FEMS Microbiology Ecology 35 (2001) 123^128

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Phylogenetic characterization of the blue lamentous bacterial community from an Icelandic geothermal spring
e Favre c , Michaele Cermola c , Cristina Danielle Takacs a , Marissa Ehringer b , Rene Gudmundur Eggertsson d , Astridur Palsdottir e , Anna-Louise Reysenbach a Y *
b a Department of Biology, Portland State University, Portland, OR, USA Human Medical Genetics Program, University of Colorado Health Sciences Center, Denver, CO, USA c International Institute of Genetics and Biophysics, Consiglio Nazionale delle Ricerche, Naples, Italy d svegur 12, Reykjavik, Iceland Institute of Biology, University of Iceland, Grensa e Institute for Experimental Pathology, University of Iceland, Keldur, Reykjavik, Iceland

Received 11 September 2000; received in revised form 30 November 2000; accepted 30 November 2000

Abstract
Molecular phylogenetic analysis of a blue filamentous community from an alkaline thermal spring (79^83C) in Iceland revealed that the blue filaments were affiliated with the Aquificales. The dominant sequence type, pIce1, was most closely related to a sequence (SRI-48) found in a white filamentous community from a separate Icelandic thermal spring and the pink filaments (EM17) from Yellowstone National Park. Fluorescent in situ hybridization with clone-specific oligonucleotide probes showed that the sample analyzed was essentially a monoculture of a single phylotype. 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
Keywords : Thermophile; Phylogeny; S-layer; Aquicales

In the past decade, knowledge of microbial diversity of high temperature ecosystems has expanded substantially [1^8]. This increased knowledge is largely due to the application of molecular phylogenetic techniques to microbial ecology, in particular the analysis of phylogenetically informative macromolecules such as small subunit rDNA (16S rDNA) genes. For example, analysis of the archaeal and bacterial diversity of Obsidian Pool, Yellowstone National Park (YNP), yielded a plethora of novel sequence types (`phylotypes'), which substantially expanded the crenarchaeal and bacterial 16S rDNA sequence database and led to the proposal of a third kingdom within the archaeal domain [3]. Similarly, the phylogenetic diversity of the conspicuous pink lamentous community associated with the outow of Octopus Pool, YNP was determined. This organism had intrigued researchers for many years, and attempts by independent researchers to enrich for this organism were unsuccessful. Phylogenetic analysis of the pink lamentous organism (EM17) revealed it to be a

* Corresponding author. Tel. : +1 (503) 725-3864; Fax: +1 (503) 725-8570; E-mail: reysenbacha@pdx.edu

close relative of the organisms in culture, Aquifex pyrophilus and Hydrogenobacter thermophilus, and it was identied in situ using uorescent oligonucleotide probes (FISH, [2]). Thermocrinis ruber, a close relative of EM17 (98.7%), subsequently was isolated from Octopus Spring [9]. Additionally, studies of white and yellow lamentous sulfur mats in Icelandic and Japanese thermal springs detected several phylotypes within the Aquicales that were closely related to organisms detected in YNP geothermal springs [7,10]. Another lamentous community, which has a blue coloration when submerged, is associated with many of the neutral-alkaline geothermal springs in Iceland. These organisms have also been targets for cultivation attempts that have yet to be successful. In this study we identied the phylogeny of the blue lamentous community and characterized the prevalent S-layer associated with these organisms. Samples were collected from the fast owing outow of Haegindi and Fluidir Springs in western Iceland for electron microscopy and molecular phylogenetic analysis. The laments were present in the stream between 79^83C. The pH of the water was 8.8 and the conductance was 0.46 mS cm32 . Samples were xed in 2%

0168-6496 / 01 / $20.00 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 6 4 9 6 ( 0 0 ) 0 0 1 1 9 - 7

