	ACKNOWLEDGMENTS

 
This study was supported by NASA Astrobiology Institute and DOE Joint Genome Institute, U.S. Department of Energy.

We thank James Tiedje, Debora Rodrigues, Monica Ponder, and Vera S. Soina for some of the Exiguobacterium isolates used in this study. We are grateful to David A. Gilichinsky for support and feedback throughout this work, Eric Altermann for assistance with the phylogenetic tree constructions, and Edward Lanwermeyer for help with editing the manuscript.

	FOOTNOTES

 
* Corresponding author. Mailing address: Department of Food Science, North Carolina State University, Schaub Hall, Room 339, Raleigh, NC 27695. Phone: (919) 513-4373. Fax: (919) 513-0014. E-mail: tavishni{at}ncsu.edu.

	REFERENCES

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Altschul, S. F., W. Gish, W. Miller, E. W. Myers, and D. J. Lipman. 1990. Basic local alignment search tool. J. Mol. Biol. 215:403-410.[CrossRef][Medline]
2. Bogdanova, E. S., I. A. Bass, L. S. Minakhin, M. A. Petrova, S. Z. Mindlin, A. A. Volodin, E. S. Kalyaeva, J. M. Tiedje, J. L. Hobman, N. L. Brown, and V. G. Nikiforov. 1998. Horizontal spread of mer operons among gram-positive bacteria in natural environments. Microbiology 144:609-620.[Abstract]
3. Brambilla, E., H. Hippe, A. Hagelstein, B. J. Tindall, and E. Stackebrandt. 2001. 16S rDNA diversity of cultured and uncultured prokaryotes of a mat sample from Lake Fryxell, McMurdo Dry Valleys, Antarctica. Extremophiles 5:23-33.[CrossRef][Medline]
4. Chandler, M., and J. Mahillon. 2002. Insertion sequences revisited, p. 305-366. In N. L. Graig, R. Craigie, M. Gellert, and A. M. Lambowitz (ed.), Mobile DNA II, vol. 1. ASM Press, Washington, D.C.
5. Cole, J. R., B. Chai, T. L. Marsh, R. J. Farris, Q. Wang, S. A. Kulam, S. Chandra, D. M. McGarrell, T. M. Schmidt, G. M. Garrity, and J. M. Tiedje. 2003. The Ribosomal Database Project (RDP-II): previewing a new autoaligner that allows regular updates and the new prokaryotic taxonomy. Nucleic Acids Res. 31:442-443.[Abstract/Free Full Text]
6. Collins, M. D., B. M. Lund, J. A. E. Farrow, and K. H. Schleifer. 1983. Chemotaxonomic study of an alkalophilic bacterium, Exiguobacterium aurantiacum gen. nov., sp. nov. J. Gen. Microbiol. 129:2037-2042.
7. Farrow, J. A. E., S. Wallbanks, and M. D. Collins. 1994. Phylogenetic interrelationships of round-spore-forming bacilli containing cell-walls based on lysine and the non-spore-forming genera Caryophanon, Exiguobacterium, Kurthia, and Planococcus. Int. J. Syst. Bacteriol. 44:74-82.[Abstract/Free Full Text]
8. Fruhling, A., P. Schumann, H. Hippe, B. Straubler, and E. Stackebrandt. 2002. Exiguobacterium undae sp. nov. and Exiguobacterium antarcticum sp. nov. Int. J. Syst. Evol. Microbiol. 52:1171-1176.[Abstract]
9. Gilichinsky, D. 2002. Permafrost as a microbial habitat, p. 932-956. In G. Briton (ed.), Encyclopedia of environmental microbiology. John Wiley & Sons, Inc., New York, N.Y.
10. Knudston, K. E., E. J. Haas, P. C. Iwen, W. C. Ramaley, and R. F. Ramaley. 2001. Characterization of a gram-positive, non-spore-forming Exiguobacterium-like organism isolated from a Western Colorado (USA) hot spring. Abstr. Annu. Meet. Am. Soc. Microbiol. 2001, I-92, p. 30.
11. Kumar, S., K. Tamura, and M. Nei. 2004. MEGA3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief. Bioinform. 5:150-163.[Abstract/Free Full Text]
12. Mahillon, J., and M. Chandler. 1998. Insertion sequences. Microbiol. Mol. Biol. Rev. 62:725-774.[Abstract/Free Full Text]
13. Miteva, V. I., P. P. Sheridan, and J. E. Brenchley. 2004. Phylogenetic and physiological diversity of microorganisms isolated from a deep Greenland glacier ice core. Appl. Environ. Microbiol. 70:202-213.[Abstract/Free Full Text]
14. Pewe, T. 1995. Permafrost and frozen soils, p. 172-759. In Encyclopedia Britannica, vol. 20. Encyclopedia Britannica, Inc., New York, N.Y.
15. Reiter, B., U. Pfeifer, H. Schwab, and A. Sessitsch. 2002. Response of endophytic bacterial communities in potato plants to infection with Erwinia carotovora subsp. atroseptica. Appl. Environ. Microbiol. 68:2261-2268.[Abstract/Free Full Text]
16. Rivkina, E., D. Gilichinsky, S. Wagener, J. Tiedje, and J. McGrath. 1998. Biogeochemical activity of anaerobic microorganisms from buried permafrost sediments. Geomicrobiol. J. 15:187-193.
17. Rozen, S., and H. Skaletsky. 2000. Primer3 on the WWW for general users and for biologist programmers. Methods Mol. Biol. 132:365-386.[Medline]
18. Rutherford, K., J. Parkhill, J. Crook, T. Horsnell, P. Rice, M. A. Rajandream, and B. Barrell. 2000. ARTEMIS: sequence visualization and annotation. Bioinformatics 16:944-945.[Abstract/Free Full Text]
19. Saitou, N., and M. Nei. 1987. The maximum-likelihood method for molecular phylogeny. Jpn. J. Genet. 62:547-548.
20. Saitou, N., and M. Nei. 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4:406-425.[Abstract]
21. Shi, T., R. H. Reeves, D. A. Gilichinsky, and E. I. Friedmann. 1997. Characterization of viable bacteria from Siberian permafrost by 16S rDNA sequencing. Microb. Ecol. 33:169-179.[CrossRef][Medline]
22. Soina, V. 2002. Personal communication.
23. Thompson, J. D., D. G. Higgins, and T. J. Gibson. 1994. Clustal-W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22:4673-4680.[Abstract/Free Full Text]
24. Vishnivetskaya, T., S. Kathariou, J. McGrath, D. Gilichinsky, and J. M. Tiedje. 2000. Low-temperature recovery strategies for the isolation of bacteria from ancient permafrost sediments. Extremophiles 4:165-173.[CrossRef][Medline]
25. Vorobyova, E., V. Soina, M. Gorlenko, N. Minkovskaya, N. Zalinova, A. Mamukelashvili, D. Gilichinsky, E. Rivkina, and T. Vishnivetskaya. 1997. The deep cold biosphere: facts and hypothesis. FEMS Microbiol. Rev. 20:277-290.[CrossRef]

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