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Fig. 1. The blue laments : (a) carbon-coated rod showing the S-layer, 26 000U, (b) carbon-coated rod partially missing its outer envelope, which revealed the underlying paracrystalline layer, 35 000U, (c) section of a blue lament, 80 000U, (d) section of a blue lament, 250 000U, (e) dominant form, 60 000U, (f) short large rod, 60 000U, (g) thin lament, 60 000U, (h) PTA negative stain of the S-layer, 90 000U. Arrow depicts S-layer, arrowhead = outer envelope.

glutaraldehyde for electron microscopy or xed in ethanol :PBS (80:20; PBS contains per l 8 g NaCl, 0.2 g KCl, 1.44 g Na2 HPO4 , and 0.24 g KH2 PO4 , pH 7.4) and stored at 320C for uorescence in situ microscopy (FISH). DNA extraction samples were frozen at 380C. For the electron microscopy, approximately 30 Wl of the glutaraldehyde-xed laments were placed on a formvarcarbon-coated grid for 10 s. The grids were stained either

with 1% phosphotungstic acid (PTA) for 1 min or coated with carbon at an angle of 45. Additional samples were prepared for freezing and freeze substitution. The pelleted, xed laments were touched onto a 2.3 mm copper grid (40 mesh), which was laid between two specimen carriers for freeze-fracture preparations (Balzers-Furstenstum, Lichtenstein). The carriers were placed into liquid propane and then quickly transferred to liquid nitrogen. The

Fig. 2. Phylogenetic analysis of the major phylotype (pIce1) from the blue lament community. The phylogenetic tree was determined by maximum likelihood analysis of conserved regions of the 16S rRNA gene. The scale bar represents 0.1 xed mutations per nucleotide position. Numbers at nodes represent bootstrap values for that node (based on 100 bootstrap resamplings). Sequence accession numbers are given in parentheses.

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carriers were separated and stored for 48 h at 380C in dry acetone containing 2% OsO4 . The samples were then slowly warmed up to room temperature, washed out of the holders and o the grids, and embedded in Polybed 812 (Polyscience Inc. Warrington). Ultrathin sections were prepared on a Reichert OMU2 microtome and stained with uranyl citrate and lead citrate. The sections were viewed with a Siemens E102 electron microscope, which revealed the laments to be long and variable in length (often more than 10 Wm), with a diameter of approximately 0.38 Wm. Some very long and thin laments as well as a few shorter broader organisms were present in all samples viewed. The presence of a paracrystalline layer (S-layer) was observed on many cells. The S-layer was visible only if the more external bacterial envelope was lost as shown in Fig. 1b, c, d. Even if the external amorphous envelope was only partially destroyed, the underlying S-layer was apparent (Fig. 1b). The outer envelope was easily detached from the S-layer (Fig. 1c, d). The ultrastructure and lattice periodicity (27 nm as determined at high magnication) of the S-layer was identical on all morphotypes and samples examined. The hexagonal lattice of the S-layer is illustrated in Fig. 1h. Community DNA was extracted from samples stored at 380C and two sets of primers were used to specically amplify bacterial and archaeal 16S rDNA by PCR [2]. No archaeal 16S rRNA genes were amplied using the 4F and 1492RPL primers [8]. Bacterial products were cloned into pBluescript KS3 (Stratagene, La Jolla, CA, USA) and clones containing inserts of the appropriate size (1^1.5 kb) were identied by agarose gel electrophoresis of small-scale plasmid preparations. Forty insert-containing clones were analyzed by single nucleotide (ddT-terminated) sequencing patterns [11] and unique clone types (phylotypes) were identied. Four unique bacterial clone types were detected by single nucleotide (ddT) sequencing patterns of the plasmid preparations: 36 of pIce1 (90%), one of pIce2 (2.5%), one of pIce3 (2.5%), and two of pIce4 (5%). To ascertain the diversity of the bacterial clones, approximately 700^800 nucleotides of each type were sequenced by the Sanger dideoxy chain termination method using a suite of forward and reverse primers to sequence both strands [8]. Sequences were later conrmed by automated sequencing using a SequiTherm long-read cycle sequencing kit-LC (Epicentre Technologies) and a Licor sequencer. The secondary structure of each of the clones was analyzed to verify the integrity of the sequence and to identify evidence of chimeras or PCR-induced artifacts. The 16S rRNA sequences were aligned by hand with a subset of bacterial and archaeal 16S rRNA sequences obtained from the Ribosomal Database Project (RDP, [12]). The alignments were based on the established secondary structure and conserved sequence regions of the 16S rRNA, thus ensuring only homologous regions were compared. Comparisons of the sequences revealed less than 1% sequence dierence between the phylotypes. The sec-

ondary structures of each phylotype did not reveal any inconsistencies that may have resulted from PCR-induced artifacts. Because pIce1 represented the dominant phylotype, both strands of the entire gene were sequenced (GenBank accession number AF301907). Of the approximately 1500 nucleotides, 1212 bases were used in the phylogenetic analysis. The phylogenetic position of the blue laments was explored using parsimony, nearest neighbor, and maximum likelihood analyses using PAUP (4.0). Tree topologies did not dier among the analyses tested and maximum likelihood was used to construct our phylogenetic tree. Corresponding bootstrap proportions were estimated using fastDNAml [13]. The phylogenetic analysis of the pIce1 sequence placed the phylotype within the deeply-branching Aquicales group (Fig. 2). Two lineages within the Aquicales are shown in the maximum likelihood tree: the `black bacteria-EX-H1' and Aquifex lineages. These two groups and the general arrangement of the tree are rmly supported by bootstrap values from 100 maximum likelihood trees. Lower, but still signicant bootstrap values resulted within the two Aquicales lineages, and placed pIce1 as a close relative of SRI-48 (98.9%), a phylotype from a geothermal sulfur spring in Grensdalur, Iceland [10]. The closest relative to these two Iceland phylotypes was EM17 (97.2%), a phylotype detected in the pink laments from Octopus Spring, Yellowstone National Park [2]. Additionally, pIce1 was closely related to Hydrogenobacter thermophilum (96.5%), Hydrogenobacter subterranea (96.4%), Calderobacterium hydrogenophilum (96%), and SS5H1 (95.3%). SS5H1 is a Calderobacterium recently isolated from Calcite Springs, Yellowstone National Park, capable of growth at 70C by aerobic hydrogen oxidation (Takacs and Reysenbach, unpublished results). In order to establish that the PCR products were representative of the blue lamentous community a uorescently labeled oligonucleotide probe was designed that was specic for the pIce phylotypes and was complementary to 1474^1447 (Escherichia coli numbering) region of the 16S rRNA (5P-GTCCCCTGCCTCCCTTGC-3P. The samples were hybridized with the pIce probe, as well as bacterial, universal, and control probes, and viewed as previously described [2]. All organisms hybridized with the pIce(Fig. 3) and bacterial-specic probes. No hybridization occurred with the archaeal-specic probes or the negative RNA-like probe (data not shown). The observed sequence variation appears to be an emerging trend in microbial community analyses where a particular phylotype is represented by a cluster of variants rather than a single rRNA [14,15]. Dierences among the phylotypes may depict variation among closely related populations within the community or may represent multiple 16S rRNA gene copies with dierent sequences within the same organism. The two Iceland clones (pIce1 and SRI-48) are closely related to EM17, and were obtained from very similar, yet geographically distant environments. This is reminiscent of

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Fig. 3. Photomicrograph (1000U) of the blue lament community probed with an rRNA probe specic for the four phylotypes (pIce1^4) determined by molecular cloning. All cells examined hybridized with the pIce- and bacteria-specic probes. A negative control and an archaeal probe did not hybridize (not shown).

the often quoted `Everything is everywhere, the milieu selects' [16], and further illustrates how similar environments select similar physiological types. Near-neutral terrestrial geothermal springs are emerging as a niche that is dominated by members of the Aquicales that are closely related based on their 16S rRNA sequences [2,6^10]. However, it is possible that analysis of other genes will indicate that these populations are distinct [17]. Based on the diversity detected by electron microscopy, the analysis of the clones, and the in situ hybridization study, the blue lamentous community appeared to be essentially a monoculture at the time of collection, comprised of one dominant phylotype that may be represented by several variants or `ecotypes' [14]. Filamentous bacterial communities from other near-neutral geothermal springs (e.g. the black laments [8] and the pink laments

[2]) are also dominated by a single phylotype. However, clone libraries of the pink, black, and white/yellow [7,10] laments revealed a greater bacterial diversity than detected in this study. Relative diversity dierences among the communities may be attributed to the stream ow or temporal variation of the community structure. The blue lamentous bacteria represent another example of an Aquicales community from a near-neutral-alkaline sulfur hot spring. All known isolates within this order are capable of hydrogen oxidation with oxygen as the electron acceptor. The majority of the Aquicales are chemolithotrophs. Presumably, the various lamentous communities dier with respect to color owing to ambient geochemical dierences and consequently, physiological activity. These organisms are conspicuous members of near-neutral geothermal springs, and as a group probably

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C.D. Takacs et al. / FEMS Microbiology Ecology 35 (2001) 123^128 Novel division level bacterial diversity in a Yellowstone hot spring. J. Bacteriol. 180, 366^376. Yamamoto, H., Hiraishi, A., Kato, K., Chiura, H.X., Maki, Y. and Shimizu, A. (1998) Phylogenetic evidence for the existence of novel thermophilic bacteria in hot spring sulfur-turf microbial mats in Japan. Appl. Environ. Microbiol. 64, 1680^1687. Reysenbach, A.L., Ehringer, M. and Hershberger, K. (2000) Microbial diversity at 83C in Calcite Springs, Yellowstone National Park: another environment where the Aquicales and `Korarchaeota' coexist. Extremophiles 4, 61^67. Huber, R., Eder, W., Heldwein, S., Wanner, G., Huber, H., Rachel, R. and Stetter, K.O. (1998) Thermocrinis ruber gen. nov., sp. nov., a pink-lament-forming hyperthermophilic bacterium isolated from Yellowstone National Park. Appl. Environ. Microbiol. 64, 3576^ 3583. Skirnisdottir, S., Hreggvidsson, G.O., Hjorleifsdottir, S., Marteinsson, V.T., Petursdottir, S.K., Holst, O. and Kristjansson, J.K. (2000) Inuence of sulde and temperature on species composition and community structure of hot spring microbial mats. Appl. Environ. Microbiol. 66, 2835^2841. Sekar, V. (1987) A rapid screening procedure for the identication of recombinant bacterial clones. BioTechniques 5, 11^13. Maidak, B.L. et al. (2000) The RDP (Ribosomal Database Project) continues. Nucleic Acids Res. 28, 173^174. Olsen, G.J., Matsuda, H., Hagstrom, R. and Overbeek, R. (1994) fastDNAml: a tool for construction of phylogenetic trees of DNA sequences using maximum likelihood. Comput. Appl. Biosci. 10, 41^ 48. Ward, D.M. (1998) A natural species concept for prokaryotes. Curr. Opin. Microbiol. 1, 271^277. Moore, L.R., Rocap, G. and Chisholm, S.W. (1998) Physiology and molecular phylogeny of coexisting Prochlorococcus ecotypes. Nature 393, 464^467. Baas Becking, L.G.M. (1934) Geobiologie of Inleiding tot de Milieukunde. W.P. Stockum and Zoon, N.V., The Hague. Garczarek, L., Hess, W.R., Holtzendor, J., van der Staay, G.W. and Partensky, F. (2000) Multiplication of antenna genes as a major adaptation to low light in a marine prokaryote. Proc. Natl. Acad. Sci. USA 97, 4098^4101.

contribute signicantly to the productivity of high temperature terrestrial ecosystems. Because of their phylogenetic position and importance in the ecology of thermal springs, determining the diversity, distribution, and physiology of members of the Aquicales may be instrumental in understanding the early evolution of life on Earth. Acknowledgements The authors thank Norman Pace for his support. This research was partially funded by an NSF grant to A.-L.R. (OCE 99-96160). References
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